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The transport of methylglyoxal bis(guanylhydrazone) and its role in the development of resistance in murine leukemia L1210 cells

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Title:
The transport of methylglyoxal bis(guanylhydrazone) and its role in the development of resistance in murine leukemia L1210 cells
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Kanthawatana, Sukanya, 1959-
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x, 94 leaves : ill. ; 29 cm.

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Cell growth ( jstor )
Cells ( jstor )
Chlorides ( jstor )
Incubation ( jstor )
Ions ( jstor )
Mitochondria ( jstor )
Polyamines ( jstor )
Radiocarbon ( jstor )
Sodium ( jstor )
Viability ( jstor )
Biological Transport, Active ( mesh )
Dissertations, Academic -- Pharmacology and Therapeutics -- UF ( mesh )
Leukemia L1210 ( mesh )
Mitoguazone ( mesh )
Pharmacology and Therapeutics thesis Ph.D ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1989.
Bibliography:
Bibliography: leaves 89-93.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Sukanya Kanthawatana.

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THE TRANSPORT OF METHYLGLYOXAL BIS(GUANYLHYDRAZONE)
AND ITS ROLE IN THE DEVELOPMENT OF RESISTANCE
IN MURINE LEUKEMIA L1210 CELLS










BY

SUKANYA KANTHAWATANA, M.D.


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


1989




THE TRANSPORT OF METHYLGLYOXAL BIS(GUANYLHYDRAZONE)
AND ITS ROLE IN THE DEVELOPMENT OF RESISTANCE
IN MURINE LEUKEMIA L1210 CELLS
BY
SUKANYA KANTHAWATANA, M.D.
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
1989


ACKNOWLEDGMENTS
I would like to express my appreciation to my advisor and supervisory
committee chairman, Dr. Allen H. Neims, for the guidance and encouragement
he provided me on this project and would like to extend my appreciation to the
fellow member of our laboratory: Rita Bortell, Lynn Raynor, Daniel Danso, Mary
Ann Kelly and Fan Xie. I am also grateful to the other members of my
supervisory committee, Dr. Raymond J. Bergeron, Dr. Lai C. Garg, Dr. Michael
S. Kilberg, Dr. Edwin M. Meyer, Dr. Thomas C. Rowe and Dr. Bruce R. Stevens,
for their committment.
This dissertation is dedicated to my parents, Mr. Tongsuk and Mrs. Tongkam
Kanthawatana.


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
Specific Aims 1
Background 1
2 MATERIALS AND GENERAL METHODS 8
Cell Culture 8
Cell Counts 8
Cell Viability 9
Clonogenic Assay 9
Transport of MGBG 9
Stability of MGBG 11
Protein Assay 12
Statistical Analysis 12
3 TRANSPORT OF MGBG AND ITS EFFECT ON SENSITIVE
L1210 CELLS 13
Effects of MGBG on Growth, Viability and Clonogenicity 13
Characterization of the Initial Transport of MGBG 14
Intracellular Accumulation of MGBG During the First
20 Hours of Exposure 14
Kinetic Characterization of the Initial Transport Process 18
Inhibition of MGBG Uptake by Spermidine, Spermine
and Putrescine 22
Effect of Extracellular pH on the Initial Uptake of MGBG 22
i i i


Efflux of MGBG 23
Countertransport of Unlabelled MGBG 23
Effect of Various Cations on the Uptake of MGBG 29
Sodium-dependency of MGBG uptake 29
Ability of various cations to substitute for sodium in
uptake of MGBG 32
Effects of lonophores on the Uptake of MGBG 38
Effect of Inhibitors of Energy Metabolism on the Uptake
of MGBG 38
Intracellular Distribution of MGBG 45
Uptake of MGBG by Isolated Mitochondria 45
Selective Release of Cytosolic Constituents by Digitonin 46
Effect of Growth Rate on MGBG Transport 47
4 TRANSPORT OF MGBG AND DRUG RESISTANCE 54
Concentration of MGBG in L1210 Cells During 20 Days of
Drug Exposure 54
Kinetic Characterization of MGBG Transport as
Resistance Develops 58
Selection of L1210 Cells Highly Resistant to MGBG 62
Transport Properties of the Resistant Cells 66
Response of Multiple Drug Resistant (MDR) Cells to MGBG 66
Correlation between Transport and Resistance 73
5 DISCUSSION 77
6 SUMMARY AND FUTURE DIRECTIONS 86
REFERENCES 89
BIOGRAPHICAL SKETCH 94
IV


LIST OF TABLES
Table gage
3-1 Comparison between the calculated and observed
intracellular concentrations of MGBG during the first
20 hours of exposure 21
3-2 Comparison between the observed rates of MGBG uptake
at different extracellular pH's with rates calculated on
the basis of transport of MGBG 2 + only 26
3-3 Effects of various ions on MGBG uptake in L1210 cells 40
3-4 Effects of potassium and choline chloride on the sodium-
dependent MGBG uptake in L1210 cells 41
3-5 Effects of ionophores on MGBG uptake in L1210 cells 42
3-6 Uptake of MGBG by isolated mitochondria 48
4-1 Effects of MGBG on the intracellular content of polyamines
and putrescine in L1210 cells 57
4-2 Kinetics of the uptake of MGBG in untreated L1210 cells,
cells that had "recovered" after exposure to MGBG,
and a resistant subclone 65
v


LIST OF FIGURES
Figure page
1-1 Structure cf polyamines, related compounds and
inhibitors of polyamine synthesis 7
3-1 Effects of various concentrations of MGBG on the
proliferation of murine L1210 cells 15
3-2 Effects of various concentrations of MGBG on the viability
of murine L1210 cells overtime 16
3-3 Effect of MGBG on the clonogenicity of L1210 cells 17
3-4 Accumulation of MGBG by L1210 cells during the first
20 hours of exposure 19
3-5 Lineweaver-Burk plot of MGBG uptake 20
3-6 Competitive inhibition of MGBG uptake by spermidine,
spermine and putrescine in control L1210 cells 24
3-7 Effects of medium pH on the initial uptake of MGBG 25
3-8 Efflux of MGBG 27
3-9 Effect of temperature on the efflux of MGBG 28
3-10 Effect of MGBG preloading on the uptake of labelled MGBG.. 30
3-11 Effect of medium concentration of MGBG on the efflux
of labelled MGBG 31
3-12 Sodium-dependency of MGBG uptake in L1210 cells 33
3-13 Relationship between MGBG concentration and the rate of
MGBG uptake in the presence or absence of sodium 34
3-14 Lineweaver-Burk plot of the sodium-dependent MGBG
uptake in L1210 cells 35
3-15 The relationship between MGBG concentration and the rate
v i


of MGBG uptake in sodium chloride, choline chloride,
and mannitol 36
3-16 Lineweaver-Burk plot of MGBG uptake in sodium chloride,
choline chloride and mannitol 37
3-17 Effects of various ions on MGBG uptake in L1210 cells 39
3-18 Effects of inhibitors of energy metabolism on MGBG uptake.... 43
3-19 Effects of inhibitors of energy metabolism on MGBG uptake.... 44
3-20 MGBG remaining in the cell pellet after permeabilization of
the plasma membrane by digitonin 49
3-21 Growth curve of L1210 cells 51
3-22 Viability of L1210 cells as a function of growth phase 52
3-23 Uptake of MGBG as a function of growth rate 53
4-1 Intracellular content of MGBG upon prolonged exposure to
the drug 56
4-2 Influx of MGBG before and after the peak intracellular
concentration of drug had been attained 59
4-3 Efflux of MGBG before and after the peak intracellular
concentration of drug had been attained 60
4-4 Influx, efflux and apparent steady-state intracellular content
of MGBG on day 6 of exposure to drug 61
4-5 Competitive inhibition of MGBG uptake by spermidine,
spermine and putrescine in L1210 cells exposed to MGBG
for 3 weeks 63
4-6 Uptake of MGBG in the "recovered" L1210 cells 64
4-7 Effects of various concentrations of MGBG on growth of
MGBG-resistant L1210 cells 67
4-8 Effects of various concentrations of MGBG on viability of
MGBG-resistant L1210 cells 68
4-9 Effects of various concentrations of MGBG on growth of
MGBG-resistant L1210 cells after one month of
drug-free period 69
4-10 Effects of various concentrations of MGBG on viability of
v i i


MGBG-resistant L1210 cells after one month of drug-free
period 70
4-11 Accumulation of MGBG in the MGBG-resistant L1210 cells 71
4-12 Competitive inhibition of MGBG uptake by spermidine, spermine
and putrescine in MGBG-resistant L1210 cells 72
4-13 Comparison of MGBG dose-response curve of parental
drug-sensitive (AuxBi) and multidrug-resistant (CHRC5)
CHO cell lines 74
4-14 Correlation between intracellular content of MGBG and the
rate of cell proliferation in various type of L1210 cells 76
v i i i


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
THE TRANSPORT OF METHYLGLYOXAL BIS(GUANYLHYDRAZONE)
AND ITS ROLE IN THE DEVELOPMENT OF RESISTANCE
IN MURINE LEUKEMIA L1210 CELLS
By
Sukanya Kanthawatana, M.D.
December 1989
Chairman: Allen H. Neims, M.D., Ph.D.
Major Department : Pharmacology and Therapeutics
When murine leukemia L1210 cells were exposed to methylglyoxal
bisfguanylhydrazone] (MGBG), drug concentrations less than 0.3 pM had no
effect on cell proliferation, and concentrations greater than 5 pM were lethal
except in subclones of cells selected for resistance. Intermediate
concentrations decreased the rate of cell growth temporarily. L1210 cells
accumulated MGBG avidly, and this accumulation was crucial for cytotoxicity
and the development of resistance. My studies focused on the transport
process, and most were conducted with 0.3 5 pM MGBG. With 1 pM MGBG in
the medium, the apparent intracellular concentration of MGBG reached
approximately 2000 pM in 24 hours. Transport of MGBG seemed to be energy
dependent and carrier-mediated with saturable kinetics characterized by a Vmax
and Km in the presence of sodium, of 296 + 19 pmole/106 cells/hour and 4.72 +


0.169 pM, respectively. The rate of transport depended on temperature,
extracellular pH and cell proliferation. Inhibitors of energy metabolism,
ionophores which dissipate the plasma membrane electrochemical gradient,
and the replacement of sodium ions by other cations, all significantly decreased
the rate of accumulation of MGBG. Naturally-occurring polyamines, spermidine
and spermine, and putrescine competed with MGBG for uptake.
Within 48 hours of exposure to 1 pM MGBG, the rate of growth of L1210 cells
had decreased substantially. Despite the continued presence of MGBG in the
medium, the intracellular concentration of MGBG decreased to approximately
500 pM by day 6 and even further to 100 400 pM well in advance of the full
recovery of cell proliferation by 2 weeks. The development of resistance during
this time was due to a decrease in MGBG influx; the rate of efflux remained
unchanged. In addition, an MGBG-resistant subclone of cells also exhibited a
deficiency in MGBG transport. In both types of resistant cells, as well as in
previously untreated cells, the relative but not absolute affinity of the transporter
for MGBG, spermidine, spermine and putrescine were constant. In all cells, an
apparent intracellular concentration of MGBG greater than 400 pM was
associated with toxicity.
Resistance to MGBG was not associated with multiple drug resistance or
expression of P-glycoprotein.
x


CHAPTER 1
INTRODUCTION
Specific Aims
Methylglyoxal bis[guanylhydrazone] (MGBG) has been used clinically in the
treatment of cancer for several years. MGBG accumulates quite extensively in
sensitive targets like mouse leukemia L1210 cells. I have observed that even a
relatively small decrease in the extent of accumulation of MGBG results in
resistance to the antiproliferative and cytotoxic actions of the drug. My
dissertation focuses on the transport of MGBG. In general terms, I propose to
1. characterize the transport and accumulation of MGBG in sensitive
( previously untreated) L1210 cells.
2. define the mechanism(s) of resistance to MGBG that develops quickly
(days) when L1210 cells are exposed to sublethal concentrations of MGBG.
3. define the mechanism(s) of resistance to MGBG in a clone of L1210 cells
selected for resistance to MGBG (resistance persists even when the cells are
grown in drug-free medium).
Background
The structure of MGBG is depicted in Figure 1-1. The figure also includes
structures of putrescine, spermidine, spermidine, N-acetylspermidine and
alpha-difluoromethyl ornithine (DFMO), a well-known inhibitor of ornithine
decarboxylase. The bis[guanylhydrazone] represent a chemical family of
compounds in which terminal amidine groups are separated by variable
aromatic or aliphatic structures frequently containing interposed nitrogen
1


2
groups. Although a number of these compounds posses significant antitumor
activity, they differ substantially in their mode of action and in their ability to
interfere with various biochemical pathways. For example, the antileukemic
action of the aromatic bisfguanylhydrazone], of which 4,4'-diacetyldiphenylurea
bis[guanylhydrazone] is probably the best studied example, has been
correlated with the ability of these compounds to bind to calf thymus DNA and to
inhibit DNA-dependent DNA polymerase (Dave £lai., 1977). The mode of
action of the aliphatic compounds is much less understood. Unlike the aromatic
bis[guanylhydrazone], MGBG and other aliphatic analogs bind weakly to DNA
and do not inhibit the activity of DNA polymerase (Dave et a]., 1977).
Early clinical trials revealed that MGBG had very impressive antiproliferative
activity, particularly against acute myelocytic leukemia (Carbone elai., 1964,
Freireich, el a]., 1962, Levin fila}., 1965, Porterslal., 1979, and Regelson and
Holland, 1963). At optimal dose levels (150 mg/m2 daily), MGBG alone
produced complete remission in about 45% of patients (Levin el Si-. 1965).
This drug can also be used in combination with other chemotherapeutic drugs,
such as 6-mercaptopurine, and remission rates of 35 - 45% have been reported
among leukemia patient (Boiron glai., 1965 and Weil ela[., 1969). MGBG was
also found to be effective in the treatment of some solid tumors, including
Hodgkin's disease (46% responsive) and non-Hodgkin's lymphoma (37%
responsive). One of the interesting approaches in the use of MGBG was its
sequential administration after DFMO (alpha-difluoromethyl ornithine), an
inhibitor of polyamine biosynthesis.
Despite being introduced into clinical use as an anticancer agent about 30
years ago, the molecular basis for MGBG's activity remains obscure. Until
recently, the mechanism of action of MGBG was thought to be related to its
various relationships with the biological polyamines. MGBG has some similarity


