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Effects of polyamine analogs on mitochondrial DNA and cell growth

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Title:
Effects of polyamine analogs on mitochondrial DNA and cell growth
Creator:
Bortell, Rita Bellis, 1953-
Publication Date:
Language:
English
Physical Description:
vii, 157 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Cell division ( jstor )
Cell growth ( jstor )
Cells ( jstor )
DNA ( jstor )
Enzymes ( jstor )
Genomes ( jstor )
Mitochondria ( jstor )
Mitochondrial DNA ( jstor )
Polyamines ( jstor )
Viability ( jstor )
Cell Division ( mesh )
DNA, Mitochondrial -- drug effects ( mesh )
Dissertations, Academic -- Pharmacology and Therapeutics -- UF ( mesh )
Pharmacology and Therapeutics thesis Ph.D ( mesh )
Polyamines ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 145-156.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Rita Bellis Bortell.

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University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
000983932 ( ALEPH )
20379902 ( OCLC )
AEW0099 ( NOTIS )
AA00006106_00001 ( sobekcm )

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Full Text









EFFECTS OF POLYAMINE ANALOGS
ON MITOCHONDRIAL DNA
AND CELL GROWTH





By

RITA BELLIS BORTELL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1987




EFFECTS OF POLYAMINE ANALOGS
ON MITOCHONDRIAL DNA
AND CELL GROWTH
By
RITA BELLIS BORTELL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987


This dissertation is dedicated to David Alan Bortell, for the support and
encouragement he gave me during this endeavor.


ACKNOWLEDGMENTS
I would like to thank my supervisor and mentor, Dr. Allen Neims, for his
careful guidance of my graduate training. I also want to thank the members of
my committee: Drs. Stephen Baker, William Hauswirth, Margaret James,
Warren Ross, and Thomas Rowe. I would like to extend my appreciation to the
fellow members of our laboratory: Lynn Raynor, Gurmit Singh, Lori Lim, Debra
Stinson, Sukanya Kanthawatana, Daniel Danso, and Mary Anne Kelly. I
particularly want to thank Mike Ingeno, Bonnie O'Brien, David Bortell, Debra,
and Lynn for the generous help they gave me. Special thanks go to Shari
McArdle, with whom I most closely shared the graduate school experience. A
final debt of gratitude goes to Dr. Allen Neims and Lynn Raynor, who greatly
enriched my training and made the experience fun.
in


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
ABSTRACT vi
INTRODUCTION 1
LITERATURE REVIEW
Mitochondrial DNA (MtDNA) 3
Nuclear/Mitochondrial Regulation 9
Polyamines 24
MGBG (Methylglyoxal bis[guanylhydrazone]) 31
Specific Aims 43
MATERIALS AND METHODS
Cell Culture 45
Cell Counts 45
Determination of IC50 46
Cell Viability 46
Clonogenic Assay 46
Cell Size 47
Flow Cytometry 47
Rhodamine 123 Uptake 47
[14C]-MGBG Stock Solution Preparation 48
Uptake Protocol 48
Cloning of MtDNA Probe 48
Nick Translation of MtDNA Probe 50
Gel Electrophoresis 50
Southern Blot 51
Dot Blot 51
Hybridization with [35s]-labeled MtDNA Probe 52
RESULTS
Characterization of L1210 Cells 53
Effects of Polyamine Analogs on Cell Division and MtDNA
Accumulation 59
Resistance to MGBG and Recovery MtDNA Synthesis and Copy
Number 84
Effects of MtDNA Content on Mitochondrial and Cell Functions .... 11 1
Uptake of MGBG 113
DISCUSSION
Determination of the MtDNA Copy Number in L1210 Cells 123
Decrease in Cell Growth and MtDNA/Cell with MGBG and DES
Exposure 124
Mitochondrial Function During MGBG Exposure 127


Increased MtDNA/Cell with Low Concentrations of MGBG 129
Recovery of MtDNA Replication 130
Implications for Nuclear/Mitochondrial Interactions 132
SUMMARY 141
REFERENCES 145
BIOGRAPHICAL SKETCH 157
v


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EFFECTS OF POLYAMINE ANALOGS
ON MITOCHONDRIAL DNA
AND CELL GROWTH
By
Rita Beilis Bortell
December 1987
Chairman: Allen H. Neims
Major Department: Pharmacology and Therapeutics
The average mouse leukemia L1210 cell contains 1450 copies of
mitochondrial DNA (mtDNA). Somehow this number is maintained during
exponential growth. We have studied the effects of the polyamine analogs,
methylglyoxal bis[guanylhydrazone] (MGBG) and diethylspermine (DES), in
order to explore this relationship because appropriate doses of the drugs
selectively Inhibit mtDNA replication without causing a decrease in cell
viability, at least in the case of MGBG.
Exponentially-growing L1210 cells were exposed to 1.0-3.3 pM MGBG
for 1-31 days. Cells were collected on various days for dot blot quantitation of
mtDNA by hybridization of cell lysate to a [35s]-labeled, full-length mouse
mtDNA probe. At >1.3 pM MGBG, mtDNA replication was completely inhibited
for variable times depending on dose (64 hr at 3.3 pM). Since cell division
continued, albeit somewhat slower, the mtDNA content per cell was diluted
with each cell division. Whereas cell size decreased when the mtDNA copy
v 1


number per cell was low, the potential-dependent uptake of rhodamine 123
uptake Into mitochondria was not affected by MGBG treatment.
Diethylspermine had similar initial effects on mtDNA accumulation and
cell growth. With exposure to either drug, the amount of mtDNA in viable cells
never decreased below 10% of the control value because cell division
ceased. Although the effects of DES on mtDNA accumulation were not
reversible, mtDNA synthesis of MGBG-exposed cells resumed after 3 days.
When mtDNA synthesis resumed, the mtDNA content per cell doubled every
12 hours whether or not drug had been removed from the medium. The fact
that this mtDNA synthesis occurred when the rate of cell division was at its
lowest resulted in repletion of the cellular content of mtDNA. When the mtDNA
was nearly repleted, the rate of cell division recovered to the control value of
12 hours. The observation that the doubling of mtDNA in the virtual absence
of cell division required the same 12 hours suggests that replication of mtDNA
may proceed at its maximal rate in untreated exponentially-growing L1210
cells and actually limit how rapidly these cells can divide.
The mechanism of recovery of mtDNA synthesis in these cells was not
due to genetic mutation, but rather to a phenotypic adaptation. The L1210
cells initially concentrated [14C]-MGBG more than 1000-fold relative to the
medium. When mtDNA synthesis resumed, the apparent intracellular
concentration of the drug had begun to decrease to a new steady state value,
although the concentration was still higher than that earlier associated with
inhibition. The mtDNA copy number of cells continuously exposed to MGBG
had returned to the control value at 31 days.
vii


INTRODUCTION
The number of copies of mitochondrial DNA (mtDNA) seems to be
characteristic for a given cell type, whether that cell is dividing or not (1,2). It
can be appreciated that maintenance of the characteristic copy number in
dividing cells is especially complex. For this amount of mtDNA per cell to be
maintained, the mtDNA must double with each cell division. Because the
nucleus and mitochondria are distinct organelles, it is not understood how this
regulation occurs.
This dissertation focuses on the basic question of how the characteristic
copy number of mitochondrial DNA (mtDNA) is maintained in dividing cells.
My basic approach is to perturb rapidly dividing cells with a drug or compound
which selectively inhibits mtDNA replication and use dot blot analysis and
measurements of cell number to follow the resultant effects on the copy
number of mtDNA and on cell division.
The drug I chose to use was methylglyoxal bis(guanylhydrazone), or
MGBG. This drug is cationic, metabolically stable, and was developed as a
polyamine congener (3). It has been used clinically as an antiproliferative
agent and to treat trypanosomiasis (4, 5). I selected this drug because it
displayed two important characteristics. First, it inhibits mtDNA replication
without damaging the mtDNA at concentrations which initially have only a
minimal effect on nuclear DNA replication (6, 7). Second, preliminary studies
in our laboratory suggested that long-term treatment (up to 31 days) of L1210
cells with MGBG did not substantially affect cell viability. Consequently, this
1


2
drug could be used to study the cell's response, overtime, to differential
perturbation of mtDNA and cell replication.


LITERATURE REVIEW
The literature review will be divided into four sections. They are 1) an
overview of the literature on mtDNA; 2) a more detailed review of the
interaction between the nuclear and mitochondrial genetic systems; 3) a brief
survey of polyamine research; and 4) a synopsis of the literature on MGBG
and related polyamine analogs. This chapter will conclude with a summary of
the specific goals of my research.
Mitochondrial DNA
Mitochondria are the organelles of eukaryotic cells which are
responsible for oxidative phosphorylation, among other functions. According
to the endosymbiotic theory of the evolutionary origin of the eukaryotic cell, the
mitochondrion evolved from a free-living prokaryote, probably the purple
nonsulfur bacteria (Rhodospirillaceae) (8), which was incorporated into an
ancestral eukaryotic cell (9). Prior to this, the protoeukaryote had depended
on fermentation for the production of ATP. In time the prokaryote suffered a
progressive loss of autonomy: its rate of proliferation became linked with that
of the host cell, many of its biosynthetic capabilities were lost or taken over by
the host, and its metabolic functions became integrated with that of the host.
This theory implies that the inner mitochondrial membrane and its
invaginations (the cristae) are homologous with the plasma membrane of
present-day bacteria, whereas the outer mitochondrial membrane is derived
from the protoeukaryote.
3


4
Mitochondria are self-replicating, arising only by growth and division of
preexisting mitochondria (10). The existence of DNA within mitochondria was
first observed by Nass and Nass in 1963 (11). Mitochondrial DNA is a double-
stranded circular genome (Figure 1); exceptions are the linear mtDNA
molecules of the ciliated protists Tetrahvmena and Paramecium (10). The
genome's two strands contain different proportions of purines, and thus can be
separated by density gradient centrifugation into heavy strand (H-strand) and
light strand (L-strand). Immediately after replication of mtDNA, the two
daughter molecules exist in a relaxed circular state. Within one hour under
normal conditions, however, a mitochondrial topoisomerase introduces
approximately 100 supercoils into each molecule (12).
This supercoiled form may subsequently be converted into a
displacement loop (D-loop) form which is unique to the mitochondrial genome.
This triplex D-loop structure is formed by the synthesis of a short daughter H-
strand that remains stably associated with the parental closed circle; the
parental H-strand is consequently displaced as a single strand. The D-loop
region varies in length between different species, and sometimes within the
same animal (13). In general, the D-loop is approximately 1000 base pairs
(bp) in length (14).
Mitochondrial DNA of human, murine, and bovine species has been
completely sequenced and consists of approximately 16,000 bp (14). In
addition, these sequences have been completely mapped for restriction sites
(15) and locations of promoters for replication and transcription of both heavy
and light strands of mtDNA (16,17). The coding regions of the genome have
been mapped, and the majority of the proteins (12 of 13) and transfer RNAs
(tRNAs) (16 of 22), and both ribosomal RNAs (rRNAs) are coded for by the H-
strand (Figure 1). The only noncoding areas of mammalian mtDNA are a short


5
Figure 1. Schematic of human mtDNA. Hatched segments show the extent of
genes for known proteins, filled segments show the genes for
transfer RNA's, and crossed segments show the genes for
ribosomal RNAs.


6
sequence outside the D-loop region which encompasses the origin of L-strand
replication, and the D-loop region which contains the promoter sites for H-
strand replication and both H- and L-strand transcription (14).
There are two current models for mitochondrial transcription in
mammalian cells. One model proposes that transcription begins near the
origin of replication and continues as a single polycistronic transcript around
the entire genome, but with a high probablity of premature termination
immediately following the 12S-16S region (18). The alternative model
suggests that there are two promoters for the initiation of RNA synthesis on the
H-strand near the origin of replication (19, 20). Transcription from one site
circumscribes the entire mitochondrial genome, while transcription from the
other site terminates after the two rRNA cistrons which are immediately
downstream. Once transcription has occurred, the polycistronic precursors
are cleaved into mature messenger RNAs (mRNAs), probably before the
genome is completely transcribed (21). Since there are very few nucleotides
between adjacent genes on mammalian mtDNA (and sometimes none), the
signals for RNA processing may not be specific sequences £er s£ (22). Most
of the mRNAs and both rRNAs are flanked by tRNA genes, and the secondary
structure of these tRNAs in the transcript may provide the signals for cleavage
by the RNA processing enzyme (23).
The mitochondrial genome is thought to be associated with the inner
membrane at its matrix face (24). Molecules of mtDNA are frequently seen to
be attached to fragments of membrane or proteinaceous material after gentle
lysis of the organelles (9). In some cases there is good evidence that such
associations are not artifacts; for example, in one study it was shown that
membrane fragments were always attached to approximately the same


7
position on HeLa cell mtDNA (10). Thus, it is probable that mtDNA molecules
are attached to organelle membranes at least some of the time.
Apart from this attachment, isolated organelle genomes generally are
not complexed with histones or other proteins in the manner of true
chromosomes in the nucleus; exceptions are the highly condensed mtDNA
molecules of the slime molds which appear to be complexed with a basic
protein (25), and the mtDNA of Xenopus oocytes, which can be isolated in
association with protein in the form of structures resembling the nucleosomes
of chromatin (10). Also, recent evidence suggests that the displaced single
strands (D-loop) of mtDNA, but not the double-stranded segments of the
genome, are coated with DNA-binding proteins apparently unique to mtDNA
(26, 27). These proteins are thought to play a critical role in maintaining the
integrity of these triplex replication loops.
In mammals, the mitochondrial genome codes for 13 proteins and
some, but not all, of the components necessary for protein synthesis; this
includes two rRNAs and 22 tRNAs (28). The known human mtDNA-coded
proteins include 3 subunits of cytochrome oxidase, one of ATPase, 8 of NADH
dehydrogenase, and one of cytochrome b (29, 30). Antibodies raised to
synthetic peptides predicted from the DNA sequence have been used to
establish the existence of translated products from all previously unassigned
reading frames (URFs) (31). Attardi and his coworkers (32) recently identified
the last URF of human mtDNA as a subunit of NADH dehydrogenase. All of
the mtDNA-coded proteins are integral components o the inner mitochondrial
membrane and play key roles in oxidative phosphorylation. It follows,
therefore, that the mitochondrial genetic system is indispensable for the
biogenesis of the aerobic energy-generating system of all eukaryotic cells.


8
Whereas mtDNA codes for 13 mitochondrial polypeptides, the
organelle contains over 270 different proteins (29, 33). The remaining
polypeptides, which account for approximately 90% of total mitochondrial
protein, are encoded in nuclear DNA and synthesized on cytoplasmic
ribosomes. Available evidence indicates that each of these proteins contains
a leader sequence enriched in basic (positively-charged) amino acids which
directs the protein to the mitochondria, possibly to the point of adherence
between the inner and outer membranes (34). The protein is then imported
into mitochondria via a set of processes dependent on the existence of a
mitochondrial membrane potential (35).
If the majority of mitochondrial proteins are imported from the
cytoplasm, then why do cells expend more than a hundred nuclear genes just
to allow the mitochondrial genetic system to transcribe and translate about a
dozen polypeptides in situ? Why, in fact, does the mitochondrial genome
retain the genes for any of the particular proteins that it has? It has been
suggested that mitochondrial translation products are so hydrophobic that they
are best made m siiu (36). However, other hydrophobic proteins are
successfully transported in the cell (37), and some mitochondrially coded
proteins are as hydrophilic as some soluble proteins (38).
Alternatively, it may be that all eukaryotes are in the evolutionary
process of transferring mitochondrial genes to the nucleus. The fact that the
more highly evolved eukaryotes have the smallest mitochondrial genome is
consistent with this idea. The mammalian mitochondrial genome has about
16,000 kb; yeast mtDNA is 5 times longer largely because 1) it has more
genes than mammalian mtDNA, 2) several genes have introns, and 3) it
contains long noncoding stretches between genes (14). This finding suggests
that there has been a dynamic net flux of mitochondrial genetic material into


9
the nucleus (39). Indeed, firm evidence has emerged that mtDNA can be
stably integrated into nuclear genomes (40). Rearranged parts of the
mitochondrial genes var 1, cob/box and orl/rep have been found in the nuclear
genome of yeast (41). Homologues of mtRNA genes occur in locust nuclei
(42) and mtDNA sequences have been reported in sea urchin (43) and rat
nuclear DNA (44). In addition, transposition of mtDNA into nuclear sequences
may be more than just an evolutionary phenomenon, as this process has been
reported to occur as a normal part of senescence in Podospora (45). Recent
evidence, however, suggests that this latter observation may be artifactual
(46). In any event, it seems increasingly likely that normal or abnormal
mtDNA, released into the cytosol during mitochondrial breakdown or the
normal organelle fusion/segregation cycles, could become incorporated into
nuclear DNA (or vice versa).
However, a possible limitation in the transfer of functional genes from
the mitochondrion to the nucleus is the slightly different genetic code used by
the two systems. One of the triplicate codons in mitochondria codes for
tryptophan, but is recognized as a stop codon in the nucleus (47). The 13
critical protein-encoding genes that remain in the mammalian mitochondia
have such a codon (48). This would make further transfer of usable genes to
the nucleus very improbable.
Nuclear/Mitochondrial Regulation
Regulation of Mitochondrial Enzymes in Differentiated Cells
Because most of the mitochondrial proteins are encoded in nuclear
genes and synthesized on cytoplasmic ribosomes, interactions between the
nucleus and mitochondria must play an important role in mitochondrial
regulation. Respiratory enzymes and mitochondrial membranes are normally


synthesized and/or degraded in a coordinated fashion as the proportions of
the various components are characteristic for a given cell (49). The content of
mitochondrial enzymes in mammalian cells is regulated by several
environmental conditions including oxygen tension and hormones (50, 51,
52). One major question is whether the mitochondrial proteins, or even just
those of the inner membrane, are regulated individually or as a set. Because
various mitochondrial components have distinct turnover times and different
degrees of nuclear and mitochondrial control including specific genomic
expressions, individual proteins appear to be individually regulated (53, 54).
But, as discussed below, there are exceptions to this general rule.
During hypoxic incubation of mouse lung macrophages and rat skeletal
muscle L8 cells, the activities of various mitochondrial enzymes (both nuclear
and mitochondrially coded) decreased about 50-60% in tandem with no
statistical difference between the various activities (55). The kinetics of this
decrease and of the recovery of activity after reexposure to normoxia were
also similar for the different enzyme activities. This suggests that, at least in
certain cases, mitochondrial enzymes can be regulated as a unit.
It is known that nuclear gene expression is frequently regulated at the
transcriptional level (56); however, the level(s) at which mtDNA gene
expression is controlled is unknown. Some reports have addressed this
question from the point of view of gene dose or mtDNA copy number, I.e. is the
increase or decrease in mtDNA gene expression proportional to changes in
the content of mtDNA? In the experiment described above, although there
were uniform decreases in mitochondrial enzyme activities after exposure to
hypoxia, there was no change in the mtDNA copy number, a result that speaks
against regulation of mitochondrial gene expression only by gene dosage.


Another experiment which sought to answer whether mtDNA gene
dosage controls gene expression was conducted by Williams (57). He
determined the concentrations of mtDNA, mitochondrial rRNA, and
cytochrome b mRNA (a mitochondrial gene product) in normal versus
electrically-stimulated rabbit striated muscles. This tissue was chosen
because the oxidative capacity of mammalian striated muscles can vary nearly
10-fold, a finding which presumably reflects major differences in the
expression of genes (including mtDNA) that encode enzymes of oxidative
metabolism (58). After electrical stimulation of the muscle, mtDNA, mtRNA,
and the mitochondrially coded gene product (cytochrome b) were observed to
vary in direct proportion to the oxidative capacity of the tissue. Because the
expression of mitochondrial genes in mammalian striated muscle was
proportional to their copy number, these results do support the hypothesis that
amplification of the mitochondrial genome relative to chromosomal DNA, i.e.
gene dosage, was responsible for the enhanced expression of mitochondrial
genes in highly oxidative tissues (57).
This interpretation, of course, differs from that of the hypoxia experiment
described above. The different conclusions reached in the two experiments
may relate to the different tissues and/or animals used. Also, the differences
may reflect the different experimental designs; e.g., there were no changes in
mtDNA copy number after 4 days of hypoxia, but neither were there changes
in mtDNA content after the same period of muscle stimulation (the changes
occurred after 7 days). In any event, the regulation of mitochondrial gene
expression in differentiated cells, to date, is not well understood.
Regulation of Mitochondrial Enzymes in Transformed Cells
The regulation of mitochondrial gene expression in transformed
mammalian cells is also not well understood (59). The pioneering manometric


studies of Warburg in the 1920s (60) first revealed that tumor cells are
defective in respiration and have abnormally high rates of aerobic glycolysis.
Warburg suggested that the cancer cell originates from heritable injury to the
mitochondria in that the respiratory system of all cancer cells was thought to
be damaged (61, 62). By 1960, this hypothesis had lost much of its impact
because elevated rates of glycolysis could not be associated in any consistent
manner with changes in the structure or function of the mitochondrion (63, 64).
Nevertheless, numerous publications in the last two decades have
catalogued a wide range of abnormalities in tumor cell mitochondria. For
example, tumor cells apparently contain fewer mitochondria than normal cells
and the organelles in the tumor cells may be structurally aberrant (65, 66).
Many deficiencies of energy-linked functions have also been reported, and
they include reduced respiratory control (ADP-stumulated state 3 respiration)
(67). Inner membrane alterations are also seen in tumor mitochondria, such
as changes in the amount and properties of ATPase (68). The levels of
mitochondrial-associated enzymes such as hexokinase are also different in
tumor cells (69, 70). It is possible that these complex multiple changes in
membrane characteristics result from rearrangements of mtDNA (71) or from
topological aberrations of the organellar genome (72). Indeed, aberrations of
mtDNA (mainly associated with an increase in the percentage of dimers and
catenanes) have been found in tumor cells (73).
Relationship Between Mitochondrial Protein and Gene Dosage
Almost all eukaryotic cells contain many molecules of mtDNA (10). The
exceptions are a few fungi and more than a thousand species of protozoa that
have no mitochondria (46). Moreover, under given metabolic conditions,
different cells appear to have a characteristic mitochondrial and mtDNA
content (1). How is this regulated? There have been attempts to relate


mtDNA content to the number of mitochondria or the mass of mitochondrial
protein isolated. The studies reviewed by Nass (74) and by Borst and Kroon
(75) indicate a yield of 0.2 to 1.8 pg of mtDNA per mg of mitochondrial protein,
or a content of roughly 2 to 10 mtDNA molecules per organelle, depending
upon the animal cell or tissue examined. Thus, multiple mitochondrial
genomes are packaged into each mitochondrion, and there are as few as one
mitochondrion to more than several thousand mitochondria per cell,
depending on the type.
However, these older studies were somewhat suspect because it was
difficult to selectively assay mtDNA, given the need to first separate it from a
several hundred-fold excess of nuclear DNA. Consequently, Bogenhagen
and Clayton (1) studied the relationship of mtDNA content to mitochondrial
volume in cells lacking the ability to incorporate exogenous thymidine into
nuclear DNA (because they had lost the major cellular thymidine kinase, TK-).
Such cell lines retain a mitochondrial-specific activity which allows mtDNA
labeling with exogenous radioactive thymidine. Stereological analysis (76) of
thin sections of two of these cell types (LMTK- and LDTK-) revealed that both
contained 51% of the total cell volume within mitochondria. Even after
considering the larger mitochondrial content of HeLaTK- cells (7% of cell
volume), the mtDNA content of HeLaTK' cells was at least four times larger
than that of either TK' L cell line. Thus, the mtDNA content did not simply
relate to the volume of mitochondria. This result was consistent, however, with
the qualitative observation that the mitochondria of HeLaTK' cells were
uniformly more well developed (i.e. contained more cristae per unit volume)
than the mitochondria of L cells.


Cell Division and Regulation of MtDNA Copy Number
As noted above, an added degree of complexity is that a given cell type
seems to maintain a characteristic copy number of mtDNA even when dividing
(1). This implies that the mtDNA content must double before each cell
division. Yet, the replication of mtDNA in dividing cells is not linked to the S
phase of the cell cycle, rather it apparently occurs continuously throughout the
cell cycle (77, 78). In addition, mtDNA may replicate in the absence of nuclear
DNA replication, as in cellular hypertrophy (24).
How is the mtDNA copy number regulated during cell division? For the
copy number to be maintained during exponential cell growth, the number of
mtDNA molecules in a culture, N, must parallel the increase in cell number,
N=N0ex1, where N0 is the number of molecules present at t=0, and t equals the
cell generation time when N/N0=2 (79). However, the rates of mitochondrial
and nuclear DNA synthesis may each be affected differently by exposure to
vanous chemicals, thereby conceivably altering the mtDNA copy number. The
antiproliferative agent, MGBG, is one such compound. It inhibits mtDNA
synthesis at concentrations which have little effect on nuclear DNA synthesis.
This and other aspects of the drug will be considered in more detail in a later
section.
The phenanthridine dye, ethidium bromide, is a well-known inhibitor of
mtDNA replication and transcnption (80, 81). When human VA2-B cells were
treated with 20 ng/ml ethidium bromide, a progressive dilution of the mtDNA
content, down to 10% of control cell values after three doublings, has been
reported (82). This large reduction is consistent with a logarithmic dilution in
the number of preexisting mtDNA molecules per cell, which in the absence of
any new synthesis or turnover should result, after 3 cell doublings, in a level
which is 1/8 or about 12% of normal. As yet, no study dealing with the long-


term effect of the drug on the mtDNA content of mammalian cells in culture has
been reported, mainly because ethidium bromide limits the growth capacity of
these cells to between 3 and 4 cell generations (82, 83).
In contrast to other vertebrate cells studied so far, chicken embryo
fibroblasts have been found to be inherently resistant to the growth-inhibitory
effect of ethidium bromide when supplied with exogenous pyrimidine
nucleosides or nucleotides (84, 85). Long-term ethidium bromide treatment
resulted in chicken embryo fibroblast cells which had no measurable mtDNA
and which were respiration deficient. This phenotype was maintained
whether or not the cells were transferred to drug-free medium (86). This was
the first and, as yet, only demonstration that vertebrate cells of the rho
phenotype (such as in Saccharomvces cerevisiae) can proliferate in culture.
Study of growth parameters indicated that no lag or adaptation period was
necessary for pyrimidine-supplemented chick cell populations to proliferate in
the presence of ethidium bromide (87).
Whereas ethidium bromide inhibits mtDNA synthesis more than nuclear
DNA synthesis, the reverse is true for treatment of cultured mammalian cells
with either 5-fluorodeoxyuridine or methotrexate (88). These drugs inhibit the
enzyme thymidylate synthetase, so that thymidine must be supplied
exogenously. Certain cells (such as LMTK-) lack the major cellular thymidine
kinase but do contain a genetically distinct mitochondrial enzyme.
Consequently, when these cells are treated with methotrexate and supplied
with exogenous thymidine, only the mitochondria can synthesize DNA. When
the concentration of exogenous thymidine is >20 p.M, the rate of mtDNA
synthesis is at least 50% of the control rate while nuclear DNA synthesis is
<4% of control. The resultant effect on the mtDNA copy number was not
measured, however.


"Natural" Homoplasmy and/or Heteroplasmv of MtDNA
The genotype of mtDNA tends to be the same within a given cell; i.e.,
most cells are homoplasmic (that is, they carry only one type of mtDNA). In
addition, the mitochondrial genotype between different tissues of a single
individual generally appears to be the same (89). This is somewhat surprising
since it has been suggested that mtDNA evolves at a rate 10-fold faster than
that of single-copy nuclear DNA (90, 91). Heteroplasmy has been observed in
only a few species, and usually analyzed only in a single individual (92, 93).
This seeming disparity between the rapid rate of mtDNA mutation and the
observation that single variant forms of the mitochondrial genome appear to
dominate the cell population of a single individual (94) can be explained partly
by the fact that mammalian oocyte mitochondria are derived from only a small
number of progenitors (see below) (95).
Unlike nuclear DNA, mtDNA is inherited maternally in mammals, i.e.,
only the egg contributes mtDNA to the zygote. The fate of paternal
mitochondrial genomes remains obscure (96, 97). There is 100 times more
mtDNA in follicular oocytes than in somatic cells, suggesting at least a 100-
fold amplification during oogenesis (98, 99, 100). Because there is no
additional mtDNA synthesis after fertilization until about the 64-cell blastocyst
stage, there is a rapid dilution of the mtDNA copy number to 1-2 per
mitochondrion in zona-encased oocytes. A higher content of mitochondrial
genomes per organelle is probably not recovered until after early cell divisions
of the embryo (101). The eventual mitochondrial genotype of the individual is
derived from only the few of these 64 cells that serve to form the growing
embryo. This may explain, in part, the seemingly incongruous reports that
mtDNA has a rapid rate of mutation and yet most individuals are homoplastic.


In addition, while alleles of nuclear genes segregate only at meiosis,
mitochondrial genes can "segregate" during mitotic divisions of the eukaryotic
cell. This vegetative segregation may explain how an initially heteroplasmic
cell can give rise to homoplasmic daughter cells during somatic cell division
(102). How might this occur? The "average" haploid yeast cell, for example,
contains about 50 copies of the mtDNA molecules and thus about 50 copies of
each mitochondrial gene (103). A mutational event, such as for resistance to
erythromycin (an inhibitor of mitochondrial protein synthesis) is unlikely to
affect more than one copy of a gene at a time and would therefore result
initially in a cell with 1 mutant and 49 wild type alleles. This heteroplasmic cell
eventually produces homoplasmic mutant progeny cells in which all mtDNA
molecules carry the mutant allele. Experiments have shown that seiection
plays the major role in determining the fate of erythromycin-resistant genomes
in the presence of erythromycin on a nonfermentable carbon source, i.e. under
"selective" conditions. This was primarily intracellular selection, acting in the
absence of cell division to make cells homoplasmic for the erythromycin-
resistant allele. Hydroxyurea, which inhibits nuclear and mtDNA synthesis
(104), blocked this selection. The simplest interpretation is that selection
requires mtDNA synthesis.
In addition, intercellular selection may play a role in determining the
fate of new erythromycin-resistant mutations in that cells with many copies of
the mutant allele may begin to grow and divide, while homoplasmic
erythromycin-sensitive cells or cells with few copies of the erythromycin-
resistant allele cannot divide. Although the mechanism for the intracellular
selection of mutant alleles in this case was unknown, there is some evidence,
at least for the petite mutation in yeast, that the presence of certain nucleotide
sequences and other features of the mutant genome may endow it with a


replicative advantage over the wild type genome, the expression of which can
become suppressed (105).
Segregation of MtDNA in Hybrids and Cvbrids
To further explore how a given mtDNA genotype is selected for
replication and/or expression, numerous experiments with interspecies
hybrids (cell/cell) and cybrids (cell/cytoplasm) have been performed (106,
107). Ideally, the two parental lines are different enough for the DNA, RNA,
and protein from the hybrid (or cybrid) cell mitochondria to be characterized as
belonging to one or both species. Analysis of the mtRNA and protein can
determine whether the two types of mtDNA function completely independently
or interact in a complementary fashion, or whether only one genome is
responsible for all of the mitochondrially coded products.
Segregation patterns of mtDNA in cybrids and hybrids have been
divided into 3 categories, namely 1) segregation of foreign mtDNA (or
chromosome-dependent segregation); 2) stochastic segregation; and 3)
segregation of host mtDNA (108, 109). In interspecies hybrids, such as
human-mouse hybrids, when there is loss of chromosomes from one parental
cell, the mtDNA from that parental cell is also lost (chromosome-dependent
segregation) (110, 111). These observations suggest that an incompatibility
exists between different species of nuclear and mitochondrial genomes and is
responsible for the "segregation of foreign mtDNA" (112).
In contrast, in intraspecies cybrids, there should be no incompatibility
between nuclear and mitochondrial genomes. Mitochondrial DNA of
cytoplasts can be propagated in the host cells and both parental mtDNAs are
found to be codominant at an early stage after cybrid isolation (108).
However, after cultivation for several months, some individual cells in the
population of cybrids were observed to have a different ratio of parental


mtDNA (112). A similar phenomenon occurs in mouse-rat hybrids in which
both parental chromosomes are stably retained (109). In these cases, since
there is no selective pressure from the nuclear genomes on either parental
mitochondrial genomes, both parent mtDNAs segregate randomly into
daughter cells and may, in a sufficient number of divisions, become pure for
either parental type of mtDNA (112). This type of mtDNA segregation is
named "stochastic segregation."
Although stochastic segregation of mtDNA should occur in intraspecies
cybrids, in human cybnds constructed by fusion of tumorigenic HeLaTG cells
with cytoplasts of normal primary fibroblasts, host cell mtDNA (HeLaTG
mtDNA) was segregated from the cybrids (113). If stochastic segregation
occurred, some subclones of the cybrids should have contained HeLaTG
mtDNA, but this was never observed. Thus, there must be some differences
between the mitochondrial genomes of HeLaTG cells and fibroblasts that are
responsible for the preferential segregation of HeLaTG mtDNA. This
segregation pattern is referred to as "segregation of host mtDNA."
Recombination of MtDNA and/or its Encoded Products
Recently, metabolic complementation between mitochondria in somatic
cell hybrids or cybrids was suggested from the observation that
chloramphenicol (CAP)-sensitive mtDNA was maintained in cells even when
they were cultivated continuously for a long time with CAP (114, 115). Oliver
and Wallace (116) showed that a marker polypeptide encoded on CAP-
sensitive mtDNA could be synthesized in the presence of CAP. These data
were interpreted to mean that mitochondrial cooperation arises from
mitochondrial fusion or intermitochondnal exchange of mRNA, a finding which
suggests, in turn, that mitochondrial genomes may interact with each other
and form recombinant molecules.