3
to spermidine (Hamilton and La Placa, 1968), competes with spermidine and
spermine for uptake into the cell (Dave and Caballes, 1973, Porter giai., 1981
and 1982), strongly inhibits the polyamine biosynthetic enzyme, S-
adenosylmethionine decarboxylase (Holtta ei aL, 1973, Williams-Ashman and
Schenone, 1972) and inhibits the polyamine degradative enzyme, diamine
oxidase (Holtta el aL, 1973). It was the inhibition of S-adenosylmethionine
decarboxylase by MGBG that was thought initially to be responsible for the
antiproliferative activity of the drug. In cells treated with MGBG, the
concentration of putrescine increases whereas spermidine and spermine
decrease slowly as a consequence of pathway blockade. The observation that
spermidine prevented the antiproliferative effects of MGBG (Mihich, 1965), led
initially to the conclusion that this effect on the naturally-occurring polyamines
was important. The action of spermidine is now attributed more to competition
for cellular uptake than to replenishment of polyamine pools. A number of
laboratories (Holtta et aL, 1979, 1981, Newton and Abdel-Monem, 1977,
Pleshkewych §igL, 1982, Seppanen glaL, 1980) have been unable to correlate
these changes in polyamine pool size with inhibition of cell growth. Moreover,
DFMO, a highly specific inhibitor of polyamine biosynthesis, is cytostatic
(Mamont §i aL, 1978), whereas MGBG is cytotoxic, a finding that suggests
different mechanisms of action for the two drugs.
On the basis of considerable ultrastructural and biochemical evidence, there
is now sufficient cause to believe that the mitochondrion may, in fact, be the
target responsible for the antiproliferative activity of MGBG. Indeed, 4, 4'-
diacetyldiphenylurea-bis[guanylhydrazone], an aromatic bis[guanylhydrazone]
with potent antiproliferative activity, causes profound ultrastructural damage to
mitochondria (Mikles-Robertson gigL, 1981) but has no effect on polyamine
biosynthesis (Corti et gl., 1974). Ultrastructural studies in a variety of cell types


4
treated in vitro (Mikles-Robertson al aL, 1979, Pathakgta!-. 1977, and
Wiseman alai-, 1980) or in vivo (Pleshkewych alai-, 1980 and Porter el ai-,
1979) with MGBG reveal that mitochondria were selectively and significantly
damaged.
In addition, the mitochondria were functionally impaired by MGBG. The
metabolic effects of MGBG include 1) decreased rates of oxidation of pyruvate
(Pleshkewych alai-, 1980) and long chain fatty acids (Nikula alai-, 1984 and
Brady eiai-, 1987) ; 2) a decrease in the intracellular concentration of ATP with
concommitant increases in ADP concentration (Pine and DiPaolo, 1966 and
Regelson and Holland, 1963) and lactate production (Porter alai-, 1982) ; and
3) a decreased rate of incorporation of acetate into lipid (Pine and DiPaolo,
1966).
A study of the effects of MGBG on isolated rat liver mitochondria (Byczkowski
alai-, 1981) demonstrated that at drug concentrations comparable to those
attained intracellularly, MGBG significantly inhibits state 4 respiration, but has
less of an effect on either state 3 or uncoupled respiration. Byczkowski al ai-
speculated that 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. Since MGBG is a cation under physiological conditions, it might
accumulate at or within the inner membrane because of its negative potential.
Similar selective binding characteristics have been noted for other cationic
compounds. Rhodamine dyes that are positively charged stain mitochondria
specifically, whereas uncharged rhodamines and the negatively charged dye,
fluorescein, do not. It is possible that MGBG neutralizes the net negative surface
potential of the mitochondrial inner membrane, perhaps by binding to
phospholipids. This might influence cation binding and/or transport and
secondarily affect mitochondrial bioenergetics (Toninello alai-, 1988). In any


5
case, the exact mechanism by which MGBG influences mitochondrial
metabolism is unknown. It is likewise not known whether or not these actions
play a role in the effects of MGBG on mitochondrial DNA replication described
below.
MGBG inhibits the replication of mitochondria DNA (mtDNA) at
concentrations of drug that do not seem to interfere with the replication of
nuclear DNA (Feuerstein elaj., 1979). Drug-induced inhibition of mtDNA
replication is likely to occur shortly after exposure to MGBG (Nass, 1984). Our
laboratory has observed that the decrease in cellular content of mtDNA with
time can be accounted for by lack of replication coupled with dilution of the
residual genome upon repetitive cellular division (Bortell, 1987). These results
were obtained in our laboratory with use of dot-blot hybridization methods
involving whole cell lysates and 35S labelled full-length mouse mtDNA probe.
Although we know little about the mechanism(s) by which MGBG adversely
affects the ultrastructure, bioenergetics and replication of mitochondria, it does
seem likely that these actions relate to the antiproliferative and cytotoxic effects
of the drug and many of its congeners.
MGBG seems to accumulate in dividing cells by utilizing the facilitated
carrier mechanism for spermidine (Dave and Caballes, 1973, Porter el ai-.
1981). Seppanen eiai. (1981) found that MGBG uptake is critically dependent
on the growth rate of tumor cells (i.e., slowly dividing cells transport less MGBG
than rapidly dividing cells). Moreover, Mikles-Robertson el a[. (1979) found that
MGBG cytotoxicity also correlates with growth rate. Athough several
descriptions of the transport of MGBG have been published, the process is still
poorly defined. MGBG uptake is thought to involve an energy dependent
saturable carrier (Porter el al., 1982; Field el al., 1964), in that cells concentrate
the drug, and concentration gradients across the plasma membrane as high as


6
1000-fold have been reported. Polyamine and/or MGBG transport has been
studied in different cell lines and tissues including neuroblastoma (Rinehart and
Chen, 1984), fibroblast (Pohjanpelto, 1976), Ehrlich ascites carcinoma
(Seppanen glai-, 1980,1981,1982 ; Seppanen, 1981), mammary gland
explants (Kano and Oka, 1976), slices of rat lung and other tissues
(Gordonsmith elal-. 1985; Smith and Wyatt, 1981), and rat lung perfused in situ
(Reynolds et al., 1985). In general, it appears that putrescine, spermidine,
spermine, and MGBG are transported by common membrane transport systems
which are distinguishable from other defined systems (e.g., for various amino
acids). The transport is saturable and temperature-dependent, with maximal
rates evident at 37C. MGBG transport is inhibited by uncouplers of oxidative
phosphorylation and certain respiratory poisons, dependent on the proliferative
status of the cells and, in some instances, subject to hormonal regulation.
Whether polyamine/MGBG transport actually occurs via the same mechanisms
in all types of cells in any particular organism is also unclear. The development
of more efficacious drugs in this class of compounds seems to depend on
further understanding of the mechanism of transport, and, perhaps more
importantly, how the transport process is regulated.


7
H2N(CH2)4NH2 H2N(CH2)3NH(CH2)4NH2
putrescine spermidine
H2N(CH2)3NH(CH2)4NH(CH2)3NH2
spermine
CH3CONH(CH2)3NH(CH2)4NH2
N1-acetylspermidine
NH CH3 NH
II I II
h2ncnhn=c-ch=nnhcnh2
MGBG
chf2
I
H2N(CH2)3C-NH2
I
COOH
DFMO
FIGURE 1-1 : Structure of polyamines, related compounds and
inhibitors of polyamine synthesis.


CHAPTER 2
MATERIALS AND GENERAL METHODS
Cell Culture
Murine L1210 cells (obtained from American Type Culture Collection,
Rockville, Maryland) were allowed to grow in suspension in 25 or 75 cm2
canted-neck tissue culture flasks (Fisher Scientific) in an incubator (National
Appliance Company, Portland, Oregon) in an atmosphere of air: CO2 (95 : 5,
v/v) at 37C. Cells were incubated in Roswell Park Memorial Institute (RPMI)
1640 medium (CellgroR, GIBCO Laboratories) supplemented with 10 % heat-
inactivated horse serum (GIBCO Laboratories), 16 mM HEPES (3-[N-
morpholino] propanesulphonic acid) and 8 mM MOPS (N-2-hydroxyethyl-
piperazine-N'-2-ethanesulphonic acid), at a final pH of 7.40. All chemicals were
obtained from Sigma Chemical Co. unless otherwise specified. Cells were
maintained in logarithmic growth by reseeding an aliquot of cells into fresh
medium every two days to a starting concentration of 7 x 104 cells per ml.
Cell Counts
Aliquots (100 or 200 pi) of cells were diluted in 10 ml of Hematall diluent
(Fisher Scientific). Cell number was determined by electronic particle analysis
(Coulter Counter, Model ZF, Coulter Electronics, Hialeah, FL). Two aliquots
were counted from each flask, and the mean was determined. Because cells
were reseeded every two days, a calculation was devised to plot cell
accumulation as a single, continuous line over the the entire period of an
experiment. Cumulative counts were calculated as:
8


9
cone, of viable cells (n+1)
Cumulative count(n+1) = Cumulative count (n) X
cone, of viable cells (n)
The population doubling time of the cells was determined as follows: N =
Noe+kt; where t = time, No Is the number of cells present at t = 0, k = In 2/
doubling time, and the doubling time Is the value of t when N = 2No.
Cell Viability
Trypan blue dye (Eastman Kodak, Rochester, NY) at a final concentration of
0.06% was added to 100 pi aliquots of cells. 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 examined by
phase contrast microscopy (Ernst Leitz, Wetzlar, Germany) at 200X
magnification. Percent viability of cells was determined as follows:
number of cells excluding trypan blue dye x 100
% viability =
total number of cells
Clonoaenic Assay
Colony-forming ability of L1210 cells was determined by growth in soft agar
(Chu and Fischer, 1968). Pre-autoclaved Agar Noble (DIFCO Laboratories,
Detroit, Ml) was admixed at 37C with RPMI 1640 medium containing 20% heat-
inactivated horse serum to achieve 1mg agar/ml final concentration A
4 ml aliquot of the prepared medium was transferred into each cloning tube.
L1210 cells from selected experitments were counted and resuspended into
cloning tubes over the concentration range of 100 to 10,000 cells per tube.
Cloning tubes were cooled down at 0 4C for 15 minutes prior to incubation at
37C. Colony counts were determined 2 weeks after the start of incubation.
Transport of MGBG
The assay of MGBG influx and efflux was based on the use of methylglyoxal-
bis[14C]guanylhydrazone ([14C] MGBG) (specific activity 92.8 pCi/mg or 25.6


mCi/mmole, Amersham). A stock solution of 1 mM [14C] MGBG was prepared
aseptically. Aliquots of this stock solution were added to the incubation medium
to obtain the indicated final concentration of drug.
Murine L1210 cells were exposed to various concentrations of [14C] MGBG
for the indicated times at 37°C and pH 7.40 unless otherwise indicated.
Incubations were terminated by centrifugation. One-ml aliquots of cell
suspensions were centrifuged at 15,000g for 10 seconds in Eppendorf tubes.
The supernatant was discarded, and the cell pellet was washed twice by
centrifugation with one ml of ice-cold medium containing 1 mM unlabelled
MGBG. The tip of each Eppendorf tube, which contained the cell pellet, was cut
off and transferred to a scintillation vial containing 10 ml of LiquiscintR (National
Diagnostics, Manville, NJ) for quantitation by liquid scintillation counting in
Beckman model LS 7000.
Cell counts and viability were obtained with use of a hemacytometer. The
diameter and volume of cells were determined by the method of Schwartz el ai.
(1983). Uniform polymeric microspheres (Polyscience, Warrington, PA) from
4.72 to 10.0 11 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 diameter and volume for each size of calibrated
microbead to obtain a standard curve.
Aliquots of 1 x106 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 and volume. The
diameter of drug-naive L1210 cells was 10.6 p and cell volume was 5.68 pi per
106 cells. There was no significant change in the size of L1210 cells during or
after an exposure to MGBG when the cells were observed under a light


microscope in the presence of standard beads. The apparent intracellular
concentration of [14C] MGBG (pM) in all experiments was calculated assuming
a volume of 5.68 pi per 106 cells. In studies of efflux, cells were preincubated in
[14C] MGBG as indicated in subsequent chapters.
Preliminary experiments had revealed that 1) the rate of accumulation of
MGBG by cells was constant for at least 20 minutes; and 2) although 1 mM
unlabelled MGBG was routinely included in the wash of cells, its presence did
not make much difference.
Stability of fHCI MGBG
It is generally believed that MGBG does not undergo biotransformation in
higher animals (Warrell and Burchenal, 1983), in part because no metabolites
of MGBG have been detected in urine, feces and various tissues and no
radioactive carbon dioxide was expired after in vivo administration of [14C]
MGBG. Nonetheless, we further established the stability of [14C] MGBG upon
incubation with L1210 cells by thin layer chromatography. L1210 cells were
exposed to [14C] MGBG for 24 and 48 hours. Aliquots of cell suspension were
taken, centrifuged and the supernatant was then removed. The cell pellet was
washed twice at 0 - 4°C with media containing 1 mM unlabelled MGBG, then
resuspended in deionized water to lyse the cells. Trichloroacetic acid was
added to a final concentration of 10% and the precipitated protein was removed
by centrifugation. Aliquots of supernatant were spotted on precoated thin layer
plates of Silica Gel 60 F254 (E. Merck, Darmstadt, Germany). Chromatograms
were developed with 50% n-butanol, 30% water and 20% glacial acetic acid.
Unlabelled MGBG was located by fluorescence quenching, and its Rf was found
to be 0.49 - 0.53. [14C] MGBG was located by autoradiography with use of
Kodak SB Film, and its migration was indistinguishable from that of authentic
MGBG. The only radioactivity observed after 24 and 48 hours of incubation of


[14C] MGBG with L1210 cells was that of the drug itself. This observation
confirmed that [14C] MGBG was stable and was not metabolized by the cells.
Protein Assay
Protein was measured by the method of Lowry el aK (1951) with use of
bovine serum albumin as standard.
Statistical Analysis
The two-tailed Student t-test was used to analyze comparisons; the
significance level was set at P < 0.05. Lineweaver-Burk graphs were evaluated
by least squares regression analysis.