20
To date, however, there is no direct evidence to suggest that
recombination of mtDNA occurs, at least in mammals. Recombination of
mammalian mtDNA has been examined using mouse X rat and mouse X
hamster somatic cell hybrids and rat cybrids (117, 118). Genetic and physical
analyses showed that the mtDNAs of the hybrids and cybrids were simple
mixtures of the two parental mtDNAs. These observations suggest that, in
contrast to the case with yeast or plant mtDNA, recombination of mammalian
mtDNA occurs rarely, if at all.
Effects of MtDNA on Expression of Nuclear DNA
Saccharomvces cerevisiae cells seem to respond to the quality and
quantity of mtDNA and modulate the levels of nuclear-encoded RNAs,
perhaps as a means of intergenomic regulation (119). These results suggest
a hitherto undescribed type of nuclear-mitochondrial interaction whereby
nuclear DNA sequences can respond to the state of the mitochondrial
genome. For example, the ability of yeast cells to utilize certain sugars varies
in different petites (119), suggesting that the expression of some pathways of
sugar utilization can in some way be influenced by different, defective
mitochondrial genomes. Another related observation is that a linear, double-
stranded DNA killer plasmid from Kluvveromvces lactis. when introduced into
Saccharomvces cerevisiae. is unstable in both rho+ and mit- cells, but is stable
in rho petites (120). The abundance of nuclear-encoded RNAs is increased
in one or more of these respiratory-deficient petites, as if these cells were
attempting to compensate for their respiratory-deficient defect or for the
particular mtDNA lesion that they harbor. Perhaps a decreased mtDNA copy
number stimulates the transcription of nuclear genes necessary for mtDNA
synthesis.


How might expression of some nuclear genes be affected differently in
these respiratory-deficient cells? One possibility is that a mitochondrial gene
product is exported and functions outside the organelle, for example, as a
negative (or positive) regulatory element (119, 121). According to this model,
control of expression of some nuclear genes would be maintained as long as
cells contained a functional mitochondrial genome. Upon conversion to
petites, however, the cells could no longer synthesize the putative regulator
since petites have lost the ability to carry out mitochondrial protein synthesis,
and they lack most, and in some cases all, of the mitochondrial transcripts
found in rho+ or mif cells. Although protein or RNA export from mitochondria
has not been demonstrated directly, there are some indications that such
export may occur.
For example, spoliation in MATalpha/MATa diploids of
Saccharomvces cerevisiae cells is accompanied by a novel pattern of protein
synthesis, as shown by the disappearance of some mitotic' polypeptides and
by the appearance of a new set of 'meiotic' polypeptides (103). Inhibition of
mitochondrial protein synthesis by erythromycin caused the disappearance of
several meiotic polypeptides within one hour. Fractionation of extracts of the
cytosolic and mitochondrial components showed that those proteins that were
sensitive to erythromycin were localized within the cytosol and that they were
also sensitive to cycloheximide, an indication that they were not mitochondrial
translational products. However, in a mitochondrially inherited erythromycin-
resistant mutant, which had altered mitochondrial ribosomes, neither
sporulation nor in vivo protein synthesis were affected by erythromycin (122).
This mutant synthesized all of the meiotic polypeptides under sporulation
conditions in the presence of erythromycin, ruling out the possibility that the
drug could act at the level of the cytosolic protein synthesis system during


22
sporulation. This evidence suggests that mitochondrial protein synthesis is
needed for the expression of some nuclear genes during sporulation.
There is now evidence that mitochondrial gene products may regulate
nuclear gene expression in mammals as well. This comes primarily from the
observations that a particular antigen, although specified in part by a gene in
the major histocompatibility complex, is maternally inherited (123). This
maternally-transmitted antigen (Mta) is a murine cell-surface molecule whose
phenotype is determined solely by that of the mother (124). Sex-linked
transmission is ruled out because the phenotypes of Fi males and females in
a single litter are identical (125). The Mta phenotype cannot be modified by
fostering, embryo transfer or transfer of bone marrow cells to lethally irradiated
mice, making the involvement of a conventional infectious agent unlikely
(123). This interpretation is further supported by the finding that the maternal
lineage of the Mta+ phenotype is stable for at least 11 generations of
backcrossing to Mta- males (126). The expression of Mta in somatic cell
hybrids requires functional mitochondria from the Mta+ parent cell line.
Pretreatment of the Mta+ parent with the mitochondrial poison Rhodamine 6G
(127, 128) resulted in hybrids which were Mta-, or diminished in Mta
expression. This is the first evidence for mitochondrial control of the
expression of a cell membrane molecule in eukaryotes. Whereas Mta is
encoded by a structural gene in the major histocompatibility complex, its
expression seems to be controlled by a mtDNA gene product.
The techniques of somatic cell genetics also provide a powerful tool for
investigating whether mitochondrial genomes play a role in the activation
and/or suppression of the phenotypic expression of tumorigenicity. Several
investigators have shown that tumorigenicity can sometimes be suppressed
by fusing tumor cells with cytoplasts from nontumorigenic cells (113, 129).


23
However, there have been no reports of the induction of tumorigenicity in
nontumorigenic cells by the addition of cytoplasts from tumorigenic cells (130,
131). These findings suggest that cytoplasmic elements and/or mitochondria!
genomes may sometimes contribute to the suppression of tumorigenicity (but
this is not consistently true).
Partitioning of Regulatory Molecules
The notion of partitioning of regulatory molecules between the
mitochondria and the nucleus is not restricted, of course, to products of the
mitochondrial genome. There is now good evidence for the partitioning of
some nuclear gene products between the mitochondria and
extramitochondrial compartments (132, 133). Thus, nuclear-encoded
regulatory factors could partition between the mitochondria and the nucleus,
and their relative distributions could be determined by the presence or
absence, or the amounts, of mtDNA (or RNA) sequences. This mechanism
would allow for regulation of nuclear gene expression in response to the
quality as well as the quantity of mtDNA or RNA sequences. In that way, the
nuclear genome could "sense" the amount or kind of mtDNA in the cell.
Indeed, Clayton elal. (134) have recently shown that a135-nucleotide RNA
species, not encoded in the mitochondrial genome, is the RNA moiety
necessary for activity of the site-specific endoribonuclease involved in primer
RNA metabolism in mammalian mitochondria. This finding implies transport of
a nucleus-encoded RNA, essential for organelle DNA replication, to the
mitochondrial matrix.


24
Polvamines
All organisms contain significant amounts of the polyamines,
spermidine and spermine, and their precursor, putrescine. In mammals the
concentrations of these aliphatic amines vary considerably between different
cell types (135). Thus, cells heavily involved in polynucleotide and protein
synthesis such as proliferating or protein secreting cells contain large amounts
of polyamines (in the mM range), while concentrations in metabolically less
active cells are lower (136). Polyamines are cations at physiological pH and,
in vitro, can bind through ionic forces to the negatively-charged groups of
nucleic acids, proteins and phospholipids (137, 138). Polyamines can also
form hydrogen bonds, and the aliphatic hydrocarbon part of their structure may
allow some interactions with hydrophobic environments such as those
occurring in membranes. These properties could contribute to the multiple
actions attributed to the polyamines in many biological processes.
The biosynthetic pathway for putrescine, spermidine and spermine in
mammalian cells has been well established (Figure 2). Two 'decarboxylases'
and two 'aminopropyltransferases' are involved. The two decarboxylases are
of particular interest as they are present in mammalian cells in very small
amounts, have very short half-lives, and are highly inducible (137). Because
of these properties the decarboxylases can regulate polyamine synthesis and
enable cells to respond to a variety of stimuli with a rapid increase in
polyamine levels.
Ornithine decarboxylase (ODC) is a pyridoxal phosphate-dependent
enzyme (136). It is thought to play the key role in polyamine synthesis, and its
half-life of about 10 minutes is the shortest reported for an enzyme in
mammalian cells (139). Ornithine decarboxylase is present in very small
amounts in quiescent cells, and its activity increases rapidly and dramatically


25
COOH
I
H2N-(CH2)3-CH-NH2
Ornithine
C02
1
M/
H2N-(CH2)4-NH2
Putrescine
S-Adenosylmethionine
C02^e
Decarboxylated
S-Adenosylmethionine
(D-SAM)
^ Methylthioadenosine
(MTA)
H2N-(CH2)3-NH-(CH2)4-NH2
Spermidine
D-SAM
MTA
V
H2N-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2
Spermine
Figure 2. Polyamine synthesis in mammalian cells. Enzymes involved are:
1) ornithine decarboxylase; 2) S-adenosylmethionine
decarboxylase; 3) spermidine synthase; and 4) spermine synthase.


26
within a few hours of exposure to trophic stimuli such as hormones, certain
drugs, and growth factors (140, 141, 142).
S-Adenosylmethionine decarboxylase (SAM-DC) also has a relatively
short half-life (~1 hr) and has the added pecularity that it utilizes as cofactor
covalently bound pyruvate instead of pyridoxal phosphate, the usual cofactor
for decarboxylases (136). Like ODC, it is present in mammmallan tissues in
very small amounts, and its activity is regulated by many hormones and other
growth-promoting stimuli. Once decarboxylated, S-adenosylmethionine is
committed to polyamine production in that no other substantial reactions
utilizing decarboxylated S-adenosylmethionine are known. The
decarboxylation of S-adenosylmethionine is the rate-limiting step in
spermidine formation.
The transfer of an aminopropyl group from decarboxylated S-adenosyl
methionine to putrescine is catalyzed by spermidine synthase, one of the
aminopropyltransferases. The other aminopropyl group needed to convert
spermidine into spermine, is also derived from decarboxylated S-
adenosylmethionine, a reaction catalyzed by the second
aminopropyltransferase, spermine synthase (136). As the activities of
spermidine synthase and spermine synthase are much higher than those of
the two decarboxylases, putrescine and decarboxylated S-
adenosylmethionine concentrations within the cell tend to be very low in
comparison to spermine and spermidine (137).
An active transport system for the polyamines distinct from those for
amino acids has been found in ail cells tested to date (136). The activity of this
system is increased in polyamine-depleted cells, and it can maintain cellular
polyamine levels in the presence of very low extracellular concentrations. The
role of the transport system under normal circumstances is unclear, however,


27
because intracellular synthesis is used to provide polyamines, and
extracellular polyamine concentrations are usually low. The transport system
can also take up other basic substances, including drugs like MGBG; this will
be discussed in greater detail in the next section. In addition, some cultured
cells efflux polyamines when cell growth has been restricted by inhibitors,
confluence, or lack of growth factors (136).
The first step in the catabolism of these polycations is acetylation (143).
The degradation of spermine into spermidine or of spermidine into putrescine,
processes which reverse the normal biosynthetic pathway for polyamines,
involves the sequential activity of two enzymes, N1-acetyltransferase and
polyamine oxidase (Figure 2). Putrescine may be acetylated and eventually
oxidized in a reaction catalyzed by monoamine oxidase, or directly oxidized in
a reaction catalyzed by diamine oxidase, which is induced by growth stimuli.
Polyamines and their acetylated derivatives can also be oxidized in a reaction
catalyzed by plasma amine oxidase (137).
Evidence from studies on polyamine-deficient mutant cells and on
drugs that inhibit polyamine biosynthesis indicates that polyamines are
necessary for cell division and differentiation (136, 144). Indeed, proliferating
tissues are particularly rich in polyamines and their biosynthetic enzymes
(145). Although it is apparent that polyamines play important biological roles,
their exact function(s) is unclear. Polyamines have a wide spectrum of effects,
particularly in cell-free systems. Such actions include control of the initiation
of translation, stimulation of ribosomal subunit association, stabilization of
tRNA and DNA structure, and stimulation of RNA and DNA synthesis (135).
Polyamines have also been shown to enhance reactions catalyzed by DNA
polymerase (146), RNA polymerase (147), polynucleotide ligase and kinase
(148), and gyrase (149).


28
In vitro, polyamines bind preferentially to double-stranded nucleic
acids. They bind in the minor groove of the double helix and, by interacting
with phosphate groups, stabilize the double helix (147). A high concentration
of polyamines induces condensation of nucleic acids. This association with
DNA may represent a major function of polyamines, involving both molecular
stabilization and control of gene expression (150). However, it is prudent to
recognize that many of these actions may only reflect the fact that polyamines
interact with acidic macromolecules in vitro and that physiological relevancy
has not been established.
The polyamines, spermine and spermidine, stimulate the rate of
phosphorylation of selected proteins by several types of cyclic nucleotide-
independent protein kinases (151, 152). For example, spermine and
spermidine increase the activity of cytosolic and nuclear type II casein kinases
(153). The enhancement of certain of these protein kinase reactions by
polycations seems to relate primarily to their interaction with the protein
substrate, yielding more favorable conformations for phosphorylation by the
protein kinase, rather than by an effect on the enzyme £er se (152).
Polyamines have also been shown to inhibit certain enzyme reactions.
For example, physiological concentrations of spermine completely inhibit
phosphorylation by nuclear protein kinase Nl (151). Polyamines, especially
spermine, inhibit cardiac phospholipid-sensitive, calcium-dependent protein
kinase and myosin light chain kinase, a calmodulin sensitive protein kinase
(154). Even protein phosphatases, including a type isolated from heart, may
be influenced by polyamines with the effect dependent on the substrate used
(155).
Moreover, spermidine or spermine, at near-physiological
concentrations of 1.5 mM, stimulates by 8-fold the phosphorylation of


29
phosphoinisitol (PI) in membranes from A431 cells. The reaction product is
almost exclusively PI-4-phosphate (156). Also, spermidine or spermine can
increase by 4-fold the rate of hydrolysis of synthetic phospholipid analogs by
phospholipase A2 (157). In addition, polyamines are substrates for
transglutaminases, are precursors of gamma-aminobutyric acid, and uncouple
the parietal cell H+, K+-ATPase system (135). Polyamines also appear to be
second messengers in mediating Ca++ fluxes and neurotransmitter release in
potassium-depolarized synaptosomes (158).
Addition of micromolar concentrations of polyamines to cultured
myocardial cells results in an increase of cGMP and a parallel decrease of
cAMP levels (137). Conversely, depletion of intracellular polyamines by
polyamine synthesis inhibitors leads to an accumulation of cAMP and a
depletion of cGMP (159). These effects are mediated by modifications of the
activities of the enzymes involved in cyclic nucleotide metabolism, i.e.
adenylate and guanylate cyclase and cAMP- and cGMP-phosphodiesterase.
Considering that cGMP and cAMP are usually regarded as positive and
negative signals, respectively, for the induction of cell growth, the property of
polyamines to affect the metabolism of the two nucleotides in opposite
directions correlates well with their suggested role in cell growth (137).
Polyamines also have many actions on mitochondria. Mitochondria
can "buffer" cytosolic free Ca++ concentrations, but the physiological
significance of this uptake has been questioned because it is triggered only by
Ca+T concentrations that exceed those found in normal cells. In the presence
of physiological concentrations of Mg++, the extramitochondrial free Ca++
which the mitochondria will buffer is 0.6 to 1 pM (160), but the physiological
free Ca++ concentration in the cytosol is only 0.15 to 0.25 pM (161, 162). It
now appears that spermine, which had been lost upon permeabilization of


30
cells, will restore the sensitivity of mitochondria to external Ca++ (160).
Spermine both increases the rate and affinity of Ca++ uptake into mitochondria
and decreases the set-point to which isolated mitochondria buffer free Ca++
concentration. Spermine stimulates Ca++ uptake by mitochondria but not by
microsomes (163). The half maximally effective concentration of spermine (50
(iM) is in the range of physiological concentrations of this polyamine in the
cell. Spermidine is 5 times less potent; putrescine is inactive. The stimulation
of mitochondrial Ca++ uptake by spermine is inhibited by Mg++ in a
concentration-dependent manner. Spermine is thus an activator of the
mitochondrial Ca++ umporter, and Mg++ is an antagonist. Spermine also
inhibits the efflux of Ca*+ from mitochondria induced by ruthenium red.
Polyamines may thus confer to the mitochondria an important role in the
regulation of the free Ca++ concentration in the cytoplasm as well as in the
mitochondrial matrix.
At physiological concentrations spermine may also be transported into
rat liver mitochondrial matnx space, provided that mitochondria are energized
and inorganic phosphate is available for concurrent transport (164).
Furthermore, mersalyl, a known inhibitor of phosphate transport, prevents both
spermine uptake and release. Magnesium ions inhibit the transport of
spermine, conceivably by competing for the same binding sites on the
mitochondrial membrane.
Fixed negative charges of anionic sites have previously been proposed
as possible binding sites for large organic cations within the mitochondrial
membranes (165). These binding sites are probably composed mainly of
ionized phospholipids and may be involved in transport of small cations and
anions across the mitochondrial membrane. The phospholipid-containing


binding sites are reportedly localized mainly at the outer face of inner
mitochondrial membrane.
In 1960, Tabor (166) reported that low concentrations of spermine and
spermidine inhibited swelling of mitochondria in hypotonic media. More
recently, polyamines were shown to improve the respiratory control and
prevent loss of control in heat-aged mitochondria, protect oxidative
phosphorylation, and prevent a fall in the mitochondrial membrane potential
(167, 168, 169). At concentrations comparable to those attained
intracellularly, the polyamines inhibited state 4 respiration of rat liver
mitochondria, but they had much less effect on state 3 or uncoupled
respiration (I25 = 7.5 and 7.0 mM for spermidine and spermine, respectively)
(167). Electron microscopy revealed that polyamines caused the outer
mitochondrial compartment to collapse, bringing the inner and outer
membranes into apparent contact with one another (167). Recent evidence
suggests that the import of nuclear-coded proteins into mitochondria occurs at
sites where the inner and outer mitochondrial membranes are juxtaposed to
one another (170). It seems possible, therefore, that polyamines may affect
this process.
MGBG (Methylglvoxal bisiauanvlhydrazone])
History and Climcal Utility
MGBG has been recognized as a potent antiproliferative agent since
1958 when Freedlander and French (171) reported that it inhibited the growth
of L1210 leukemia cells m mice. Clinical studies disclosed that MGBG (also
known as methyl-GAG) could induce remissions in some patients with acute
myelocytic leukemia, malignant lymphoma, and certain other neoplasms (4,
5). MGBG has also been used in the treatment of trypanosomiasis (172).


32
However, MGBG accumulated to extremely high levels In certain
normal cells (6, 173), and the consequent severe toxicity set practical
limitations on its use (3). Mucositis was the dose-limiting toxicity in Phase Ml
clinical trials (4). Fatigue, anorexia, mild nausea and vomiting, myalgias, and
neuropathy were also seen. The subacute toxicity of MGBG involved both
proliferative (gastrointestinal, bone marrow, and lymphoid) and
nonproliferative (hepatic, renal, and cardiac) tissues (174). However, more
recent investigations involving variation in dosage schedules and use in
combination with other anti-proliferative agents have rekindled interest in the
clinical use of MGBG (3, 175). Because no metabolites of MGBG have ever
been detected in urine, feces, or various tissues, and virtually no radioactive
CO2 was expired after in vivo administration of [14C]-MGBG, it is believed that
MGBG does not undergo biotransformation in higher animals (175).
Relationship to Polvamines
French and his colleagues were the first to point out that MGBG had
structural resemblances to spermidine (Figure 3), and they suggested that
MGBG might interfere with the biological functions of this naturally-occurring
polyamine (3). MGBG is a basic molecule consisting of 2 aminoguanidine
groups separated by an aliphatic chain. MGBG has pKa's of about 7.5 and 9.2
at 25C (3), presumably due to resonance stabilization of the protonated forms
of the aminoguanidine moieties at both ends of the molecule. Spermidine,
whose molecular contours are quite similar to those of MGBG in its most
extended conformations, has pKa values of about 8.4, 9.8 and 10.8 at 25C
(3).
MGBG is a potent (<1 jiM) competitive inhibitor of S-adenosyl-
methionine decarboxylase (SAM-DC), which is a key enzyme in the
biosynthesis of the polyamines, spermidine and spermine (176, 177).


Spermidine
NH
H2N-C-NH-
HCH3 NH
=C-C=N-NH-C-NH2
MGBG
Figure 3. Structures of spermidine and its analog, MGBG.


34
However, the inhibitory effects of MGBG on polyamine biosynthesis in animals
are transient because of a striking secondary increase in SAM-DC. It is
hypothesized that binding of MGBG to the enzyme may stabilize it, and hence
prolong the normal one hour half-life of SAM-DC (178).
MGBG is also a potent noncompetitive inhibitor of diamine oxidase
(179), an enzyme involved in putrescine degradation. In addition, MGBG
causes an increase in the activities of ornithine decarboxylase (ODC), which
synthesizes putrescine, and in spermidine/spermine N1-acetyl-transferase
(SAT), an enzyme which participates in the degradation of spermine and
spermidine to putrescine (176). The increases of both ODC and SAT activities
provoked by MGBG in cultured human erythroid leukemia K562 cells were
blocked by treatment with cycloheximide, a finding that suggests that the
increase in enzyme activity required synthesis of protein. The putrescine
content in cells treated with MGBG increased 20-fold, whereas the levels of
spermidine and spermine were depressed by 35 and 50%, respectively. The
marked increase m putrescine levels presumably resulted from inhibition of
SAM-DC and diamine oxidase, and induction of ODC and spermidine and
spermine acetyltransferases. All of these factors act to increase putrescine
levels.
Thus, m cells treated with MGBG, putrescine pools increase while those
of spermidine and spermine decrease slowly (180). Nonetheless, a number of
laboratories (181, 182, 183) have been unable to correlate these changes in
polyamine concentration with inhibition of cell growth. It seems that the
antiproliferative effect produced by MGBG correlates much better with the
intracellular MGBG concentrations than with the depletion of polyamines (183,
184). The ability of spermidine to prevent the antiproliferative effects of MGBG,
initially a critical observation in linking MGBG to polyamines, is now attributed


to competition for cellular uptake rather than replenishment of MGBG-depleted
spermidine pools (185). Moreover, difluoromethylornithine (DFMO), a highly
specific inhibitor of polyamine biosynthesis, is cytostatic (186), whereas MGBG
is cytotoxic, a result suggesting that the two drugs may have different
mechanisms of action.
Effects on Mitochondria
Indeed, another pharmacological action for MGBG has been identified.
In 1966, Pine and DiPaolo (187) found that MGBG and certain other cationic
compounds inhibited the respiration of ascites L1210 cells, and uncoupled
phosphorylation in isolated mitochondria. In common with 2,4-dinitrophenol,
the drugs selectively inhibited acetate incorporation into lipid and, to some
extent, reduced the cellular pools of ATP. In a different study, pyruvate
oxidation, another measure of mitochondrial function, was significantly
decreased 4 hours after exposure of ascites L1210 leukemia cells to MGBG;
growth inhibition did not occur until after 10-12 hours (185). Depletion of
spermidine pools to levels comparable to those attained during DFMO-
induced cytostasis did not occur even after 24 hours of exposure. Also, in
studies with intact A30 cells, the rate of oxygen uptake in MGBG-treated cells
was decreased to as low as 10% of control (188). The degree of inhibition of
oxygen consumption was dependent on the concentration of MGBG. When
spermidine was added to the MGBG-containing medium, however, respiratory
activity was restored and approached that of normal cells.
Pathak i si. (189) have demonstrated that exposure of cultured
leukemia L1210 cells to 0.1 to 10 pM MGBG resulted in a concentration-
dependent inhibition of cellular proliferation which became apparent after
about 1 to 2 generation times (12 to 24 hr). Ultrastructural examination of cells
exposed to at least 1.0 pM MGBG for 24 hr revealed extensive swelling of


36
mitochondria, deterioration and eventual loss of cristae and increase in matrix
density. This ultrastructural damage was selective for the mitochondria; there
were no concomitant changes in nuclear ultrastructure (190). In addition, the
mitochondrial damage preceded growth inhibition by about 12 hr and did not
immediately affect cell viability. Because damage was apparent in all of the
mitochondria of each affected cell, the organelle population was probably
homogeneous in its sensitivity to the drug. Moreover, exposure to 10 pM
MGBG for 24 hr caused all of the cells to be affected. Whether the
mitochondrial structural damage was associated with functional deterioration
and whether the damage was reversible were not demonstrated.
Similar damage has been observed in ascites L1210 cells treated in
vivo with a single dose (75 mg/kg) of either MGBG or ethidium bromide (190).
After 24 hr, the mitochondria were swollen, lost their inner structure, and, in the
case of treatment with ethidium bromide, developed numerous electron
densities within the matrix. Analysis of the nucleotide pools of these cells by
HPLC revealed that treatment with either MGBG or ethidium bromide depleted
the ATP pools to 52 and 16% of control, respectively. The overall adenylate
energy charge of the cell (1/2[ADP+2ATP] + [AMP+ADP+ATP]) was also
reduced to 68 and 58% of control, respectively. The measurement of ATP
levels suggested that, whereas MGBG caused substantial mitochondrial
damage, the resultant energy depletion ger s£ was not sufficient to account for
growth inhibition (191).
When MGBG-treated ceils were harvested, washed, and reinoculated
into untreated mice, all cells reattained nearly normal ultrastructure after 48 hr,
whereas ethidium bromide-treated cells did not, even after 96 hr (190).
Analysis of the nucleotide pools of the MGBG-treated cells indicated that the
mitochondria had recovered their functional capabilities as well. The


37
adenylate energy charge for these cells was essentially the same as that for
untreated cells. Also, the cells previously treated with MGBG displayed
unaltered leukemogenicity in that the animals died at nearly the same time as
they did after inoculation with untreated cells.
MGBG also causes ultrastructural damage to mitochondria in several
other murine and human cell types including P288 mouse leukemia, L-cell
mouse fibroblast, C3H/10T1/2 mouse embryo fibroblast, and NALM-1 human
chronic myelocytic leukemia cells (176). Interestingly, the onset of
mitochondrial damage in these cell lines related inversely to the generation
time of the particular cell lines. This relationship between mitochondrial
damage and the rate of cell proliferation was tested in two separate cell
systems (174). In cultures of human lymphocytes stimulated with
phytohemagglutinin, only those cells undergoing blastogenesis were bound to
be affected by MGBG. Similarly, MGBG treatment of confluent and
subconfluent cultures of C3H/10T1/2 mouse embryo fibroblasts affected only
the dividing, subconfluent cells.
The effects of MGBG on isolated rat liver mitochondria have also been
studied (191). At drug concentrations comparable to those attained
intracellularly, MGBG significantly inhibited state 4 respiration (125=6 mM), but
had less of an inhibitory effect on state 3 or uncoupled respiration (125=16-20
mM). This may be due to the fact that in the absence of ADP (state 4),
mitochondria generate a significant electrochemical gradient across their
inner membrane. By election micrcscopy, MGBG-treated mitochondria
appeared swollen, and the spaces between cristae membranes or inner and
outer membranes were collapsed, obliterating the intermembrane space
(191).