CHAPTER 3
TRANSPORT OF MGBG AND ITS EFFECT ON SENSITIVE L1210 CELLS
This chapter deals with 1) the effects of varying doses of MGBG on the
proliferation, viability and clonogenicity of L1210 cells; 2) the characterization
of the processes involved in the transport of MGBG into and out of these cells
upon first exposure to the drug; 3) an assessment of the intracellular
distribution of the MGBG, especially with regard to possible accumulation within
mitochondria; and 4) the relationship between transport of MGBG and the rate
of cell growth.
Effects of MGBG on Growth. Viability and Clonoaenicitv
To determine the effects of MGBG on cell growth and viability, L1210 cells in
logarithmic growth were incubated continuously in 0 to 20 pM drug at 37C, pH
7.40. Cells were reseeded every two days in fresh medium containing MGBG.
Cell count and viability were determined daily. The cumulative count of viable
cells is depicted in Figure 3-1, and the percent of total cells that were viable is
presented in Figure 3-2. Untreated cells exhibit a doubling time of
approximately 9 hours.
MGBG at concentrations as high as 20 pM had minimal effect on cell growth
or viability for the first few days of exposure. Our laboratory (Bortell, 1987) has
reported that the cellular content of mt DNA decreases markedly during this
time. The decrease was found to reflect dilution of the mitochondrial DNA due
to lack of its replication coupled with continuing cell division. Thereafter cell
13


growth was inhibited by MBGB in a dose-dependent manner, perhaps because
of the depletion of mt DNA. At MGBG concentrations between 0.3 and 1.0 pM,
the growth rates were markedly inhibited for about 10 days after which recovery
to rates virtually identical to untreated cells was observed. At these doses,
viability was also adversely affected in a concentration-dependent manner, but
it did not fall below 60%. Viability recovered to the control value of nearly 100%
when growth rate recovered. At the intermediate dose of 5.0 pM MGBG, the
percent of viable cells decreased to a plateau of about 40%, and recovery of
growth had not occurred by day 17 of treatment. The cytotoxic affect of MGBG
was apparent when the medium contained 20 pM MGBG. Cell viability
decreased progressively, and no living cells could be detected after day eight.
Further experiments were performed to determine the effects of MGBG on
clonogenicity of murine L1210 cells in soft agar. L1210 cells were pretreated
with different concentrations of MGBG (0-10 pM) for 24 or 72 hours before
reseeding in drug-free RPMI 1640 medium containing 1 gm/ml Agar Noble.
MGBG inhibited the clonogenicity of L1210 cells in soft agar in a concentration-
dependent manner (Fig 3-3). The inhibitory effect of MGBG on the
clonogenicity of L1210 cells was evident as early as 24 hours after MGBG
exposure, whereas the cell viability was unaffected at that time.
Characterization of the Initial Transport of MGBG
Intracellular Accumulation of MGBG During the First 20 Hours of Exposure
Although we have measured the intracellular content of MGBG over a few
weeks of incubation (see below), this portion of the proposal focuses on the first
20 hours of incubation. It is during this time that the L1210 cells accumulated
large amounts of MGBG. By 20 hours, the replication of mitochondrial DNA has
been inhibited, but the rate of cell growth and cell viability have remained
unaffected.


cumulative cell count
15
time (days)
FIGURE 3-1 : Effects of various concentrations of MGBG on the proliferation of
murine L1210 cells. Cells in logarithmic growth were incubated
continuously in 0 20 pM MGBG and were reseeded every two days in
fresh medium containing MGBG. Cell counts were determined by
electronic particle analysis (Coulter Counter). Cumulative cell counts were
calculated and cell accumulation was plotted as a single continuous line
over the entire period of an experiment.


% viability
16
FIGURE 3-2 : Effects of various concentrations of MGBG on the viability of
murine L1210 cells over time. Cells in logarithmic growth were incubated
continuously in 0 - 20 pM MGBG and were reseeded every two days in
fresh medium containing MGBG. Cell viability was determined with use of
the trypan blue dye exclusion method.


clonogenicity (% control)
17
|iM MGBG
o 72hr in MGBG
* 24hr in MGBG
FIGURE 3-3 : Effects of MGBG on the clonogenicity of L1210 cells. L1210 cells
were exposed to different concentrations of MGBG (0-10 pM) for 24 or 72
hours prior to reseeding in RPMI 1640 medium containing 1 mg/ml Agar
Noble. Clonogenicity was determined by an ability of cells to form colonies in
agar after 2 weeks of incubation, and values are expressed as percent control.


Cells in logarithmic growth were incubated in various concentrations of
[14C] MGBG at 37 C, pH 7.40. At various times, aliquots of cells were
harvested and washed at 0 C by centrifugation. Although the routine washing
procedure involves the use of unlabelled 1 mM MGBG, initial experiments
revealed that little added radiolabel was displaced from the cells by the carrier.
In addition, the rate of efflux of radiolabel from the cells is sufficiently slow even
at 37C to permit this approach to separation of cells from incubation medium.
The results are presented in Figure 3-4. MGBG accumulated within cells at
reasonably constant rates dependending on the initial extracellular MGBG
concentration. It is apparent in Figure 3-4 that the apparent intracellular
concentration of MGBG exceeded that of the medium by about 1000-fold within
12 hours.
Kinetic Characterization of the Initial Uptake Process
The initial uptake of MGBG occurs by a saturable process. To determine Km
and Vmax, L1210 cells in logarithmic growth were reseeded at a density of 3 x
105 cells per ml in prewarmed (37C) media containing different concentrations
of [14C] MGBG at pH 7.40. "Intracellular" radioactivity was determined 20
minutes later (see Chapter 2 for details of centrifugation and washing of the
cells). As noted above, all of the radioactivity, even 48 hours after the start of
incubation, remained stable as [14C] MGBG. The amount of drug in cells was
expressed as pmole per 106 cells. Four separate experiments were conducted.
Lineweaver-Burk plots were constructed (Figure 3-5), and Km and Vmax were
determined by linear regression. The Km and Vmax for uptake of MGBG at 37C
were 4.72 0.17 pM and 296 19 pmole/106 cells/hour, respectively.
Interestingly, the amount of intracellular [14C] MGBG over the first 20 hours of
exposure to the drug (Figure 3-4) could be predicted with use of the kinetic
parameters determined over 20 minutes (Table 3-1). Given the volumes of cells


|iM pellet [MGBG]
19
time (hrs.)
FIGURE 3-4 : Accumulation of MGBG in L1210 cells during the first 20 hours of
exposure. Cells in logarithmic growth were incubated in 0.1 - 5 pM [14C]
MGBG. At various times, aliquots of cells were harvested and cell pellet was
washed twice by centrifugation with use of medium containing 1 mM MGBG.
Cell counts and viability were obtained with use of a hemacytometer and the
results were expressed as apparent intracellular concentrations of [14C]
MGBG (pM).


1/pmole MGBG/milion cells/hr.
20
1/jiM [MGBG]
FIGURE 3-5 : Lineweaver-Burk plot of MGBG uptake in control L1210 cells. L1210
cells in logarithmic growth were reseeded at a density of 3 x 105 cells per ml
in media containing different concentrations of [14C] MGBG. Intracellular
radioactivity was determined 20 minutes later and the amount of drug in cells
was expressed as pmole [14C] MGBG per 106 cells. The Km and Vmax were
determined by linear regression of the Lineweaver-Burk Plot.


TABLE 3-1 : Comparison between the calculated and observed intracellular concentrations of MGBG during
the first 20 hours of exposure.
hrs. after Intracellular concentration of [14-C] MGBG (pMI
incubation 0.1 pM 0.5 uM 0.93 uM 4.8 pM
calculated observed calculated observed calculated observed calculated observed
5.7
60
38.9 (1.5)
271.0
289.8 (11.3)
487.8
561.5 (34.5)
1488.9
1420.5
(49.1)
13.3
141.3
131.7 (9.6)
637.6
735.0 (32.9)
1147.6
1374.9 (79.5)
3503.1
3423.5
(224)
20.1
169.5
164.8 (10.1)
960.8
1110 (120)
1729.2
1946.5 (136)
5074.5
3944.5
(263)
Note : The intracellular concentration of MGBG at various times was calculated with use of Michaelis-Menten Equation
( v = Vmax[S]/Km + [S]), using the predetermined values for the Vmax and Km of 296 ± 19 pmole/106 cells/hour and 4.72
±_0.169 pM, respectively. Efflux was ignored in the calculations. Each observed value represents the mean + SEM.


22
and of the incubation media, the change in extracellular concentrations of
MGBG over 20 hours did not exceed 25% of the initial concentration. Indeed,
except for the lowest initial concentration of drug, the decrease in extracellular
[MGBG] was considerably less than 25%.
Experiments were also conducted at 0 4C. Rates of uptake of 10 pM
MGBG at 0 4C was less than 10% of that seen at 37C. This result would be
anticipated if the uptake of MGBG depended on active transport.
Inhibition of MGBG Uptake bv Spermidine. Spermine and Putrescine
L1210 cells were incubated in medium containing different concentrations of
[14C] MGBG (1.2 58.6 pM) with or without 10 pM spermidine, spermine or
putrescine. The incubation was carried out for 20 minutes at 37 C. Uptake of
MGBG was inhibited competitively by spermidine, spermine and putrescine
(Figure 3-6). This result implies that MGBG and the naturally occurring
polyamines, spermidine, spermine and putrescine, share a common transport
mechanism. The K¡'s of spermidine, spermine and putrescine for the inhibition
of MGBG uptake were 0.524 0.03, 0.582 0.029 and 3.74 0.029 pM,
respectively.
Effect of Extracellular pH on the Initial Uptake of MGBG
Logarithmically-growing L1210 cells were exposed to [14C] MGBG (2.0 pM)
for 0 to 10 minutes at 37C over the range of extracellular pH between 7.0 and
7.7. The rates of uptake of MGBG were strongly influenced by pH An increase
in extracellular hydrogen ion concentration enhanced the rate of uptake of [14C]
MGBG. Similar results were observed when the experiments were conducted
for 0 to 75 minutes with 0.8 and 8.5 pM [14C] MGBG (Figure 3-7). MGBG is a
weak base with pKa's of about 7.5 and 9.2 at 25C (Williams-Ashman and
Seidenfeld, 1986). At physiological pH, MGBG exists primarily as a mixture of
monovalent and divalent cations. The results depicted in Figure 3-7 suggest


23
that it is the divalent form of MGBG (MGBG 2+) that is transported into L1210
cells. Some evidence for this idea derived from consideration of the competitive
inhibition of MGBG transport by the naturally occurring polyamines. For
example, spermidine, the extended molecular conformation of which is quite
similar to that of MGBG, has pKa values of about 8.4, 9.8 and 10.8 at 25C
(Williams-Ashman and Seidenfeld, 1986), and exists almost exclusively as a
mixture of divalent and trivalent cations at physiological pH. The hypothesis that
MGBG2+ is the substrate for transport is supported further by the good
agreement between the observed and calculated values of MGBG uptake
presented in Table 3-2. The calculations were based on Henderson-
Hasselbach computations of the concentrations of MGBG2+ at different pH's
and on the assumption that only MGBG2+ is transported.
Efflux of MGBG
The following experiments were designed to study the characteristics of
efflux of MGBG from L1210 cells. Logarithmically-growing L1210 cells were
exposed to 1.5 pM [14C] MGBG for four hours before centrifugation and
resuspension in drug-free medium. The apparent intracellular concentration of
MGBG was about 400 pM at the time of resuspension. The efflux of radioactivity
with time is depicted in Figure 3-8. Efflux occurs by a process with
approximately first-order characteristics with a half-life of about three hours.
Experiments were also conducted at 0 4C. The results reveal that the efflux
of MGBG at this low temperature was minimal (Figure 3-9), which would be
anticipated if the efflux of MGBG depended on active transport.
Countertransport of Unlabelled MGBG
To explore the possibility that MGBG transport is mediated by an antiport
carrier, the effect of preloading the cells with unlabelled MGBG on the
subsequent rate of uptake of [14C] MGBG was assessed. L1210 cells were


1/pmole MGBG/10e6 cells/hr.
24
control
10 nM spd
d lO^Mspm
10|iMputres
FIGURE 3-6 : Competitive inhibition of MGBG uptake by spermidine, spermine and
putrescine in control L1210 cells. Control L1210 cells in logarithmic growth
were reseeded at a density of 3 x 105 cells per ml in media containing
different concentrations of [14C] MGBG (1.7 80.6 pM) in the presence or
absence of 10 pM spermidine (spd), spermine (spm) or putrescine (putres).
The uptake of [14C] MGBG at 37C was assessed after 20 minutes
incubation. The K¡'s of spermidine, spermine and putrescine for the uptake
of MGBG were 0.524 0.03, 0.582 0.029 and 3.74 0.029 pM,
respectively.


|iM pellet [MGBG]
25
medium
pH
a
6.90

7.10
a
7.36
o
7.50

7.75
a
8.35
FIGURE 3-7 : Effect of extracellular pH on the initial uptake of MGBG.
Logarithmically-growing L1210 cells were exposed to [14C] MGBG (0.8
pM) for 0 75 minutes over the range of extracellular pH between 6.90
and 8.35. Intracellular radioactivity was determined at various times (0 -
75 minutes) after the exposure.