38
Since the inhibition of mitochondrial respiration in isolated
mitochondria was prevented by potassium cations and enhanced by
valinomycin, the drug may compete for potassium-binding sites, possibly
membrane phosphoplipids (191). Pretreatment of mitochondria with MGBG
protected against the nonspecific swelling effects of Triton X-100, and the
electrophoretic mobility of mitochondria was markedly slowed by MGBG.
Overall, the data suggest that MGBG neutralizes the net negative surface
potential of isolated mitochondria by binding to sites (possibly phospholipids)
at the inner mitochondrial membrane; any subsequent interference with cation
binding and/or transport may result in inhibition of bioenergetic functions.
Fixed negative charges of anionic sites have been proposed as
possible binding sites for large organic cations within the mitochondrial
membranes (165). Neutralization of this surface potential with compounds
such as polyamines can affect many mitochondrial activities, as well as
ultrastructure. Similar or identical binding sites at the inner mitochondrial
membrane have been suggested for binding certain bis[guanylhydrazones]
(191). Moreover, it has been shown that cationic drugs need not penetrate
across inner mitochondrial membranes because binding sites are probably
fixed at the outer surface (165). In addition, the effects of MGBG on isolated rat
liver mitochondria are readily prevented or reversed by polyamines, and these
interactions are also affected by the mitochondrial transmembrane potential
(192). Magnesium cations enhance the protective action of polyamines.
These data indicate that competition exists between MGBG and polyamines
for low affinity, negatively-charged binding sites at the outer surface of inner
mitochondria! membranes
Evidence which implicates mitochondrial damage as important in the
antiproliferative effects of MGBG comes from studies of other polyamine


analogs. For example, 4,4'-diacetyldlphenylurea-bis(guanylhydrazone) is an
aromatic bls-(guanylhydrazone) with potent antiproliferative activity. The drug
causes profound ultrastructural damage to mitochondria (174), but has no
effect on polyamine biosynthesis (193). Similarly, ultrastructural damage also
precedes detectable inhibition of cell growth (189). Damage to mitochondrial
ultrastructure is selective for proliferating cells. This has been observed
among cultured cells (174) and more recently in the intestinal epithelium
(194), a site of MGBG toxicity, where damage only occurs to mitochondria of
the dividing crypt cells but not to the nondividing villous cells. Although uptake
studies were not done, Diala el al. (195) made the interesting observation that
wild-type yeast are growth-inhibited by MGBG while petite mutants, lacking
mitochondria, are not. Growth inhibition by MGBG was antagonized by
spermidine, but neither the polyamine nor MGBG had any effect on growth
inhibition by ethidium bromide. MGBG had little or no effect on cell viability
throughout the 2-day exposure.
While the numerous effects of MGBG on mitochondria provide
substantial indication that these actions are responsible for the
antiproliferative activity of the drug, this relationship has not yet been
established. Several workers have demonstrated that normal and malignant
cells possess high-affinity and saturable transport systems that promote
intracellular accumulation of putrescine, spermidine, spermine, and certain
agents such as MGBG (196, 197). Because a saturable carrier is involved, the
system seems to be specific for certain structural features of the polyamines
and their analogs. In general, it appears that putrescine, spermidine,
spermine, and MGBG are transported by common membrane transport
systems that are: 1) distinguishable from other known transport systems (e.g.
for various amino acids); 2) saturable and temperature-dependent, with


40
maximal rates at 37C; 3) inhibited by uncouplers of oxidative phosphorylation
and certain respiratory poisons; and 4) dependent on the proliferative and, in
some instances, hormonal status of the cells (198). Like the polyamine-
biosynthetic enzymes, the transport system seems to be highly regulated,
responding rapidly to polyamine antimetabolites such as DFMO, an
irreversible inhibitor of ODC, and MGBG, an inhibitor of SAM-DC (199, 200,
201). Agents such as MGBG, which utilize the polyamine transport system,
may be concentrated intracellularly more than 1000-fold relative to the
medium (196, 202).
Seppanen et aL (203) have found that cells can concentrate MGBG
some 600- to 1500-fold so that, following exposure to 5-10 pM drug, the
internal concentration is 4-6 mM. A minimum intracellular concentration of 0.5
to 1 mM was required for growth inhibition to occur. Seppanen el aL (204)
also found that MGBG uptake is critically dependent on the growth rate of
tumor cells (i.e., slowly dividing cells transport less MGBG than rapidly dividng
cells). Mikles-Robertson eiaL (174) found that MGBG cytotoxicity also
correlated with cell growth rate.
Recently, mutants of human fibroblast VA2 cells have been developed
which are 10- to 20-fold more resistant to the growth inhibitory effects of MGBG
than the parent line (205, 206). The variants took up MGBG to a similar extent
as wild type during short-term incubations (less than 5 min), accumulated less
drug during longer incubations (30-120 min), and more readily lost MGBG
when shifted to drug-free medium, results which might indicate decreased
intracellular binding of MGBG. In the absence or presence of MGBG, the
overall pools of pclyamines were not appreciably changed from those of the
wild type. Pyruvate oxidation was not significantly inhibited in the mutants
when grown in the presence of MGBG, but was dramatically reduced in wild


type cells. Furthermore, the mitochondrial ultrastructure of the drug-resistant
cell lines was essentially unaffected by culture In MGBG. The resistant cells
took up about 40% less MGBG than their sensitive counterparts. This
difference is considered small among transport mutants (203). Drug
resistance was not cytoplasmically transmitted by cytoplast cell fusion for any
of the four sublines, suggesting that the genes responsible for resistance were
likely of nuclear rather than mitochondrial origin (206). Defective polyamine
and MGBG transport was also found in mutant Chinese hamster ovary and rat
myoblast cells that were resistant to MGBG (207).
Because of the inhibition of cell growth by MGBG, many studies have
addressed the effects of the drug on DNA synthesis. Unlike aromatic
bisguanylhydrazones, which bind effectively to nuclear DNA, MGBG binds
only weakly to DNA and has little effect on DNA polymerase alpha or
thymidine kinase (198, 208). Addition of MGBG to HeLa S3 suspension
cultures caused a decrease in incorporation of [3H]thymidine into nuclear
DNA, but only after putrescine levels had increased and spermidine and
spermine levels had decreased. Since the rate of DNA chain elongation was
only reduced slightly by MGBG, decreased levels of spermidine and spermine
might lead to a decrease in the number of replication units active in DNA
synthesis within each cell.
Effects on MtDNA
The effects of MGBG on mtDNA synthesis have also been Investigated
because of the mitochondrial damage elicited by this drug. In exponentially-
growing L1210 leukemia cells prelabeled with [14C]thymidine, the
incorporation of [3H]thymidine into mtDNA was selectively inhibited (55% of
control) when studied 5 hr after exposure of cells to 10 pM MGBG (7).
Incorporation of label into nuclear DNA, however, was not affected until 8 hr;


the rate of nuclear DNA synthesis was 60% of control after 12 hr. After 16 hr of
exposure, the rates of nuclear and mtDNA synthesis were both about 40% of
control values. Exposure of L1210 cells to 1 pM MGBG resulted in a mtDNA
synthesis rate 60% of control. At this concentration, growth inhibition was 90%
of control after 18 hr of exposure.
Dye-CsCI gradients of mtDNA indicated that the inhibition of synthesis
occurred in replicative forms of circular DNA (7). Uptake studies excluded the
possibility of drug interference with cellular uptake of thymidine.
Ultrastructural studies revealed a very close correlation between the dose-
response curve for mitochondrial damage and that for MGBG inhibition of
mtDNA synthesis. This correlation suggests a close relationship between
ultrastructural damage and inhibition of mtDNA synthesis, but the possibility
exists that both are epiphenomena of another action of the drug.
A more detailed study of the effects of MGBG on mtDNA synthesis in the
Syrian hamster tumor cell line was done by Nass (6). The fate of mtDNA both
during (24-48 hr) and following (7 hr to 7 days) MGBG treatment was
monitored by ultrastructural, pulse-labeling, and restriction cleavage methods.
MGBG (50 pM) selectively inhibited mtDNA replication prior to significant
inhibiton of nuclear DNA synthesis (73 versus 19%, respectively, at 16 hr).
The drug induced structural alterations, without substantial degradation, of the
closed circular form of mtDNA. Importantly, D-loop strand (7S) initiation was
completely abolished, but mtDNA strands already initiated were able to
complete the circle of replication.
Electron microscopy revealed selective ultrastructural damage to the
mitochondria (including swelling, loss of cristae and matrix components, and
dense inclusions) in up to 96% of cells, while nuclei appeared normal.
Quantitative assays of the uptake and retention of rhodamine 123 in these


MGBG-treated cells revealed that the mitochondrial membrane potential was
maintained. After removal of MGBG, mtDNA resumed replicative active, and
damaged mitochondria recovered near-normal ultrastructure within 1 to 2
days.
Specific Aims
Compounds which, at certain doses, inhibit mtDNA replication more than
cell division would be predicted to deplete the characteristic mtDNA copy
number with each successive cell division. Preliminary studies in our
laboratory with mouse leukemia L1210 cells treated chronically with MGBG at
concentrations which initially inhibited mtDNA replication did decrease the
mtDNA copy number. These studies also demonstrated that there are doses
at which long term survival in MGBG is possible. I wanted to determine if there
was a minimal copy number of mtDNA which cells would tolerate and whether
a new steady state level of mtDNA would be established with chronic MGBG
treatment. I also wished to explore in detail the relative contributions of the
rates of mtDNA replication and cell division to the regulation of the mtDNA
copy number during MGBG treatment. And, finally, I endeavored to determine
how mitochondrial and/or cell function was affected as a consequence of a
change in the mtDNA copy number.
The specific aims of this research were the following.
1) Determine the degree to which mtDNA replication and cell division
are coupled. That is, will cells continue to divide even though their mtDNA
replication is inhibited, thus halving the amount of mtDNA per cell with each
division?
2) It so, determine the minimum copy number of mtDNA per cell which
these cells will tolerate.


44
3) Once the copy number of mtDNA is diluted by MGBG treatment,
determine whether these cells establish a new steady state with respect to the
content of mtDNA, or return to their characteristic copy number.
4) Determine the relative contributions of the mtDNA doubling rate and
the cell doubling rate to the regulation of the mtDNA copy number in MGBG-
treated cells.
5) Determine if mitochondrial function is normal or impaired
proportionally to the mtDNA copy number, at least as measured by rhodamine
123 uptake.


MATERIALS AND METHODS
Cell Culture
Mouse leukemia L1210 cells were grown as suspension cultures in 25
or 75 cm2, canted-neck tissue culture flasks (Fisher Scientific). The cells were
maintained in an incubator (National Appliance Company, Portland, Oregon)
at 37C, and the flasks were kept tightly capped. The medium used was RPMI
1640 supplemented with 16 mM HEPES (3-[N-morpholino] propanesulfonic
acid), 8 mM MOPS (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid),
2.0 g/L NaHC03, and10% fetal bovine serum. The starting pH was 7.4. Stock
solutions of MGBG and diethylspermine (DES) were made to concentrations
of 1 mM in water. All ingredients were obtained through Sigma Chemical Co
(except DES, gift of R. Bergeron). The medium and drugs were sterilized by
0.2 (i filtration (Fisher Scientific). Cells were maintained in exponential growth
by reseeding every two days into fresh medium; the starting concentration was
1 x1 o5 cells/ml.
Cell Counts
Aliquots (200 pi) of cells to be counted were diluted in 10 ml of Hematall
diluent (Fisher Scientific). The cell number was determined by electronic
particle analysis (Coulter Counter, Model Zf, Coulter Electronics, Hialeah, FL).
Two aliquots of cells were counted from each flask and the mean determined.
Because the cells were passed every two days, a calculation was
devised to plot the counts as a single, continuous line over the entire time
course of an experiment. Cumulative counts were calculated simply as
Cumulative count(n+1) = Cumulative count(n) x Concentration Qf C.SliS,(Ptl)
Conce rtraton of viable oel!s(n)


46
The mean population doubling time of the cells was determined as
follows: N=N0ext, where t=time, N0 is the number of cells present at t=0, and
the doubling time is the value of t when N=2N0.
Determination of ICt;n
The IC50 was defined as the concentration of compound necessary to
decrease cell growth to 50% of control growth at a given time. The percentage
of control growth was determined as follows:
[final cell number (treated group) initial inoculum] X 100
final cell number (control group) initial inoculum
The IC50 of MGBG was determined for the first 2 days of treatment.
Cell Viability
Trypan blue (Eastman Kodak, Rochester, NY) was added to 100 pi
aliquots of cells to a final concentration of 0.06%. The cells were mixed well,
and a 10 pi aliquot was transferred to a hemacytometer (Reichert Scientific
Instruments, Buffalo, NY). At least 100 cells per sample were counted with a
phase contrast microscope (Ernst Leitz, Wetzlar, Germany) at 200X
magnification. The percent viability of the cells was determined as follows:
number of cells excluding trypan blue X 100
total number of cells
Clonoaenic Assay
Cells from selected MGBG treatments were counted and diluted to a
concentration of 4 cells/ml. Aliquots of 100 pi were transferred to triplicate 96-
well culture plates (Fisher Scientific) and incubated at 37C for 1 week. The
culture plates were then examined with an inverse phase microscope (Zeiss,
West Germany) for numbers of wells which had colonies of cells. Groups of
>50 ceiis/weil were considered as having been cloned from a single viable


47
cell. The percent cloning efficiency of the drug-treated cells was determined
as follows:
number of wells with colonies (treated group) X 100
number of wells with colonies (control group)
Cell Size
The diameter of the cells was determined directly by the method of
Schwartz £lai. (209). Uniform polymeric microspheres (Polyscience,
Warrington, PA) from 4.72 to 10.0 p in diameter were diluted in Hematall, and
the particle size measured electronically with a FACS Analyzer (Becton-
Dickinson, Sunnyvale, CA) with the amplifier in the log mode. The peak
channel number was plotted against the corresponding calibrated diameter for
each size of microbead to obtain a calibration curve.
Aliquots of 1x106 cells were collected, pelleted, and resuspended in
0.5 ml Hematall for analysis. The peak channel number for the cells was
plotted on the calibration curve to obtain the approximate cell diameter. The
volume of the cells was then calculated from this diameter.
Flow Cvtometrv
Flow cytometric analysis of nuclear DNA content was done with a
RATCOM flow cytometer (RATCOM Inc., Miami, FL) interfaced with a
microcomputer (IBM-XP). Aliquots of 1x106 cells were resuspended in 0.5 ml
Hematall and were stained with 4',6-diamidino-2-phenylindole (210). The
distribution of nuclear DNA content among the cells was plotted and
integrated by computer.
Rhodamine 123 Uptake
Aliquots of 1x106 cells were pelleted and resuspended in 100 pi of
serum-free medium. Rhodamine 123 (2-[6-amino-3-imino-3H-xanthen-9-yl]-
benzoic acid methyl ester) (Sigma Chemical) was added to each aliquot to


48
give a final concentration of 1 (ig/ml (211). The samples were incubated at
37C for 10 min and washed once with medium. The stained cells were
observed under an epifluorescence microscope (Zeiss) at 400X magnification.
The excitation wavelength was 485 nm.
Preparation ofM4C|-MGBG Stock Solution
A stock solution of 1 mM [14C]-MGBG (specific activity 26 mCi/mmol,
Amersham) was prepared aseptically. Aliquots of this stock were added to
media to obtain the indicated final concentration of drug.
Uptake Protocol
Cells were incubated with [14C]-MGBG at concentrations ranging from
1.0 to 3.3 (iM. At the end of each incubation period, an appropriate aliquot of
cells was transferred to a 15-ml tube, centrifuged at 650g for 3 minutes, and
the supernatant was decanted. To remove any labeled drug bound to the
outside of the cell, the cell pellet was washed twice with 4C media containing
1 mM unlabeled MGBG. The cell pellet was resuspended in 1 ml of distilled
water, transferred to a scintillation vial, and the amount of [14C]-label
quantitated by scintillation counting.
Cloning of MtDNA Probe
The 16,295 base pair, full-length murine mtDNA was inserted into the
Sac I site of pSP64 and transfected into E. coll strain HB101 (gift of W.
Hauswirth). A 20 |il inoculate of E. coli containing the recombinant plasmid
was aseptically transferred to 250 ml of sterilized Luria broth containing 2.5 g
Bacto-tryptone, 1.25 g Bacto-yeast extract (Difco Laboratories, Detroit, Ml), 2.5
g sodium chloride, pH 7.5, and 25 mg ampicillin (Sigma Chemical). The
bacteria were incubated for 16 hrs in a 37C shaking incubator (New
Brunswick Scientific, Edison, NJ) in a 1 L sterilized flask to maximize aeration.


The bacteria were transferred to six 50-ml tubes and centrifuged at
2650g for 15 min at 4C. Pellets were resuspended in 0.5 ml sterile buffer
(25% sucrose, 50 mM Tris, pH 8.0) and combined in a 50 ml centrifuge tube.
Five mg of egg white lysozyme (Calbiochem, La Jolla, CA) was added and,
after mixing, the material was allowed to incubate at room temperature for 5
min. One ml of sterile Na2EDTA (0.25 M, pH 8.0) was added, and the
suspension was allowed to incubate at room temperature for 2 min. Eight ml
of a buffer/detergent/ (50 mM Tris and 62.5 mM Na2EDTA, pH 8.0; that was
sterilized before addition of 2% Triton X-100) was added and mixed gently for
2-5 min at room temperature until the viscosity increased markedly. The
mixture was then centrifuged immediately at 31,500g for 25 min at 12C.
The supernatant was transferred to a 50 ml centrifuge tube, the volume
measured, and an equal volume placed in each of 2 ultracentrifuge tubes.
Cesium chloride (0.9 g/ml of supernatant) (BRL Scientific, Gaithersburg, MD)
was added and mixed by inverting the tube. Ethidium bromide (0.5 mg/ml of
supernatant) was added, mixed, and protected from light in all further steps.
The tubes were balanced and centrifuged at 92,000g for 36-40 hrs at 20C.
Bands of material which fluoresced upon illumination at 360 nm were
located with a hand-held UV light (Ultra-Violet Products, Inc., San Gabriel,
CA). The bands were, starting at the top, protein, Form II DNA (nicked
circular), Form I DNA (closed circular), and RNA. Form I DNA was collected
and transferred to a centrifuge tube. The ethidium bromide was removed by
repeated extraction with isopropanol:5M NaC! (1:1) until tne isopropanci was
completely transparent two consecutive times. For each ml of solution, 2.6 ml
of distilled water and 7.2 ml of 20C 95% ethanol were added. The mixture
was then placed in an ethanol/dry ice bath for 30 min and centrifuged at
9000g for 30 min at 4C. The pellet was rinsed with 70% ethanol and vacuum


50
dried for 30 min. The final pellet was resuspended in 200 pi of TE buffer (10
mM Tris-HCI containing 1 mM Na2EDTA, pH 8.0).
The molecular weight and concentration of DNA was estimated by gel
electrophoresis of the sample and comparison to marker DNAs, the
concentration and molecular weight of which were known.
Nick Translation of MtDNA Probe
To label the mtDNA probe with [35S] nucleotides, 1 pg of the cloned
probe was added to 20 pM each of dCTP, dGTP, and dTTP, 50 pCi of
deoxyadenosine 5'(alpha [35S]thio)triphosphate (specific activity 1145
Ci/mmole, Amersham, Arlington Heights, IL), and a mixture of polymerase I
and DNase I (0.2 units and 0.2 ng, respectively) (BRL Scientific) in a final
volume of 50 pi. The mixture was allowed to incubate at 15-17C for 90 min.
Stop solution (30 mM Na2EDTA) was added, and the mixture put on ice.
The nick translated products were loaded onto a Sephadex G-50
column (Pharmacia Inc., Piscataway, NJ) which had been washed previously
with TE buffer and loaded with 3 pg yeast tRNA. The column was centrifuged
at 400g for 4 min and the eluate collected in a 1.5 ml Eppendorf tube.
Gel Electrophoresis
Gels were prepared with 0.7% agarose (Bio-Rad Laboratories,
Richmond, CA) in TBE buffer (1 M Tris, 0.5 M borate, and 50 mM Na2EDTA,
pH 8.0). Molecular weight markers were the Hind III fragments of lambda virus
DNA and a supercoiled pBR322 ladder (BRL Scientific). Tracking dye
(0.025% bromcphenol blue in 2% glycerol) was added to all samples.
Samples were electrophoresed for 150 minutes at 100 volts; the running
buffer was TBE. The gels were stained in the dark with 1 pg/m! ethidium
bromide for 30 min. The DNA was visualized at 360 nm on a UV light box
(Ultra-Violet Products). The gel was photographed under UV light with a


Polaroid camera (F-stop 8, exposure 4 sec) and Type 57 film (Polaroid Corp.,
Cambridge, MA).
Southern Blot
After electrophoresis, the gel was soaked in 100 ml 0.5 M NaOH in 1.5
M NaCI for 1 hr to denature the DNA. The gel was neutralized with 100 ml 1 M
Tris (pH 8.0) in 1.5 M NaCI for 1 hr. The gel was then soaked in 100 ml 10X
SSC (20X SSC = 3 M NaCI, 0.3 M sodium citrate, pH 7.0) for 15 min. The gel
was blotted onto nitrocellulose filters (Bio-Rad Laboratories) by the method of
Southern (212). The filters were incubated at 85C in a vacuum oven
(National Appliance Company) for 2-3 hrs. Immobilized DNA was hybridized
with the full-length mouse mtDNA probe inserted in the Sac I site of pSP64
and labeled by nick translation with [35s]-labeled dATP described above.
Dot Blot
Mitochondrial DNA was quantitated by dot blot analysis of whole cell
lysate by a modification of the method of Barker et aL (213). Aliquots of 0.5-
2.0x1 o5 cells were placed in 1.5 ml Eppendorf tubes. The cells were
sedimented by centrifugation for 10 sec at 13,000g in a microfuge (Fisher
Scientific). The supernatant was decanted, and the cells were resuspended in
4 pi of buffer (250 mM sucrose, 10 mM Tris, and 1 mM Na2EDTA, pH 7.4).
Cells were then incubated in a mixture of 2% sodium dodecyl sulfate, 5 pg
proteinase K (Sigma Chemical), and 100 units RNase T1 (BRL Scientific) in a
final volume of 7.5-8.5 pi. The initial incubation was for 15 min at 37C, a
procedure which allowed degradation of up to 4 pg of added yeast tRNA
without inactivation of the RNase T1 by the proteinase K (data not shown).
This was followed by incubation for an additional 2-3 hr at 50C.
The cell lysate was then treated with 0.1 M NaOH to denature DNA, and
the mixture was applied to nitrocellulose filters with a dot blot apparatus


52
(BioRad Laboratories). Immobilized DNA was hybridized with the full-length
mouse mtDNA probe inserted in the Sac I site of pSP64 and labeled by nick
translation with [35S]-labeled dATP described above. The dots were
visualized by autoradiography (SB-5 X-ray film, Eastman Kodak). To assure
equal size of the dots, nitrocellulose paper was cut out with a hole punch, and
quantitated by scintillation counting.
Hybridization with f33S]-labeled MtDNA Probe
Nitrocellulose paper from the Southern or dot blot was placed in a
plastic Seal-a-meal bag (Sears) and a pre-hybridization solution was added
(30 ml 6X SSC, 0.6 ml 50X Denhardt's solution [1% w/v Ficoll 400,
polyvinylpyrrolidone, and bovine serum albumin in distilled water], and 0.9 mg
denatured yeast tRNA). The bag was sealed and allowed to incubate for 8 hrs
at 68C.* The pre-hybridization solution was then drained, and the
hybridization solution added (10 ml 6X SSC, 0.2 ml 50X Denhardt's solution,
0.1 mg denatured yeast tRNA, and 50 jil denatured [35S]-labeled mtDNA).
The bag was resealed and incubated 18-24 hrs at 68C.
The hybridization solution was drained into a radioactive waste
container, and the blot was removed. The blot was washed first in 100 ml of
6X SSC for 2 hrs at 68C, and then in 100 ml of 2X SSC for another 2 hrs at
68C. The blot was placed between 2 sheets of filter paper and dried in a
vacuum oven (National Appliance Company) for 15 min at 85C. The blot was
placed under autoradiographic film (SB-5, Eastman Kodak) in a film cassette.
The film was exposed for 15-20 hrs and developed witn an X-ray film
processor (Konishiroku Photo Co., Japan).


RESULTS
Characterization of L1210 Cells
Growth Characteristics of Untreated L1210 Cells
To determine the growth characteristics of the untreated murine
leukemia L1210 cell line, cells were seeded to a starting concentration of
2.5x104 cells/ml and incubated at 37 for 5 days without reseeding or adding
fresh medium. The exponential growth, or log phase, of these cells
encompassed a concentration range from 2.5x104 to 2x106 cells/ml (Figure
4). The plateau phase of cell growth occurred at >2x106 cells/ml. Viability, as
measured by trypan blue exclusion, was >98% in log-phase cells but
progressively declined to 40% by 6 days without reseeding. The mean
population doubling time for the log-phase cells under these conditions was
11 hrs. For all further experiments L1210 cells were maintained in
exponential, or log phase, growth by reseeding every 2 days into fresh
medium and with fresh drug, where appropriate.
Coov Number of MtDNA in Untreated L1210 Cells
In order to reliably and accurately assay the mtDNA content of L1210
cells, we found it necessary to routinely incorporate an external (and
occasionally, an internal) standard in the dot blot procedure. The plasmid
pSP64, which had served as the vector for cloning of the mouse mtDNA, was
used as the standard because 1) its quantity (in terms of DNA) is easy to
assess and 2) it hybridizes to the radioiabeiied, nick-transiated probe because
the probe contains the appropriate complementary sequences. This meant


Figure 4. Cell counts/ml of L1210 cells grown in suspension culture for 6 days
without reseeding. Starting concentration of the cells was
2.5x104/ml. Growth was exponential for 3 days, and plateau phase
was reached at 2x106 cells/ml.


Days
Cell counts/ml
O O o o
4* Cl a -g
99


56
that only one probe (containing both pSP64 and mtDNA sequences) was
required for hybridization, a procedure that minimized variation between the
degree of hybridization of the standard and the mtDNA. A standard curve (ng
DNA vs cpm's [counts per minute]) was generated for each dot blot
experiment. The copy number for mtDNA in L1210 cells was calculated from
this experimental value for amount of mtDNA in a given number of cells and
the molecular weight (107 daltons) of murine mtDNA.
Certain other assumptions were validated in development of this dot
blot. First, the nick-translated probe (containing both plasmid and mouse
mtDNA) hybridized equivalently to both the plasmid standard and cellular
mtDNA, a necessary condition for direct comparison of the counts. Second,
we confirmed that the cell lysate £er se did not interfere with hybridization. It
was found that varying mixtures of cell lysate and plasmid yield the expected
additive value for DNA which hybridized to the probe. And, finally,
radiolabelled, nick-translated pSP64 plasmid itself did not hybridize with
cellular DNA.
For determination of the mtDNA copy number, known quantities (1-6
ng) of plasmid and known aliquots (1.0-6.0x105) of L1210 cells were added to
a dot blot apparatus and hybridized with a [35s]-labeled full-length murine
mtDNA probe inserted into pSP64. The extent of hybridization of probe to
mtDNA was proportional to aliquot size at least over the range of 1.0x10^ to
6.0x105 cells (Figure 5). This asynchronous population of L1210 cells
contained 1450 copies of mtDNA per cell; the mean standard deviation of 3
experiments was 13%.


Figure 5. The amount (ng) of mtDNA in increasing quantities of exponentially-
growing L1210 cells. The amount of mtDNA was measured by dot blot
analysis, and was linear over the range of 0-6x105 cells. Each point
represents the mean value of 3 separate experiments; the mean
standard deviation of the 3 experiments was 13%.


Cell no. X 10
Amount of mtDNA (ng)
i
cn
o ro a> oo o ro
8 g


59
Effects of Polvamine Analogs on Cell Division and MtDNA Accumulation
Dose-Ranging Experiments with MGBG
Concentration response for MGBG. Because the intent of these
experiments was to use MGBG to modulate the mtDNA content of L1210 cells
without causing overt cell toxicity, an MGBG concentration response
experiment was done to define the concentration range at which MGBG
inhibited mtDNA accumulation without causing a substantial decrease in cell
viablity. The initial experiment encompassed MGBG concentrations from 0.1
to 100 |iM in the medium. Cell counts and viability were determined daily for
two days.
Cell growth over this 2-day period was inhibited by MGBG in a
concentration-dependent manner (Figure 6). After 2 days of incubation with
MGBG, aliquots of cells from each treatment were lysed and the mtDNA
quantitated. When the concentration of MGBG in the medium was <1.0 (iM,
there was no effect on the content of mtDNA per cell (Table 1). At MGBG
concentrations >3.3 pM, mtDNA content per cell was decreased compared to
control, but cell viability was greatly reduced, especially at MGBG
concentrations >10 pM. Furthermore, 55% of cells treated with 10 pM MGBG
for 2 days had lost the ability to form clones (compared to untreated controls).
The IC5o's of MGBG for inhibiting cell growth at 24 and 48 hrs of
treatment were 10 and 2 pM, respectively.
Decreased mtDNA content at 1.8 to 3.3 uM MGBG. Based on these
previous results, the MGBG concentration range was narrowed to 1.0 to 3.3
pM for all further experiments. These concentrations provided optimal
differentiation of the effects of MGBG on cell growth and
rrnnimol
vviiii 11 In in i iui
overt cellular toxicity. Although MGBG had discernible effects at
concentrations <1.3 pM (see below), attention will first be directed to results


Figure 6. Cell counts/ml of L1210 cells exposed to 0-100 pM MGBG for 0-2
days. Starting concentration of the cells was 7.2x104/ml. Cell
counts were done in duplicate for each experiment; each point
represents the mean value of 2 separate experiments.


Cell counts/ml
- control
0.10 pM
-o- 0.33
1.0
-* 3.3 (iM
-o 10 jiM
-* 33 |iM
100 (iM
ON
Days MGBG treatment


62
Table 1. Effects of MGBG on the relative amount of mtDNA/cell and the
percent viability in L1210 cells exposed to 0-100 pM MGBG for 2
days.
Treatment
(pM MGBG)
MtDNA/Cell
(% Control)
% Cell Viability
Control
100
99
0.10
105
98
0.33
94
99
1.0
95
98
3.3
23
96
10
26
80
33
*
75
100

60
The mtDNA was determined 8 times and the cell viability once for each
experiment; each point represents the mean value of 2 different experiments.
not done.


63
obtained with MGBG in the range of 1.8 to 3.3 (iM, concentrations at which
there are consistent and striking effects on mtDNA accumulation. Although the
cells were exposed in these experiments to MGBG for 18 days, our initial focus
involves the first 3 days of MGBG treatment because mtDNA synthesis
became resistant to MGBG after that time. This phenomenon will be explored
in more detail in a later section.
Cells were incubated with 1.8 to 3.3 pM MGBG and reseeded every 2
days with fresh medium and drug. At these concentrations of MGBG, cell
growth was inhibited in a concentration-dependent manner, i.e. the greater the
dose, the greater the degree of inhibition (Figure 7). However, this difference
was small for the first 3 days of exposure. Conversely, the effect of MGBG on
mtDNA accumulation did not show a gradation in degree of inhibition, but
rather in the duration of inhibition (Figure 8). Incubation of L1210 cells with
1.8, 2.5, or 3.3 pM MGBG prevented accumulation of mtDNA for 1,2, and 3
days, respectively.
Because cell growth continued at a rate which was relatively faster than
the accumulation of mtDNA, a decrease in the amount of mtDNA per cell can
be anticipated with each cell division. The amount of mtDNA per cell did
decrease progressively to only 10% of the control value upon treatment with
2.5 or 3.3 pM MGBG (Figure 9). The time course of this decrease was
consistent with a complete inhibition of mtDNA accumulation coupled with
continued cell division (see theoretical curve in Figure 9), i.e., the amount of
mtDNA per cell was halved with each division.
Determination of mtDNA degradation by MGBG. The inhibition of
mtDNA accumulation by MGBG may reflect inhibition of synthesis, increased
degradation, or both. Consequently, mtDNA was isolated from MGBG treated
cells to determine if there was increased mtDNA damage relative to control.


Figure 7. Cumulative cell counts for L1210 cells exposed to 0-3.3 pM MGBG for
0-3 days. Cells were reseeded every 2 days with fresh media and
drug. Cell counts were done in duplicate for each experiment; each
point represents the mean value of 4 separate experiments.


Cumulative cell counts
-a- control
1.8(iM
2.5 )iM
3.3 (iM
o\
Days MGBG treatment


Figure 8. MtDNA accumulation for L1210 cells exposed to 0-3.3 pM MGBG for 0-
3 days. Cells were reseeded every 2 days with fresh media and drug.
The mtDNA was determined 8 times and the cell counts twice for
each experiment; each point represents the mean value of 4 separate
experiments.


MtDNA accumulation
a>
o
<
Z
a
E
x
(0
c
3
o
o
O
control
1.8 (iM
2.5 nM
-o- 3.3 |iM
o\
Days MGBG treatment


Figure 9. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-3.3 pM MGBG for 0-3 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 4 separate
experiments.


140
120
100
80
60
40
20
0
control
-+ 1.8 |iM
2.5
-o- 3.3 [iM
theoretical*
O
VO
T
1
T
2
3
Days MGBG treatment


70
Mitochondrial DNA from equal numbers of control or MGBG treated cells was
isolated and separated into Forms I, II, or III (supercoiled, nicked circular, or
linear, respectively) by gel electrophoresis. The gel was transferred to a
nitrocellulose filter, and the filter was hybridized with a [35S]-labeled mtDNA
probe.
Autoradiography of the blot showed that there was no substantial
change in the ratio of intact and nicked forms upon exposure of cells to 1.0 or
3.3 |iM MGBG. However, the total amount of mtDNA at 1.0 pM had decreased
and at 3.3 pM this decrease was marked. At a higher dose (10 pM) than those
used in the mtDNA accumulation experiments, there was a decrease in the
intact form and a corresponding increase in the linear form of mtDNA.
Interestingly, however, the total content of mtDNA was more than at the 3.3 pM
treatment. This suggests that damaged mtDNA was not degraded and that
cell growth was so inhibited at 10 pM MGBG that the mtDNA copy number was
diluted less than at 3.3 pM. Taken together, these results are compatible with
the hypothesis that the decrease in mtDNA content, at least in the 1.0 to 3.3
pM range, reflected a lack of synthesis rather than increased degradation.
Effects of Diethylspermine (DES) on Cell Growth and MtDNA
We tested another polyamine analog to determine if it might have
similar effects on mtDNA accumulation and cell growth. This work was done
in collaboration with Dr. Ray Bergeron and Mike Ingeno in the College of
Pharmacy. Diethylspermine (DES, a gift of R. Bergeron, is spermine with ethyl
substituents on each of the 2 terminal nitrogen atoms) was made to a stock
solution of 1 mM. The L1210 cells were reseeded with fresh medium and drug
every 2 days, and incubated with 0, 0.1, 1.0, or 10 pM DES for 6 days. The
growth rates of cells exposed to 0.1 pM DES were the same as control (data
not shown). Cells exposed to 1.0 and 10 pM DES for 1 day had normal


growth, but their growth was inhibited after 2 days, and was slowest from 4-6
days (Figure 10). Cell viability (as measured by trypan blue exclusion) was
>90% over the 6-day period.
The mtDNA accumulation was not affected in cells exposed to 0.1 pM
DES (data not shown). However, the mtDNA accumulation of cells exposed to
1.0 or 10 pM DES was substantially inhibited and showed no signs of
recovery during the 6-day exposure to DES (Figure 11). Whereas the cells
exposed to 0.1 pM DES had a normal content of mtDNA, the mtDNA content of
cells exposed to 1.0 or 10 pM DES decreased to 10-15% of control by 3 days
(Figure 12). The mtDNA content remained at this low level through day 6 of
DES exposure.
Effects of Low Doses of MGBG
Increased mtDNA content at 1.0 to 1.3 uM MGBG. The results obtained
with MGBG concentrations <1.8 pM were interesting. After 1 day of treatment
with 1.0 or 1.3 pM MGBG, there was a slight inhibition of mtDNA accumulation
relative to control (Figure 13), in general agreement with the decreasing
duration of inhibition with decreasing MGBG concentration (Figure 8).
Thereafter, the rate of mtDNA accumulation equaled that of control. The rate
of cell growth was slightly inhibited by 24 hrs but, unlike mtDNA accumulation,
continued to be slightly inhibited throughout the 6-day exposure (Figure 14).
By dot blot analysis, 1.0 and 1.3 pM MGBG caused a 12 and 25% decrease,
respectively, in the content of mtDNA per cell at 24 hrs (Figure 15). However,
after this initial decrease, the amount of mtDNA per cell increased and by 4
days actually exceeded the control value. This increase resulted from a
normai rate of mtDNA accumulation in cells that were slightly growth-inhibited.
When the mtDNA content per cell was followed for 18 days at these
concentrations of MGBG, the mtDNA copy number never exceeded 155% of


Figure 10. Cumulative cell counts for L1210 cells exposed to 0-10 pM DES
for 0-3 days. Cells were reseeded every 2 days with fresh media
and drug. Cell counts were done in duplicate for each experiment;
each point represents the mean value of 2 separate experiments.


Cumulative cell counts
control
1 (iM
-o- 10 |iM
u>
Days DES treatment


Figure 11. MtDNA accumulation for L1210 cells exposed to 0-10 pM DES for
0-3 days. Cells were reseeded every 2 days with fresh media and
drug. The mtDNA was determined 8 times and the cell counts twice
for each experiment; each point represents the mean value of 2
separate experiments.


Days DES treatment
MtDNA accumulation
[Cell counts X (%mtDNA/cell)]
o o o o o
tn o -sj oo co
* i *
o
O T= O
T= S =
^ O
SL


Figure 12. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-10 jiM DES for 0-6 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 2 separate
experiments.


120
100
80
60
40
20
0
-o- control
1 |iM
- 10 nM
i 1 1 1 1 1
1 2 3 4 5 6
Days DES treatment


Figure 13. MtDNA accumulation for L1210 cells exposed to 0-1.3 pM MGBG for
0-6 days. Cells were reseeded every 2 days with fresh media and
drug. The mtDNA was determined 8 times and the cell counts twice
for each experiment; each point represents the mean value of 4 or 2
separate experiments for 1.0 or 1.3 pM, respectively.


MtDNA accumulation
a>
o
z
o
X

c
3
O
O
CD
o
0 1 2 3 4 5 6
control
1.0 |^M
-o- 1.3 pM
si
VO
Days MGBG treatment


Figure 14. Cumulative cell counts for L1210 cells exposed to 0-1.3 pM MGBG
for 0-6 days. Cells were reseeded every 2 days with fresh media
and drug. Cell counts were done in duplicate for each experiment;
each point represents the mean value of 4 or 2 separate experiments
for 1.0 or 1.3 pM, respectively.


Cumulative cell counts
0 1 2 3 4 5 6
Days MGBG treatment
+ I -Si


Figure 15. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-1.3 pM MGBG for 0-6 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 4 or 2 separate
experiments for 1.0 or 1.3 pM, respectively.


160
140
120
100
80
60
40
20
0
control
-* 1.0|iM
-- 1.3 (iM
oo
LO
Days MGBG treatment


84
control. Thus, even though the rates of mtDNA accumulation and cell growth
at low concentrations of MGBG were similar, there were subtle differences that
caused the mtDNA content per cell to vary between 75% of control early in
exposure to 155% of control after several days.
Effects on mtDNA of decreased cell growth due to serum depletion.
Because of the unexpected increase in mtDNA per cell following incubation
with low concentrations of MGBG, an experiment was done to determine if this
increase was a nonspecific effect of decreased cell growth. If mtDNA
replication and cell division are not tightly coupled, then it can be
hypothesized that an increased mtDNA copy number may result from any
condition which causes decreased cell growth. To test this idea, mtDNA
content was measured in L1210 cells incubated in media containing limiting
amounts of serum. Decreases in serum concentration slowed cell growth in a
proportional way; the mtDNA content per cell at 3 days, however, was not
different from control (Figure 16).
Resistance to MGBG and Recovery of MtDNA Synthesis and Copy Number
Resistance to MGBG
Resistance of mtDNA synthesis to MGBG. As mentioned earlier, mtDNA
synthesis in L1210 cells resumed 1,2, or 3 days after the start of exposure to
1.8, 2.5, or 3.3 pM MGBG, respectively. This recovery occurred despite the
continued presence of drug in the medium, and suggested that the cell was
able to overcome the initial effects of MGBG on mtDNA accumulation. In
addition, mtDNA synthesis, once resumed, occurred at the same rate,
regardless of dose of MGBG (Figure 17). Surprisingly, this rate of mtDNA
accumulation was virtually equal to that seen in untreated, growing cells--in
spite of the fact that the treated cells exhibited a decreased growth rate over
this time period (Figure 18).


Figure 16. The mtDNA/cell (expressed as % control) of L1210 cells incubated
for 3 days in media supplemented with different percentages of
serum. Cells were reseeded on day 2 with fresh media and the
appropriate amount of serum. The mtDNA was determined 8 times
for each experiment; each point represents the mean value of 2
separate experiments. Ten percent serum represents the control
value.


% Serum
% Control mtDNA
98
120


Figure 17. MtDNA accumulation for L1210 cells exposed to 0-3.3 pM MGBG for
0-6 days. Cells were reseeded every 2 days with fresh media and
drug. The mtDNA was determined 8 times and the cell counts twice
for each experiment; each point represents the mean value of 4 or 2
separate experiments for 1.8 and 2.5, or 3.3 pM, respectively.


MtDNA accumulation
o
z
Q
X
V)
c
Z3
o
u
o
O
-o- control
1.8 |jM
2.5 (jM
-o- 3.3 nM
oo
oo
Days MGBG treatment


Figure 18. Cumulative cell counts for L1210 cells exposed to 0-3.3 pM MGBG
for 0-6 days. Cells were reseeded every 2 days with fresh media
and drug. Cell counts were done in duplicate for each experiment;
each point represents the mean value of 2 or 4 separate experiments
for 1.8 and 2.5, or 3.3 pM, respectively.


Cumulative cell counts
0 1 2 3 4 5 6
control
1.8 (iM
-o- 2.5 |iM
-* 3.3 nM
NO
o
Days MGBG treatment


In contrast to the quantal (on/off) effect of MGBG on mtDNA
accumulation, cell growth exhibited a graded response to varying doses of
MGBG (Figure 18). Cell growth was inhibited in a concentration-dependent
and time-of-treatment-dependent manner.
The net effect of these different rates of mtDNA accumulation and cell
growth was to modulate the mtDNA copy number of these cells (Figure 19).
As noted earlier, there was an initial decrease in the copy number of mtDNA
due to complete inhibition of mtDNA accumulation for up to 3 days. After that,
the copy number increased towards the control value because of the
combined effects of the recovery of mtDNA synthesis and a marked decrease
in the cell growth rate.
Effects of transfer of treated cells to a MGBG-free medium. This result,
in which mtDNA accumulation recovered, but at a rate which never occurred
faster than the 12-hr doubling time of control cells, was unexpected in view of
the reported observation that the mtDNA genome can replicate in vitro in
about 1 hr (214). One possibility was that this 12-hr doubling time
coincidentally represented a continued, but lesser, inhibition of mtDNA
replication by MGBG. To rule out this possibility, a study was done in which
L1210 cells were treated with MGBG and then transferred to drug-free medium
at the time at which recovery of mtDNA synthesis regularly occurred. If the
drug continued to cause partial inhibition of mtDNA synthesis, then those cells
transferred to drug-free medium should have a faster rate of mtDNA
accumulation than those remaining in drug.
The L1210 cells were treated with 3.3 pM MGBG for 4 days. At that
time, haif the ceiis were maintained ai 3.3 pM while the other half were
transferred to drug-free medium for an additional 4 days. Cell counts and dot
blot analysis of mtDNA content were done during the initial exposure to


Figure 19. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-3.3 jiM MGBG for 0-6 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 4 or 2 separate
experiments for 1.8 and 2.5, or 1.3 pM, respectively. The bars for
the 3.3 pM group represent 1 standard deviation.


Full Text

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81,9(56,7< 2) )/25,'$


UNIVERSITY OF FLORIDA
3 1262 08554 4053


EFFECTS OF POLYAMINE ANALOGS
ON MITOCHONDRIAL DNA
AND CELL GROWTH
By
RITA BELLIS BORTELL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987

This dissertation is dedicated to David Alan Bortell, for the support and
encouragement he gave me during this endeavor.

ACKNOWLEDGMENTS
I would like to thank my supervisor and mentor, Dr. Allen Neims, for his
careful guidance of my graduate training. I also want to thank the members of
my committee: Drs. Stephen Baker, William Hauswirth, Margaret James,
Warren Ross, and Thomas Rowe. I would like to extend my appreciation to the
fellow members of our laboratory: Lynn Raynor, Gurmit Singh, Lori Lim, Debra
Stinson, Sukanya Kanthawatana, Daniel Danso, and Mary Anne Kelly. I
particularly want to thank Mike Ingeno, Bonnie O'Brien, David Bortell, Debra,
and Lynn for the generous help they gave me. Special thanks go to Shari
McArdle, with whom I most closely shared the graduate school experience. A
final debt of gratitude goes to Dr. Allen Neims and Lynn Raynor, who greatly
enriched my training and made the experience fun.
in

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
ABSTRACT vi
INTRODUCTION 1
LITERATURE REVIEW
Mitochondrial DNA (MtDNA) 3
Nuclear/Mitochondrial Regulation 9
Polyamines 24
MGBG (Methylglyoxal bis[guanylhydrazone]) 31
Specific Aims 43
MATERIALS AND METHODS
Cell Culture 45
Cell Counts 45
Determination of IC50 46
Cell Viability 46
Clonogenic Assay 46
Cell Size 47
Flow Cytometry 47
Rhodamine 123 Uptake 47
[14C]-MGBG Stock Solution Preparation 48
Uptake Protocol 48
Cloning of MtDNA Probe 48
Nick Translation of MtDNA Probe 50
Gel Electrophoresis 50
Southern Blot 51
Dot Blot 51
Hybridization with [35s]-labeled MtDNA Probe 52
RESULTS
Characterization of L1210 Cells 53
Effects of Polyamine Analogs on Cell Division and MtDNA
Accumulation 59
Resistance to MGBG and Recovery MtDNA Synthesis and Copy
Number 84
Effects of MtDNA Content on Mitochondrial and Cell Functions .... 11 1
Uptake of MGBG 113
DISCUSSION
Determination of the MtDNA Copy Number in L1210 Cells *\2ó
Decrease in Cell Growth and MtDNA/Cell with MGBG and DES
Exposure 124
Mitochondrial Function During MGBG Exposure 127

Increased MtDNA/Cell with Low Concentrations of MGBG 129
Recovery of MtDNA Replication 130
Implications for Nuclear/Mitochondrial Interactions 132
SUMMARY 141
REFERENCES 145
BIOGRAPHICAL SKETCH 157
v

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EFFECTS OF POLYAMINE ANALOGS
ON MITOCHONDRIAL DNA
AND CELL GROWTH
By
Rita Beilis Bortell
December 1987
Chairman: Allen H. Neims
Major Department: Pharmacology and Therapeutics
The average mouse leukemia L1210 cell contains 1450 copies of
mitochondrial DNA (mtDNA). Somehow this number is maintained during
exponential growth. We have studied the effects of the polyamine analogs,
methylglyoxal bis[guanylhydrazone] (MGBG) and diethylspermine (DES), in
order to explore this relationship because appropriate doses of the drugs
selectively Inhibit mtDNA replication without causing a decrease in cell
viability, at least in the case of MGBG.
Exponentially-growing L1210 cells were exposed to 1.0-3.3 pM MGBG
for 1-31 days. Cells were collected on various days for dot blot quantitation of
mtDNA by hybridization of cell lysate to a [35s]-labeled, full-length mouse
mtDNA probe. At >1.3 pM MGBG, mtDNA replication was completely inhibited
for variable times depending on dose (64 hr at 3.3 pM). Since cell division
continued, albeit somewhat slower, the mtDNA content per cell was diluted
with each cell division. Whereas cell size decreased when the mtDNA copy
v 1

number per cell was low, the potential-dependent uptake of rhodamine 123
uptake Into mitochondria was not affected by MGBG treatment.
Diethylspermine had similar initial effects on mtDNA accumulation and
cell growth. With exposure to either drug, the amount of mtDNA in viable cells
never decreased below 10% of the control value because cell division
ceased. Although the effects of DES on mtDNA accumulation were not
reversible, mtDNA synthesis of MGBG-exposed cells resumed after 3 days.
When mtDNA synthesis resumed, the mtDNA content per cell doubled every
12 hours whether or not drug had been removed from the medium. The fact
that this mtDNA synthesis occurred when the rate of cell division was at its
lowest resulted in repletion of the cellular content of mtDNA. When the mtDNA
was nearly repleted, the rate of cell division recovered to the control value of
12 hours. The observation that the doubling of mtDNA in the virtual absence
of cell division required the same 12 hours suggests that replication of mtDNA
may proceed at its maximal rate in untreated exponentially-growing L1210
cells and actually limit how rapidly these cells can divide.
The mechanism of recovery of mtDNA synthesis in these cells was not
due to genetic mutation, but rather to a phenotypic adaptation. The L1210
cells initially concentrated [14C]-MGBG more than 1000-fold relative to the
medium. When mtDNA synthesis resumed, the apparent intracellular
concentration of the drug had begun to decrease to a new steady state value,
although the concentration was still higher than that earlier associated with
inhibition. The mtDNA copy number of cells continuously exposed to MGBG
had returned to the control value at 31 days.
vii

INTRODUCTION
The number of copies of mitochondrial DNA (mtDNA) seems to be
characteristic for a given cell type, whether that cell is dividing or not (1,2). It
can be appreciated that maintenance of the characteristic copy number in
dividing cells is especially complex. For this amount of mtDNA per cell to be
maintained, the mtDNA must double with each cell division. Because the
nucleus and mitochondria are distinct organelles, it is not understood how this
regulation occurs.
This dissertation focuses on the basic question of how the characteristic
copy number of mitochondrial DNA (mtDNA) is maintained in dividing cells.
My basic approach is to perturb rapidly dividing cells with a drug or compound
which selectively inhibits mtDNA replication and use dot blot analysis and
measurements of cell number to follow the resultant effects on the copy
number of mtDNA and on cell division.
The drug I chose to use was methylglyoxal bis(guanylhydrazone), or
MGBG. This drug is cationic, metabolically stable, and was developed as a
polyamine congener (3). It has been used clinically as an antiproliferative
agent and to treat trypanosomiasis (4, 5). I selected this drug because it
displayed two important characteristics. First, it inhibits mtDNA replication
without damaging the mtDNA at concentrations which initially have only a
minimal effect on nuclear DNA replication (6, 7). Second, preliminary studies
in our laboratory suggested that long-term treatment (up to 31 days) of L1210
cells with MGBG did not substantially affect cell viability. Consequently, this
1

2
drug could be used to study the cell's response, overtime, to differential
perturbation of mtDNA and cell replication.

LITERATURE REVIEW
The literature review will be divided into four sections. They are 1) an
overview of the literature on mtDNA; 2) a more detailed review of the
interaction between the nuclear and mitochondrial genetic systems; 3) a brief
survey of polyamine research; and 4) a synopsis of the literature on MGBG
and related polyamine analogs. This chapter will conclude with a summary of
the specific goals of my research.
Mitochondrial DNA
Mitochondria are the organelles of eukaryotic cells which are
responsible for oxidative phosphorylation, among other functions. According
to the endosymbiotic theory of the evolutionary origin of the eukaryotic cell, the
mitochondrion evolved from a free-living prokaryote, probably the purple
nonsulfur bacteria (Rhodospirillaceae) (8), which was incorporated into an
ancestral eukaryotic cell (9). Prior to this, the protoeukaryote had depended
on fermentation for the production of ATP. In time the prokaryote suffered a
progressive loss of autonomy: its rate of proliferation became linked with that
of the host cell, many of its biosynthetic capabilities were lost or taken over by
the host, and its metabolic functions became integrated with that of the host.
This theory implies that the inner mitochondrial membrane and its
invaginations (the cristae) are homologous with the plasma membrane of
present-day bacteria, whereas the outer mitochondrial membrane is derived
from the protoeukaryote.
3

4
Mitochondria are self-replicating, arising only by growth and division of
preexisting mitochondria (10). The existence of DNA within mitochondria was
first observed by Nass and Nass in 1963 (11). Mitochondrial DNA is a double-
stranded circular genome (Figure 1); exceptions are the linear mtDNA
molecules of the ciliated protists Tetrahvmena and Paramecium (10). The
genome's two strands contain different proportions of purines, and thus can be
separated by density gradient centrifugation into heavy strand (H-strand) and
light strand (L-strand). Immediately after replication of mtDNA, the two
daughter molecules exist in a relaxed circular state. Within one hour under
normal conditions, however, a mitochondrial topoisomerase introduces
approximately 100 supercoils into each molecule (12).
This supercoiled form may subsequently be converted into a
displacement loop (D-loop) form which is unique to the mitochondrial genome.
This triplex D-loop structure is formed by the synthesis of a short daughter H-
strand that remains stably associated with the parental closed circle; the
parental H-strand is consequently displaced as a single strand. The D-loop
region varies in length between different species, and sometimes within the
same animal (13). In general, the D-loop is approximately 1000 base pairs
(bp) in length (14).
Mitochondrial DNA of human, murine, and bovine species has been
completely sequenced and consists of approximately 16,000 bp (14). In
addition, these sequences have been completely mapped for restriction sites
(15) and locations of promoters for replication and transcription of both heavy
and light strands of mtDNA (16,17). The coding regions of the genome have
been mapped, and the majority of the proteins (12 of 13) and transfer RNAs
(tRNAs) (16 of 22), and both ribosomal RNAs (rRNAs) are coded for by the H-
strand (Figure 1). The only noncoding areas of mammalian mtDNA are a short

5
Figure 1. Schematic of human mtDNA. Hatched segments show the extent of
genes for known proteins, filled segments show the genes for
transfer RNA's, and crossed segments show the genes for
ribosomal RNA’s.

6
sequence outside the D-loop region which encompasses the origin of L-strand
replication, and the D-loop region which contains the promoter sites for H-
strand replication and both H- and L-strand transcription (14).
There are two current models for mitochondrial transcription in
mammalian cells. One model proposes that transcription begins near the
origin of replication and continues as a single polycistronic transcript around
the entire genome, but with a high probablity of premature termination
immediately following the 12S-16S region (18). The alternative model
suggests that there are two promoters for the initiation of RNA synthesis on the
H-strand near the origin of replication (19, 20). Transcription from one site
circumscribes the entire mitochondrial genome, while transcription from the
other site terminates after the two rRNA cistrons which are immediately
downstream. Once transcription has occurred, the polycistronic precursors
are cleaved into mature messenger RNAs (mRNAs), probably before the
genome is completely transcribed (21). Since there are very few nucleotides
between adjacent genes on mammalian mtDNA (and sometimes none), the
signals for RNA processing may not be specific sequences ger s£ (22). Most
of the mRNAs and both rRNAs are flanked by tRNA genes, and the secondary
structure of these tRNAs in the transcript may provide the signals for cleavage
by the RNA processing enzyme (23).
The mitochondrial genome is thought to be associated with the inner
membrane at its matrix face (24). Molecules of mtDNA are frequently seen to
be attached to fragments of membrane or proteinaceous material after gentle
lysis of the organelles (9). In some cases there is good evidence that such
associations are not artifacts; for example, in one study it was shown that
membrane fragments were always attached to approximately the same

7
position on HeLa cell mtDNA (10). Thus, it is probable that mtDNA molecules
are attached to organelle membranes at least some of the time.
Apart from this attachment, isolated organelle genomes generally are
not complexed with histones or other proteins in the manner of true
chromosomes in the nucleus; exceptions are the highly condensed mtDNA
molecules of the slime molds which appear to be complexed with a basic
protein (25), and the mtDNA of Xenopus oocytes, which can be isolated in
association with protein in the form of structures resembling the nucleosomes
of chromatin (10). Also, recent evidence suggests that the displaced single
strands (D-loop) of mtDNA, but not the double-stranded segments of the
genome, are coated with DNA-binding proteins apparently unique to mtDNA
(26, 27). These proteins are thought to play a critical role in maintaining the
integrity of these triplex replication loops.
In mammals, the mitochondrial genome codes for 13 proteins and
some, but not all, of the components necessary for protein synthesis; this
includes two rRNAs and 22 tRNAs (28). The known human mtDNA-coded
proteins include 3 subunits of cytochrome oxidase, one of ATPase, 8 of NADH
dehydrogenase, and one of cytochrome b (29, 30). Antibodies raised to
synthetic peptides predicted from the DNA sequence have been used to
establish the existence of translated products from all previously unassigned
reading frames (URFs) (31). Attardi and his coworkers (32) recently identified
the last URF of human mtDNA as a subunit of NADH dehydrogenase. All of
the mtDNA-coded proteins are integral components oí the inner mitochondrial
membrane and play key roles in oxidative phosphorylation. It follows,
therefore, that the mitochondrial genetic system is indispensable for the
biogenesis of the aerobic energy-generating system of all eukaryotic cells.

8
Whereas mtDNA codes for 13 mitochondrial polypeptides, the
organelle contains over 270 different proteins (29, 33). The remaining
polypeptides, which account for approximately 90% of total mitochondrial
protein, are encoded in nuclear DNA and synthesized on cytoplasmic
ribosomes. Available evidence indicates that each of these proteins contains
a leader sequence enriched in basic (positively-charged) amino acids which
directs the protein to the mitochondria, possibly to the point of adherence
between the inner and outer membranes (34). The protein is then imported
into mitochondria via a set of processes dependent on the existence of a
mitochondrial membrane potential (35).
If the majority of mitochondrial proteins are imported from the
cytoplasm, then why do cells expend more than a hundred nuclear genes just
to allow the mitochondrial genetic system to transcribe and translate about a
dozen polypeptides in situ? Why, in fact, does the mitochondrial genome
retain the genes for any of the particular proteins that it has? It has been
suggested that mitochondrial translation products are so hydrophobic that they
are best made m (36). However, other hydrophobic proteins are
successfully transported in the cell (37), and some mitochondrially coded
proteins are as hydrophilic as some soluble proteins (38).
Alternatively, it may be that all eukaryotes are in the evolutionary
process of transferring mitochondrial genes to the nucleus. The fact that the
more highly evolved eukaryotes have the smallest mitochondrial genome is
consistent with this idea. The mammalian mitochondrial genome has about
16,000 kb; yeast mtDNA is 5 times longer largely because 1) it has more
genes than mammalian mtDNA, 2) several genes have introns, and 3) it
contains long noncoding stretches between genes (14). This finding suggests
that there has been a dynamic net flux of mitochondrial genetic material into

9
the nucleus (39). Indeed, firm evidence has emerged that mtDNA can be
stably integrated into nuclear genomes (40). Rearranged parts of the
mitochondrial genes var 1, cob/box and orl/rep have been found in the nuclear
genome of yeast (41). Homologues of mtRNA genes occur in locust nuclei
(42) and mtDNA sequences have been reported in sea urchin (43) and rat
nuclear DNA (44). In addition, transposition of mtDNA into nuclear sequences
may be more than just an evolutionary phenomenon, as this process has been
reported to occur as a normal part of senescence in Podospora (45). Recent
evidence, however, suggests that this latter observation may be artifactual
(46). In any event, it seems increasingly likely that normal or abnormal
mtDNA, released into the cytosol during mitochondrial breakdown or the
normal organelle fusion/segregation cycles, could become incorporated into
nuclear DNA (or vice versa).
However, a possible limitation in the transfer of functional genes from
the mitochondrion to the nucleus is the slightly different genetic code used by
the two systems. One of the triplicate codons in mitochondria codes for
tryptophan, but is recognized as a stop codon in the nucleus (47). The 13
critical protein-encoding genes that remain in the mammalian mitochondia
have such a codon (48). This would make further transfer of usable genes to
the nucleus very improbable.
Nuclear/Mitochondrial Regulation
Regulation of Mitochondrial Enzymes in Differentiated Cells
Because most of the mitochondrial proteins are encoded in nuclear
genes and synthesized on cytoplasmic ribosomes, interactions between the
nucleus and mitochondria must play an important role in mitochondrial
regulation. Respiratory enzymes and mitochondrial membranes are normally

synthesized and/or degraded in a coordinated fashion as the proportions of
the various components are characteristic for a given cell (49). The content of
mitochondrial enzymes in mammalian cells is regulated by several
environmental conditions including oxygen tension and hormones (50, 51,
52). One major question is whether the mitochondrial proteins, or even just
those of the inner membrane, are regulated individually or as a set. Because
various mitochondrial components have distinct turnover times and different
degrees of nuclear and mitochondrial control including specific genomic
expressions, individual proteins appear to be individually regulated (53, 54).
But, as discussed below, there are exceptions to this general rule.
During hypoxic incubation of mouse lung macrophages and rat skeletal
muscle L8 cells, the activities of various mitochondrial enzymes (both nuclear
and mitochondrially coded) decreased about 50-60% in tandem with no
statistical difference between the various activities (55). The kinetics of this
decrease and of the recovery of activity after reexposure to normoxia were
also similar for the different enzyme activities. This suggests that, at least in
certain cases, mitochondrial enzymes can be regulated as a unit.
It is known that nuclear gene expression is frequently regulated at the
transcriptional level (56); however, the level(s) at which mtDNA gene
expression is controlled is unknown. Some reports have addressed this
question from the point of view of gene dose or mtDNA copy number, I.e. is the
increase or decrease in mtDNA gene expression proportional to changes in
the content of mtDNA? In the experiment described above, although there
were uniform decreases in mitochondrial enzyme activities after exposure to
hypoxia, there was no change in the mtDNA copy number, a result that speaks
against regulation of mitochondrial gene expression only by gene dosage.

Another experiment which sought to answer whether mtDNA gene
dosage controls gene expression was conducted by Williams (57). He
determined the concentrations of mtDNA, mitochondrial rRNA, and
cytochrome b mRNA (a mitochondrial gene product) in normal versus
electrically-stimulated rabbit striated muscles. This tissue was chosen
because the oxidative capacity of mammalian striated muscles can vary nearly
10-fold, a finding which presumably reflects major differences in the
expression of genes (including mtDNA) that encode enzymes of oxidative
metabolism (58). After electrical stimulation of the muscle, mtDNA, mtRNA,
and the mitochondrially coded gene product (cytochrome b) were observed to
vary in direct proportion to the oxidative capacity of the tissue. Because the
expression of mitochondrial genes in mammalian striated muscle was
proportional to their copy number, these results do support the hypothesis that
amplification of the mitochondrial genome relative to chromosomal DNA, i.e.
gene dosage, was responsible for the enhanced expression of mitochondrial
genes in highly oxidative tissues (57).
This interpretation, of course, differs from that of the hypoxia experiment
described above. The different conclusions reached in the two experiments
may relate to the different tissues and/or animals used. Also, the differences
may reflect the different experimental designs; e.g., there were no changes in
mtDNA copy number after 4 days of hypoxia, but neither were there changes
in mtDNA content after the same period of muscle stimulation (the changes
occurred after 7 days). In any event, the regulation of mitochondrial gene
expression in differentiated cells, to date, is not well understood.
Regulation of Mitochondrial Enzymes in Transformed Cells
The regulation of mitochondrial gene expression in transformed
mammalian cells is also not well understood (59). The pioneering manometric

studies of Warburg in the 1920s (60) first revealed that tumor cells are
defective in respiration and have abnormally high rates of aerobic glycolysis.
Warburg suggested that the cancer cell originates from heritable injury to the
mitochondria in that the respiratory system of all cancer cells was thought to
be damaged (61, 62). By 1960, this hypothesis had lost much of its impact
because elevated rates of glycolysis could not be associated in any consistent
manner with changes in the structure or function of the mitochondrion (63, 64).
Nevertheless, numerous publications in the last two decades have
catalogued a wide range of abnormalities in tumor cell mitochondria. For
example, tumor cells apparently contain fewer mitochondria than normal cells
and the organelles in the tumor cells may be structurally aberrant (65, 66).
Many deficiencies of energy-linked functions have also been reported, and
they include reduced respiratory control (ADP-stumulated state 3 respiration)
(67). Inner membrane alterations are also seen in tumor mitochondria, such
as changes in the amount and properties of ATPase (68). The levels of
mitochondrial-associated enzymes such as hexokinase are also different in
tumor cells (69, 70). It is possible that these complex multiple changes in
membrane characteristics result from rearrangements of mtDNA (71) or from
topological aberrations of the organellar genome (72). Indeed, aberrations of
mtDNA (mainly associated with an increase in the percentage of dimers and
catenanes) have been found in tumor cells (73).
Relationship Between Mitochondrial Protein and Gene Dosage
Almost all eukaryotic cells contain many molecules of mtDNA (10). The
exceptions are a few fungi and more than a thousand species of protozoa that
have no mitochondria (46). Moreover, under given metabolic conditions,
different cells appear to have a characteristic mitochondrial and mtDNA
content (1). How is this regulated? There have been attempts to relate

mtDNA content to the number of mitochondria or the mass of mitochondrial
protein isolated. The studies reviewed by Nass (74) and by Borst and Kroon
(75) indicate a yield of 0.2 to 1.8 pg of mtDNA per mg of mitochondrial protein,
or a content of roughly 2 to 10 mtDNA molecules per organelle, depending
upon the animal cell or tissue examined. Thus, multiple mitochondrial
genomes are packaged into each mitochondrion, and there are as few as one
mitochondrion to more than several thousand mitochondria per cell,
depending on the type.
However, these older studies were somewhat suspect because it was
difficult to selectively assay mtDNA, given the need to first separate it from a
several hundred-fold excess of nuclear DNA. Consequently, Bogenhagen
and Clayton (1) studied the relationship of mtDNA content to mitochondrial
volume in cells lacking the ability to incorporate exogenous thymidine into
nuclear DNA (because they had lost the major cellular thymidine kinase, TK-).
Such cell lines retain a mitochondrial-specific activity which allows mtDNA
labeling with exogenous radioactive thymidine. Stereological analysis (76) of
thin sections of two of these cell types (LMTK* and LDTK-) revealed that both
contained 5±1% of the total cell volume within mitochondria. Even after
considering the larger mitochondrial content of HeLaTK- cells (7% of cell
volume), the mtDNA content of HeLaTK- cells was at least four times larger
than that of either TK' L cell line. Thus, the mtDNA content did not simply
relate to the volume of mitochondria. This result was consistent, however, with
the qualitative observation that the mitochondria of HeLaTK' cells were
uniformly more well developed (i.e. contained more cristae per unit volume)
than the mitochondria of L cells.

Cell Division and Regulation of MtDNA Copy Number
As noted above, an added degree of complexity is that a given cell type
seems to maintain a characteristic copy number of mtDNA even when dividing
(1). This implies that the mtDNA content must double before each cell
division. Yet, the replication of mtDNA in dividing cells is not linked to the S
phase of the cell cycle, rather it apparently occurs continuously throughout the
cell cycle (77, 78). In addition, mtDNA may replicate in the absence of nuclear
DNA replication, as in cellular hypertrophy (24).
How is the mtDNA copy number regulated during cell division? For the
copy number to be maintained during exponential cell growth, the number of
mtDNA molecules in a culture, N, must parallel the increase in cell number,
N=N0ex1, where N0 is the number of molecules present at t=0, and t equals the
cell generation time when N/N0=2 (79). However, the rates of mitochondrial
and nuclear DNA synthesis may each be affected differently by exposure to
vanous chemicals, thereby conceivably altering the mtDNA copy number. The
antiproliferative agent, MGBG, is one such compound. It inhibits mtDNA
synthesis at concentrations which have little effect on nuclear DNA synthesis.
This and other aspects of the drug will be considered in more detail in a later
section.
The phenanthridine dye, ethidium bromide, is a well-known inhibitor of
mtDNA replication and transcnption (80, 81). When human VA2-B cells were
treated with 20 ng/ml ethidium bromide, a progressive dilution of the mtDNA
content, down to 10% of control cell values after three doublings, has been
reported (82). This large reduction is consistent with a logarithmic dilution in
the number of preexisting mtDNA molecules per cell, which in the absence of
any new synthesis or turnover should result, after 3 cell doublings, in a level
which is 1/8 or about 12% of normal. As yet, no study dealing with the long-

term effect of the drug on the mtDNA content of mammalian cells in culture has
been reported, mainly because ethidium bromide limits the growth capacity of
these cells to between 3 and 4 cell generations (82, 83).
In contrast to other vertebrate cells studied so far, chicken embryo
fibroblasts have been found to be inherently resistant to the growth-inhibitory
effect of ethidium bromide when supplied with exogenous pyrimidine
nucleosides or nucleotides (84, 85). Long-term ethidium bromide treatment
resulted in chicken embryo fibroblast cells which had no measurable mtDNA
and which were respiration deficient. This phenotype was maintained
whether or not the cells were transferred to drug-free medium (86). This was
the first and, as yet, only demonstration that vertebrate cells of the rho°
phenotype (such as in Saccharomvces cerevisiae) can proliferate in culture.
Study of growth parameters indicated that no lag or adaptation period was
necessary for pyrimidine-supplemented chick cell populations to proliferate in
the presence of ethidium bromide (87).
Whereas ethidium bromide inhibits mtDNA synthesis more than nuclear
DNA synthesis, the reverse is true for treatment of cultured mammalian cells
with either 5-fluorodeoxyuridine or methotrexate (88). These drugs inhibit the
enzyme thymidylate synthetase, so that thymidine must be supplied
exogenously. Certain cells (such as LMTK-) lack the major cellular thymidine
kinase but do contain a genetically distinct mitochondrial enzyme.
Consequently, when these cells are treated with methotrexate and supplied
with exogenous thymidine, only the mitochondria can synthesize DNA. When
the concentration of exogenous thymidine is >20 p.M, the rate of mtDNA
synthesis is at least 50% of the control rate while nuclear DNA synthesis is
<4% of control. The resultant effect on the mtDNA copy number was not
measured, however.