26
TABLE 3-2 : Comparison between the observed rates of MGBG uptake at
different extracellular pH's with rates calculated on the basis of transport
of MGBG2+ only.
Extracellular pH [MGBG1 +]/[MGBG2+]
m/m
calculated v
(pM/min.)
observed v
(pM/min.)
At 0.8 pM[14C] MGBG
6.90
0.16/0.64
1.04
1.07
7.10
0.23/0.57
0.92
1.08
7.36
0.34/0.46
0.77
0.72
7.50
0.40/0.40
0.67
0.34
7.75
0.64/0.34
0.58
0.28
8.35
0.70/0.10
0.18
0.10
At 8.5 pM [14C] MGBG
6.90
1.70/6.79
5.12
5.06
7.10
2.42/6.08
4.89
5.65
7.36
3.57/4.93
4.43
4.22
7.50
4.25/4.25
4.11
2.52
7.75
5.44/3.06
3.41
1.48
8.35
7.45/1.05
1.58
0.96
Note : L1210 cells were exposed to 0.8 and 8.5 pM [14C] MGBG in RPMI 1640 medium
which was adjusted to different pH's with use of concentrated HCI or NaOH. Cells were
incubated at 37 C and aliquots of cell suspension were taken intermittently during the
course of exposure to determine pellet radioactivity. Observed values of the uptake
rate of [14C] MGBG were then compared to the values calculated with an assumption
that the Km and Vmax of MGBG uptake (4.72 pM and 8.68 pM per min., respectively)
were unchanged at different pH's but apply only to MGBG2+. The Henderson-
Hassenbach equation (pH = pKa + log [MGBG1+]/[MGBG2+] was used to calculate an
availability of divalent form of MGBG and the Michaelis-Menten equation (v = Vmax
[S]/Km + [S]), to predict the intracellular accumulation of [14C] MGBG if only the
divalent form of MGBG was transported into L1210 cells.


(iM pellet [MGBG]
27
time after crossover (hrs.)
FIGURE 3-8 : Efflux of MGBG. Logarithmically-growing L1210 cells were exposed to
1.5 (iM [14C] MGBG for 4 hours on the first day (day 0) of incubation before
centrifugation and resuspension in drug-free medium. Intracellular
radioactivity was determined at various times (0 48 hours) after
resuspension.


|iM pellet [MGBG]
28
FIGURE 3-9 : Effect of temperature on the efflux of MGBG. L1210 cells at 3 x 105
cells per ml were incubated in medium containing 5 pM [14C] MGBG for
1.5 hours at 37 C. Cell pellet was washed once with drug-free medium at
0 4 C before subsequent resuspension in drug-free medium, and the
cell suspension then was incubated at 0 4 C or 37 C. Intracellular
radioactivity was determined for 0 12 hours after the resuspension.


29
exposed to 5 pM MGBG for two hours before centrifugation and resuspension
into medium containing 1 pM [14C] MGBG. The apparent intracellular
concentration of nonradioactive MGBG would be expected to be about 400 pM
at the time of resuspension. The results shown in Figure 3-10 revealed that
preloading the cells with MGBG did enhance modestly the uptake of [14C]
MGBG.
We also explored the effects of various extracellular concentrations of MGBG
on the rate of efflux of [14C] MGBG from preloaded L1210 cells (Figure 3-11).
No stimulation of efflux was noted; when the extracellular concentration of
MGBG was 1 mM, some inhibition of efflux was observed.
In summary, the presence of unlabelled MGBG inside cells stimulated the
uptake of [14C] MGBG, but the presence of unlabelled MGBG in the medium did
not stimulate the efflux of [14C] MGBG. Interpretation of this observation is quite
complex because of the presence of naturally occurring polyamines in these
L1210 cells.
Effects of Various Cations on the Uptake of MGBG
Sodium-dependency of MGBG uptake
The accumulation of [14C] MGBG (4.0 pM) by L1210 cells was determined,
at 0, 5, 15 and 30 minutes in an Earle's balanced salt solution (EBSS) which
contains NaCI (116 mM). This rate of accumulation was compared to
incubations in which NaCI was replaced iso-osmotically by choline choride
(Figure 3-12). The results suggest that the uptake of MGBG by L1210 cells is
sodium dependent. Other experiments were done to further establish this
conclusion. L1210 cells were exposed to different concentrations of [14C]
MGBG (0.5 - 30 pM) in 10 mM HEPES-TRIS buffer, pH 7.40, which contains
either 100 mM NaCI or choline chloride. The results shown in figure 3-13
revealed an MGBG concentration dependent enhancement of [14C] MGBG in


pM pellet [MGBG]
30
preloaded MGBG (pM)
Q none
5.0
o 10.0
o 20.0
FIGURE 3-10 : Effect of MGBG preloading on the uptake of labelled MGBG. L1210
cells were exposed to 0 20 pM MGBG for two hours before centrifugation
and resuspension into a medium containing 10 pM [14C] MGBG. Intracellular
radioactivity was determined for 0-120 minutes after resuspension.


|iM pellet [MGBG]
31
medium [MGBG]
□—
â–  none
â–  10 nM
• 100 |iM
o
1 mM
FIGURE 3-11 : Effect of medium concentration of MGBG on the efflux of labelled
MGBG. L1210 cells were exposed to 1.0 pM [14C] MGBG for one hour
before centrifugation, and the cell pellet was resuspended into medium
containing MGBG (10 pM - 1 mM). Intracellular radioactivity was
determined for 0 - 90 minutes after resuspension.


32
the presence of NaCI. To determine kinetics of the sodium-dependent process,
uptake in choline chloride was subtracted from uptake in the presence of NaCI.
A Lineweaver-Burk plot of the sodium-dependent uptake as a function of [14C]
MGBG concentration was linear (Figure 3-14). These results indicated that the
sodium-dependent component of uptake of MGBG was a single transport
system with a Km and Vmax of 2.10 0.08 pM and 226.8 18.0 pmole/106
cells/hour, respectively. Michaelis-Menten calculations revealed that at an
extracellular MGBG concentration of 1 pM, which is cytotoxic, the sodium
dependent transport of MGBG accounts for more than 90 % of total uptake.
Results of experiments in which both sodium and choline are replaced by
mannitol are interesting. Uptake of [14C] MGBG in10 mM HEPES-TRIS buffer,
pH 7.40 containing 200 mM mannitol was surprisingly high (Figure 3-15).
However, Lineweaver-Burk plots of MGBG uptake as a function of MGBG
concentration in the presence of mannitol, like choline chloride but unlike
sodium chloride, revealed a low affinity process (high Km).
Ability of various cations to substitute for sodium in uptake of MGBG
To determine the cation specificity of MGBG uptake, sodium chloride was
replaced by a variety of monovalent cation chloride salts in incubations of
L1210 cells with [14C] MGBG (Figure 3-17 and Table 3-3). Choline and lithium
maintained rates of uptake of MGBG about 2/3 that of sodium. Cesium and
potassium were less effective in that the observed uptakes ranged from only 7
to 24 % of sodium controls. Table 3-4 depicts experiments in which MGBG
uptake was assessed in the presence of varying mixtures of sodium, potassium
and choline chloride. The detrimental effect of potassium, even in comparison
to choline, is again apparent.
The specificity toward chloride was also examined. Substitution of sodium
chloride with sodium gluconate had no effect on MGBG uptake. Replacement of


% control uptake rate
33
FIGURE 3-12: Sodium dependency of MGBG uptake in L1210 cells. The uptake of
[14C] MGBG ( 4.0 (iM) in logarithmic-growing L1210 cells was measured in an
Earle's balanced salt solution in which NaCI was iso-osmotically replaced by
varying amount (0, 25, 50, 75 and 100%) of choline chloride. In each case,
the uptake of MGBG was assessed at 0, 5,15 and 30 minutes. The rate of
uptake at 100% (116 mM) NaCI was set at 100% and the other rates were
plotted against the fractional content of sodium. Each point represents the
average of three separate experiments, with triplicate measurements in each
experiment.


MGBG in pellet (|i.M/30 minutes)
34
FIGURE 3-13 : Relationship between MGBG concentration and the rate of uptake
of MGBG in the presence or absence of sodium. L1210 cells were
exposed to different concentrations of [14C] MGBG (0.5 - 30 pM) for 30
minutes in 10 mM HEPES-TRIS buffer, containing either 100 mM NaCI or
100 mM choline chloride.


1/d uptake NaCI-Choline Cl (pM/30 minutes)
35
o 1/d uptake
FIGURE 3-14 : Lineweaver-Burk plot of the sodium-dependent MGBG uptake
in L1210 cells. Sodium-dependent uptake of MGBG refers to the
diference between uptake in the presence of sodium chloride and
choline chloride as indicated in Figure 3-13.


MGBG in pellet (pM/30 minutes)
36
600
500
400
300
200
100
0
0 1 0 20 30 40
MGBG concentration (p.M)
NaCI
Choline Cl
Mannitol
FIGURE 3-15 : The relationship between MGBG concentration and the rate of
MGBG uptake in sodium chloride, choline chloride and mannitol. Cells
were incubated for 30 minutes in [14C] MGBG in 10 mM HEPES-TRIS
buffer, pH 7.40, containing 100 mM NaCI, 100 mM choline chloride or
200 mM mannitol.


1/pmole/milMon cells/hr.
37
NaCI
Choline Cl
Mannitol
FIGURE 3-16 : Lineweaver-Burk plot of MGBG uptake in sodium chloride,
choline chloride and mannitol. The data are presented in Figure 3-15.
Vmax of MGBG uptake in NaCI, choline chloride and mannitol were
362.4 19.7, 450.6 31.6, 2372.8 103 pmole/106 cells/hour with Km
of 9.34 + 1.04, 61.5 2.52 and 50.7 + 2.10 pM, respectively.


38
chloride ion with the relatively impermeable anion, sulfate, did not affect the
initial (up to 10 minutes) uptake of MGBG, but did decrease uptake thereafter.
Effect of lonoohores on the Uptake of MGBG
Two classes of ionophores, mobile ion carriers and channel formers, with
different cation specificities, were evaluated with regard to their effect on MGBG
uptake. L1210 cells were preincubated with the ionophore (5 pM final
concentration) for 20 minutes in Earle's balanced salt solution before [14C]
MGBG was added to achieve the final concentration of 6 pM. The results are
shown in table 3-5. Among the ionophores tested, gramicidin A was the most
effective inhibitor of MGBG uptake. It is a channel-forming ionophore and does
not discriminate between sodium and potassium ions. Therefore, it can
dissipate both sodium and potassium electrochemical potential differences
across the plasma membrane. In contrast, valinomycin, a neutral ionophore
which is specific for the potassium ion did not significantly affect the uptake of
MGBG. Effects of monensin, an ionophore specific for sodium ion, and A23187,
which is specific for calcium ion (but also allows an entry of sodium and
potassium ions), were intermediate. These results support the notion that a
sodium electrochemical gradient across the plasma membrane is important in
attaining maximal uptake of MGBG.
Effect of Inhibitors of Energy Metabolism on the Uptake of MGBG
The effect of metabolic inhibitors on the uptake of [14C] MGBG was assessed
in order to gain some insight into the energetics of the transport process. The
inhibitors included antimycin A (an inhibitor of cytochrome reductase), 2, 4-
dinitrophenol (2, 4-DNP, an uncoupler of oxidative phosphorylation), and
potassium cyanide (KCN, an inhibitor of cytochrome oxidase). The effects of
these inhibitors of mitochondrial energetics (0.01 mM antimycin A, 0.1 mM 2,4-
DNP and 0.1 mM KCN) on drug uptake were studied by incubating L1210 cells


pM pellet [MGBG]
39
â–  NaCI
0 KCI
ED Choline Cl
0 LICI
â–¡ CsCI
â–  Sod. gluconate
Ü Sod. sulfate
time (minutes)
FIGURE 3-17 : Effects of various ions on MGBG uptake in L1210 cells.
Incubations were performed in 10 mM HEPES-TRIS buffer, pH 7.40,
containing 100 mM NaCI, KCI, choline Cl, LiCI, CsCI, Na2SC>4 or Na
gluconate. Mannitol was also added in the buffer to achieve 100 mM
concentration.


40
TABLE 3-3 : Effects of various ions on MGBG uptake in L1210 cells.
salt
10 min. uotake
30 min. uDtake
pM
%
pM
%
NaCI
39.2 ±1.2
100 a
124.7 ±5.2
100 a
KCI
7.0 ±1.2**
17.8
9.26 ±0.7**
7.4
Choline Cl
26.2 ± 1.5*
66.8
71.5 ± 1.3*
57.3
LiCI
26.7 ± 1.9*
68.1
74.0 ±4.4
59.3
CsCI
9.3 ±0.3**
23.7
29.3 ±1.2*
23.5
Na gluconate
35.8 ± 1.8
91.3
126.3 ±3.5
101.3
Na2SÜ4
34.7 ±0.9
88.5
67.9 ±1.61*
54.4
Note : All salts were included at a concentration of 100 mM (except Na2SC>4, 50 pM) in
the medium containing 100 mM mannitol and 10 mM HEPES-TRIS solution, pH
7.40. Results are mean + SE of four determinations of three separate experiments.
The final concentration of [14C] MGBG in the incubating medium was 3.0 pM. a The
uptake in NaCI was defined as 100 % (** p < 0.01, * p < 0.05).


41
TABLE 3-4 : Effects of potassium and choline chloride on the sodium-
dependent MGBG uptake in L1210 cells.
EBSS treatment
percent
100 % NaCI
100a
50 % NaCI : 50 % choline Cl
81.0
100 % choline chloride
57.3
50 % NaCI : 50 % KCI
58.6
100 % KCI
7.4
50 % KCI : 50 % choline Cl
42.6
Note : The transport rates of [14C] MGBG (3-4 |xM) in logarithmically-growing L1210
cells were measured, respectively, for 30 - 35 minutes in an Earle's balanced salt
solution where NaCI was displaced iso-osmotically by choline chloride and/or KCI (100
% NaCI corresponds to 116 mM). At the end of incubation period, cells were washed
and cell-associated radioactivity was determined as described in " Materials and
General Methods The data represents the average of two separated experiments,
with quadruplicate measurements in each experiment. a The uptake in 100 % NaCI
was defined as 100 %.


42
TABLE 3-5 : Effects of ionophores on MGBG uptake in L1210 cells.
Addition
Cation Specificity
MGBG Uptake
(|iM/20minutes)
% Inhibition
None
24.1 ±1.45
0
Valinomycin
K+
23.5 ± 1.05
2.5
Monensln
Na+
19.0 ± 0.65*
21.0
Gramicidin A
Na+, K+
10.0± 0.45**
58.3
A23187
Ca++
16.4 ± 0.4*
32.1
Note : L1210 cells were preincubated with the individual ionophores (5 jxM) for 20
minutes in Earle's balanced salt solution prior to an addition of [14C] MGBG (6
(xM final concentration). MGBG uptake was measured at 0, 20 and 40 minutes
after adding MGBG. Pellet radioactivity was determined as described earlier.
Each value represents the mean ± SEM, of three separate experiments, each
conducted in triplicate (* p < 0.05, ** p < 0.01).


pellet [MGBG]
43
control
E3 antimycln A
m 2,4-DNP
KCN
time (min.)
FIGURE 3-18 : Effects of inhibitors of energy metabolism on MGBG uptake.
L1210 cells were pretreated with 0.01 mM Antimycin A, 0.1 mM 2,4-
DNP or 0.1 mM KCN in PBS solution for 20 minutes, prior to addition
of [14C] MGBG (5 pM final concentration). The cells were incubated in
[14C] MGBG for the indicated times. Values are expressed as
intracellular concentrations of MGBG (jiM).


% control uptake
44
Antimycln A
0 2,4-DNP
El KCN
time (min.)
FIGURE 3-19 : Effects of inhibitors of energy metabolism on MGBG uptake.
L1210 cells were pretreated with 0.01 mM Antimycin A, 0.1 mM 2,4-
DNP or 0.1 mM KCN in PBS solution for 20 minutes, prior to addition
of [14C] MGBG (5 pM final concentration). The cells were incubated in
[14C] MGBG for the indicated times. Values are expressed as percent
of control (no inhibitor).


45
with the inhibitors for 20 minutes at 37C prior to the addition of [14C] MGBG.
These uptake studies were performed in the phosphate-buffered saline without
glucose to minimizing energy derived from glycolysis. The uptake of drug at 15,
30 and 60 minutes is expressed as an intracellular concentration of MGBG (pM)
in Figure 3-18 and as a percentage of control uptake in Figure 3-19. All are
corrected for rapid binding. The results reveal a significant decrease in the
accumulation of [14C] MGBG in the cells pretreated with the metabolic inhibitors
(antimycin 2,4-DNP > KCN). The data suggest a requirement of energy for
the drug uptake.
Intracellular Distribution of MGBG
I was particularly interested in exploring whether or not the striking
intracellular accumulation of MGBG reflected accumulation of the drug into
mitochondria. Two factors prompted this interest: 1) the cationic nature of
MGBG; and 2) the effects of MGBG on mitochondrial structure, function and
replication. Attempts to preload the cells with [14C] MGBG and then isolate
mitochondria were not pursued vigorously because the procedure involved in
the preparation of a mitochondrial fraction from these cells would predispose to
redistribution of radiolabel. Rather, two other experimental approaches were
taken: 1) uptake of [14C] MGBG by isolated L1210 mitochondria; and 2) digitonin
permeabilization of the plasma membrane of cells preloaded with [14C] MGBG.
Uptake of MGBG bv Isolated Mitochondria
Exponentially-growing L1210 cells were incubated with 0.1% w/v digitonin
in dimethylsulfoxide (DMSO) until approximately 70% of the cells no longer
excluded trypan blue. Cells were then washed once with a solution containing
250 mM sucrose, 2 mM EDTA and bovine serum albumin (1 mg/ml) at pH 7.40.
The cell pellet was immediately homogenized on ice with a 2 ml tapered glass
tissue grinder until more than 90% of the cells were ruptured. The homogenate


46
was centrifuged at 1000g for 10 minutes. The resulting supernatant fraction
was centrifuged at 16,000g for 15 minutes to obtain the mitochondrial fraction.
The respiratory control ratio (RCR) or the state 3/state 4 respiratory ratio of
the mitochondrial fraction was 2.0 to 2.5; with intact cells the RCR was 3.0 to
3.5. Oxygen consumption was measured with an oxygen electrode with
succinate as substrate in an incubation medium containing 150 mM sucrose, 25
mM glycylglycine, 40 mM KCI, and bovine serum albumin (1 mg/ml).
Mitochrondrial pellets were resuspended in 2.5 pM [14C] MGBG in RPMI
1640 medium at 37C, pH 7.40. Radioactivity in the mitochondrial pellet was
measured after 0, 0.5, 1,2, and 3 hours of incubation. The mitochrondrial pellet
was collected by centrifugation and washed once in medium containing 1 mM
unlabelled MGBG before recentrifugation. The uptake of [14C] MGBG by
mitochondria (in pmoles per mg protein) is presented in Table 3-4. The
concentration of [14C] MGBG in the mitochondrial pellet even after three hours of
exposure to 2.5 pM drug is less than 2.5 pM (for cells, about 15 pmoles per mg
protein is equivalent to 2.5 pM MGBG). Furthermore, mitochondrial uptake is
not likely to be the driving force for sequestration of MGBG within the cell
because the observed rate of uptake is only about 1% of that predicted by the
kinetic constants determined by experiments with intact cells.
Selective Release of Cytosolic Constituents by Diqitonin
Because of its low content of cholesterol, the inner membrane of
mitochondria is more resistant than the plasma membrane to disruption by
digitonin. I used this difference to assess whether or not the MGBG that had
accumulated within L1210 cells was predominately localized in mitochondria;
rhodamine 123 was used as a positive control since it is well established that
this cationic dye is highly concentrated within mitochondria (Johnson £t al-.
1981).


L1210 cells were exposed to 0.5 pM [14C] MGBG for 2 to 8 days. In addition
to determination of the apparent intracellular concentration of MGBG, one-ml
aliquots of cells were admixed gently in an ice bath with enough 0.1% (w/v)
digitonin in DMSO to cause 90 to 95% of the cells to become permeable to
trypan blue. The [14C] MGBG remaining in the pellet of the intact and
permeabilized cells was compared (Figure 3-20). Less than 6% of the drug in
the intact cells was recovered in the pellet of cells with disrupted plasma
membranes.
Concurrent experiments with rhodamine 123 provided evidence that a
compound that is accumulated by mitochondria remained within the cell after
this treatment with digitonin. One million 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 at the
final concentration of 1 pg/ml. The samples were incubated at 37°C for 10
minutes and washed once by centrifugation. The stained cells were observed
under a Zeiss epifluorescence microscope and were virtually indistinguishable
(in terms of mitochondrial fluorescence at an excitation wavelength of 485 nm)
from cells not disrupted by digitonin.
The experiments with both isolated mitochondria and digitonin lead me to
conclude that the large accumulation of MGBG within the cells involved active
transport of MGBG across the plasma membrane, some degree of intracellular
binding, and passive equilibration with the various intramitochondrial
compartments.
Effect of Growth Rate of L1210 cells on MGBG Transport
L1210 cells were seeded at an initial concentration of 3 x 104 cells/ml and
incubated at 37°C for five days without reseeding or adding fresh medium. The
cells divided exponentially until their concentration reached 1.8 x 106 cells/ml


48
TABLE 3-6 : Uptake of MGBG by isolated mitochondria a.
Time of incubation in
MGBG (hrs)
Mitochondrial MGBG
(pmole/mg protein)
% of expected intracellular
MGBG b
0.5
0.50 0.02*
0.36
1
3.83 0.04
1.35
2
1.94 0.07
0.35
3
2.68 0.06
0.32
Note : a The mitochondrial fraction obtained from L1210 cells was incubated in 2.5 |iM
[14C] MGBG for 0 3 hrs. The radioactivity in the mitochondrial pellet was determined
after two washes with ice-cold 1 mM MGBG. The results were expressed as pmole
mitochondrial [14C] MGBG/mg protein (* mean + SEM ).
b I assumed for this calculation that the uptake of MGBG by L1210 cells was due
totally to accumulation of the drug in mitochondria. The kinetic constants obtained from
experiments with intact cells were used to predict rates of uptake by mitochondria. It
can be seen that the actual rates of uptake were only about 1 % of the prediction.


pellet cpm/million cells
49
g control
gg digitonized
* p < 0.0001
Time (days)
FIGURE 3-20 : MGBG remaining in the cell pellet after permeabilization of the
plasma membrane by digitonin. L1210 cells were exposed to 0.5 pM [14C]
MGBG for 2 to 8 days. Prior to determination of the apparent intracellular
concentration of MGBG, one-ml aliquots of cells were admixed gently in an
ice bath with enough 0.1% (w/v) digitonin in dimethylsulfoxide until
approximately 95% of the cells became permeable to trypan blue dye. The
[14C] MGBG remaining in the pellet of the intact and permeabilized cells
was determined and compared.


50
(Figure 3-21). Plateau growth ensued when the concentration of cells reached
2 x106 cells/ml. Cell viability exceeded 95% when the cells were in logarithmic
growth, but it declined progressively to 40% by the fifth day of incubation when
the cells were at plateau (Figure 3-22).
Cells in logarithmic growth were compared to those in early plateau with
regard to transport of MGBG. L1210 cells at the two growth rates were
reseeded in fresh media containing 0.5 pM [14C] MGBG at starting
concentrations of 1 x 106 and 2 x 106 cells/ml, respectively. The intracellular
content of radiolabel was determined at 0, 24 and 48 hours after the start of
incubation at 37C, pH 7.40. The accumulation of MGBG was higher in the
logarithmically-growing cells (Figure 3-22). Kinetic experiments with cells just
entering plateau phase revealed that the decrease in uptake of MGBG in these
cells reflected more on an increase in Km (9.04 0.34 pM, P < 0.01) than a
decrease in Vmax (0.271 0.019 nmoles/106 cells/hour) relative to cells in
exponential growth.


cumulative cell count
51
FIGURE 3-21 : Growth curve of L1210 cells. L1210 cells were seeded at an initial
concentration of 3 x 104 cells/ml and incubated for 5 days without reseeding
or adding fresh medium. Cell counts were determined by electronic particle
analysis (Coulter Counter).


% cell viability
52
FIGURE 3-22 : Viability of L1210 cells as a function of growth phase. L1210 cells
were seeded at an initial concentration of 3 x 104 cells/ml and incubated for 5
days without reseeding or adding fresh medium. Cell viability was
determined with use of Trypan Blue dye exclusion method.


|j.M pellet [MGBG]
53
FIGURE 3-23 : Uptake of MGBG as a function of growth rate. Exponentially
growing L1210 cells were seeded at initial concentrations of 0.1 - 2.0 x 106
cells/ml to attain different subsequent rate of growth. In addition, cells
already in the plateau phase were seeded at 2.0 x 106 cells/ml. All cells
were exposed to fresh medium containing 0.5 pM [14C] MGBG at starting
concentrations of 1 x 10^, 3 x 10^ and 2 x 10^ cells per ml. The intracellular
content of radiolabel was determined at 0, 24, and 48 hours after the start of
incubation.


CHAPTER 4
TRANSPORT OF MGBG AND DRUG RESISTANCE
When L1210 cells were treated with MGBG at concentrations that were low
enough (< 5 pM) to allow survival of some cells, the residual cells reacquired
the control rate of proliferation within one to three weeks (Figures 3-1 and 3-2)
despite the continued presence of MGBG in the medium. As will be
documented below, this resistance seems to reflect a decreased
intracellular/extracellular concentration ratio of MGBG. This Chapter describes
the changes that account for this decrease in the intracellular accumulation of
MGBG. As discussed in Chapter 3, MGBG had accumulated in cells for the first
20 hours of exposure to drug in accord with a saturable process with a Vmax of
296 19 pmole/106 cells/hour and a Km of 4.72 0.17 pM. Our focus now
turns to subsequent 19 days of exposure.
Concentration of MGBG in L1210 Cells Purina 20 Davs of Drug Exposure
This experiment resembles that depicted in Figure 3-1 in which the effects of
varying concentrations of MGBG on L1210 cell growth were ascertained except
that the current experiment incorporated the use of [14C] MGBG to allow
intermittent measurement of radiolabel in the cell pellet. Logarithmically-
growing L1210 cells were reseeded every two days at a density of 8 x 104
cells/ml in media at 37C, pH 7.40 containing 0.1 to 5.0 pM [14C] MGBG.
MGBG, 0.1 pM, has minimal, if any, effect on growth or viability. MGBG, 5.0 pM,
is occasionally compatible with long-term cell survival. The intracellular content
54


55
of [14C] MGBG was determined intermittently (Figure 4-1). The apparent
intracellular concentration of MGBG depended both on the concentration of
drug added to the medium and on the duration of exposure. At 1.0 pM MGBG in
the medium, the intracellular concentration of drug reached 2000 pM about 24
hours after the start of incubation (as described in Chapter 3). Despite
continued presence of the drug in the medium (including reseeding the cells
into fresh drug every 48 hours), the intracellular concentration of MGBG
decreased to 500 pM by day 6 and 100 pM by day 20. The same pattern
applied to all doses of MGBG as long as they were high enough to affect cell
growth; peak intracellular concentrations occurred between 24 and 72 hours
after the start of the incubation and decreased thereafter. When the
concentration of MGBG was too low to have a detectable effect on the growth
rate or viability of L1210 cells, no such maximum was seen.
Although it is tempting to attribute, or at least relate, this decrease in
intracellular content of MGBG to the decrease in growth rate that occurs a few
days after the start of exposure to MGBG, the rate of cell proliferation returned to
the control value within two weeks of exposure to MGBG (Figure 3-1), a time
when the intracellular/extracellular ratio of MGBG is still decreasing.
Intracellular content of putrescine, spermidine and spermine in L1210 cells
was determined with use of high performance liquid chromatography (HPLC) in
our laboratory by M. Kelly (Table 4-1). By 48 hours of exposure in 0.5 pM
MGBG, the intracellular content of putrescine increased while the levels of
spermidine and spermine had decreased. This result was anticipated because
MGBG is an effective inhibitor of S-adenosylmethionine decarboxylase. By day
20, when rate of cell proliferation had recovered, the intracellular
concentrations of polyamines had also returned to control.


|xM pellet [MGBG]
56
FIGURE 4-1 : Intracellular content of MGBG upon prolonged exposure to the drug.
Logarithmically-growing L1210 cells were reseeded every two days at a
density of 8 x 104 cells/ml in media containing 0.1 to 5.0 pM [14C] MGBG.
The intracellular content of [14C] MGBG was determined periodically.


57
TABLE 4-1 :
: Effects of MGBG on the intracellular content of polyamines and
putrescine in L1210 cells.
Treatment
Intracellular content (pmole/million cells, mean ± SEMI
Putrescine Spermidine Spermine Total
Control
282 ±6 2577 ±81 612 ± 17 3483 ±120
Cells treated with 0.5 pM MGBG for 48 hours
1838 ± 49** 1334 ±39* 344 ± 35* 3574 ± 82
Cells treated with 0.5 pM MGBG for 20 days (after recovery)
244 ±27 2494 ±201 537 ± 26 3275 ± 249
Note : Intracellular polyamine and putrescine content was determined with use of high
performance liquid chromatography ; ** p < 0.01, * p < 0.05 when compared to the
control.