"Natural" Homoplasmy and/or Heteroplasmv of MtDNA
The genotype of mtDNA tends to be the same within a given cell; i.e.,
most cells are homoplasmic (that is, they carry only one type of mtDNA). In
addition, the mitochondrial genotype between different tissues of a single
individual generally appears to be the same (89). This is somewhat surprising
since it has been suggested that mtDNA evolves at a rate 10-fold faster than
that of single-copy nuclear DNA (90, 91). Heteroplasmy has been observed in
only a few species, and usually analyzed only in a single individual (92, 93).
This seeming disparity between the rapid rate of mtDNA mutation and the
observation that single variant forms of the mitochondrial genome appear to
dominate the cell population of a single individual (94) can be explained partly
by the fact that mammalian oocyte mitochondria are derived from only a small
number of progenitors (see below) (95).
Unlike nuclear DNA, mtDNA is inherited maternally in mammals, i.e.,
only the egg contributes mtDNA to the zygote. The fate of paternal
mitochondrial genomes remains obscure (96, 97). There is 100 times more
mtDNA in follicular oocytes than in somatic cells, suggesting at least a 100-
fold amplification during oogenesis (98, 99, 100). Because there is no
additional mtDNA synthesis after fertilization until about the 64-cell blastocyst
stage, there is a rapid dilution of the mtDNA copy number to 1-2 per
mitochondrion in zona-encased oocytes. A higher content of mitochondrial
genomes per organelle is probably not recovered until after early cell divisions
of the embryo (101). The eventual mitochondrial genotype of the individual is
derived from only the few of these 64 cells that serve to form the growing
embryo. This may explain, in part, the seemingly incongruous reports that
mtDNA has a rapid rate of mutation and yet most individuals are homoplastic.

In addition, while alleles of nuclear genes segregate only at meiosis,
mitochondrial genes can "segregate" during mitotic divisions of the eukaryotic
cell. This vegetative segregation may explain how an initially heteroplasmic
cell can give rise to homoplasmic daughter cells during somatic cell division
(102). How might this occur? The "average" haploid yeast cell, for example,
contains about 50 copies of the mtDNA molecules and thus about 50 copies of
each mitochondrial gene (103). A mutational event, such as for resistance to
erythromycin (an inhibitor of mitochondrial protein synthesis) is unlikely to
affect more than one copy of a gene at a time and would therefore result
initially in a cell with 1 mutant and 49 wild type alleles. This heteroplasmic cell
eventually produces homoplasmic mutant progeny cells in which all mtDNA
molecules carry the mutant allele. Experiments have shown that seiection
plays the major role in determining the fate of erythromycin-resistant genomes
in the presence of erythromycin on a nonfermentable carbon source, i.e. under
"selective" conditions. This was primarily intracellular selection, acting in the
absence of cell division to make cells homoplasmic for the erythromycin-
resistant allele. Hydroxyurea, which inhibits nuclear and mtDNA synthesis
(104), blocked this selection. The simplest interpretation is that selection
requires mtDNA synthesis.
In addition, intercellular selection may play a role in determining the
fate of new erythromycin-resistant mutations in that cells with many copies of
the mutant allele may begin to grow and divide, while homoplasmic
erythromycin-sensitive cells or cells with few copies of the erythromycin-
resistant allele cannot divide. Although the mechanism for the intracellular
selection of mutant alleles in this case was unknown, there is some evidence,
at least for the petite mutation in yeast, that the presence of certain nucleotide
sequences and other features of the mutant genome may endow it with a

replicative advantage over the wild type genome, the expression of which can
become suppressed (105).
Segregation of MtDNA in Hybrids and Cvbrids
To further explore how a given mtDNA genotype is selected for
replication and/or expression, numerous experiments with interspecies
hybrids (cell/cell) and cybrids (cell/cytoplasm) have been performed (106,
107). Ideally, the two parental lines are different enough for the DNA, RNA,
and protein from the hybrid (or cybrid) cell mitochondria to be characterized as
belonging to one or both species. Analysis of the mtRNA and protein can
determine whether the two types of mtDNA function completely independently
or interact in a complementary fashion, or whether only one genome is
responsible for all of the mitochondrially coded products.
Segregation patterns of mtDNA in cybrids and hybrids have been
divided into 3 categories, namely 1) segregation of foreign mtDNA (or
chromosome-dependent segregation); 2) stochastic segregation; and 3)
segregation of host mtDNA (108, 109). In interspecies hybrids, such as
human-mouse hybrids, when there is loss of chromosomes from one parental
cell, the mtDNA from that parental cell is also lost (chromosome-dependent
segregation) (110, 111). These observations suggest that an incompatibility
exists between different species of nuclear and mitochondrial genomes and is
responsible for the "segregation of foreign mtDNA" (112).
In contrast, in intraspecies cybrids, there should be no incompatibility
between nuclear and mitochondrial genomes. Mitochondrial DNA of
cytoplasts can be propagated in the host cells and both parental mtDNAs are
found to be codominant at an early stage after cybrid isolation (108).
However, after cultivation for several months, some individual cells in the
population of cybrids were observed to have a different ratio of parental

mtDNA (112). A similar phenomenon occurs in mouse-rat hybrids in which
both parental chromosomes are stably retained (109). In these cases, since
there is no selective pressure from the nuclear genomes on either parental
mitochondrial genomes, both parent mtDNAs segregate randomly into
daughter cells and may, in a sufficient number of divisions, become pure for
either parental type of mtDNA (112). This type of mtDNA segregation is
named "stochastic segregation.”
Although stochastic segregation of mtDNA should occur in intraspecies
cybrids, in human cybnds constructed by fusion of tumorigenic HeLaTG cells
with cytoplasts of normal primary fibroblasts, host cell mtDNA (HeLaTG
mtDNA) was segregated from the cybrids (113). If stochastic segregation
occurred, some subclones of the cybrids should have contained HeLaTG
mtDNA, but this was never observed. Thus, there must be some differences
between the mitochondrial genomes of HeLaTG cells and fibroblasts that are
responsible for the preferential segregation of HeLaTG mtDNA. This
segregation pattern is referred to as "segregation of host mtDNA."
Recombination of MtDNA and/or its Encoded Products
Recently, metabolic complementation between mitochondria in somatic
cell hybrids or cybrids was suggested from the observation that
chloramphenicol (CAP)-sensitive mtDNA was maintained in cells even when
they were cultivated continuously for a long time with CAP (114, 115). Oliver
and Wallace (116) showed that a marker polypeptide encoded on CAP-
sensitive mtDNA could be synthesized in the presence of CAP. These data
were interpreted to mean that mitochondrial cooperation arises from
mitochondrial fusion or intermitochondnal exchange of mRNA, a finding which
suggests, in turn, that mitochondrial genomes may interact with each other
and form recombinant molecules.

20
To date, however, there is no direct evidence to suggest that
recombination of mtDNA occurs, at least in mammals. Recombination of
mammalian mtDNA has been examined using mouse X rat and mouse X
hamster somatic cell hybrids and rat cybrids (117, 118). Genetic and physical
analyses showed that the mtDNAs of the hybrids and cybrids were simple
mixtures of the two parental mtDNAs. These observations suggest that, in
contrast to the case with yeast or plant mtDNA, recombination of mammalian
mtDNA occurs rarely, if at all.
Effects of MtDNA on Expression of Nuclear DNA
Saccharomvces cerevisiae cells seem to respond to the quality and
quantity of mtDNA and modulate the levels of nuclear-encoded RNA’s,
perhaps as a means of intergenomic regulation (119). These results suggest
a hitherto undescribed type of nuclear-mitochondrial interaction whereby
nuclear DNA sequences can respond to the state of the mitochondrial
genome. For example, the ability of yeast cells to utilize certain sugars varies
in different petites (119), suggesting that the expression of some pathways of
sugar utilization can in some way be influenced by different, defective
mitochondrial genomes. Another related observation is that a linear, double-
stranded DNA killer plasmid from Kluvveromvces lactis. when introduced into
Saccharomvces cerevisiae. is unstable in both rho+ and mit* cells, but is stable
in rho° petites (120). The abundance of nuclear-encoded RNAs is increased
in one or more of these respiratory-deficient petites, as if these cells were
attempting to compensate for their respiratory-deficient defect or for the
particular mtDNA lesion that they harbor. Perhaps a decreased mtDNA copy
number stimulates the transcription of nuclear genes necessary for mtDNA
synthesis.

How might expression of some nuclear genes be affected differently in
these respiratory-deficient cells? One possibility is that a mitochondrial gene
product is exported and functions outside the organelle, for example, as a
negative (or positive) regulatory element (119, 121). According to this model,
control of expression of some nuclear genes would be maintained as long as
cells contained a functional mitochondrial genome. Upon conversion to
petites, however, the cells could no longer synthesize the putative regulator
since petites have lost the ability to carry out mitochondrial protein synthesis,
and they lack most, and in some cases all, of the mitochondrial transcripts
found in rho+ or mif cells. Although protein or RNA export from mitochondria
has not been demonstrated directly, there are some indications that such
export may occur.
For example, sporulation in MATalpha/MATa diploids of
Saccharomvces cerevisiae cells is accompanied by a novel pattern of protein
synthesis, as shown by the disappearance of some ’mitotic' polypeptides and
by the appearance of a new set of 'meiotic' polypeptides (103). Inhibition of
mitochondrial protein synthesis by erythromycin caused the disappearance of
several ’meiotic’ polypeptides within one hour. Fractionation of extracts of the
cytosolic and mitochondrial components showed that those proteins that were
sensitive to erythromycin were localized within the cytosol and that they were
also sensitive to cycloheximide, an indication that they were not mitochondrial
translational products. However, in a mitochondrially inherited erythromycin-
resistant mutant, which had altered mitochondrial ribosomes, neither
sporulation nor in vivo protein synthesis were affected by erythromycin (122).
This mutant synthesized all of the meiotic polypeptides under sporulation
conditions in the presence of erythromycin, ruling out the possibility that the
drug could act at the level of the cytosolic protein synthesis system during

22
sporulation. This evidence suggests that mitochondrial protein synthesis is
needed for the expression of some nuclear genes during sporulation.
There is now evidence that mitochondrial gene products may regulate
nuclear gene expression in mammals as well. This comes primarily from the
observations that a particular antigen, although specified in part by a gene in
the major histocompatibility complex, is maternally inherited (123). This
maternally-transmitted antigen (Mta) is a murine cell-surface molecule whose
phenotype is determined solely by that of the mother (124). Sex-linked
transmission is ruled out because the phenotypes of Fi males and females in
a single litter are identical (125). The Mta phenotype cannot be modified by
fostering, embryo transfer or transfer of bone marrow cells to lethally irradiated
mice, making the involvement of a conventional infectious agent unlikely
(123). This interpretation is further supported by the finding that the maternal
lineage of the Mta+ phenotype is stable for at least 11 generations of
backcrossing to Mta- males (126). The expression of Mta in somatic cell
hybrids requires functional mitochondria from the Mta+ parent cell line.
Pretreatment of the Mta+ parent with the mitochondrial poison Rhodamine 6G
(127, 128) resulted in hybrids which were Mta-, or diminished in Mta
expression. This is the first evidence for mitochondrial control of the
expression of a cell membrane molecule in eukaryotes. Whereas Mta is
encoded by a structural gene in the major histocompatibility complex, its
expression seems to be controlled by a mtDNA gene product.
The techniques of somatic cell genetics also provide a powerful tool for
investigating whether mitochondrial genomes play a role in the activation
and/or suppression of the phenotypic expression of tumorigenicity. Several
investigators have shown that tumorigenicity can sometimes be suppressed
by fusing tumor cells with cytoplasts from nontumorigenic cells (113, 129).

23
However, there have been no reports of the induction of tumorigenicity in
nontumorigenic cells by the addition of cytoplasts from tumorigenic cells (130,
131). These findings suggest that cytoplasmic elements and/or mitochondria!
genomes may sometimes contribute to the suppression of tumorigenicity (but
this is not consistently true).
Partitioning of Regulatory Molecules
The notion of partitioning of regulatory molecules between the
mitochondria and the nucleus is not restricted, of course, to products of the
mitochondrial genome. There is now good evidence for the partitioning of
some nuclear gene products between the mitochondria and
extramitochondrial compartments (132, 133). Thus, nuclear-encoded
regulatory factors could partition between the mitochondria and the nucleus,
and their relative distributions could be determined by the presence or
absence, or the amounts, of mtDNA (or RNA) sequences. This mechanism
would allow for regulation of nuclear gene expression in response to the
quality as well as the quantity of mtDNA or RNA sequences. In that way, the
nuclear genome could "sense" the amount or kind of mtDNA in the cell.
Indeed, Clayton elaL (134) have recently shown that a135-nucleotide RNA
species, not encoded in the mitochondrial genome, is the RNA moiety
necessary for activity of the site-specific endoribonuclease involved in primer
RNA metabolism in mammalian mitochondria. This finding implies transport of
a nucleus-encoded RNA, essential for organelle DNA replication, to the
mitochondrial matrix.

24
Polvamines
All organisms contain significant amounts of the polyamines,
spermidine and spermine, and their precursor, putrescine. In mammals the
concentrations of these aliphatic amines vary considerably between different
cell types (135). Thus, cells heavily involved in polynucleotide and protein
synthesis such as proliferating or protein secreting cells contain large amounts
of polyamines (in the mM range), while concentrations in metabolically less
active cells are lower (136). Polyamines are cations at physiological pH and,
in vitro, can bind through ionic forces to the negatively-charged groups of
nucleic acids, proteins and phospholipids (137, 138). Polyamines can also
form hydrogen bonds, and the aliphatic hydrocarbon part of their structure may
allow some interactions with hydrophobic environments such as those
occurring in membranes. These properties could contribute to the multiple
actions attributed to the polyamines in many biological processes.
The biosynthetic pathway for putrescine, spermidine and spermine in
mammalian cells has been well established (Figure 2). Two 'decarboxylases'
and two 'aminopropyltransferases' are involved. The two decarboxylases are
of particular interest as they are present in mammalian cells in very small
amounts, have very short half-lives, and are highly inducible (137). Because
of these properties the decarboxylases can regulate polyamine synthesis and
enable cells to respond to a variety of stimuli with a rapid increase in
polyamine levels.
Ornithine decarboxylase (ODC) is a pyridoxal phosphate-dependent
enzyme (136). It is thought to play the key role in polyamine synthesis, and its
half-life of about 10 minutes is the shortest reported for an enzyme in
mammalian cells (139). Ornithine decarboxylase is present in very small
amounts in quiescent cells, and its activity increases rapidly and dramatically

25
COOH
I
H2N-(CH2)3-CH-NH2
Ornithine
C02
1
\i/
H2N-(CH2)4-NH2
Putrescine
S-Adenosylmethionine
C02^e
Decarboxylated
S-Adenosylmethionine
(D-SAM)
Methylthioadenosine
(MTA)
H2N-(CH2)3-NH-(CH2)4-NH2
Spermidine
D-SAM
MTA
V
H2N-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2
Spermine
Figure 2. Polyamine synthesis in mammalian cells. Enzymes involved are:
1) ornithine decarboxylase; 2) S-adenosylmethionine
decarboxylase; 3) spermidine synthase; and 4) spermine synthase.

26
within a few hours of exposure to trophic stimuli such as hormones, certain
drugs, and growth factors (140, 141,142).
S-Adenosylmethionine decarboxylase (SAM-DC) also has a relatively
short half-life (~1 hr) and has the added pecularity that it utilizes as cofactor
covalently bound pyruvate instead of pyridoxal phosphate, the usual cofactor
for decarboxylases (136). Like ODC, it is present in mammmalian tissues in
very small amounts, and its activity is regulated by many hormones and other
growth-promoting stimuli. Once decarboxylated, S-adenosylmethionine is
committed to polyamine production in that no other substantial reactions
utilizing decarboxylated S-adenosylmethionine are known. The
decarboxylation of S-adenosylmethionine is the rate-limiting step in
spermidine formation.
The transfer of an aminopropyl group from decarboxylated S-adenosyl¬
methionine to putrescine is catalyzed by spermidine synthase, one of the
aminopropyltransferases. The other aminopropyl group needed to convert
spermidine into spermine, is also derived from decarboxylated S-
adenosylmethionine, a reaction catalyzed by the second
aminopropyltransferase, spermine synthase (136). As the activities of
spermidine synthase and spermine synthase are much higher than those of
the two decarboxylases, putrescine and decarboxylated S-
adenosylmethionine concentrations within the cell tend to be very low in
comparison to spermine and spermidine (137).
An active transport system for the polyamines distinct from those for
amino acids has been found in ail cells tested to date (136). The activity of this
system is increased in polyamine-depleted cells, and it can maintain cellular
polyamine levels in the presence of very low extracellular concentrations. The
role of the transport system under normal circumstances is unclear, however,

27
because intracellular synthesis is used to provide polyamines, and
extracellular polyamine concentrations are usually low. The transport system
can also take up other basic substances, including drugs like MGBG; this will
be discussed in greater detail in the next section. In addition, some cultured
cells efflux polyamines when cell growth has been restricted by inhibitors,
confluence, or lack of growth factors (136).
The first step in the catabolism of these polycations is acetylation (143).
The degradation of spermine into spermidine or of spermidine into putrescine,
processes which reverse the normal biosynthetic pathway for polyamines,
involves the sequential activity of two enzymes, N1-acetyltransferase and
polyamine oxidase (Figure 2). Putrescine may be acetylated and eventually
oxidized in a reaction catalyzed by monoamine oxidase, or directly oxidized in
a reaction catalyzed by diamine oxidase, which is induced by growth stimuli.
Polyamines and their acetylated derivatives can also be oxidized in a reaction
catalyzed by plasma amine oxidase (137).
Evidence from studies on polyamine-deficient mutant cells and on
drugs that inhibit polyamine biosynthesis indicates that polyamines are
necessary for cell division and differentiation (136, 144). Indeed, proliferating
tissues are particularly rich in polyamines and their biosynthetic enzymes
(145). Although it is apparent that polyamines play important biological roles,
their exact function(s) is unclear. Polyamines have a wide spectrum of effects,
particularly in cell-free systems. Such actions include control of the initiation
of translation, stimulation of ribosomal subunit association, stabilization of
tRNA and DNA structure, and stimulation of RNA and DNA synthesis (135).
Polyamines have also been shown to enhance reactions catalyzed by DNA
polymerase (146), RNA polymerase (147), polynucleotide ligase and kinase
(148), and gyrase (149).

28
In vitro, polyamines bind preferentially to double-stranded nucleic
acids. They bind in the minor groove of the double helix and, by interacting
with phosphate groups, stabilize the double helix (147). A high concentration
of polyamines induces condensation of nucleic acids. This association with
DNA may represent a major function of polyamines, involving both molecular
stabilization and control of gene expression (150). However, it is prudent to
recognize that many of these actions may only reflect the fact that polyamines
interact with acidic macromolecules in vitro and that physiological relevancy
has not been established.
The polyamines, spermine and spermidine, stimulate the rate of
phosphorylation of selected proteins by several types of cyclic nucleotide-
independent protein kinases (151, 152). For example, spermine and
spermidine increase the activity of cytosolic and nuclear type II casein kinases
(153). The enhancement of certain of these protein kinase reactions by
polycations seems to relate primarily to their interaction with the protein
substrate, yielding more favorable conformations for phosphorylation by the
protein kinase, rather than by an effect on the enzyme £er se (152).
Polyamines have also been shown to inhibit certain enzyme reactions.
For example, physiological concentrations of spermine completely inhibit
phosphorylation by nuclear protein kinase Nl (151). Polyamines, especially
spermine, inhibit cardiac phospholipid-sensitive, calcium-dependent protein
kinase and myosin light chain kinase, a calmodulin sensitive protein kinase
(154). Even protein phosphatases, including a type isolated from heart, may
be influenced by polyamines with the effect dependent on the substrate used
(155).
Moreover, spermidine or spermine, at near-physiological
concentrations of 1.5 mM, stimulates by 8-fold the phosphorylation of

29
phosphoinisitol (PI) in membranes from A431 cells. The reaction product is
almost exclusively PI-4-phosphate (156). Also, spermidine or spermine can
increase by 4-fold the rate of hydrolysis of synthetic phospholipid analogs by
phospholipase A2 (157). In addition, polyamines are substrates for
transglutaminases, are precursors of gamma-aminobutyric acid, and uncouple
the parietal cell H+, K+-ATPase system (135). Polyamines also appear to be
second messengers in mediating Ca++ fluxes and neurotransmitter release in
potassium-depolarized synaptosomes (158).
Addition of micromolar concentrations of polyamines to cultured
myocardial cells results in an increase of cGMP and a parallel decrease of
cAMP levels (137). Conversely, depletion of intracellular polyamines by
polyamine synthesis inhibitors leads to an accumulation of cAMP and a
depletion of cGMP (159). These effects are mediated by modifications of the
activities of the enzymes involved in cyclic nucleotide metabolism, i.e.
adenylate and guanylate cyclase and cAMP- and cGMP-phosphodiesterase.
Considering that cGMP and cAMP are usually regarded as positive and
negative signals, respectively, for the induction of cell growth, the property of
polyamines to affect the metabolism of the two nucleotides in opposite
directions correlates well with their suggested role in cell growth (137).
Polyamines also have many actions on mitochondria. Mitochondria
can "buffer" cytosolic free Ca++ concentrations, but the physiological
significance of this uptake has been questioned because it is triggered only by
Ca+T concentrations that exceed those found in normal cells. In the presence
of physiological concentrations of Mg++, the extramitochondrial free Ca++
which the mitochondria will buffer is 0.6 to 1 pM (160), but the physiological
free Ca++ concentration in the cytosol is only 0.15 to 0.25 pM (161, 162). It
now appears that spermine, which had been lost upon permeabilization of

30
cells, will restore the sensitivity of mitochondria to external Ca++ (160).
Spermine both increases the rate and affinity of Ca++ uptake into mitochondria
and decreases the set-point to which isolated mitochondria buffer free Ca++
concentration. Spermine stimulates Ca++ uptake by mitochondria but not by
microsomes (163). The half maximally effective concentration of spermine (50
(iM) is in the range of physiological concentrations of this polyamine in the
cell. Spermidine is 5 times less potent; putrescine is inactive. The stimulation
of mitochondrial Ca++ uptake by spermine is inhibited by Mg++ in a
concentration-dependent manner. Spermine is thus an activator of the
mitochondrial Ca++ umporter, and Mg++ is an antagonist. Spermine also
inhibits the efflux of Ca*+ from mitochondria induced by ruthenium red.
Polyamines may thus confer to the mitochondria an important role in the
regulation of the free Ca++ concentration in the cytoplasm as well as in the
mitochondrial matrix.
At physiological concentrations spermine may also be transported into
rat liver mitochondrial matnx space, provided that mitochondria are energized
and inorganic phosphate is available for concurrent transport (164).
Furthermore, mersalyl, a known inhibitor of phosphate transport, prevents both
spermine uptake and release. Magnesium ions inhibit the transport of
spermine, conceivably by competing for the same binding sites on the
mitochondrial membrane.
Fixed negative charges of anionic sites have previously been proposed
as possible binding sites for large organic cations within the mitochondrial
membranes (165). These binding sites are probably composed mainly of
ionized phospholipids and may be involved in transport of small cations and
anions across the mitochondrial membrane. The phospholipid-containing

binding sites are reportedly localized mainly at the outer face of inner
mitochondrial membrane.
In 1960, Tabor (166) reported that low concentrations of spermine and
spermidine inhibited swelling of mitochondria in hypotonic media. More
recently, polyamines were shown to improve the respiratory control and
prevent loss of control in heat-aged mitochondria, protect oxidative
phosphorylation, and prevent a fall in the mitochondrial membrane potential
(167, 168, 169). At concentrations comparable to those attained
intracellularly, the polyamines inhibited state 4 respiration of rat liver
mitochondria, but they had much less effect on state 3 or uncoupled
respiration (I25 = 7.5 and 7.0 mM for spermidine and spermine, respectively)
(167). Electron microscopy revealed that polyamines caused the outer
mitochondrial compartment to collapse, bringing the inner and outer
membranes into apparent contact with one another (167). Recent evidence
suggests that the import of nuclear-coded proteins into mitochondria occurs at
sites where the inner and outer mitochondrial membranes are juxtaposed to
one another (170). It seems possible, therefore, that polyamines may affect
this process.
MGBG fMethylglvoxal bisiauanvlhydrazone])
History and Climcal Utility
MGBG has been recognized as a potent antiproliferative agent since
1958 when Freedlander and French (171) reported that it inhibited the growth
of L1210 leukemia ceils m mice. Clinical studies disclosed that MGBG (also
known as methyl-GAG) could induce remissions in some patients with acute
myelocytic leukemia, malignant lymphoma, and certain other neoplasms (4,
5). MGBG has also been used in the treatment of trypanosomiasis (172).

32
However, MGBG accumulated to extremely high levels in certain
normal cells (6, 173), and the consequent severe toxicity set practical
limitations on its use (3). Mucositis was the dose-limiting toxicity in Phase Ml
clinical trials (4). Fatigue, anorexia, mild nausea and vomiting, myalgias, and
neuropathy were also seen. The subacute toxicity of MGBG involved both
proliferative (gastrointestinal, bone marrow, and lymphoid) and
nonproliferative (hepatic, renal, and cardiac) tissues (174). However, more
recent investigations involving variation in dosage schedules and use in
combination with other anti-proliferative agents have rekindled interest in the
clinical use of MGBG (3, 175). Because no metabolites of MGBG have ever
been detected in urine, feces, or various tissues, and virtually no radioactive
CO2 was expired after in vivo administration of [14C]-MGBG, it is believed that
MGBG does not undergo biotransformation in higher animals (175).
Relationship to Polvamines
French and his colleagues were the first to point out that MGBG had
structural resemblances to spermidine (Figure 3), and they suggested that
MGBG might interfere with the biological functions of this naturally-occurring
polyamine (3). MGBG is a basic molecule consisting of 2 aminoguanidine
groups separated by an aliphatic chain. MGBG has pKa's of about 7.5 and 9.2
at 25°C (3), presumably due to resonance stabilization of the protonated forms
of the aminoguanidine moieties at both ends of the molecule. Spermidine,
whose molecular contours are quite similar to those of MGBG in its most
extended conformations, has pKa values of about 8.4, 9.8 and 10.8 at 25°C
(3).
MGBG is a potent (<1 jiM) competitive inhibitor of S-adenosyl-
methionine decarboxylase (SAM-DC), which is a key enzyme in the
biosynthesis of the polyamines, spermidine and spermine (176, 177).

Spermidine
NH
H2N-C-NH-
HCH3 NH
=C-C=N-NH-C-NH2
MGBG
Figure 3. Structures of spermidine and its analog, MGBG.

34
However, the inhibitory effects of MGBG on polyamine biosynthesis in animals
are transient because of a striking secondary increase in SAM-DC. It is
hypothesized that binding of MGBG to the enzyme may stabilize it, and hence
prolong the normal one hour half-life of SAM-DC (178).
MGBG is also a potent noncompetitive inhibitor of diamine oxidase
(179), an enzyme involved in putrescine degradation. In addition, MGBG
causes an increase in the activities of ornithine decarboxylase (ODC), which
synthesizes putrescine, and in spermidine/spermine N1 -acetyl-transferase
(SAT), an enzyme which participates in the degradation of spermine and
spermidine to putrescine (176). The increases of both ODC and SAT activities
provoked by MGBG in cultured human erythroid leukemia K562 cells were
blocked by treatment with cycloheximide, a finding that suggests that the
increase in enzyme activity required synthesis of protein. The putrescine
content in cells treated with MGBG increased 20-fold, whereas the levels of
spermidine and spermine were depressed by 35 and 50%, respectively. The
marked increase m putrescine levels presumably resulted from inhibition of
SAM-DC and diamine oxidase, and induction of ODC and spermidine and
spermine acetyltransferases. All of these factors act to increase putrescine
levels.
Thus, m cells treated with MGBG, putrescine pools increase while those
of spermidine and spermine decrease slowly (180). Nonetheless, a number of
laboratories (181, 182, 183) have been unable to correlate these changes in
polyamine concentration with inhibition of cell growth. It seems that the
antiproliferative effect produced by MGBG correlates much better with the
intracellular MGBG concentrations than with the depletion of polyamines (183,
184). The ability of spermidine to prevent the antiproliferative effects of MGBG,
initially a critical observation in linking MGBG to polyamines, is now attributed

to competition for cellular uptake rather than replenishment of MGBG-depleted
spermidine pools (185). Moreover, difluoromethylornithine (DFMO), a highly
specific inhibitor of polyamine biosynthesis, is cytostatic (186), whereas MGBG
is cytotoxic, a result suggesting that the two drugs may have different
mechanisms of action.
Effects on Mitochondria
Indeed, another pharmacological action for MGBG has been identified.
In 1966, Pine and DiPaolo (187) found that MGBG and certain other cationic
compounds inhibited the respiration of ascites L1210 cells, and uncoupled
phosphorylation in isolated mitochondria. In common with 2,4-dinitrophenol,
the drugs selectively inhibited acetate incorporation into lipid and, to some
extent, reduced the cellular pools of ATP. In a different study, pyruvate
oxidation, another measure of mitochondrial function, was significantly
decreased 4 hours after exposure of ascites L1210 leukemia cells to MGBG;
growth inhibition did not occur until after 10-12 hours (185). Depletion of
spermidine pools to levels comparable to those attained during DFMO-
induced cytostasis did not occur even after 24 hours of exposure. Also, in
studies with intact A30 cells, the rate of oxygen uptake in MGBG-treated cells
was decreased to as low as 10% of control (188). The degree of inhibition of
oxygen consumption was dependent on the concentration of MGBG. When
spermidine was added to the MGBG-containing medium, however, respiratory
activity was restored and approached that of normal cells.
Pathak ¡¿iai. (189) have demonstrated that exposure of cultured
leukemia L1210 cells to 0.1 to 10 pM MGBG resulted in a concentration-
dependent inhibition of cellular proliferation which became apparent after
about 1 to 2 generation times (12 to 24 hr). Ultrastructural examination of cells
exposed to at least 1.0 pM MGBG for 24 hr revealed extensive swelling of

36
mitochondria, deterioration and eventual loss of cristae and increase in matrix
density. This ultrastructural damage was selective for the mitochondria; there
were no concomitant changes in nuclear ultrastructure (190). In addition, the
mitochondrial damage preceded growth inhibition by about 12 hr and did not
immediately affect cell viability. Because damage was apparent in all of the
mitochondria of each affected cell, the organelle population was probably
homogeneous in its sensitivity to the drug. Moreover, exposure to 10 pM
MGBG for 24 hr caused all of the cells to be affected. Whether the
mitochondrial structural damage was associated with functional deterioration
and whether the damage was reversible were not demonstrated.
Similar damage has been observed in ascites L1210 cells treated in
vivo with a single dose (75 mg/kg) of either MGBG or ethidium bromide (190).
After 24 hr, the mitochondria were swollen, lost their inner structure, and, in the
case of treatment with ethidium bromide, developed numerous electron
densities within the matrix. Analysis of the nucleotide pools of these cells by
HPLC revealed that treatment with either MGBG or ethidium bromide depleted
the ATP pools to 52 and 16% of control, respectively. The overall adenylate
energy charge of the cell (1/2[ADP+2ATP] + [AMP+ADP+ATP]) was also
reduced to 68 and 58% of control, respectively. The measurement of ATP
levels suggested that, whereas MGBG caused substantial mitochondrial
damage, the resultant energy depletion ger s£ was not sufficient to account for
growth inhibition (191).
When MGBG-treated ceils were harvested, washed, and reinoculated
into untreated mice, all cells reattained nearly normal ultrastructure after 48 hr,
whereas ethidium bromide-treated cells did not, even after 96 hr (190).
Analysis of the nucleotide pools of the MGBG-treated cells indicated that the
mitochondria had recovered their functional capabilities as well. The

37
adenylate energy charge for these cells was essentially the same as that for
untreated cells. Also, the cells previously treated with MGBG displayed
unaltered leukemogenicity in that the animals died at nearly the same time as
they did after inoculation with untreated cells.
MGBG also causes ultrastructural damage to mitochondria in several
other murine and human cell types including P288 mouse leukemia, L-cell
mouse fibroblast, C3H/10T1/2 mouse embryo fibroblast, and NALM-1 human
chronic myelocytic leukemia cells (176). Interestingly, the onset of
mitochondrial damage in these cell lines related inversely to the generation
time of the particular cell lines. This relationship between mitochondrial
damage and the rate of cell proliferation was tested in two separate cell
systems (174). In cultures of human lymphocytes stimulated with
phytohemagglutinin, only those cells undergoing blastogenesis were bound to
be affected by MGBG. Similarly, MGBG treatment of confluent and
subconfluent cultures of C3H/10T1/2 mouse embryo fibroblasts affected only
the dividing, subconfluent cells.
The effects of MGBG on isolated rat liver mitochondria have also been
studied (191). At drug concentrations comparable to those attained
intracellularly, MGBG significantly inhibited state 4 respiration (125=6 mM), but
had less of an inhibitory effect on state 3 or uncoupled respiration (125=16-20
mM). This may be due to the fact that in the absence of ADP (state 4),
mitochondria generate a significant electrochemical gradient across their
inner membrane. By election micrcscopy, MGBG-treated mitochondria
appeared swollen, and the spaces between cristae membranes or inner and
outer membranes were collapsed, obliterating the intermembrane space
(191).