58
Kinetic Characterization of MGBG Transport as Resistance Develops
An experiment was designed to compare rates of influx and efflux of MGBG
before and after the peak intracellular concentration had been attained. The
cells were studied 4 hours and 156 hours (6.5 days) after the start of incubation
in 1.5 pM MGBG. The experiment was designed in this way in order to compare
transport in cells with an intracellular concentration of MGBG of about 400 pM
on the increase (4 hours) and on the decrease (156 hours). To be able to study
both influx and efflux in the same sets of cells, the L1210 cells were incubated
in either radioactive or non radioactive MGBG and crossed over at 4 hours or
156 hours. For example, to study efflux after exposure to MGBG for 156 hours,
cells were incubated in 1.5 pM [14C] MGBG for 156 hours at which time the
intracellular concentration of [14C] MGBG is about 400 pM. The cells were then
collected, washed quicky, and reseeded in fresh medium containing 1.5 pM
nonradioactive MGBG and release of label into the medium was followed.
The results depicted in Figures 4-2 and 4-3 reveal that the rate of influx of
MGBG had decreased more than two fold between 4 and 156 hours whereas
the rate of efflux remained unchanged. Within 156 hours of MGBG exposure,
the rate of influx was approximately the same as the rate of efflux (Figure 4-4),
i.e. steady-state had nearly been obtained. This observation explains the
relative stability of intracellular concentrations of MGBG thereafter.
A more detailed kinetic analysis was obtained with cells that had been
exposed to 0.5 pM MGBG for one month. After one month of such treatment, the
Km for MGBG uptake at 37C, pH 7.40 had increased from 4.7 0.2 to 24.9
1.4 pM (P < 0.01) and the Vmax had decreased from 296 19 to 142 13
pmole/106 cells/hour (P < 0.01). If the cells were incubated in 1.0 pM MGBG,
the calculated rate of influx of drug would be less than 10% of what it had been
during the first 20 hours of exposure. These changes in the kinetic properties of


pmole MGBG/0.1 million cells
59
hrs. after crossover
— Day 0 influx
*— Day 6 influx
FIGURE 4-2 : Influx of MGBG before and after the peak intracellular concentration of
drug had been attained. L1210 cells were incubated in 1.5 pM MGBG for 4 or
156 hours before centrifugation, washing, and resuspension in fresh medium
containing 1.5 pM [14C] MGBG. The intracellular content of [14C] MGBG was
determined periodically from 0 - 26 hours after crossover.


pmole MGBG/0.1 million cells
60
hrs. after crossover
a Day 0 efflux
- Day 6 efflux
FIGURE 4-3 : Efflux of MGBG before and after the peak intracellular concentration of
drug had been attained. L1210 cells were incubated in 1.5 pM [14C] MGBG
for 4 or 156 hours before centrifugation, washing, and resuspension in fresh
medium containing 1.5 pM MGBG. The intracellular content of [14C] MGBG
was determined intermittently for 0 26 hours after crossover.


(iM pellet [MGBG]
61
D6 influx
D6 efflux
D6 Total
time (hours) after crossover
Figure 4-4 : Influx, efflux and apparent steady-state intracellular content of MGBG on
day 6 of exposure to drug. L1210 cells were incubated in 1.5 pM MGBG or
[14C] MGBG for 156 hours before centrifugation, washing and resuspension
in fresh medium containing 1.5 pM [14C] MGBG or MGBG, respectively. The
intracellular (pellet) content of t14C] MGBG was determined intermittently for
0 26 hours after crossover. The "D6 Total" curve (p) represents the sum of
intracellular radioactive MGBG obtained from the influx and efflux
experiments.


62
the influx process can explain the gradual decrease in the intracellular content
of MGBG that precedes the recovery of control rates of proliferation.
In Chapter 3, data were presented concerning the relative capacities of
putrescine, spermidine and spermine to compete with MGBG for uptake. Since
the kinetic characteristics of MGBG uptake had changed upon emergence of
resistance, I explored whether or not the K¡'s for putrescine, spermidine and
spermine had changed also, in order to gain some insight into the nature of the
altered transport system. The results are presented in Figure 4-5. The Kfs for
each compound had increased in rough proportion to the increase in Km for
MGBG. This finding suggests that the apparent substrate specificity of the
transport system in resistant cells had not changed.
When these cells, namely, those exposed to 0.5 pM MGBG for one month,
were transferred to drug-free medium, they did not reacquire the capacity of
untreated cells to accumulate MGBG even after incubation in drug-free medium
for 20 days (Figure 4-6). Kinetic studies were conducted with cells that had
been grown in drug-free medium for one month (Table 4-2). By this time Vmax
had returned to control value (321 19 pmole/106 cells/hour) but Km had
remained increased (11.57 pM; P < 0.01 compared to control cells.
Selection of L1210 Cells Highly Resistant to MGBG
Another approach that I have taken to explore the mechanism of resistance
to MGBG involved the selection of L1210 cells presumably resistant on a
genetic basis. L1210 cells were incubated in 5 pM MGBG for two weeks and
then reseeded in medium containing 10 pM MGBG. A clone of cells retained
striking resistance to MGBG even when grown in drug-free medium for months.
When grown in drug-free medium, these resistant cells divided slowly, with a
doubling time nearly twice that of untreated cells (16 to 20 vs. 8 to 10 hours).
The dose-response curves for the resistant line of cells with MGBG are


1/pmole MGBG/10e6 cells/hr.
63
â–¡ control
• 25 p.M spd
â–  25 p.M spm
o 25 (iM putres
FIGURE 4-5 : Competitive uptake inhibition of MGBG uptake by spermidine,
spermine and putrescine in L1210 cells exposed MGBG for 3 weeks.
L1210 cells which were exposed to 0.5 pM MGBG for 3 weeks, were kept in
drug-free media for 1 week prior to this study. The cells were incubated in
the media containing different concentrations of [14C] MGBG (4.0 -117.5
pM) with or without 25 pM spermidine, spermine or putrescine. The Kfs of
spermidine, spermine and putrescine for inhibition of the uptake of MGBG
were 7.9 + 0.3, 12.0 + 0.4 and 20.9 + 0.7 pM, respectively.


|iM pellet [MGBG]
64
drug-free period
o— 20 days
•— 12 days
b— 3 days
—o— control
drug-naive
Time (min.)
FIGURE 4-6 : Uptake of MGBG in the " recovered " L1210 cells. L1210 cells that
had been exposed to 0.5 pM MGBG for one month, were transferred to
drug-free medium for 3 - 20 days. These cells were subsequently reseeded
in a medium containing [14C] MGBG (20 pM). and the uptake of drug was
followed for 75 minutes.


65
TABLE 4-2 : Kinetics of the uptake of MGBG in untreated L1210 cells, cells that
had "recovered" after exposure to MGBG, and a resistant subclone.
Cell Type and Treatment
Km (pM)
(mean SEM)
Vmax (pmole/106 cells/hr.
(mean SEM)
Control
4.72 0.17
296 19
Cells exposed to 0.5 pM
MGBG for 30 days and then
- 2 days drug-free
24.87 1.36*
142 13*
-1 month drug-free
11.57 1.63*
321 19
MGBG-resistant subclone
- 3 days drug-free
16.18 0.52*
134 15*
- 2 month drug-free
5.14 0.48
145 14*
Note : p < 0.01 (vs control)


66
presented in Figure 4-7 (growth) and Figure 4-8 (viability). Logarithmically-
growing resistant cells were seeded at a starting concentration of 1.05 x 105
cells/ml in media containing 0 to 30 pM MGBG. The cells were reseeded into
fresh medium containing MGBG 2 days later. In comparison to the curves
depicted in Figures 3-1 and 3-2 with untreated L1210 cells, these cells exhibit
striking resistance to MGBG. Even after growth in drug-free medium for six
weeks, resistance to MGBG was retained (Figures. 4-9 and 4-10).
Transport Properties of the Resistant Cells
When these resistant cells were exposed to 2.0 to 6.7 pM [14C] MGBG at
37C, pH 7.40, they accumulated significantly less MGBG than did control
L1210 cells (Figure 4-11). This decreased accumulation correlated with an
increased Km (16.2 0.5 compared to 4.7 0.2 pM; P < 0.01) and a decreased
Vmax (134 15 compared to 296 19 pmole/106 cells/hour; P < 0.01). After six-
weeks of growth in drug-free medium, Vmax remained low (145 14 pmole/106
cells/hour), the cell doubling time remained long, but Km had returned to the
control value (5.1 0.4 pM).
Once again, the relative affinities toward putrescine, spermidine and
spermine of the transport system in resistant cells, as assessed by K¡'s for
inhibition of MGBG uptake, remained unchanged (Figure 4-12).
Response of multiple drug resistant (MDFO cells to MGBG
Two Chinese hamster ovary (CHO) cell lines were generously provided by
Dr. Gurmit Singh (McMaster University, Ontario, Canada). The parental line
(Aux Bi) is drug sensitive. The derived line (CHRC5) exhibits multiple drug
resistant and contains P-glycoprotein. The selection of CHRC5 is described by
Ling and Thompson in1974. Briefly, cross-resistance to a variety of antitumor
antibiotics in these CHO cell line was induced by exposure to increasing
concentrations of colchicine. This pleotropic or multiple drug, resistance (MDR)


cumulative cell count
67
Time (hrs.)
FIGURE 4-7 : Effects of various concentrations of MGBG on growth of MGBG-
resistant L1210 cells. Logarithmically-growing resistant cells (see text)
were transferee! to drug-free medium for 3 days prior to the study. The
cells were reseeded at a starting concentration of 1.05 x 105 cells/ml in
media containing 0 - 30 pM MGBG. The cells were maintained in
logarithmic growth by reseeding into fresh medium containing MGBG at 2
days.


% cell viability
68
Time (hrs.)
MGBG (p.M)
—□— none
—* 1-0
—a— 10.0
—o 20.0
—m— 30.0
FIGURE 4-8 : Effects of various concentrations of MGBG on viability of MGBG-
resistant L1210 cells. See Figure 4-7: viability was assessed by the
Trypan Blue dye exclusion method.


cumulative cell count
69
Time (days)
FIGURE 4-9 : Effects of various concentrations of MGBG on growth of the MGBG-
resistant L1210 cells after one month of growth in drug-free medium.
MGBG-resistant cells (see text) were maintained in drug-free medium for
one month prior to the study. Cells in logarithmic growth were seeded in
media containing 0 50 (iM MGBG. Cell counts were determined daily
with use of a Coulter Counter and the cells were reseeded into fresh
media with or without drug at 2 days.


% cell viability
70
Time (days)
FIGURE 4-10 : Effects of various concentrations of MGBG on viability of the
MGBG-resistant L1210 cells after one month of drug-free period. Cell
viability was determined daily with use of the Trypan Blue dye exclusion
method (see Figure 4-9).


|iM pellet [MGBG]
71
Time (hrs.)
FIGURE 4-11 : Accumulation of MGBG in the MGBG-resistant L1210 cells. The
resistant cells (see text) were maintained in a drug-free medium for 3
weeks prior to the study. The logarithmically-growing cells were seeded in
various concentrations of [14C] MGBG for 0 48 hours and the intracellular
content of [14C] MGBG was determined at the times indicated. For
comparison, a previously untreated L1210 cell exposed to 1 pM MGBG
would be expected to exhibit an intracellular concentration of MGBG of
about 2000 pM by 24 hours.


1/pmole MGBG/10e6 cells/hr.
72
Q control
25 |iM spd
25 |iM spm
25 (iM putres
FIGURE 4-12 : Competitive inhibition of MGBG uptake by spermidine, spermine
and putrescine in MGBG-resistant L1210 cells. MGBG-resistant L1210 cells
were kept in drug-free medium for 1 week prior to this study. The cells were
incubated in the media containing different concentrations of [14C] MGBG
(4.0 117.5 pM) with or without 25 pM spermidine, spermine or putrescine.
The K¡'s of spermidine, spermine and putrescine for inhibition of the uptake
of MGBG were 2.68 0.09, 2.51 0.08 and 9.33 + 0.10 pM, respectively.


73
was associated with the presence of a 170 KDa membrane glycoprotein
(Juliano and Ling, 1976). This protein is termed the P-glyco prote in because of
its association with the plasma membrane. It behaves as an efflux pump and
hence, decreases intracellular accumulation of antineoplastic agents in
resistant cells. Both CHO cell lines were grown in alpha-modified essential
medium (alpha-MEM) with L-glutamine, ribonucleosides and
deoxyribonucleosides (GIBCO Laboratory). The medium was supplemented
with 10% fetal bovine serum (FBS), streptomycin and penicillin. The cells were
grown in Petri dishes and incubated at 37°C in 5% CO2 : 95% air. The sensitive
and MDR CHO cell lines were exposed to different concentrations of MGBG (0 -
10 |iM) for 24 to 72 hours. The initial cell number plated was such that the
control cells would not reach confluence before day 3, when the inhibitory effect
of the drug was evident. After 46 or 72 hours of MGBG exposure, the medium
was removed and the cells were trypsinized. Trypsinization was stopped by
adding two volumes of medium containing 10% FBS. The dishes were gently
shaken to dislodge the cells and repipetted to break up any clumps before
counting the cells by a Coulter Counter. Cell viability was determined with use
of trypan blue dye exclusion method.
Due to the fact that the doubling time of the Aux B1 cells is shorter than the
CHrC5 cells, the sensitivities to MGBG of these two cell lines were compared
after exposure for approximately 3 doubling times (46 and 72 hours for Aux B1
and CHrC5 cells, respectively). The results depicted in Figure 4-13 reveal that
there is no difference in the sensitivity of the CHRCs and the Aux B1 to the
antiproliferative activity of MGBG. This finding suggests that different
mechanisms of resistance exist in the MDR and the MGBG-resistant cells.
Correlation between Transport and Resistance
It is important to ask whether or not "all" of the resistance to MGBG that I


% control growth
74
- Aux B1 day2
* CHR C5 day3
MGBG (|iM)
FIGURE 4-13: Comparison of MGBG dose-response curve of parental drug-
sensitive (Aux B1) and multidrug resistant (CHRC5) cell lines. The two CHO
cell lines were exposed to different concentrations of MGBG (0-10 pM) for
0 3 days (2 days for Aux Bi cell line and 3 days for CHRC5 cell line). Cell
counts were determined by electronic particle analysis (Coulter Counter)
and cell viability was determined with use of the Trypan Blue exclusion
method. The results are expressed as percent control growth (no drug).


75
observed in various experiments, can be attributed to changes in transport of
MGBG (i.e., in the accumulation of drug within cells). I have attempted to
approach this question by comparing untreated cells, "adapted" cells (0.5 pM
MGBG for one month) and selected cells (as defined above) with regard to
intracellular concentration of MGBG and rates of growth. There was a linear
relationship (r = 0.91) between intracellular concentration of MGBG at 24 hours
and growth rate over the following day, regardless of the type of cell involved
(Figure 4-14). in other words, the data from all cells fit the same line. Growth
rates between 0 and 48 hours have also been analyzed with similar results.
These results indicate that virtually all of the resistance that develops can be
explained by changes in transport and not by some intrinsic change in
sensitivity to MGBG within cell.


% control growth (24-48 hrs.)
76
100
80
60
40
20
0
|iM pellet [MGBG] by 24 hrs.
L1210 cells
control
o 0.5 pM exp
h selected resist
FIGURE 4-14 : Correlation between intracellular content of MGBG and the rate of
cell proliferation in various type of L1210 cells. Untreated L1210 cells,
L1210 cells that "recovered" after exposure to 0.5 pM MGBG, and MGBG-
resistant subclone (see text), were exposed to various concentrations of
[14C] MGBG for 0 48 hours. The intracellular content of [14C] MGBG was
determined at 24 hours. Cell counts were determined at 0, 1 and 2 days
with use of a hemacytometer, and cell viability was determined by Trypan
Blue dye exclusion method.


CHAPTER 5
DISCUSSION
Discussion will focus on: 1) the mechanism of MGBG transport, and 2)
resistance of L1210 cells to MGBG.
Mechanism of MGBG Transport
As reported earlier (Field £ia!., 1964, Dave and Caballes, 1973, and Porter
1981), MGBG was found to accumulate extensively in the cell pellet. The
extensive accumulation of MGBG we observed, was not due to surface binding
since the radiolabelled MGBG in the cell pellet could not be displaced by 1 mM
MGBG. The fact that nongrowing L1210 cells do not accumulate MGBG also
makes significant adsorption of MGBG to the cell surface most improbable as an
important quantitative factor in accumulation. All of our experiments suggested
that the major gradient in MGBG concentration was across the plasma
membrane. Accumulation of MGBG inside the cells was time-dependent, and
the rate of accumulation was linear with time for more than 20 hours.
Interestingly, accumulation of the drug over 20 hours could be predicted by
kinetic constants from 20-minute experiment (Table 3-1).
Reported effects of MGBG on mitochondrial structure and bioenergetic
functions were described in Chapter 1. Even though mitochondria seem to be
an important site of MGBG's action, studies with use of isolated mitochondria
and digitonin revealed that mitochondria were not the driving force for extensive
77


78
accumulation of MGBG inside the cells. In other words, there was no
suggestion of an appreciable gradient of MGBG between cytosol and
mitochondrial compartments. Most of the intracellular drug was likely to be in
the cytosol. The actual free or unbound concentration of MGBG in the cytosol or
mitochondria remains unknown. MGBG is a weak base with pKa's of about 7.5
and 9.2 at 25 C (Wiliams-Ashman and Seidenfeld, 1986). At physiological
pH, MGBG exists primarily as a mixture of monovalent and divalent cations, and
a large number and wide variety of binding sites likely exist intracellularly. As
noted above, the accumulation of MGBG was linear for at least 20 hours; this
result suggests the binding of MGBG to intracellular binding sites. Our
experiments with digitonin to permeabilize the cell membrane, however,
revealed an immediate release of the labelled drug with cytosolic components,
a result which suggests that MGBG was loosely bound to the intracellular
binding sites at least those associated with membranes or organells. Efflux of
MGBG was close to first order (Figure 3-8), another finding which implied that
most of the intracellular MGBG was either loosely bound or dissociated from
binding sites at an appreciable rate. In addition, because MGBG is relatively
poorly protonated at physiological pH compared to the natural polyamines,
MGBG would not likely bind as tightly as spermidine or spermine to anionic
polynucleotides and phospholipids or other intracellular molecules. The
extremely low abundance of S-adenosylmethionine decarboxylase
(AdoMetDC), diamine oxidase and spermidine/spermine N'-acetyltransferase
which are high affinity binding sites for MGBG, would preclude these enzyme
from binding more than a negligible fraction of the MGBG that is found within the
cells (Williams-Ashman and Seidenfeld et a!., 1986).
As mentioned earlier, MGBG is positively charged under physiological
conditions. It is unlikely that MGBG enters the cells by simple diffusion. Indeed,


79
the influx of MGBG exhibits saturation kinetics with apparent Km's of 2.10 + 0.08
and 9.34 + 1.04 pM, for the sodium-dependent and total processes,
respectively. Thus, it seems that the uptake of MGBG involves a carrier with
reasonably high affinity for MGBG. This carrier is likely also to be involved in
the transport of the naturally occurring polyamines, spermidine and spermine,
as well as putrescine, in that each of these compounds competed with MGBG
for uptake. This competition had been recognized previously (Dave and
Caballes, 1973, Janne §t aL, 1978, Porter el al., 1981, Williams-Ashman and
Pegg, 1981, and Heby, 1981). Studies of countertransport also support the
notion of a carrier molecule. Interpretation of this experiment, however, is
complex because of the existence of naturally occurring polyamines within the
cells. The rates of uptake of MGBG were strongly influenced by pH (Figure 3-6).
An increase in extracellular hydrogen ion concentration enhanced the rate of
uptake of MGBG. These results suggested that the divalent form of MGBG
(MGBG2+) was the actual substrate for transport into the cells. This hypothesis
was supported further by the good agreement between the observed rate of
uptake and a rate predicted using values of MGBG2+ concentration based on
Henderson-Hasselbach computations (Table 3-2).
Even though normal and malignant cells in active growth could accumulate
MGBG (Field el al., 1964, Kramer el ai-, 1985), an intracellular concentration
2000 fold higher than that in the medium concentration was observed in our
studies (Figures 3-4 and 4-1). This result does not distinguish active transport
from facilitated diffusion coupled with intracellular binding. To pursue that
issue, studies were conducted to determine whether energy was required for
the accumulation of MGBG. The uptake of MGBG at low temperature (0 4C)
was only about 10% of that of uptake at 37C. Preincubation of cells with
inhibitors of energy metabolism (antimycin A, KCN and 2,4-DNP) decreased


80
and 2,4-DNP) decreased intracellular accumulation of MGBG. These results
would be anticipated if energy was required for the accumulation of the drug. In
addition, efflux of MGBG was also affected by temperature. At 0 4 C, the
efflux of MGBG was barely detectable. It is often difficult to define the energy
requirement for active transport because of the potential nonspecific effects of
inhibitors of ATP synthesis.
The transport of sugars, amino acids, and numerous other solutes in many
cell types is observed to be driven against a chemical potential gradient by
cotransport with sodium ion down its electrochemical potential gradient. The
free energy dissipated by sodium ion movement down this gradient is coupled
with the increase in free energy associated with concentration of the substrate
within the cell. In various systems of this type, the carrier is proposed to be a
two-site molecule; one site accommodating sodium ion and the other
simultaneously accomodating the transported substrate (Schafer and Barfuss,
1986). We found that transport of MGBG in L1210 cells was sodium-dependent.
The sodium-dependent uptake process was consistent with a single transport
system in that the Lineweaver-Burk plot for sodium-dependent uptake was
linear. The dependency of MGBG uptake on sodium was rather specific
because the uptake of MGBG decreased when sodium was replaced by
choline, lithium, or especially, potassium or cesium. Even though there was a
decrease in the uptake of MGBG when sodium was replaced by choline, there
was a substantial amount of the intracellular accumulation of the drug. A
comparison of kinetics in these circumstances revealed that the presence of
sodium ion did not affect Vmax but did decrease Km or increase affinity toward
MGBG. The effect of sodium was therefore particularly important at low (pM)
concentrations of MGBG.
If the sodium electrochemical gradient is involved in MGBG transport,


81
dissipation of this gradient with specific antibiotic ionophores should inhibit
uptake of MGBG. The results summarized in Table 3-5 suggested that this was
indeed the case. The ion selectivity of ionophores is a combined function of the
energy required for desolution of the ion and the liganding energy obtained on
complexation (Simon and Morf, 1973 and Eisenman el a!., 1968). Valinomycin
which exhibits 10,000 : 1 preference for K+ (radius, r = 1.33 A) over Na+ (r =
0.95 A) in both biological and model system (Moore and Pressman, 1964,
Pressman, 1965, 1968, 1976), has no effect on the intracellular accumulation of
MGBG in L1210 cells. Monensin, which is more specific for Na+ than K+,
exhibited a moderate inhibition of MGBG accumulation whereas Gramicidin A, a
channel-forming ionophore which does not discriminate between Na+ and K+,
was the most potent inhibitor of MGBG uptake. The inhibitory effect of monensin
and gramicidin A implied the requirement of an electrochemical potential
gradient and the possible co-transport of Na+ with MGBG to enter the cells.
Although there was no direct evidence to support the cotransport of sodium ion
with MGBG in our studies, the fact that MGBG accumulated more than 2000-fold
within the cell, and the inhibitory effects of sodium ionophores, were strongly
suggestive. A23187 also caused a decrease in intracellular MGBG
accumulation. This compound is a carboxylic ionophore which possesses a
high selectivity for divalent over monovalent ions (Pfeiffer el a)., 1974), allows
Ca++ to enter cells (Reed and Lardy, 1972, Pressman, 1973, Pressman and
deGuzman, 1974,1975) and is capable of stimulating various Ca++-dependent
reactions without disturbing preexisting balances of Na+ and K+. The inhibitory
effect of A23187 on MGBG uptake might be due to the biological effects of high
intracellular level of Ca++. A23187 also transports Mg++, but gradients of this
ion across biological membranes seldom participate in biological control
(Pressman, 1976). Interpretation of the specific effects of ionophores is very


82
complex.
Resistance of L1210 Cells to MGBG
Studies in drug resistant tumor cell lines have identified many different
mechanisms for the development of resistance. In cell culture studies, three
major mechanisms of genetic resistance have been delineated and
characterized. These mechanisms include overproduction of the drug target
(e.g., gene amplification, Schimke el al, 1983, Rice el al., 1987, Somfai-Relle et
ah, 1984, and Jones, 1984), reduced drug permeability ( Mandel and Flintoff,
1978, Sirca et ai-, 1987, Rodrigues eta}.,1987, Waud, 1987, and Kraker and
Moore, 1988 ) and altered target interaction of the drug (Herman et a}., 1979,
Schabel el 2l-> 1982, Seebergl aL, 1982, Hunt el ai-. 1983, and Horns el ai-,
1983). Murine leukemia L1210 cells in our studies acquired a drug resistant
phenotype to MGBG during exposure to sublethal concentrations of drug. Our
data suggest that a decrease in drug uptake was responsible for this resistance.
We found that the influx rate of MGBG in L1210 cells started to decrease
significantly within a few days of exposure whereas the rate of efflux remained
unchanged. A separate subclone of genetically stable MGBG-resistant L1210
cells also exhibited a deficiency in drug transport which was characterized by a
significant decrease in Vmax and an increase in Km for MGBG transport. In both
"adapted" cells and the MGBG-resistant subclone, as well as in previously
untreated cells, an apparent intracellular concentration of MGBG less than 400
pM was required for toxicity. A similar quantitative relationship between the
intracellular concentration of MGBG 24 hours after exposure and the growth
rate over the following day was observed for all types of L1210 cells. It is
therefore unlikely that an intracellular response to a given concentration of
MGBG plays a significant role in the development of resistance. MGBG had
been reported to be metabolically inert (Warrel and Burchenal, 1983), and we


83
have confirmed that finding.
Two sites of MGBG action have received most attention with regard to its
antiproliferative activity: mitochondria and polyamine metabolism (inhibition of
AdoMetDC). Of these, as mentioned earlier, it appears that drug effects related
to mitochondrial integrity correlate best with the antiproliferative action of MGBG
in various cell lines (Pleshkewych et a|., 1980 Mikles-Robertson et a|., 1979
and 1981, Corti £la|., 1974, Pathak ei£-, 1977, Wiseman gl ah, 1980 and 1983,
and Porter el al., 1979). In general, drug effects on parameters related to
polyamine metabolism fail to correlate with the antiproliferative action of MGBG.
In addition, there is no simple correlation between direct inhibition of AdoMetDC
and antileukemic activity in vivo for a series of MGBG analogs (Mihich, 1975,
Corti nial-, 1974, and Porter el a!-. 1981). Prolonged exposure to MGBG
actually increased activity of AdoMetDC. This might be related to an increase in
the enzyme synthesis (Pegg alai-. 1973, Pegg, 1984, and Fillingame and
Morris, 1973) or its stabilization (Williams-Ashman and Seidenfield, 1986).
I explored the relationship between multiple drug (MDR) resistance and
resistance to MGBG. Studies of Chinese hamster ovary (CHO) cells that were
resistant to the drug, colchicine (Ling and Thompson, 1974 and Ling, 1975),
revealed a specific alteration of the plasma membrane in multidrug resistance.
Drug-resistant cells contained a unique glycoprotein that was absent in the
drug-sensitive cells (Juliano and Ling, 1976). This glycoprotein was large in
size (molecular weight, about 170,000) and was named P-glycoprotein for its
association with the apparent permeability barrier at the plasma membrane to
drugs that were included in multidrug resistance. Further evidence suggested
that P-glycoprotein functions as a drug efflux pump (Gros el ai-, 1986 and Chen
£ia!-. 1986). Interestingly, resistance of MDR cells to colchicine, adriamycin
and other agents can be reversed by exposure to calcium channel-blocking