38
Since the inhibition of mitochondrial respiration in isolated
mitochondria was prevented by potassium cations and enhanced by
valinomycin, the drug may compete for potassium-binding sites, possibly
membrane phosphoplipids (191). Pretreatment of mitochondria with MGBG
protected against the nonspecific swelling effects of Triton X-100, and the
electrophoretic mobility of mitochondria was markedly slowed by MGBG.
Overall, the data suggest that MGBG neutralizes the net negative surface
potential of isolated mitochondria by binding to sites (possibly phospholipids)
at the inner mitochondrial membrane; any subsequent interference with cation
binding and/or transport may result in inhibition of bioenergetic functions.
Fixed negative charges of anionic sites have been proposed as
possible binding sites for large organic cations within the mitochondrial
membranes (165). Neutralization of this surface potential with compounds
such as polyamines can affect many mitochondrial activities, as well as
ultrastructure. Similar or identical binding sites at the inner mitochondrial
membrane have been suggested for binding certain bis[guanylhydrazones]
(191). Moreover, it has been shown that cationic drugs need not penetrate
across inner mitochondrial membranes because binding sites are probably
fixed at the outer surface (165). In addition, the effects of MGBG on isolated rat
liver mitochondria are readily prevented or reversed by polyamines, and these
interactions are also affected by the mitochondrial transmembrane potential
(192). Magnesium cations enhance the protective action of polyamines.
These data indicate that competition exists between MGBG and polyamines
for low affinity, negatively-charged binding sites at the outer surface of inner
mitochondria! membranes
Evidence which implicates mitochondrial damage as important in the
antiproliferative effects of MGBG comes from studies of other polyamine

analogs. For example, 4,4'-diacetyldlphenylurea-bis(guanylhydrazone) is an
aromatic bls-(guanylhydrazone) with potent antiproliferative activity. The drug
causes profound ultrastructural damage to mitochondria (174), but has no
effect on polyamine biosynthesis (193). Similarly, ultrastructural damage also
precedes detectable inhibition of cell growth (189). Damage to mitochondrial
ultrastructure is selective for proliferating cells. This has been observed
among cultured cells (174) and more recently in the intestinal epithelium
(194), a site of MGBG toxicity, where damage only occurs to mitochondria of
the dividing crypt cells but not to the nondividing villous cells. Although uptake
studies were not done, Diala el al. (195) made the interesting observation that
wild-type yeast are growth-inhibited by MGBG while petite mutants, lacking
mitochondria, are not. Growth inhibition by MGBG was antagonized by
spermidine, but neither the polyamine nor MGBG had any effect on growth
inhibition by ethidium bromide. MGBG had little or no effect on cell viability
throughout the 2-day exposure.
While the numerous effects of MGBG on mitochondria provide
substantial indication that these actions are responsible for the
antiproliferative activity of the drug, this relationship has not yet been
established. Several workers have demonstrated that normal and malignant
cells possess high-affinity and saturable transport systems that promote
intracellular accumulation of putrescine, spermidine, spermine, and certain
agents such as MGBG (196, 197). Because a saturable carrier is involved, the
system seems to be specific for certain structural features of the polyamines
and their analogs. In general, it appears that putrescine, spermidine,
spermine, and MGBG are transported by common membrane transport
systems that are: 1) distinguishable from other known transport systems (e.g.
for various amino acids); 2) saturable and temperature-dependent, with

40
maximal rates at 37°C; 3) inhibited by uncouplers of oxidative phosphorylation
and certain respiratory poisons; and 4) dependent on the proliferative and, in
some instances, hormonal status of the cells (198). Like the polyamine-
biosynthetic enzymes, the transport system seems to be highly regulated,
responding rapidly to polyamine antimetabolites such as DFMO, an
irreversible inhibitor of ODC, and MGBG, an inhibitor of SAM-DC (199, 200,
201). Agents such as MGBG, which utilize the polyamine transport system,
may be concentrated intracellularly more than 1000-fold relative to the
medium (196, 202).
Seppanen et aL (203) have found that cells can concentrate MGBG
some 600- to 1500-fold so that, following exposure to 5-10 pM drug, the
internal concentration is 4-6 mM. A minimum intracellular concentration of 0.5
to 1 mM was required for growth inhibition to occur. Seppanen el aL (204)
also found that MGBG uptake is critically dependent on the growth rate of
tumor cells (i.e., slowly dividing cells transport less MGBG than rapidly dividng
cells). Mikles-Robertson eiaL (174) found that MGBG cytotoxicity also
correlated with cell growth rate.
Recently, mutants of human fibroblast VA2 cells have been developed
which are 10- to 20-fold more resistant to the growth inhibitory effects of MGBG
than the parent line (205, 206). The variants took up MGBG to a similar extent
as wild type during short-term incubations (less than 5 min), accumulated less
drug during longer incubations (30-120 min), and more readily lost MGBG
when shifted to drug-free medium, results which might indicate decreased
intracellular binding of MGBG. In the absence or presence of MGBG, the
overall pools of pclyamines were not appreciably changed from those of the
wild type. Pyruvate oxidation was not significantly inhibited in the mutants
when grown in the presence of MGBG, but was dramatically reduced in wild

type cells. Furthermore, the mitochondrial ultrastructure of the drug-resistant
cell lines was essentially unaffected by culture In MGBG. The resistant cells
took up about 40% less MGBG than their sensitive counterparts. This
difference is considered small among transport mutants (203). Drug
resistance was not cytoplasmically transmitted by cytoplast cell fusion for any
of the four sublines, suggesting that the genes responsible for resistance were
likely of nuclear rather than mitochondrial origin (206). Defective polyamine
and MGBG transport was also found in mutant Chinese hamster ovary and rat
myoblast cells that were resistant to MGBG (207).
Because of the inhibition of cell growth by MGBG, many studies have
addressed the effects of the drug on DNA synthesis. Unlike aromatic
bisguanylhydrazones, which bind effectively to nuclear DNA, MGBG binds
only weakly to DNA and has little effect on DNA polymerase alpha or
thymidine kinase (198, 208). Addition of MGBG to HeLa S3 suspension
cultures caused a decrease in incorporation of [3H]thymidine into nuclear
DNA, but only after putrescine levels had increased and spermidine and
spermine levels had decreased. Since the rate of DNA chain elongation was
only reduced slightly by MGBG, decreased levels of spermidine and spermine
might lead to a decrease in the number of replication units active in DNA
synthesis within each cell.
Effects on MtDNA
The effects of MGBG on mtDNA synthesis have also been Investigated
because of the mitochondrial damage elicited by this drug. In exponentially-
growing L1210 leukemia cells prelabeled with [14C]thymidine, the
incorporation of [3H]thymidine into mtDNA was selectively inhibited (55% of
control) when studied 5 hr after exposure of cells to 10 pM MGBG (7).
Incorporation of label into nuclear DNA, however, was not affected until 8 hr;

the rate of nuclear DNA synthesis was 60% of control after 12 hr. After 16 hr of
exposure, the rates of nuclear and mtDNA synthesis were both about 40% of
control values. Exposure of L1210 cells to 1 pM MGBG resulted in a mtDNA
synthesis rate 60% of control. At this concentration, growth inhibition was 90%
of control after 18 hr of exposure.
Dye-CsCI gradients of mtDNA indicated that the inhibition of synthesis
occurred in replicative forms of circular DNA (7). Uptake studies excluded the
possibility of drug interference with cellular uptake of thymidine.
Ultrastructural studies revealed a very close correlation between the dose-
response curve for mitochondrial damage and that for MGBG inhibition of
mtDNA synthesis. This correlation suggests a close relationship between
ultrastructural damage and inhibition of mtDNA synthesis, but the possibility
exists that both are epiphenomena of another action of the drug.
A more detailed study of the effects of MGBG on mtDNA synthesis in the
Syrian hamster tumor cell line was done by Nass (6). The fate of mtDNA both
during (24-48 hr) and following (7 hr to 7 days) MGBG treatment was
monitored by ultrastructural, pulse-labeling, and restriction cleavage methods.
MGBG (50 pM) selectively inhibited mtDNA replication prior to significant
inhibiton of nuclear DNA synthesis (73 versus 19%, respectively, at 16 hr).
The drug induced structural alterations, without substantial degradation, of the
closed circular form of mtDNA. Importantly, D-loop strand (7S) initiation was
completely abolished, but mtDNA strands already initiated were able to
complete the circle of replication.
Electron microscopy revealed selective ultrastructural damage to the
mitochondria (including swelling, loss of cristae and matrix components, and
dense inclusions) in up to 96% of cells, while nuclei appeared normal.
Quantitative assays of the uptake and retention of rhodamine 123 in these

MGBG-treated cells revealed that the mitochondrial membrane potential was
maintained. After removal of MGBG, mtDNA resumed replicative active, and
damaged mitochondria recovered near-normal ultrastructure within 1 to 2
days.
Specific Aims
Compounds which, at certain doses, inhibit mtDNA replication more than
cell division would be predicted to deplete the characteristic mtDNA copy
number with each successive cell division. Preliminary studies in our
laboratory with mouse leukemia L1210 cells treated chronically with MGBG at
concentrations which initially inhibited mtDNA replication did decrease the
mtDNA copy number. These studies also demonstrated that there are doses
at which long term survival in MGBG is possible. I wanted to determine if there
was a minimal copy number of mtDNA which cells would tolerate and whether
a new steady state level of mtDNA would be established with chronic MGBG
treatment. I also wished to explore in detail the relative contributions of the
rates of mtDNA replication and cell division to the regulation of the mtDNA
copy number during MGBG treatment. And, finally, I endeavored to determine
how mitochondrial and/or cell function was affected as a consequence of a
change in the mtDNA copy number.
The specific aims of this research were the following.
1) Determine the degree to which mtDNA replication and cell division
are coupled. That is, will cells continue to divide even though their mtDNA
replication is inhibited, thus halving the amount of mtDNA per cell with each
division?
2) It so, determine the minimum copy number of mtDNA per cell which
these cells will tolerate.

44
3) Once the copy number of mtDNA is diluted by MGBG treatment,
determine whether these cells establish a new steady state with respect to the
content of mtDNA, or return to their characteristic copy number.
4) Determine the relative contributions of the mtDNA doubling rate and
the cell doubling rate to the regulation of the mtDNA copy number in MGBG-
treated cells.
5) Determine if mitochondrial function is normal or impaired
proportionally to the mtDNA copy number, at least as measured by rhodamine
123 uptake.

MATERIALS AND METHODS
Cell Culture
Mouse leukemia L1210 cells were grown as suspension cultures in 25
or 75 cm2, canted-neck tissue culture flasks (Fisher Scientific). The cells were
maintained in an incubator (National Appliance Company, Portland, Oregon)
at 37°C, and the flasks were kept tightly capped. The medium used was RPMI
1640 supplemented with 16 mM HEPES (3-[N-morpholino] propanesulfonic
acid), 8 mM MOPS (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid),
2.0 g/L NaHC03, and10% fetal bovine serum. The starting pH was 7.4. Stock
solutions of MGBG and diethylspermine (DES) were made to concentrations
of 1 mM in water. All ingredients were obtained through Sigma Chemical Co
(except DES, gift of R. Bergeron). The medium and drugs were sterilized by
0.2 (i filtration (Fisher Scientific). Cells were maintained in exponential growth
by reseeding every two days into fresh medium; the starting concentration was
1 x105 cells/ml.
Cell Counts
Aliquots (200 pi) of cells to be counted were diluted in 10 ml of Hematall
diluent (Fisher Scientific). The cell number was determined by electronic
particle analysis (Coulter Counter, Model Zf, Coulter Electronics, Hialeah, FL).
Two aliquots of cells were counted from each flask and the mean determined.
Because the cells were passed every two days, a calculation was
devised to plot the counts as a single, continuous line over the entire time
course of an experiment. Cumulative counts were calculated simply as
Cumulative count(n+1) = Cumulative count(n) x Concentration Qf C.SliS,(Ptl)
Conce rtraton of viable oel!s(n)

46
The mean population doubling time of the cells was determined as
follows: N=N0ext, where t=time, N0 is the number of cells present at t=0, and
the doubling time is the value of t when N=2N0.
Determination of ICt;n
The IC50 was defined as the concentration of compound necessary to
decrease cell growth to 50% of control growth at a given time. The percentage
of control growth was determined as follows:
[final cell number (treated group) - initial inoculum] X 100
final cell number (control group) - initial inoculum
The IC50 of MGBG was determined for the first 2 days of treatment.
Cell Viability
Trypan blue (Eastman Kodak, Rochester, NY) was added to 100 pi
aliquots of cells to a final concentration of 0.06%. The cells were mixed well,
and a 10 pi aliquot was transferred to a hemacytometer (Reichert Scientific
Instruments, Buffalo, NY). At least 100 cells per sample were counted with a
phase contrast microscope (Ernst Leitz, Wetzlar, Germany) at 200X
magnification. The percent viability of the cells was determined as follows:
number of cells excluding trypan blue X 100
total number of cells
Clonoaenic Assay
Cells from selected MGBG treatments were counted and diluted to a
concentration of 4 cells/ml. Aliquots of 100 pi were transferred to triplicate 96-
well culture plates (Fisher Scientific) and incubated at 37°C for 1 week. The
culture plates were then examined with an inverse phase microscope (Zeiss,
West Germany) for numbers of wells which had colonies of cells. Groups of
>50 ceiis/weil were considered as having been cloned from a single viable

47
cell. The percent cloning efficiency of the drug-treated cells was determined
as follows:
number of wells with colonies (treated group) X 100
number of wells with colonies (control group)
Cell Size
The diameter of the cells was determined directly by the method of
Schwartz elai. (209). Uniform polymeric microspheres (Polyscience,
Warrington, PA) from 4.72 to 10.0 p in diameter were diluted in Hematall, and
the particle size measured electronically with a FACS Analyzer (Becton-
Dickinson, Sunnyvale, CA) with the amplifier in the log mode. The peak
channel number was plotted against the corresponding calibrated diameter for
each size of microbead to obtain a calibration curve.
Aliquots of 1x106 cells were collected, pelleted, and resuspended in
0.5 ml Hematall for analysis. The peak channel number for the cells was
plotted on the calibration curve to obtain the approximate cell diameter. The
volume of the cells was then calculated from this diameter.
Flow Cvtometrv
Flow cytometric analysis of nuclear DNA content was done with a
RATCOM flow cytometer (RATCOM Inc., Miami, FL) interfaced with a
microcomputer (IBM-XP). Aliquots of 1x106 cells were resuspended in 0.5 ml
Hematall and were stained with 4',6-diamidino-2-phenylindole (210). The
distribution of nuclear DNA content among the cells was plotted and
integrated by computer.
Rhodamine 123 Uptake
Aliquots of 1x106 cells were pelleted and resuspended in 100 pi of
serum-free medium. Rhodamine 123 (2-[6-amino-3-imino-3H-xanthen-9-yl]-
benzoic acid methyl ester) (Sigma Chemical) was added to each aliquot to

48
give a final concentration of 1 (ig/ml (211). The samples were incubated at
37°C for 10 min and washed once with medium. The stained cells were
observed under an epifluorescence microscope (Zeiss) at 400X magnification.
The excitation wavelength was 485 nm.
Preparation ofM4C|-MGBG Stock Solution
A stock solution of 1 mM [14C]-MGBG (specific activity 26 mCi/mmol,
Amersham) was prepared aseptically. Aliquots of this stock were added to
media to obtain the indicated final concentration of drug.
Uptake Protocol
Cells were incubated with [14C]-MGBG at concentrations ranging from
1.0 to 3.3 (iM. At the end of each incubation period, an appropriate aliquot of
cells was transferred to a 15-ml tube, centrifuged at 650g for 3 minutes, and
the supernatant was decanted. To remove any labeled drug bound to the
outside of the cell, the cell pellet was washed twice with 4°C media containing
1 mM unlabeled MGBG. The cell pellet was resuspended in 1 ml of distilled
water, transferred to a scintillation vial, and the amount of [14C]-label
quantitated by scintillation counting.
Cloning of MtDNA Probe
The 16,295 base pair, full-length murine mtDNA was inserted into the
Sac I site of pSP64 and transfected into E. coll strain HB101 (gift of W.
Hauswirth). A 20 |il inoculate of E. coli containing the recombinant plasmid
was aseptically transferred to 250 ml of sterilized Luria broth containing 2.5 g
Bacto-tryptone, 1.25 g Bacto-yeast extract (Difco Laboratories, Detroit, Ml), 2.5
g sodium chloride, pH 7.5, and 25 mg ampicillin (Sigma Chemical). The
bacteria were incubated for 16 hrs in a 37°C shaking incubator (New
Brunswick Scientific, Edison, NJ) in a 1 L sterilized flask to maximize aeration.

The bacteria were transferred to six 50-ml tubes and centrifuged at
2650g for 15 min at 4°C. Pellets were resuspended in 0.5 ml sterile buffer
(25% sucrose, 50 mM Tris, pH 8.0) and combined in a 50 ml centrifuge tube.
Five mg of egg white lysozyme (Calbiochem, La Jolla, CA) was added and,
after mixing, the material was allowed to incubate at room temperature for 5
min. One ml of sterile Na2EDTA (0.25 M, pH 8.0) was added, and the
suspension was allowed to incubate at room temperature for 2 min. Eight ml
of a buffer/detergent/ (50 mM Tris and 62.5 mM Na2EDTA, pH 8.0; that was
sterilized before addition of 2% Triton X-100) was added and mixed gently for
2-5 min at room temperature until the viscosity increased markedly. The
mixture was then centrifuged immediately at 31,500g for 25 min at 12°C.
The supernatant was transferred to a 50 ml centrifuge tube, the volume
measured, and an equal volume placed in each of 2 ultracentrifuge tubes.
Cesium chloride (0.9 g/ml of supernatant) (BRL Scientific, Gaithersburg, MD)
was added and mixed by inverting the tube. Ethidium bromide (0.5 mg/ml of
supernatant) was added, mixed, and protected from light in all further steps.
The tubes were balanced and centrifuged at 92,000g for 36-40 hrs at 20°C.
Bands of material which fluoresced upon illumination at 360 nm were
located with a hand-held UV light (Ultra-Violet Products, Inc., San Gabriel,
CA). The bands were, starting at the top, protein, Form II DNA (nicked
circular), Form I DNA (closed circular), and RNA. Form I DNA was collected
and transferred to a centrifuge tube. The ethidium bromide was removed by
repeated extraction with isopropanol:5M NaC! (1:1) until tne isopropanci was
completely transparent two consecutive times. For each ml of solution, 2.6 ml
of distilled water and 7.2 ml of 20°C 95% ethanol were added. The mixture
was then placed in an ethanol/dry ice bath for 30 min and centrifuged at
9000g for 30 min at 4°C. The pellet was rinsed with 70% ethanol and vacuum

50
dried for 30 min. The final pellet was resuspended in 200 pi of TE buffer (10
mM Tris-HCI containing 1 mM Na2EDTA, pH 8.0).
The molecular weight and concentration of DNA was estimated by gel
electrophoresis of the sample and comparison to marker DNA's, the
concentration and molecular weight of which were known.
Nick Translation of MtDNA Probe
To label the mtDNA probe with [35S] nucleotides, 1 pg of the cloned
probe was added to 20 pM each of dCTP, dGTP, and dTTP, 50 pCi of
deoxyadenosine 5'(alpha [35S]thio)triphosphate (specific activity 1145
Ci/mmole, Amersham, Arlington Heights, IL), and a mixture of polymerase I
and DNase I (0.2 units and 0.2 ng, respectively) (BRL Scientific) in a final
volume of 50 pi. The mixture was allowed to incubate at 15-17°C for 90 min.
Stop solution (30 mM Na2EDTA) was added, and the mixture put on ice.
The nick translated products were loaded onto a Sephadex G-50
column (Pharmacia Inc., Piscataway, NJ) which had been washed previously
with TE buffer and loaded with 3 pg yeast tRNA. The column was centrifuged
at 400g for 4 min and the eluate collected in a 1.5 ml Eppendorf tube.
Gel Electrophoresis
Gels were prepared with 0.7% agarose (Bio-Rad Laboratories,
Richmond, CA) in TBE buffer (1 M Tris, 0.5 M borate, and 50 mM Na2EDTA,
pH 8.0). Molecular weight markers were the Hind III fragments of lambda virus
DNA and a supercoiled pBR322 ladder (BRL Scientific). Tracking dye
(0.025% bromcphenol blue in 2% glycerol) was added to all samples.
Samples were electrophoresed for 150 minutes at 100 volts; the running
buffer was TBE. The gels were stained in the dark with 1 pg/m! ethidium
bromide for 30 min. The DNA was visualized at 360 nm on a UV light box
(Ultra-Violet Products). The gel was photographed under UV light with a

Polaroid camera (F-stop 8, exposure 4 sec) and Type 57 film (Polaroid Corp.,
Cambridge, MA).
Southern Blot
After electrophoresis, the gel was soaked in 100 ml 0.5 M NaOH in 1.5
M NaCI for 1 hr to denature the DNA. The gel was neutralized with 100 ml 1 M
Tris (pH 8.0) in 1.5 M NaCI for 1 hr. The gel was then soaked in 100 ml 10X
SSC (20X SSC = 3 M NaCI, 0.3 M sodium citrate, pH 7.0) for 15 min. The gel
was blotted onto nitrocellulose filters (Bio-Rad Laboratories) by the method of
Southern (212). The filters were incubated at 85°C in a vacuum oven
(National Appliance Company) for 2-3 hrs. Immobilized DNA was hybridized
with the full-length mouse mtDNA probe inserted in the Sac I site of pSP64
and labeled by nick translation with [35s]-labeled dATP described above.
Dot Blot
Mitochondrial DNA was quantitated by dot blot analysis of whole cell
lysate by a modification of the method of Barker et aL (213). Aliquots of 0.5-
2.0x1 o5 cells were placed in 1.5 ml Eppendorf tubes. The cells were
sedimented by centrifugation for 10 sec at 13,000g in a microfuge (Fisher
Scientific). The supernatant was decanted, and the cells were resuspended in
4 |il of buffer (250 mM sucrose, 10 mM Tris, and 1 mM Na2EDTA, pH 7.4).
Cells were then incubated in a mixture of 2% sodium dodecyl sulfate, 5 pg
proteinase K (Sigma Chemical), and 100 units RNase T1 (BRL Scientific) in a
final volume of 7.5-8.5 pi. The initial incubation was for 15 min at 37°C, a
procedure which allowed degradation of up to 4 pg of added yeast tRNA
without inactivation of the RNase T1 by the proteinase K (data not shown).
This was followed by incubation for an additional 2-3 hr at 50°C.
The cell lysate was then treated with 0.1 M NaOH to denature DNA, and
the mixture was applied to nitrocellulose filters with a dot blot apparatus

52
(BioRad Laboratories). Immobilized DNA was hybridized with the full-length
mouse mtDNA probe inserted in the Sac I site of pSP64 and labeled by nick
translation with [35S]-labeled dATP described above. The dots were
visualized by autoradiography (SB-5 X-ray film, Eastman Kodak). To assure
equal size of the dots, nitrocellulose paper was cut out with a hole punch, and
quantitated by scintillation counting.
Hybridization with f33S]-labeled MtDNA Probe
Nitrocellulose paper from the Southern or dot blot was placed in a
plastic Seal-a-meal bag (Sears) and a pre-hybridization solution was added
(30 ml 6X SSC, 0.6 ml 50X Denhardt's solution [1% w/v Ficoll 400,
polyvinylpyrrolidone, and bovine serum albumin in distilled water], and 0.9 mg
denatured yeast tRNA). The bag was sealed and allowed to incubate for 8 hrs
at 68°C.* The pre-hybridization solution was then drained, and the
hybridization solution added (10 ml 6X SSC, 0.2 ml 50X Denhardt's solution,
0.1 mg denatured yeast tRNA, and 50 jil denatured [35S]-labeled mtDNA).
The bag was resealed and incubated 18-24 hrs at 68°C.
The hybridization solution was drained into a radioactive waste
container, and the blot was removed. The blot was washed first in 100 ml of
6X SSC for 2 hrs at 68°C, and then in 100 ml of 2X SSC for another 2 hrs at
68°C. The blot was placed between 2 sheets of filter paper and dried in a
vacuum oven (National Appliance Company) for 15 min at 85°C. The blot was
placed under autoradiographic film (SB-5, Eastman Kodak) in a film cassette.
The film was exposed for 15-20 hrs and developed witn an X-ray film
processor (Konishiroku Photo Co., Japan).

RESULTS
Characterization of L1210 Cells
Growth Characteristics of Untreated L1210 Cells
To determine the growth characteristics of the untreated murine
leukemia L1210 cell line, cells were seeded to a starting concentration of
2.5x104 cells/ml and incubated at 37° for 5 days without reseeding or adding
fresh medium. The exponential growth, or log phase, of these cells
encompassed a concentration range from 2.5x104 to 2x106 cells/ml (Figure
4). The plateau phase of cell growth occurred at >2x106 cells/ml. Viability, as
measured by trypan blue exclusion, was >98% in log-phase cells but
progressively declined to 40% by 6 days without reseeding. The mean
population doubling time for the log-phase cells under these conditions was
11 hrs. For all further experiments L1210 cells were maintained in
exponential, or log phase, growth by reseeding every 2 days into fresh
medium and with fresh drug, where appropriate.
Coov Number of MtDNA in Untreated L1210 Cells
In order to reliably and accurately assay the mtDNA content of L1210
cells, we found it necessary to routinely incorporate an external (and
occasionally, an internal) standard in the dot blot procedure. The plasmid
pSP64, which had served as the vector for cloning of the mouse mtDNA, was
used as the standard because 1) its quantity (in terms of DNA) is easy to
assess and 2) it hybridizes to the radioiabeiied, nick-transiated probe because
the probe contains the appropriate complementary sequences. This meant

Figure 4. Cell counts/ml of L1210 cells grown in suspension culture for 6 days
without reseeding. Starting concentration of the cells was
2.5x104/ml. Growth was exponential for 3 days, and plateau phase
was reached at 2x106 cells/ml.

Days
Cell counts/ml
O O o o
â– f* d a -g
99

56
that only one probe (containing both pSP64 and mtDNA sequences) was
required for hybridization, a procedure that minimized variation between the
degree of hybridization of the standard and the mtDNA. A standard curve (ng
DNA vs cpm's [counts per minute]) was generated for each dot blot
experiment. The copy number for mtDNA in L1210 cells was calculated from
this experimental value for amount of mtDNA in a given number of cells and
the molecular weight (107 daltons) of murine mtDNA.
Certain other assumptions were validated in development of this dot
blot. First, the nick-translated probe (containing both plasmid and mouse
mtDNA) hybridized equivalently to both the plasmid standard and cellular
mtDNA, a necessary condition for direct comparison of the counts. Second,
we confirmed that the cell lysate £er se did not interfere with hybridization. It
was found that varying mixtures of cell lysate and plasmid yield the expected
additive value for DNA which hybridized to the probe. And, finally,
radiolabelled, nick-translated pSP64 plasmid itself did not hybridize with
cellular DNA.
For determination of the mtDNA copy number, known quantities (1-6
ng) of plasmid and known aliquots (1.0-6.0x105) of L1210 cells were added to
a dot blot apparatus and hybridized with a [35s]-labeled full-length murine
mtDNA probe inserted into pSP64. The extent of hybridization of probe to
mtDNA was proportional to aliquot size at least over the range of 1.0x10^ to
6.0x105 cells (Figure 5). This asynchronous population of L1210 cells
contained 1450 copies of mtDNA per cell; the mean standard deviation of 3
experiments was ±13%.

Figure 5. The amount (ng) of mtDNA in increasing quantities of exponentially-
growing L1210 cells. The amount of mtDNA was measured by dot blot
analysis, and was linear over the range of 0-6x105 cells. Each point
represents the mean value of 3 separate experiments; the mean
standard deviation of the 3 experiments was ±13%.

Cell no. X 10
Amount of mtDNA (ng)
i
cn
O ro -fck. CD OD o
8 g

59
Effects of Polvamine Analogs on Cell Division and MtDNA Accumulation
Dose-Ranging Experiments with MGBG
Concentration response for MGBG. Because the intent of these
experiments was to use MGBG to modulate the mtDNA content of L1210 cells
without causing overt cell toxicity, an MGBG concentration response
experiment was done to define the concentration range at which MGBG
inhibited mtDNA accumulation without causing a substantial decrease in cell
viablity. The initial experiment encompassed MGBG concentrations from 0.1
to 100 |iM in the medium. Cell counts and viability were determined daily for
two days.
Cell growth over this 2-day period was inhibited by MGBG in a
concentration-dependent manner (Figure 6). After 2 days of incubation with
MGBG, aliquots of cells from each treatment were lysed and the mtDNA
quantitated. When the concentration of MGBG in the medium was <1.0 (iM,
there was no effect on the content of mtDNA per cell (Table 1). At MGBG
concentrations >3.3 pM, mtDNA content per cell was decreased compared to
control, but cell viability was greatly reduced, especially at MGBG
concentrations >10 pM. Furthermore, 55% of cells treated with 10 pM MGBG
for 2 days had lost the ability to form clones (compared to untreated controls).
The IC5o's of MGBG for inhibiting cell growth at 24 and 48 hrs of
treatment were 10 and 2 pM, respectively.
Decreased mtDNA content at 1.8 to 3.3 uM MGBG. Based on these
previous results, the MGBG concentration range was narrowed to 1.0 to 3.3
pM for all further experiments. These concentrations provided optimal
differentiation of the effects of MGBG on cell growth and
»jni*h rrnnimol
v«iu i i i In in i iui
overt cellular toxicity. Although MGBG had discernible effects at
concentrations <1.3 pM (see below), attention will first be directed to results

Figure 6. Cell counts/ml of L1210 cells exposed to 0-100 pM MGBG for 0-2
days. Starting concentration of the cells was 7.2x104/ml. Cell
counts were done in duplicate for each experiment; each point
represents the mean value of 2 separate experiments.

Cell counts/ml
control
-+â–  0.10 pM
-o- 0.33
1.0
3.3 nM
-» 10 [iM
33 |iM
100 (iM
o\
Days MGBG treatment

62
Table 1. Effects of MGBG on the relative amount of mtDNA/cell and the
percent viability in L1210 cells exposed to 0-100 pM MGBG for 2
days.
Treatment
(pM MGBG)
MtDNA/Cell
(% Control)
% Cell Viability
Control
100
99
0.10
105
98
0.33
94
99
1.0
95
98
3.3
23
96
10
26
80
33
*
75
100
★
60
The mtDNA was determined 8 times and the cell viability once for each
experiment; each point represents the mean value of 2 different experiments.
not done.

63
obtained with MGBG in the range of 1.8 to 3.3 (iM, concentrations at which
there are consistent and striking effects on mtDNA accumulation. Although the
cells were exposed in these experiments to MGBG for 18 days, our initial focus
involves the first 3 days of MGBG treatment because mtDNA synthesis
became resistant to MGBG after that time. This phenomenon will be explored
in more detail in a later section.
Cells were incubated with 1.8 to 3.3 pM MGBG and reseeded every 2
days with fresh medium and drug. At these concentrations of MGBG, cell
growth was inhibited in a concentration-dependent manner, i.e. the greater the
dose, the greater the degree of inhibition (Figure 7). However, this difference
was small for the first 3 days of exposure. Conversely, the effect of MGBG on
mtDNA accumulation did not show a gradation in degree of inhibition, but
rather in the duration of inhibition (Figure 8). Incubation of L1210 cells with
1.8, 2.5, or 3.3 pM MGBG prevented accumulation of mtDNA for 1,2, and 3
days, respectively.
Because cell growth continued at a rate which was relatively faster than
the accumulation of mtDNA, a decrease in the amount of mtDNA per cell can
be anticipated with each cell division. The amount of mtDNA per cell did
decrease progressively to only 10% of the control value upon treatment with
2.5 or 3.3 pM MGBG (Figure 9). The time course of this decrease was
consistent with a complete inhibition of mtDNA accumulation coupled with
continued cell division (see theoretical curve in Figure 9), i.e., the amount of
mtDNA per cell was halved with each division.
Determination of mtDNA degradation by MGBG. The inhibition of
mtDNA accumulation by MGBG may reflect inhibition of synthesis, increased
degradation, or both. Consequently, mtDNA was isolated from MGBG treated
cells to determine if there was increased mtDNA damage relative to control.

Figure 7. Cumulative cell counts for L1210 cells exposed to 0-3.3 pM MGBG for
0-3 days. Cells were reseeded every 2 days with fresh media and
drug. Cell counts were done in duplicate for each experiment; each
point represents the mean value of 4 separate experiments.

Cumulative cell counts
-a- control
1.8(iM
-°m 2.5 )iM
-*â–  3.3 (iM
o\
Days MGBG treatment

Figure 8. MtDNA accumulation for L1210 cells exposed to 0-3.3 pM MGBG for 0-
3 days. Cells were reseeded every 2 days with fresh media and drug.
The mtDNA was determined 8 times and the cell counts twice for
each experiment; each point represents the mean value of 4 separate
experiments.

MtDNA accumulation
o
o
<
Z
a
■*-»
E
S*
x
(/)
â– 4-^
C
3
O
o
O
control
1.8 |iM
2.5 nM
-o- 3.3 |iM
o\
Days MGBG treatment

Figure 9. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-3.3 pM MGBG for 0-3 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 4 separate
experiments.

140
120
100
80
60
40
20
0
control
1.8 |iM
2.5
-o- 3.3 [iM
theoretical*
ON
NO
T
1
T
2
3
Days MGBG treatment

70
Mitochondrial DNA from equal numbers of control or MGBG treated cells was
isolated and separated into Forms I, II, or III (supercoiled, nicked circular, or
linear, respectively) by gel electrophoresis. The gel was transferred to a
nitrocellulose filter, and the filter was hybridized with a [35S]-labeled mtDNA
probe.
Autoradiography of the blot showed that there was no substantial
change in the ratio of intact and nicked forms upon exposure of cells to 1.0 or
3.3 (iM MGBG. However, the total amount of mtDNA at 1.0 pM had decreased
and at 3.3 pM this decrease was marked. At a higher dose (10 pM) than those
used in the mtDNA accumulation experiments, there was a decrease in the
intact form and a corresponding increase in the linear form of mtDNA.
Interestingly, however, the total content of mtDNA was more than at the 3.3 pM
treatment. This suggests that damaged mtDNA was not degraded and that
cell growth was so inhibited at 10 pM MGBG that the mtDNA copy number was
diluted less than at 3.3 pM. Taken together, these results are compatible with
the hypothesis that the decrease in mtDNA content, at least in the 1.0 to 3.3
pM range, reflected a lack of synthesis rather than increased degradation.
Effects of Diethylspermine (DES) on Cell Growth and MtDNA
We tested another polyamine analog to determine if it might have
similar effects on mtDNA accumulation and cell growth. This work was done
in collaboration with Dr. Ray Bergeron and Mike Ingeno in the College of
Pharmacy. Diethylspermine (DES, a gift of R. Bergeron, is spermine with ethyl
substituents on each of the 2 terminal nitrogen atoms) was made to a stock
solution of 1 mM. The L1210 cells were reseeded with fresh medium and drug
every 2 days, and incubated with 0, 0.1, 1.0, or 10 pM DES for 6 days. The
growth rates of cells exposed to 0.1 pM DES were the same as control (data
not shown). Cells exposed to 1.0 and 10 pM DES for 1 day had normal

growth, but their growth was inhibited after 2 days, and was slowest from 4-6
days (Figure 10). Cell viability (as measured by trypan blue exclusion) was
>90% over the 6-day period.
The mtDNA accumulation was not affected in cells exposed to 0.1 pM
DES (data not shown). However, the mtDNA accumulation of cells exposed to
1.0 or 10 pM DES was substantially inhibited and showed no signs of
recovery during the 6-day exposure to DES (Figure 11). Whereas the cells
exposed to 0.1 pM DES had a normal content of mtDNA, the mtDNA content of
cells exposed to 1.0 or 10 pM DES decreased to 10-15% of control by 3 days
(Figure 12). The mtDNA content remained at this low level through day 6 of
DES exposure.
Effects of Low Doses of MGBG
Increased mtDNA content at 1.0 to 1.3 uM MGBG. The results obtained
with MGBG concentrations <1.8 pM were interesting. After 1 day of treatment
with 1.0 or 1.3 pM MGBG, there was a slight inhibition of mtDNA accumulation
relative to control (Figure 13), in general agreement with the decreasing
duration of inhibition with decreasing MGBG concentration (Figure 8).
Thereafter, the rate of mtDNA accumulation equaled that of control. The rate
of cell growth was slightly inhibited by 24 hrs but, unlike mtDNA accumulation,
continued to be slightly inhibited throughout the 6-day exposure (Figure 14).
By dot blot analysis, 1.0 and 1.3 pM MGBG caused a 12 and 25% decrease,
respectively, in the content of mtDNA per cell at 24 hrs (Figure 15). However,
after this initial decrease, the amount of mtDNA per cell increased and by 4
days actually exceeded the control value. This increase resulted from a
normai rate of mtDNA accumulation in cells that were slightly growth-inhibited.
When the mtDNA content per cell was followed for 18 days at these
concentrations of MGBG, the mtDNA copy number never exceeded 155% of

Figure 10. Cumulative cell counts for L1210 cells exposed to 0-10 pM DES
for 0-3 days. Cells were reseeded every 2 days with fresh media
and drug. Cell counts were done in duplicate for each experiment;
each point represents the mean value of 2 separate experiments.

Cumulative cell counts
control
1 (iM
-o- 10 |iM
u>
Days DES treatment

Figure 11. MtDNA accumulation for L1210 cells exposed to 0-10 pM DES for
0-3 days. Cells were reseeded every 2 days with fresh media and
drug. The mtDNA was determined 8 times and the cell counts twice
for each experiment; each point represents the mean value of 2
separate experiments.

Days DES treatment
MtDNA accumulation
[Cell counts X (%mtDNA/cell)]
o o o o o
cn o «sj co * i *
o
O T= O
-C 2 =
S o
sz.

Figure 12. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-10 jiM DES for 0-6 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 2 separate
experiments.

120
100
80
60
40
20
0
-o- control
1 |iM
-»■ 10 nM
-j
i â–  1 â–  1 ' 1 â–  1 1
1 2 3 4 5 6
Days DES treatment

Figure 13. MtDNA accumulation for L1210 cells exposed to 0-1.3 pM MGBG for
0-6 days. Cells were reseeded every 2 days with fresh media and
drug. The mtDNA was determined 8 times and the cell counts twice
for each experiment; each point represents the mean value of 4 or 2
separate experiments for 1.0 or 1.3 pM, respectively.

MtDNA accumulation
Q)
O
z
Q
X
«
c
3
O
u
CD
O
control
1.0
-o- 1.3 pM
si
VO
Days MGBG treatment

Figure 14. Cumulative cell counts for L1210 cells exposed to 0-1.3 pM MGBG
for 0-6 days. Cells were reseeded every 2 days with fresh media
and drug. Cell counts were done in duplicate for each experiment;
each point represents the mean value of 4 or 2 separate experiments
for 1.0 or 1.3 pM, respectively.

Cumulative cell counts
0 1 2 3 4 5 6
Days MGBG treatment
+ I -Si

Figure 15. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-1.3 pM MGBG for 0-6 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 4 or 2 separate
experiments for 1.0 or 1.3 pM, respectively.

160
140
120
100
80
60
40
20
0
-o- control
-♦* 1.0(iM
-®- 1.3 |iM
oo
LO
Days MGBG treatment

84
control. Thus, even though the rates of mtDNA accumulation and cell growth
at low concentrations of MGBG were similar, there were subtle differences that
caused the mtDNA content per cell to vary between 75% of control early in
exposure to 155% of control after several days.
Effects on mtDNA of decreased cell growth due to serum depletion.
Because of the unexpected increase in mtDNA per cell following incubation
with low concentrations of MGBG, an experiment was done to determine if this
increase was a nonspecific effect of decreased cell growth. If mtDNA
replication and cell division are not tightly coupled, then it can be
hypothesized that an increased mtDNA copy number may result from any
condition which causes decreased cell growth. To test this idea, mtDNA
content was measured in L1210 cells incubated in media containing limiting
amounts of serum. Decreases in serum concentration slowed cell growth in a
proportional way; the mtDNA content per cell at 3 days, however, was not
different from control (Figure 16).
Resistance to MGBG and Recovery of MtDNA Synthesis and Copy Number
Resistance to MGBG
Resistance of mtDNA synthesis to MGBG. As mentioned earlier, mtDNA
synthesis in L1210 cells resumed 1,2, or 3 days after the start of exposure to
1.8, 2.5, or 3.3 pM MGBG, respectively. This recovery occurred despite the
continued presence of drug in the medium, and suggested that the cell was
able to overcome the initial effects of MGBG on mtDNA accumulation. In
addition, mtDNA synthesis, once resumed, occurred at the same rate,
regardless of dose of MGBG (Figure 17). Surprisingly, this rate of mtDNA
accumulation was virtually equal to that seen in untreated, growing cells-in
spite of the fact that the treated cells exhibited a decreased growth rate over
this time period (Figure 18).

Figure 16. The mtDNA/cell (expressed as % control) of L1210 cells incubated
for 3 days in media supplemented with different percentages of
serum. Cells were reseeded on day 2 with fresh media and the
appropriate amount of serum. The mtDNA was determined 8 times
for each experiment; each point represents the mean value of 2
separate experiments. Ten percent serum represents the control
value.

% Serum
% Control mtDNA
in
vO
O'
98
120

Figure 17. MtDNA accumulation for L1210 cells exposed to 0-3.3 pM MGBG for
0-6 days. Cells were reseeded every 2 days with fresh media and
drug. The mtDNA was determined 8 times and the cell counts twice
for each experiment; each point represents the mean value of 4 or 2
separate experiments for 1.8 and 2.5, or 3.3 pM, respectively.

MtDNA accumulation
o
z
Q
X
V)
c
Z3
o
o
o
O
T 1 I 1 I ' 1 1 1 ' 1 '
0 1 2 3 4 5 6
-o- control
-+â–  1.8 |jM
2.5 (jM
-o- 3.3 nM
oo
oo
Days MGBG treatment

Figure 18. Cumulative cell counts for L1210 cells exposed to 0-3.3 pM MGBG
for 0-6 days. Cells were reseeded every 2 days with fresh media
and drug. Cell counts were done in duplicate for each experiment;
each point represents the mean value of 2 or 4 separate experiments
for 1.8 and 2.5, or 3.3 pM, respectively.

Cumulative cell counts
0 1 2 3 4 5 6
-o- control
1.8 (iM
-**- 2.5 |iM
3.3 nM
VO
o
Days MGBG treatment

In contrast to the quantal (on/off) effect of MGBG on mtDNA
accumulation, cell growth exhibited a graded response to varying doses of
MGBG (Figure 18). Cell growth was inhibited in a concentration-dependent
and time-of-treatment-dependent manner.
The net effect of these different rates of mtDNA accumulation and cell
growth was to modulate the mtDNA copy number of these cells (Figure 19).
As noted earlier, there was an initial decrease in the copy number of mtDNA
due to complete inhibition of mtDNA accumulation for up to 3 days. After that,
the copy number increased towards the control value because of the
combined effects of the recovery of mtDNA synthesis and a marked decrease
in the cell growth rate.
Effects of transfer of treated cells to a MGBG-free medium. This result,
in which mtDNA accumulation recovered, but at a rate which never occurred
faster than the 12-hr doubling time of control cells, was unexpected in view of
the reported observation that the mtDNA genome can replicate in vitro in
about 1 hr (214). One possibility was that this 12-hr doubling time
coincidentally represented a continued, but lesser, inhibition of mtDNA
replication by MGBG. To rule out this possibility, a study was done in which
L1210 cells were treated with MGBG and then transferred to drug-free medium
at the time at which recovery of mtDNA synthesis regularly occurred. If the
drug continued to cause partial inhibition of mtDNA synthesis, then those cells
transferred to drug-free medium should have a faster rate of mtDNA
accumulation than those remaining in drug.
The L1210 cells were treated with 3.3 pM MGBG for 4 days. At that
time, haif the ceiis were maintained ai 3.3 pM while the other half were
transferred to drug-free medium for an additional 4 days. Cell counts and dot
blot analysis of mtDNA content were done during the initial exposure to

Figure 19. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-3.3 jiM MGBG for 0-6 days. Cells were reseeded every 2 days with
fresh media and drug. The mtDNA was determined 8 times for each
experiment; each point represents the mean value of 4 or 2 separate
experiments for 1.8 and 2.5, or 1.3 pM, respectively. The bars for
the 3.3 pM group represent ± 1 standard deviation.

140
120
100
80
60
40
20
0
â– o- control
1.8 nM
2.5 }iM
-o- 3.3 hM
u>
Days MGBG treatment

94
MGBG, and following transfer of cells to media with or without the same
concentration of MGBG. The results indicated that, after the recovery of
mtDNA synthesis, the rate of mtDNA accumulation was the same whether or
not MGBG was present (Figure 20). The cell growth rate was also virtually the
same for 2 days after the transfer. After this 2-day lag, however, the drug-free
cells divided more rapidly.
Determination if resistance to MGBG is genetic. Cell growth studies
indicated that MGBG-resistance was not due to selection of a genetically-
resistant population since at no time during drug treatment was there a
decrease in cell number, as might be expected if MGBG-sensitive cells had
died. This is in agreement with the cell viability (as measured by trypan blue
exclusion) which remained >90% throughout MGBG treatment. Also, cells
treated with 3.3 pM MGBG for 2 days formed colonies on clonogenic assay at
95% the control frequency. The results suggest that virtually all MGBG-treated
cells were able to recover and grow, not just a select group.
However, to further evaluate the possibility that resistance of mtDNA
synthesis in L1210 cells to inhibition by MGBG was due to selection of a
mutant population, previously-treated ("resistant") L1210 cells were
rechallenged with MGBG. Cells were treated for 10 days with 3.3 pM MGBG
and then transferred to drug-free media for 4 days. These cells were then
retreated with 3.3 pM MGBG. If resistance was due to genetic mutation with
subsequent selection of this population with MGBG treatment, retreated cells
should be resistant to the effects of MGBG on mtDNA replication. Also, the
relatively short duration of drug-free exposure makes the possibility of back
mutation(s) repopulating the inoculum quite unlikely.
The results of this experiment show that previously-treated cells are as
sensitive as drug-naive cells to inhibition of mtDNA synthesis by MGBG (Table

Figure 20. The accumulation of cells and mtDNA in L1210 cells exposed to 0
(control) or 3.3 pM MGBG for 4 days (day 0 to day 4). At day 4, the
cells from each group were split into 2 sub-groups. One sub-group
was continued in medium with 3.3 pM MGBG, and the other group was
transferred to drug-free medium (day 4 to day 8). Cells were
reseeded every 2 days with fresh media and the appropriate amount
of drug. The mtDNA was determined 8 times for each experiment and
the cell counts twice; each point represents the mean value of 2
separate experiments.

Cell or mtDNA accumulation
10'
10‘
10'
10'
10'
10
0
2
4
Days
control
cells-3.3 (.iM
cells-drug free
mtDNA-3.3 |iM
mtDNA-drug free
o>

97
2). This is inconsistent with a genetic change but in agreement with a
phenotypic adaptation by the cell to resist the effects of drug treatment.
Factors Affecting the Recovery of the MtDNA Copy Number
Recovery of the original mtDNA copy number. A further objective of this
research was to determine if the mtDNA copy number of L1210 cells
chronically treated with MGBG would return to the original value of 1450
copies per cell or, conversely, establish a new steady state. Thirty-one days
after the start of exposure to MGBG, the cells grown in both the 2.5 and 3.3 pM
MGBG had regained their characteristic content of 1450 mitochondrial
genomes, whereas the cells treated with <1.3 pM MGBG were within ±25% of
that value (Figure 21).
Cell growth oscillation during recovery of mtDNA copy number. To
determine the relative contributions of mtDNA accumulation and cell growth to
the recovery of the mtDNA content, the rates of each were contrasted. One
would anticipate that, no matter what the absolute rates of mtDNA synthesis
and cell division were, mtDNA synthesis must be faster, relative to cell
division, for repletion to occur. This may occur through a rate of mtDNA
synthesis greater than normal, through a slowing of cell growth, or a
combination of both.
Results described above revealed that when mtDNA synthesis resumed
after initial inhibition by MGBG, the rate of its accumulation was virtually the
same as that seen in untreated cells; this rate remained relatively constant
throughout the 18-day MGBG exposure (Figure 22). Cell growth rate, on the
other hand, was regularly variable (Figure 23). To better contrast the relatively
constant mtDNA accumulation rate and the more variable cell growth rate,
mtDNA and cell accumulation were graphed in terms of their doubling times
as a function of time of treatment (Figure 24).

98
Table 2. Effects of previous exposure to MGBG on the resistance of
mtDNA accumulation in L1210 cells to inhibition by MGBG.
Pre-treatment
Treatment
(pM MGBG)
Days of
Exposure
MtDNA/Cell
(% Control)
none
0
0
100
4
100
none
3.3
0
100
4
44
yes
3.3
0
100
4
45
Pre-treated cells were exposed to 3.3 |iM MGBG for 10 days and then
transferred to drug-free medium for 4 days. These cells and drug-naive cells
were assayed for mtDNA content to determine their baseline values (0 days of
treatment) and then exposed to 3.3 pM MGBG for 4 days.

Figure 21. The mtDNA/cell (expressed as % control) of L1210 cells after
exposure to 0-3.3 pM MGBG for 31 days. Cells were reseeded every 2
days with fresh drug and media. The mtDNA was determined 8 times
for each experiment; each point represents the mean value of 2
separate experiments.

MtDNA/cell (% control)
120
100 -
80 -
60 -
40 -
20 -
0
31 days MGBG
K3 control
E3 1.0 nM
O 1.3mM
E¿3 2.5 nM
â–¡ 3.3 (iM
100

Figure 22. MtDNA accumulation for L1210 cells exposed to 0-3.3 pM MGBG for
0-18 days. Cells were reseeded every 2 days with fresh media and
drug. The mtDNA was determined 8 times and the cell counts twice
for each experiment; each point represents the mean value of 2
separate experiments..

Days MGBG treatment
MtDNA accumulation
[Cell counts X (%mtDNA/cell)]
O
cn
o
o
o
o
CD
o
CO
Hi H
to ro o
to tn bo °
T= T= T= ~
SSSo
ZO I

Figure 23. Cumulative cell counts for L1210 cells exposed to 0-3.3 pM MGBG
for 0-18 days. Cells were reseeded every 2 days with fresh media
and drug. Cell counts were done in duplicate for each experiment;
each point represents the mean value of 2 separate experiments.

Cumulative cell counts
-c- control
1.8 mM
-*â–  2.5 mM
-o- 3.3 jiM
Days MGBG treatment
104

Figure 24. The cell and mtDNA doubling (or generation) time for L1210 cells
exposed to 0 (control) or 2.5 pM MGBG for 18 days. Cells were
reseeded every 2 days with fresh drug and media. The mtDNA was
determined 8 times for each experiment and the cell counts twice;
each point represents the mean value of 2 separate experiments.
The open boxes represent the control value for both cell and mtDNA
doubling times. For cells exposed to 2.5 pM MGBG, the diamonds
represent the cell doubling time and the closed boxes represent the
mtDNA doubling time.

Days MGBG treatment
Cell or mtDNA doubling time
-*â–  ro co .u in
o o o o o o
901

107
Cell growth rates were lowest immediately after the nadirs of mtDNA
content; this presumably allowed the mtDNA copy number to return toward
normal (Figure 25). Before the copy number had completely recovered,
however, cell growth increased toward normal so that the amount of mtDNA
per cell was once again depleted. This cycle of mtDNA depletion and
repletion occurred with a periodicity of 6 days. The first oscillation in mtDNA
content had the greatest magnitude because of the initial inhibition of mtDNA
synthesis. Subsequent oscillations were progressively muted since mtDNA
accumulation was no longer inhibited.
Thus, mtDNA accumulation and cell growth both contributed to recovery
of the mtDNA copy number, but in different ways. On one hand, mtDNA
accumulated at a relatively constant rate which never exceeded that of control
cells. Cell growth rate, on the other hand, increased or decreased in a cyclical
way so as to maintain the amount of mtDNA per cell within certain limits and
ultimately to 'fine tune' the mtDNA content back to its original 1450 copy
number.
Effect of MGBG on the cell cycle. It is possible that the repletion of
mtDNA occurred during a particular phase of the cell cycle, and that during
repletion cells were "blocked" in that phase. To test this hypothesis, cells were
analyzed by flow cytometry for arrest in a certain phase of the cell cycle.
Because large discrepancies between cell growth and rate of mtDNA
accumulation occurred between 3 and 5 days of MGBG treatment, cells were
analyzed at these times. Flow cytometric analysis of control and MGBG-
treated cells at 3 and 5 days of treatment showed that there was no change in
the distribution of nuclear DNA content among ceiis (Tabie 3). There was no
discernible block in any phase of the cell cycle; rather, the entire cell cycle was
presumably lengthened because cell growth was slowed.

Figure 25. The mtDNA/cell (expressed as % control) for L1210 cells exposed to
0-3.3 pM MGBG for 0-18 days. Cells were reseeded every 2 days
with fresh media and drug. The mtDNA was determined 8 times for
each experiment; each point represents the mean value of 2 separate
experiments.

140
120
100
80
60
40
20
0
-o- control
1.8 (iM
2.5 |iM
-o- 3.3 |iM
1 1 i â–  i â–  i i * i â–  i 1 i â– 
2 4 6 8 10 12 14 16 18
Days MGBG treatment
109

Table 3. The percent distribution within each phase of the cell cycle of
L1210 cells exposed to 0 (control) or 3.3 pM MGBG for 3 or 5 days.
Treatment
Percent of cells in each phase
GO/1 S G2
Control
45.66
46.76
7.58
3.3 pM MGBG, 3 days
46.00
46.70
7.30
3.3 pM MGBG, 5 days
45.05
48.38
6.58
The cells were reseeded every 2 days with fresh media and drug. At
the appropriate times, aliquots of 1 x 10^ cells were resuspended in Hematall,
stained with 4',6-diamidino-2-phenylindole, and analyzed by flow cytometry.

Effects of MtDNA Content on Mitochondrial and Cell Functions
Rhodamine 123 Uptake
The vital stain, rhodamine 123, is a cationic dye which accumulates
reasonably specifically in mitochondria that generate an internal negative
membrane potential (215). Consequently, rhodamine 123 uptake was
determined as a measure of mitochondrial function after exposure of cells to
MGBG. Rhodamine 123 uptake was measured throughout 6 days of MGBG
exposure to determine if this particular mitochondrial function was affected by
a decrease in the mtDNA copy number.
More than 95% of cells exposed to 1.0-3.3 pM MGBG throughout a 6-
day period of exposure were able to take up rhodamine 123 (data not shown).
This percentage was the same as for control. In addition, no gross difference
in the amount of fluorescence per cell was seen; this suggests that these
mitochondria were present in reasonably normal numbers and were
maintaining a membrane potential even at times when the amount of mtDNA
per cell was as low as 10% of control. Thus, in these experiments rhodamine
123 uptake was affected neither directly by MGBG treatment nor indirectly as a
consequence of a decreased amount of mtDNA per cell.
Cell Size
Microscopic observation of cells exposed to MGBG suggested that they
were smaller in size than untreated cells. To verify this observation, the
diameters and volumes of control and MGBG-treated cells were determined.
Although cells exposed to 1.8 pM MGBG for 2 days had the same diameter
and volume as control cells, the size of treated cells decreased by 4 days
(Table 4). The cell diameter and volume were substantiaiiy decreased in ceils
exposed for 2 or 4 days to 3.3 pM MGBG. Cells exposed to 10 pM MGBG

Table 4. The cell diameter of L1210 cells after exposure to 1.8, 3.3, or 10
pM MGBG for 0 to 4 days.
Treatment
day 0
Cell diameter (p)
day 2
day 4
1.8 pM
11.2
11.2
9.5
3.3 pM
11.2
8.8
8.9
10 pM
11.2
9.0
★
The cells were reseeded every 2 days with fresh media and drug. At
the appropriate times, aliquots of 1 x 106 cells were resuspended in Hematall
and the cell diameter measured electronically.
*not done because of a >50% loss in cell viability

showed an intermediate decrease in cell size at day 2 but were not measured
at day 4 because of poor viability.
Uptake of MGBG
Determination of Vmax and Km for MGBG Uptake
Exponentially-growing L1210 cells were incubated with [14C]-MGBG at
concentrations of 1.0, 3.3, or 10 pM. The amount of intracellular radioactivity
in 1 x106 cells was determined after 0.5 and 3.5 hr of incubation. The
apparent uptake of [14C]-MGBG is shown for three different concentrations of
drug in the media (Figure 26). The rate of uptake was linear for at least the
first 3.5 hr. The Vmax and Km for MGBG uptake into the cells were determined
from the double-reciprocal plot of the velocity of uptake versus drug
concentration (Figure 27). The Vmax was 0.25 nmole/hr x 10§ cells and the
Km was 4 pM.
Time Course of MGBG Uptake
Because inhibition of drug uptake and/or increase in drug efflux is a
common mechanism whereby cells become resistant to drug, an experiment
was done to follow the intracellular content of MGBG over a 6-day period to
ascertain whether it related to the recovery of mtDNA synthesis. The uptake of
MGBG was determined for MGBG concentrations of 1.0, 1.8, and 3.3 pM in the
media. The cells concentrated the drug approximately 1000-fold, and the
peak concentration was observed between 36 and 60 hr of treatment (Figure
28). Based on these data, the recovery of mtDNA synthesis could not simply
be explained by a decreased intracellular drug content, because mtDNA
synthesis recovered while the intracellular concentration was still high.
In addition, whereas the maximum intracelluiar conients were
reasonably proportional to the amount of drug in the medium, the
concentrations at 6 days were each about 400pM. Thus, cells from each

Figure 26. Plot of the velocity of [14C]-MGBG uptake into L1210 cells versus
drug concentration in the media. The uptake was measured after 30
minutes of incubation. Aliquots of 106 cells were pelleted and
washed with 4°C media containing 1 mM unlabeled drug. The cell
pellets were solubilized and the amount of intracellular radiolabel
was determined by scintillation counting.

S (fiM MGBG)
V (nmole MGBG uptake/hr X 10Ccells)
o O o
o io
£1 I

Figure 27. Double-reciprocal plot of the velocity of [14C]-MGBG uptake versus
drug concentration. A straight line was drawn through the points to
determine the Km (4 pM) and the Vmax (0.25nmole/hr x 106 cells).

1/s (mM)
1/v (nmole/hr X lO^cells)
O
L I I

Figure 28. Uptake of [14C]-MGBG into L1210 cells incubated with 1.0, 1.8, or 3.3
pM radiolabeled MGBG in the media. Cells were reseeded every 2
days with fresh media and radiolabeled drug.

4000
3000
2000
1000
0
-o- 1|iM
1.8|iM
-o- 3.3^M
Days MGBG treatment
119

treatment group maintained a different concentration gradient for MGBG
between the internal and external sides of the cells (i.e. between the
intracellular compartment and the medium) in order for the same intracellular
concentration to be maintained.
Uptake of MGBG in Chronicallv-treated Cells
To determine if chronically-treated cells maintained their ability to keep
intracellular MGBG concentration at -400 pM, another drug uptake study was
done. In this case L1210 cells were treated with 1.3 or 2.5 pM unlabeled
MGBG for 31 days and transferred to fresh medium with [14C]-MGBG at the
same concentrations. Uptake of labeled MGBG was determined for a 5-day
period (Figure 29). The intracellular concentration over this time course did
not exceed 600 pM; however, cells exposed to the higher drug concentration
in the medium apparently had a lower concentration of MGBG in the cells.

Figure 29. Uptake of [14C]-MGBG into L1210 cells which were previously
incubated with unlabeled MGBG for 31 days immediately preceeding
the addition of radiolabeled drug at day 0. Starting at day 0 the
cells were incubated with1.3 or 2.5 pM [14C]-MGBG in the media.
Cells were reseeded every 2 days with fresh media and radiolabeled
drug.

o
3
Days MGBG treatment
-O 1.3 |iM
2.5 (iM
T
4
5
122

DISCUSSION
The discussion of this research will be divided into the following
sections: 1) determination of the mtDNA copy number in L1210 cells; 2)
decrease in cell growth and the cellular content of mtDNA upon exposure to
MGBG or DES; 3) mitochondrial function during MGBG exposure; 4) increased
mtDNA/cell upon exposure to low concentrations of MGBG; 5) recovery of
mtDNA replication after a few days of exposure to MGBG; and 6) the
implications of these results for nuclear/mitochondrial interaction.
Determination of the MtDNA Copy Number in L1210 Cells
Most studies involving the effects of various compounds on mtDNA
utilized assays for mtDNA that required both preparation of a mitochondrial
fraction and subsequent resolution of mtDNA from contaminating nuclear
DNA by density gradient centrifugation. In our experiments, a dot blot assay
involving whole cell lysate was central to quantitating the amount of mtDNA
per cell. There were two major advantages to this technique. First, the fact
that whole cell lysate was used for the dot blot analysis eliminated any
differential recovery of mitochondria, or mtDNA, due to drug treatment.
Second, the use of a mtDNA probe for hybridization virtually eliminated
accidental inclusion of nuclear DNA in the quantitation of the amount of
mtDNA. Because mtDNA represents <1% of total DNA, contamination by even
a small percentage of nuclear DNA would add significant error to the results.
Because a species-specific mtDNA probe was used in these studies
with murine L1210 cells, stringent hybridization and wash conditions could be

124
used. This minimized non-specific binding of the probe. In addition, RNase
T1 treatment of the samples eliminated possible confounding results from
hybridization of the probe to mitochondrial RNA. Furthermore, all dot blot
conditions were carefully worked out so as to minimize experimental variability
within samples; the mean standard deviation was ±13%.
An important innovation with the dot blot assay was the use of the
plasmid vector (pSP64) as an external standard. This allowed absolute
measurements of the amount of mtDNA in addition to the relative values more
commonly obtained with the dot blot. As a result, the copy number of mtDNA
in these L1210 cells was found to be 1450 ± 188 (S.D.). This compares
favorably with the reported value of 1100 for the murine L cell (1).
Because the mtDNA copy number was determined for an asynchronous
population of L1210 cells, however, 1450 represented the mean amount of
mtDNA in cells which were in all phases of the cell cycle. Under these
circumstances, the amount of mtDNA must double between the beginning of
G1 and the end of mitosis. Because mtDNA synthesis is thought to occur
continuously at a constant rate throughout the cell cycle (77), 1450 copies
represents a '1,5X' copy number midway between the '1X' amount of a cell
entering G1 (just after mitosis) and the '2X' amount of a cell at the end of G2/M.
The mtDNA copy number of L1210 cells at the start of G1 is therefore about
967 (1450+1.5).
Decrease in Cell Growth and MtDNA/Cell with MGBG and DES Exposure
In these experiments, MGBG partially inhibited cell growth in a
concentration-dependent manner over the relatively broad concentration
range of 1.0 to 100 pM. Conversely, the concentration range over which
MGBG exhibited virtually no inhibition to complete inhibition of mtDNA
accumulation was very narrow (from 1.0 to less than 1.8 pM). At 1.8 pM MGBG

and above, mtDNA accumulation was completely inhibited for a period of 24
hr or longer. This finding is in general agreement with a previous study in
which mtDNA synthesis of L1210 cells was inhibited to 62 and 55% of control
at 1.0 and 10 pM MGBG, respectively, after 5 hrs of treatment (7). A more
complete study with a Syrian hamster tumor cell line showed that 50 pM
MGBG inhibited mtDNA synthesis by 73% at 16 hr of treatment (6). The lack of
complete inhibition of mtDNA synthesis in these studies may reflect the
method by which the mtDNA was isolated for quantitation. In both studies the
mtDNA was enriched by density gradient centrifugation, leaving open the
possibility of nuclear DNA contamination.
Further, because cell growth was progressively inhibited at MGBG
concentrations greater than those necessary to completely block mtDNA
accumulation, it follows that this cessation of mtDNA accumulation was not the
cause of the decreased cell growth rate, at least initially. It seems therefore
that MGBG had initial effects on cell growth unrelated to its inhibitory effects on
mtDNA accumulation. However, extended exposure of L1210 cells to lesser
concentrations (e.g. 1.8, 2.5, and 3.3 pM) of MGBG revealed another type of
inhibition of cell growth. In this case, cell growth at each MGBG concentration
was inhibited most after 3 days of exposure; in each case this coincided with
the time at which the amount of mtDNA/cell has reached its nadir. Although
correlational only, this result suggests that a decreased copy number of
mtDNA may cause cell growth to slow. This is an important distiction.
Whereas the rate of mtDNA accumulation may not be coordinately linked to
cell division such that inhibition of one results in the proportional inhibition of
the other, the amount of mtDNA/cell appears to be a characteristic by whi
_ K
ui i
the cell recognizes a perturbation in the nuclear/mitochondrial coordination

and regulates its rate of proliferation. The decreased cell growth rate may, in
turn, be secondary to a decreased capacity of mitochondrial function.
An additional finding of this study was that the ratio of mtDNA forms did
not change after MGBG exposure, which suggested that the drug did not act to
damage mtDNA. This finding was consistent with those of Nass (6), in which
she demonstrated no increase in the nicked or circular forms of mtDNA of
hamster tumor cells exposed to MGBG, but a complete cessation of D-loop
strand initiation within the replication origin. Her data suggest that MGBG
inhibits the initiation of mtDNA synthesis, but does not inhibit the completion of
mtDNA replication once initiated.
Both MGBG and DES exhibited very narrow concentration ranges over
which the accumulation of mtDNA was completely inhibited. Also, with both
drugs cell growth was slowed greatly when the mtDNA content per cell was
lowest. Despite these similarities between the drugs, there were important
differences. Diethylspermine does not seem to have the additional, immediate
effect on cell growth described above for MGBG over the concentration range,
1.0 to 100 (iM. This conclusion is supported by several lines of evidence.
First, MGBG inhibited cell growth at concentrations which had little
effect on mtDNA. With DES treatment, on the other hand, concentrations that
did not inhibit mtDNA accumulation did not inhibit cell growth. Second, MGBG
inhibited cell growth in a graded, concentration-dependent manner, even at
concentrations above which there was no further effect on mtDNA
accumulation. Cell growth in DES treatments, however, was initially affected
less than MGBG. After a lag, DES had a relatively sharp concentration
response for ceii growth, which correlated weil with that for mtDNA
accumulation, i.e., once mtDNA accumulation was completely inhibited,
increasing DES concentration had virtually no more effect on cell growth.

And, finally, MGBG at concentrations >3.3 jiM had acute effects on cell
viability, and the higher the dose, the lower the viability. Diethylspermine, on
the other hand, did not have an acute effect on viability; decreased viablity
only occurred after the lag which corresponded with the decreased mtDNA
copy number. Taken together, these data indicate the actions of DES are
more specific for mtDNA than are those of MGBG and that the action of DES
on cell growth is correlated closely with its actions on mtDNA.
Another property of MGBG not shared with DES was the apparent
reversibility of its effects. Mitochondrial accumulation was inhibited up to 3
days with MGBG, but mtDNA synthesis recovered thereafter and remained
unaffected by continued drug treatment for up to 31 days. Cell growth
recovered in a manner which correlated with the mtDNA copy number, i.e. cell
growth increased at times when the mtDNA copy number was highest and
decreased at times when the copy number was lowest. Cells treated with
DES, on the other hand, recovered neither a normal rate of mtDNA
accumulation nor cell growth. After depletion of the mtDNA copy number to
20% of control after 2 days of DES exposure, no further change occurred over
the next 4 days. Cell growth slowed dramatically after 2 days and remained
slow throughout the 6-day period. Cell viability was close to normal until 8
days of treatment, at which time it dropped precipitously to 40%. Thus,
although these cells did not grow, they remained viable (as measured by
trypan blue exclusion) for a relatively long period of time. This is consistent
with the idea that cells may survive if they have a certain minimum amount of
mtDNA, and L1210 cells may not survive indefinitely without dividing.
Mitochondrial Function During MGBG Exposure
An important aspect of these experiments was to try to relate
mitochondrial and cellular functions with changes in the mtDNA copy number.

128
Rhodamine 123 uptake was not affected by MGBG treatment throughout a 6-
day treatment period. This suggested that the drug itself had no direct effect
on rhodamine uptake. Also, the uptake was not affected indirectly by the
mtDNA copy number, even when the latter was decreased to 10% of normal.
Rhodamine 123 uptake depends on mitochondria maintaining a
membrane potential (negative with respect to the cell) (215). The membrane
potential in mitochondria is produced from two sources: a chemical potential
due to the difference in concentration of cations and anions across the
mitochondrial membranes, and the hydrogen ion potential generated by the
electron transport chain. The capacity of the electron transport chain may be
such that only a small fraction of total capacity is necessary for sufficient
membrane potential to concentrate the dye. It follows that rhodamine 123
uptake may not correlate directly with mitochondrial electron transport
capacity.
Because MGBG-treated cells divided most slowly shortly after the
mtDNA per cell had reached its nadir, mitochondria from these cells may
produce sufficient energy to maintain a membrane potential but not enough to
allow cell division. This idea may also apply to the effects of MGBG on cell
size, i.e., the volume and surface area of L1210 cells decreased when the
mtDNA content per cell was low. This finding was consistent with those of
Shmookler Reis and Goldstein (2). They reported that the average number of
mtDNA genomes per cell varied relatively little during the replicative lifespan
of any given normal fibroblast cell line, but increased in 5 of 6 lines by 10-50%
at late passage. When normalized to cell protein content, which also
increased Dy 3u-ou'/o in ilioue 5 lines which gamed mtDNA,
of mtDNA genomes remained constant over the period of in vitro culture.
Indeed, they suggested that mtDNA replication is not regulated by direct

coupling to that of nuclear DNA, but rather by the cell mass to be serviced by
mitochondria.
Increased MtDNA/Cell with Low Concentrations of MGBG
In our experiments the mtDNA content per cell was 87 and 75% of
normal after 24 hrs of treatment at 1.0 and 1.3 pM MGBG, respectively. This
suggested either that replication was inhibited only for a short time (<6 hr) or
that inhibition was only partial. Cell division in these treatment groups was
inhibited slightly, but the inhibition continued throughout the drug treatment,
presumably a reflection of the direct growth-inhibiting property of MGBG which
distinguished it from DES (see above). Consequently, mtDNA accumulation
was affected less by MGBG than was cell division. This resulted in less
accumulation of cells relative to mtDNA, i.e., the mtDNA copy number per cell
increased.
It was interesting that the mtDNA content never exceeded 155%,
however. While it is reasonable to think that a decreased mtDNA content
might be deleterious to the cell (possibly because of decreased mitochondrial
function), it is less obvious why a more substantial increase in mtDNA content
is not tolerated, especially since there are other cells with more than 5000
copies per cell. In any event, the property of MGBG to inhibit cell growth more
than mtDNA accumulation is in agreement with the idea that MGBG has
additional effects on cell growth unrelated to its inhibitory effect on mtDNA
accumulation. Methotrexate is another drug which inhibits the synthesis of
nuclear DNA more than that of mtDNA. In one study, methotrexate inhibited
the incorporation of labelled thymidine into nuclear DNA by 96%, compared to
an inhibition of oniy 50% in mtDNA (88). However, the effect Gn the mtDNA
copy number of this differential inhibition was not measured.

130
It might be hypothesized that an increased mtDNA copy number may
result from any condition which causes a decrease in the rate of cell growth.
To test this idea, mtDNA content was measured in L1210 cells incubated in
medium with decreasing amounts of serum. When cell growth was slowed by
serum depletion, the mtDNA content per cell remained the same as control.
This suggested that the cell is able to regulate its mtDNA copy number in
certain circumstances of decreased cell growth. Treatment with MGBG,
however, seemed to uncouple this regulation between mtDNA synthesis and
cell growth.
Recovery of MtDNA Replication
After an initial period in which mtDNA accumulation was inhibited at the
higher doses of MGBG, mtDNA synthesis resumed, even in the continued
presence of drug. Because inhibition of drug uptake and/or increased efflux of
drug are common mechanisms whereby cancer cells become resistant to
antiproliferative agents, a drug uptake study was done with [14C]-MGBG. The
apparent intracellular concentration of MGBG peaked at 36 to 60 hr, with a
~1000-fold concentration of drug in the cells relative to the medium (1.0 to 3.3
(iM in these experiments). The subsequent decline in the intracellular
concentration of MGBG related to a decrease in uptake rather than an
increase in efflux (216). Mitochondrial DNA synthesis consistently resumed
within ±12 hr of the time at which drug concentration in the cell first decreased.
At the time where mtDNA synthesis resumed, the apparent intracellular
concentration of MGBG, as measured by [14C]-MGBG content, was still high
(>1 mM). Also, this intracellular concentration represented parent compound,
as the drug is not metabolized as far as can be determined within the limits of
the sensitivities of the assays (either TLC or HPLC) (175). Since the
intracellular concentration was thus higher than that which initially inhibited

1 3 1
mtDNA accumulation, therefore this factor alone does not account for the
recovery of mtDNA synthesis. It is possible that the drug binds to
macromolecules or other compounds so that the free drug concentration
decreased with time to levels less than that needed to inhibit mtDNA
accumulation. Likewise, the drug also could have redistributed within the cell
such that the amount of drug at the purported site of mtDNA synthesis
inhibition was below the critical level needed for effect. However, drug uptake
studies with cells which were subsequently treated with digitonin to
permeabilize all but the mitochondrial membranes, suggest no special
transport of MGBG into mitochondria other than equilibration of that across the
cell's plasma membrane (216). Another possible explanation is that both the
change in transport (influx) and mtDNA synthesis are reflections of a common
third event in the emergence of phenotypic resistance to MGBG.
It was interesting that all cells continuously exposed to MGBG reached
an intracellular concentration of -400 pM by 6 days, even though the MGBG
concentrations in the medium were different (from 1.0-3.3 (iM). The
intracellular concentration of cells after 31 days of MGBG exposure was still
-400 pM, which suggests that the cells were regulating transport of MGBG so
as to maintain an intracellular steady state level of -400 pM MGBG.
Because the intracellular MGBG concentration was higher when
mtDNA synthesis recovered than at the time it was inhibited, another study
was done to determine if the rate of recovery of mtDNA synthesis would
increase if the drug was removed from the medium. When cells were
transferred to drug-free medium at the time at which mtDNA synthesis was
expected to resume even in the presence of drug, the two rates of mtDNA
reaccumulation were indistinguishable. Other experiments had established
that intracellular drug concentration does decrease more rapidly in drug-free

132
medium. This suggests that mtDNA accumulation during the recovery phase
of drug exposure is completely refractory to the presence of MGBG.
The recovery of mtDNA synthesis was not due to a genetic selection for
MGBG-resistant cells. This was demonstrated by two lines of evidence. First,
at no time during MGBG treatment did cell number decrease, as would be
expected if MGBG-resistant cells pre-empted the population while MGBG-
sensitive cells died. Also, resistant cells transferred to drug-free medium for
only 4 days, were equally sensitive to inhibition of mtDNA replication and cell
growth as were control cells upon retreatment with MGBG. This result
indicates a phenotypic, but not a genetic, type of resistance.
Implications for Nuclear/Mitochondrial Interactions
Uncoupling of Nuclear and MtDNA Replication
Because inhibition of mtDNA accumulation at 1.8 pM was virtually
complete, increasing MGBG concentration did not further inhibit the amount of
mtDNA accumulation, but it increase the duration of inhibition. Inhibition was
virtually complete for 3 days at 3.3 pM MGBG. Because MGBG at 1.8, 2.5, and
3.3 pM inhibited mtDNA accumulation more than cell division over the first 3
days of treatment, the net result was a decrease in the mtDNA content per cell.
The experimentally-determined decrease in mtDNA per cell at the two higher
concentrations was identical to the calculated rate of decrease in mtDNA
content, where inhibition of mtDNA synthesis was complete despite continued
cell division.
Thus, MGBG seems to have distinguishable effects on cell growth and
mtDNA accumulation. It is known that, unlike nuclear DNA replication, mtDNA
replication is not restricted to the S phase of the cell cycle, but occurs
throughout the cycle. In addition, mtDNA replication in MGBG-treated cells
does not seem to be coupled to the cell cycle as a whole. This was suggested

133
by the findings that at various times during MGBG treatment cell division could
occur without mtDNA replication and vice versa. Interestingly, this uncoupling
of nuclear and mtDNA synthesis occurs in certain physiologic situations. For
example, whereas differentiated cells cease to divide, mtDNA still exhibits
some degree of turnover (217). In the analogous, but opposite, situation, early
in zygote development, cell division occurs but the mtDNA does not replicate
(101).
Autonomous synthesis of nuclear and mtDNA has also been observed
to occur during exposure to various drugs. Ethidium bromide, for example, is
an inhibitor of mtDNA replication and transcription; it intercalates into DNA and
also inhibits the polymerase responsible for mtDNA replication (80). The rate
of [3H]-thymidine incorporation into mtDNA decreased to nearly zero after
VA2-B cells were exposed to ethidium bromide for one day (82). The cell
doubling rate, however, remained virtually normal for 3 to 4 doublings. The
net result was a dilution of the mtDNA content per cell to 10% of control.
Methotrexate, on the other hand, seems to uncouple nuclear and
mtDNA synthesis in the opposite direction. In studies with LMTK' cells, 10 [iM
methotrexate inhibited nuclear DNA synthesis to only 4% of the control rate,
whereas mtDNA synthesis continued at 50 to 60% of the control rate.
However, the net result on the mtDNA copy number was not measured.
The drug MGBG appears to be unique in that both situations (i.e.
inhibition of mtDNA synthesis > or < inhibition of cell growth) can be caused by
different concentrations of the drug. Thus, MGBG can either increase or
decrease the mtDNA copy number of treated L1210 cells, a phenomenon that
can be explained by this differential inhibition of cell doubling and mtDNA
accumulation following MGBG treatment. Diethylspermine, on the other hand,

134
like ethidium bromide only decreases the mtDNA content of cells at effective
doses.
Minimum MtDNA Copy Number for Dell Division/Normal Growth
In any event, even though the coupling of mtDNA and cell doubling may
be somewhat relaxed during MGBG treatment, in another sense, several
degrees of regulation were apparent. First, there seems to be a certain
minimum mtDNA copy number which is tolerated by the cell. The amount of
mtDNA in MGBG-treated L1210 cells never dropped below 10% of control.
When this level was approached, cell growth slowed dramatically such that no
further dilution of mtDNA occurred. There may be a minimum level of mtDNA
below which the cell will not divide, perhaps because of insufficient energy
secondary to decreased mitochondrial function. It is conceivable that a low
mtDNA copy number may not be sufficient for maintainence of normal ATP
production through oxidative phosphorylation. The decreased cell growth
may then reflect a lack of sufficient ATP for maximal proliferation. Alternatively,
the cell growth may have slowed due to some other factor, e.g. it remains
possible that the changes in mtDNA are an epiphenomenon.
This 10% 'limit' is also the minimum mtDNA tolerated in human VA2-B
cells treated with ethidium bromide (82). In those studies, cells removed from
drug after decreasing the mtDNA to 10% of normal were able to survive. If
drug was not removed at this point, the mtDNA copy number continued to
decrease and the cells died. Consistent with these studies which suggest that
mammalian cells may survive if their mtDNA content is not reduced below
10% of normal, the cells treated with <3.3 pM MGBG had >90% viability, even
at times when the mtDNA copy number was as low as 10% of normal, but
never lower.

135
Another example which is compatible with the idea of a minimum
amount of mtDNA (or perhaps mitochondria) involves doxycycline, an
antibiotic which inhibits mitochondrial protein synthesis. When human PC-3
and NC-65 tumor cells were exposed to doxycycline continuously, growth was
inhibited after a lag period corresponding to 5 and 9 days for PC-3 and NC-65
cells, respectively (218). The doubling time of the PC-3 cells (about 2 days)
was shorter than that of the NC-65 cells (about 4 days). It follows, therefore,
that both tumor lines can perform about 2 cell cycles in the presence of
doxycycline before cell proliferation becomes arrested. Similar results have
been observed with chloramphenicol, another inhibitor of mitochondrial
protein synthesis (219).
All of these data taken together suggest that cells tolerate a 75-90%
reduction in their normal copy number of mtDNA before any response in cell
growth is recognized. This implies two things. First, cells may normally have
more copies of mtDNA than they actually need. Second, cells are able to
recognize a depletion in their mtDNA copy number. This latter finding has at
least two different implications. If slowed growth of the cell following depletion
of its mtDNA content is a secondary response to a loss of mitochondrial
function, this implies a passive response of the cell in attenuating the dilution
of its mtDNA. Alternatively, it is conceivable that the cell may directly
recognize its mtDNA copy number, and slowed growth may reflect a response
by the cell to adjust its mtDNA copy number. These hypotheses can not be
distinguished by available information.
There is one exception to the idea of a minimum mtDNA copy number
necessary for survival. This exception is the chick embryo fibroblast, which
can be depleted of any detectable mtDNA by continuous ethidium bromide
treatment for 20 or more days (84). At this time the cells also lack cytochrome

136
oxidase and mitochondrial cytochromes, parts of which are coded for by the
mitochondrial genome. The reason for this difference between avian and
mammalian cells is unknown.
In addition to a minimum 'limit' of mtDNA necessary for survival, it may
be that there is another, higher 'cut-off of mtDNA content which is necessary
for optimal cell growth. In my experiments with MGBG and L1210 cells, when
the mtDNA copy number and, perhaps, mitochondrial function, had partially
repleted by day 6, the cell growth rate increased towards normal. It is
interesting that, for all MGBG concentrations used in these experiments, the
cell growth rate increased only after >62% of the normal mtDNA copy number
had been regained. This is very close to the 67% mtDNA copy number
predicted for untreated cells at the start of G1 (since the mtDNA content
midway through the cell cycle is defined as 100%).
Rate of MtDNA Replication Mav be Rate-Limiting for Maximal Cell Growth
Once mtDNA synthesis resumed, the mtDNA copy number (which had
been depleted to as low as 10% of normal) began to increase towards normal.
To determine the relationship between mtDNA synthesis and cell growth to
this event, the rates of each were studied separately. One would anticipate
that, no matter what the rates of mtDNA synthesis and cell division were,
mtDNA synthesis must have been relatively faster than cell division for
recovery of the mtDNA copy number to occur. This may occur through a rate
of mtDNA synthesis greater than normal, through a slowing of cell growth, or a
combination of both.
In vitro replication of the complete mitochondrial genome can occur in
one hour; another hour is required to supercoil the molecule (12). Therefore,
when mtDNA synthesis resumed, doubling of the mtDNA might have been
expected to occur in as little time as 2 hr. Consequently, an unexpected

137
finding in these experiments was that, when mtDNA synthesis resumed, the
mtDNA did not double in less than 12 hr. This is precisely the time it takes
mtDNA to double in untreated cells; i.e. there was no compensatory
accelerated replication.
This finding was not a consequence of the coordination between
mtDNA and cell doubling because mtDNA doubling was happening at a rate
faster than that of cell doubling (because the latter was slow). Also, this
finding could not be explained by a continued partial inhibition of mtDNA
accumulation by MGBG, which coincidentally resulted in a 12-hr doubling,
because cells switched to drug-free medium had the same rate of mtDNA
doubling as those continued on drug. It seems beyond coincidence that
mtDNA replicated in 12 hours, which is the normal cell doubling time of control
L1210 cells. Indeed, this finding suggests that the rate at which mtDNA
replicates may contribute to, or even determine, the maximal rate at which
these cells can divide.
A study by Wiseman and Attardi (82) supports my findings that mtDNA
cannot replicate at a rate faster than that of normal exponential growth. These
researchers reported that 20 ng/ml of ethidium bromide inhibits the mtDNA
synthesis of human VA2-B cells. The mtDNA content of these cells was
progressively diluted down to 10% of control cell values after 3 doublings.
When the cells were transferred to drug-free medium, they recovered a near¬
normal amount of mtDNA in 5 days. These and other observations were
interpreted to indicate that each human mtDNA molecule replicated on the
average 2.5 times per cell cycle during the first doublings. However, cell
growth was slowed substantially at this time. Thus, while mtDNA replicated
faster than nuclear DNA during recovery from ethidium bromide, the rate of
mtDNA replication did not exceed that of its exponential growth.

138
Recovery of the MtDNA Copy Number
Whereas mtDNA synthesis resumed at virtually the control rate, cell
doubling, on the other hand, was more variable. Cell growth for the 1.8-3.3
|iM treatments was slowest at 4 to 6 days, when the mtDNA content per cell
was at its nadir and/or recovering. The accumulation of mtDNA was not due to
a block in the cell cycle which allowed repletion of the mtDNA copy number,
since the distribution of nuclear DNA (as measured by flow cytometry) in
MGBG-treated cells at these times was no different from control.
When the mtDNA copy number and, perhaps, mitochondrial function,
had partially repleted by day 6, the cell growth rate increased towards normal.
Because the mtDNA copy number had not returned to 100% before cell
growth rate increased, the mtDNA copy number decreased to a limiting
concentration once more. The subsequent decrease in the mtDNA copy
number was less pronounced than the initial depletion during the first 3 days
of treatment since, during this later phase, mtDNA was being synthesized, the
decrease merely seeming to reflect cell division before complete repletion.
These cycles of mtDNA depletion and repletion coupled with a decrease or
increase, respectively, in the rate of cell growth had a periodicity of 6 days,
and each oscillation was progressively more muted.
Ultimately, after 31 days of MGBG treatment, the L1210 cells from the
2.5 and 3.3 |iM treatments regained their original 1450 mtDNA copy number.
This recovery depended on the rates of both cell growth and mtDNA
accumulation. We have already noted that mtDNA replication and cell division
are not tightly linked in MGBG-treated L1210 cells, yet it seemed conceivable
that the cell could regulate each one separately so as to recover the
characteristic content of mtDNA per cell. Our experiments revealed only either
no accumulation of mtDNA or accumulation at a singular fixed rate (doubling

139
time, 12 hrs) whether or not the cells were dividing. The fine tuning of mtDNA
content seemed to reflect this on-off rate coupled with a finely variable rate of
cell division, the combination of which always seemed to produce a cell with
1450 copies of mtDNA. Thus, it would appear that mtDNA replication was
unable to respond to MGBG-induced changes in the mtDNA content (i.e. there
was no compensatory acceleration in mtDNA replication), and only the cell
division rate was regulated precisely so as to regain the characteristic content
of mtDNA per cell.
Polvamines as 'Signals' in Nuclear/Mitochondrial Interactions
In addition to its antimitochondrial effects, MGBG is known to affect
polyamine metabolism. Because polyamines interact with DNA and influence
the activity of topoisomerases, at least in vitro, it is possible that MGBG may act
to alter the topology of mtDNA, thereby inhibiting its ability to replicate.
However, there was no obvious change in the forms of mtDNA, as compared
to untreated cells, by gel electrophoresis. Alternatively, because mtDNA
synthesis exhibited no gradation in response to MGBG in terms of either
inhibition or recovery, this drug may not interact directly with mtDNA to inhibit
its synthesis but, rather, may be acting at the level of a finely-tuned, putative
'signal' for mtDNA replication. This theory would fit with the 'on/off effect of
MGBG on mtDNA replication and the sharp concentration response seen for
both inhibition and recovery of mtDNA accumulation.
This hypothesis proposes that the machinery to replicate mtDNA is
'unaware' that it should replicate (the signal is 'turned off’), rather than that it is
directly inhibited from doing so. Likewise, the recovery of mtDNA synthesis is
consistent with the 'turning on' of a signal for mtDNA replication; mtDNA
synthesis in cells recovering from MGBG occurs at a constant rate which

corresponds to the rate in normally-dividing cells, even though the growth rate
of these cells is substantially slowed.
This putative signal for mtDNA replication in dividing cells may be
linked to polyamines. Consistent with this hypothesis, DES and other
polyamine analogs have been shown to inhibit mtDNA replication (220).
Polyamines are found in millimolar concentrations within the cell and function
in cell growth and proliferation. It may be that mtDNA molecules associate
with the mitochondrial membrane in a particular way which can be modulated
by polyamines. The polyamines have been shown to modulate the activity of
numerous phospholipases and phosphorylases (155). Recent work suggests
that polyamines, perhaps via their effects on these enzymes, modulate the
uptake of physiologic concentrations of calcium into mitochondria (163), which
may in turn signal mtDNA replication. Thus, MGBG and the other polyamine
analogs be act as antagonists to endogenous polyamines, e.g. the drugs may
compete with polyamines for 'receptors' on the mitochondrial membrane, or
they be act to modulate the uptake of Ca++ into the mitochondria which may,
in turn, influence mtDNA replication.

SUMMARY
We have studied the integration of mtDNA replication with cell division
during exponential growth of mouse leukemia L1210 cells. In particular, we
studied the effects of the polyamine analogs, MGBG and DES, on mtDNA
accumulation and cell growth because at appropriate doses they selectively
inhibit mtDNA replication.
The mtDNA copy number in exponentially-growing L1210 cells was
determined by dot blot analysis to be 1450 ± 188 (S. D.). Electrophoretic
separation of mtDNA from cells exposed to MGBG demonstrated that there
was no change in the ratio of intact/nicked forms compared to control.
Exposure of these cells to >1.3 pM MGBG inhibited the accumulation of
mtDNA while cell division continued. Thus, with continued drug exposure the
amount of mtDNA per cell successively decreased over several cell
generations. This demonstrates that the doubling of mtDNA is not tightly
linked with cell division.
Several parameters of cellular function were measured during
exposure of cells to MGBG. Cell size decreased shortly after the decrease in
mtDNA copy number. Although other studies have demonstrated
ultrastructural damage to mitochondria following exposure of cells to >1 pM
MGBG, the potential-dependent uptake of rhodamine 123 into mitochondria
was not affected during 6 days of 1.0-3.3 pM MGBG treatment. This suggests
either that drug treatment and/or a decreased cellular content of mtDNA has
141

142
no effect on this aspect of mitochondrial function, or that rhodamine 123
uptake is not directly proportional to mitochondrial functional capacity.
In collaboration with Dr. Raymond Bergeron and Mike Ingeno, we have
discovered that DES inhibits mtDNA accumulation. The initial effects of DES
on mtDNA accumulation and cell growth were similar to those of MGBG. After
3 days, the mtDNA synthesis of MGBG-exposed cells was no longer inhibited,
and the mtDNA copy number returned toward normal; however, the effects of
DES on mtDNA accumulation were not reversed. Also, MGBG, but not DES,
seemed to have initial effects on cell growth and viability unrelated to effects
on mtDNA. For example, >10 pM MGBG affected cell viability acutely, before
dilution of the mtDNA copy number had occurred. Also, 1.0-1.3 pM MGBG had
virtually no affect on mtDNA accumulation whereas cell growth was inhibited
slightly; the net result was an increase in the mtDNA copy number per cell.
However, this effect was not seen in cells growth-inhibited by serum depletion.
For exposure to either drug, the amount of mtDNA in viable cells never
decreased below 10% of the control value because cell division ceased. The
viability of cells exposed to MGBG remained normal, and the cellular mtDNA
began to reaccumulate after 3 day. In the case of DES, the mtDNA content
remained at 10% of control for about one week at which time the cells died.
This is consistent with the idea that cells may survive if they have a certain
minimum amount of mtDNA, but that L1210 cells may not survive indefinitely
without dividing.
Although the effects of DES on mtDNA accumulation were not
reversible, mtDNA synthesis of MGBG-exposed cells resumed after 3 days.
When mtDNA synthesis resumed, the mtDNA content per cel! doubled every
12 hours whether or not drug had been removed from the medium. This
mtDNA synthesis occurred when the rate of cell division was at its lowest, yet

143
flow cytometric analysis demonstrated that there was no block in any phase of
the cell cycle. Mitochondrial DNA synthesis in cells which were dividing very
slowly resulted in repletion of the cellular content of mtDNA. When the mtDNA
was nearly repleted, the rate of cell division recovered to the control value of
12 hours. The observation that the doubling of mtDNA in the virtual absence
of cell division required the same 12 hours suggests that replication of mtDNA
may proceed at its maximal rate in untreated exponentially-growing L1210
cells and actually limit how rapidly these cells can divide.
The mechanism of recovery of mtDNA synthesis in these cells was not
due to genetic mutation, but rather to a phenotypic adaptation. The L1210
cells initially concentrated [14C]-MGBG more than 1000-fold relative to the
incubation medium. The Km and Vmax of transport were 4 pM and 0.25
nmoles/hr x 10^ cell, respectively. When mtDNA synthesis resumed, the
apparent intracellular concentration of the drug had begun to decrease to a
new steady-state value of -400 pM, although the concentration was still higher
than that earlier associated with inhibition. This intracellular steady-state
value of -400 pM was maintained at 31 days of MGBG exposure, and the
mtDNA copy number of cells continuously exposed to MGBG had returned to
the control value at 31 days.
Future directions of this research would include determining why the
effects of these polyamine analogs on mtDNA are reversed in the case of
MGBG, but not for DES. A logical starting point would be to compare the
uptake of radiolabeied DES with that of MGBG. Because of this difference in
reversibility of effects on mtDNA, the information taken together from the study
of each drug provides a useful model to determine how changes in the cellular
content of mtDNA affect the mitochondrial protein which it encodes. Other
directions would include studying the mechanism by which the polyamine

144
analogs inhibit mtDNA replication and whether mtDNA replication may be
rate-limiting in other cells with different doubling times.

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BIOGRAPHICAL SKETCH
Rita Beilis Bortell was born in Eustis, Florida, on June 29, 1953. She
attended the University of Florida and obtained a degree in microbiology in
1975. She worked seven years as a lab technologist for Dr. George Edds in
the College of Veterinary Medicine and obtained a Masters of Science degree
under his tutelage.
In 1983, the author began working towards her Ph.D. degree in the
Department of Pharmacology and Therapeutics at the University of Florida.
157

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Allen H. Neims, Chairman
Professor of Pharmacology and
Therapeutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Jkabvi
Stephen P. Baker
Associate Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my opinion,it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree ^Doctor otPbtfoso
William W. Hauswirth
Professor of Ophthalmology and
Immunology and Medical Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
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