84
agents, a procedure which also reverses the defect in intracellular drug
accumulation (Tsuruo giaL, 1982, Rogan gt al, 1984, and Willingham elai-.
1986). Due to the fact that the efflux rate of MGBG in L1210 cells in our studies
remained unchanged after adaptation to MGBG, it is unlikely that resistance to
MGBG would be an MDR-type resistance. When parental and MDR CHO cell
lines were exposed to different concentrations of MGBG, both cell lines were
equally sensitive to the antiproliferative activity of the drug.
Our results were discussed earlier in terms of "adaptation" for the L1210
cells which had recovered within two weeks after exposure to 0.3 - 5 pM MGBG.
Several lines of evidence from our studies suggest that this resistance to MGBG
was due to adaptation of the cells to the drug, not selection of preexisting
mutant. In recent years, it has become increasingly evident that neoplasms
contain complex subpopulations of cells, each of which differs in expression of
biological properties. A concept now receiving increased attention is that drug
resistant neoplastic stem cells often arise from mutations, and that the drug-
resistant phenotype is inherited and propagated. The concept of the
development by spontaneous mutation of cancer cells that have resistance to
drugs they not yet encountered is called Goldie-Coldman hypothesis (Goldie
and Coldman, 1979). According to the Goldie-Coldman model, rates of
mutation give rise to resistance to a variety of antineoplastic agents in a variety
of mammalian tumor cells at a prevalence of 1 per 104 to 107 cells. The fact that
MGBG uptake by cells "adapted" to the presence of 0.5 pM had not returned to
the control transport rate even after removal of cells from drug for six weeks
raised the possibility that we had selected a mutant cells rather than caused an
"adaptation" of most cells. If we assume that only mutant cells can divide during
the course of MGBG exposure, the Y-intercept obtained from an extrapolation of
the MGBG dose-response curve of L1210 cells would reveal the frequency of


85
preexisting MGBG-resistant cells in overall cell population. At 0.5 pM MGBG,
this frequency was observed to be 1 in 104 cells (Figure 3-1), a value within the
estimated range denoted above. However, clonogenic experiments (Figure 3-
3) revealed that a large fraction of cells (> 60 %) was clonogenic even 72 hours
after exposure to 0.5 pM MGBG. In addition, the intracellular content of MGBG
had decreased significantly within a few days of initial exposure to the drug.
These results suggest strongly that most cells were adapting to the presence of
MGBG as opposed to, or at least in addition to, the selection of preexisting
mutants of L1210 cells that were constitutively deficient in MGBG transport. In
other words, the concept of preexisting mutant cells is unable by itself to explain
the sudden and significant decrease in the intracellular content of MGBG
within a few days after exposure to the drug. The importance of adaptive
regulation of transport is also supported by the kinetic results obtained midway
through the change in transport (after 6 days of incubation with drug). Had two
types of cells been present at this time (one with a Km of 4.72 pM and the other
with a Km of 24.87 pM for MGBG influx), I would have anticipated a nonlinear
Lineweaver-Burk plot; no hint of nonlinearlity was observed. Furthermore, a
comparison of the cells "adapted" to MGBG and those selected for resistance to
MGBG suggests that both types of cells exhibit an adaptive increase in a Km,
but a decrease in Vmax is characteristic only of the genetically stable subclone
and not of the "adapted" cells.


CHAPTER 6
SUMMARY AND FUTURE DIRECTIONS
Summary
We have studied the effect of MGBG on cell proliferation in murine leukemia
L1210 cells. Because these L1210 cells developed resistance to MGBG during
exposure, our experiments were designed to define mechanism of resistance to
MGBG in these cells and also in a clone of L1210 cells selected for resistance
to a lethal concentration of MGBG.
The important role of transport in the development of resistance to MGBG,
was first implied by a study of the apparent intracellular concentration of MGBG
for 12 days after exposure of previously untreated L1210 cells to drug. We
confirmed that MGBG accumulated quite extensively in these cells. The
apparent intracellular concentration of MGBG depended on both the
concentration of drug added to the medium and the duration of exposure. At 1
pM MGBG in the medium, the intracelllular concentration of drug reached
approximately 2000 pM about 24 hours after the addition of MGBG. Despite
continued presence of the drug in the medium, the intracellular concentration of
MGBG decreased to approximately 500 pM by day 6 and further to 100 400
pM thereafter. The same pattern applied to all doses of MGBG as long as they
were high enough to affect cell growth; peak intracellular concentrations
occurred 24 48 hours after the start of incubation and invariably decreased by
72 hours. Decrease in the intracellular accumulation of MGBG was due to a
86


87
decrease in the influx, not an increase in the efflux of MGBG. The rate of cell
proliferation returned to the control value within two weeks of exposure, a time
when the intracellular concentration of MGBG was < 400 pM. The dose-
response (cell proliferation) curve of these L1210 cells was shifted to the right
more than 10 fold. The lethal concentration of MGBG increased from 5 to about
50 pM in these "adapted" L1210 cells. Similar results were obtained in studies
with a subclone of genetically stable MGBG-resistant L1210 cells. Higher
extracellular concentrations of MGBG were required for the L1210 resistant
subclone to achieve a given intracellular concentration of MGBG. "Adapted"
L1210 cells (0.5 pM MGBG for 1 month) and MGBG-resistant subclone, as well
as untreated L1210 cells, were compared with regard to intracellular
concentration of MGBG and rate of cell growth. There was a linear relationship
between intracellular concentration of MGBG at 24 hours and rate of cell
proliferation over the following day, regardless of the type of cell involved. At
apparent intracellular concentration in excess of 400 pM, the growth rate of all
cells decreased.
Indirect methods were used to determine the intracellular distribution of
MGBG in L1210 cells. Mitochondria were unlikely to be the driving force for an
extensive intracellular accumulation of MGBG even though the effect of MGBG
was specific for mitochondria.
Transport mechanism of MGBG was also examined. The transport of MGBG
seemed to be energy dependent and carrier-mediated. It exhibited saturable
kinetics with a Vmax and Km of 296 19 pmole/106 cells/hour and 4.72 + 0.169
pM, respectively. The rate of transport depended on temperature, pH and rate
of cell proliferation. Inhibitors of energy metabolism, ionophores, and the
replacement of sodium ion by other cations, all significantly decreased the rate
of accumulation of MGBG. The experiments with ionophores suggested that


88
uptake of MGBG depended on a plasma membrane electrochemical gradient.
The facilitation of uptake by sodium ions was compatible with a process in
which sodium increased the affinity of the carrier for MGBG, but two separate
processes, sodium-dependent and sodium-independent remain possible.
Resistance to MGBG was not associated with MDR-type resistance because
the sensitivity of the parental and MDR (containing P-glycoprotein) CHO cell
lines to MGBG, was indistinguishable.
Future Directions
Since transport of MGBG is the major determinant of sensitivity, and since
the MGBG gradient occurs at the plasma membrane, future directions focus use
of membrane vesicle technique to further characterize the transport of MGBG.
There are several interesting analogies with other known transport system, e.g.
for amino acid ( Rinehart and Chen, 1984 and Karl et ai., 1989) and/or some
organic cations which was resently reported by Sobol (1989), that should be
explored. It would also be of interest to examine the sodium dependency of
MGBG transport in the two types of resistant cells, "adapted" and MGBG-
resistant subclones. Understanding of the mechanism of regulation of the
MGBG transporter may lead to a discovery of new strategies for cancer
chemotherapy, either with MGBG or with novel analogs.


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89


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PAGE 107

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


THE TRANSPORT OF METHYLGLYOXAL BIS(GUANYLHYDRAZONE)
AND ITS ROLE IN THE DEVELOPMENT OF RESISTANCE
IN MURINE LEUKEMIA L1210 CELLS
BY
SUKANYA KANTHAWATANA, M.D.
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
1989

ACKNOWLEDGMENTS
I would like to express my appreciation to my advisor and supervisory
committee chairman, Dr. Allen H. Neims, for the guidance and encouragement
he provided me on this project and would like to extend my appreciation to the
fellow member of our laboratory: Rita Bortell, Lynn Raynor, Daniel Danso, Mary
Ann Kelly and Fan Xie. I am also grateful to the other members of my
supervisory committee, Dr. Raymond J. Bergeron, Dr. Lai C. Garg, Dr. Michael
S. Kilberg, Dr. Edwin M. Meyer, Dr. Thomas C. Rowe and Dr. Bruce R. Stevens,
for their committment.
This dissertation is dedicated to my parents, Mr. Tongsuk and Mrs. Tongkam
Kanthawatana.

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT ix
CHAPTERS
1 INTRODUCTION 1
Specific Aims 1
Background 1
2 MATERIALS AND GENERAL METHODS 8
Cell Culture 8
Cell Counts 8
Cell Viability 9
Clonogenic Assay 9
Transport of MGBG 9
Stability of MGBG 11
Protein Assay 12
Statistical Analysis 12
3 TRANSPORT OF MGBG AND ITS EFFECT ON SENSITIVE
L1210 CELLS 13
Effects of MGBG on Growth, Viability and Clonogenicity 13
Characterization of the Initial Transport of MGBG 14
Intracellular Accumulation of MGBG During the First
20 Hours of Exposure 14
Kinetic Characterization of the Initial Transport Process 18
Inhibition of MGBG Uptake by Spermidine, Spermine
and Putrescine 22
Effect of Extracellular pH on the Initial Uptake of MGBG 22
i i i

Efflux of MGBG 23
Countertransport of Unlabelled MGBG 23
Effect of Various Cations on the Uptake of MGBG 29
Sodium-dependency of MGBG uptake 29
Ability of various cations to substitute for sodium in
uptake of MGBG 32
Effects of lonophores on the Uptake of MGBG 38
Effect of Inhibitors of Energy Metabolism on the Uptake
of MGBG 38
Intracellular Distribution of MGBG 45
Uptake of MGBG by Isolated Mitochondria 45
Selective Release of Cytosolic Constituents by Digitonin 46
Effect of Growth Rate on MGBG Transport 47
4 TRANSPORT OF MGBG AND DRUG RESISTANCE 54
Concentration of MGBG in L1210 Cells During 20 Days of
Drug Exposure 54
Kinetic Characterization of MGBG Transport as
Resistance Develops 58
Selection of L1210 Cells Highly Resistant to MGBG 62
Transport Properties of the Resistant Cells 66
Response of Multiple Drug Resistant (MDR) Cells to MGBG 66
Correlation between Transport and Resistance 73
5 DISCUSSION 77
6 SUMMARY AND FUTURE DIRECTIONS 86
REFERENCES 89
BIOGRAPHICAL SKETCH 94
IV

LIST OF TABLES
Table gage
3-1 Comparison between the calculated and observed
intracellular concentrations of MGBG during the first
20 hours of exposure 21
3-2 Comparison between the observed rates of MGBG uptake
at different extracellular pH's with rates calculated on
the basis of transport of MGBG 2 + only 26
3-3 Effects of various ions on MGBG uptake in L1210 cells 40
3-4 Effects of potassium and choline chloride on the sodium-
dependent MGBG uptake in L1210 cells 41
3-5 Effects of ionophores on MGBG uptake in L1210 cells 42
3-6 Uptake of MGBG by isolated mitochondria 48
4-1 Effects of MGBG on the intracellular content of polyamines
and putrescine in L1210 cells 57
4-2 Kinetics of the uptake of MGBG in untreated L1210 cells,
cells that had "recovered" after exposure to MGBG,
and a resistant subclone 65
v

LIST OF FIGURES
Figure page
1-1 Structure cf polyamines, related compounds and
inhibitors of polyamine synthesis 7
3-1 Effects of various concentrations of MGBG on the
proliferation of murine L1210 cells 15
3-2 Effects of various concentrations of MGBG on the viability
of murine L1210 cells overtime 16
3-3 Effect of MGBG on the clonogenicity of L1210 cells 17
3-4 Accumulation of MGBG by L1210 cells during the first
20 hours of exposure 19
3-5 Lineweaver-Burk plot of MGBG uptake 20
3-6 Competitive inhibition of MGBG uptake by spermidine,
spermine and putrescine in control L1210 cells 24
3-7 Effects of medium pH on the initial uptake of MGBG 25
3-8 Efflux of MGBG 27
3-9 Effect of temperature on the efflux of MGBG 28
3-10 Effect of MGBG preloading on the uptake of labelled MGBG.. 30
3-11 Effect of medium concentration of MGBG on the efflux
of labelled MGBG 31
3-12 Sodium-dependency of MGBG uptake in L1210 cells 33
3-13 Relationship between MGBG concentration and the rate of
MGBG uptake in the presence or absence of sodium 34
3-14 Lineweaver-Burk plot of the sodium-dependent MGBG
uptake in L1210 cells 35
3-15 The relationship between MGBG concentration and the rate
v i

of MGBG uptake in sodium chloride, choline chloride,
and mannitol 36
3-16 Lineweaver-Burk plot of MGBG uptake in sodium chloride,
choline chloride and mannitol 37
3-17 Effects of various ions on MGBG uptake in L1210 cells 39
3-18 Effects of inhibitors of energy metabolism on MGBG uptake.... 43
3-19 Effects of inhibitors of energy metabolism on MGBG uptake.... 44
3-20 MGBG remaining in the cell pellet after permeabilization of
the plasma membrane by digitonin 49
3-21 Growth curve of L1210 cells 51
3-22 Viability of L1210 cells as a function of growth phase 52
3-23 Uptake of MGBG as a function of growth rate 53
4-1 Intracellular content of MGBG upon prolonged exposure to
the drug 56
4-2 Influx of MGBG before and after the peak intracellular
concentration of drug had been attained 59
4-3 Efflux of MGBG before and after the peak intracellular
concentration of drug had been attained 60
4-4 Influx, efflux and apparent steady-state intracellular content
of MGBG on day 6 of exposure to drug 61
4-5 Competitive inhibition of MGBG uptake by spermidine,
spermine and putrescine in L1210 cells exposed to MGBG
for 3 weeks 63
4-6 Uptake of MGBG in the "recovered" L1210 cells 64
4-7 Effects of various concentrations of MGBG on growth of
MGBG-resistant L1210 cells 67
4-8 Effects of various concentrations of MGBG on viability of
MGBG-resistant L1210 cells 68
4-9 Effects of various concentrations of MGBG on growth of
MGBG-resistant L1210 cells after one month of
drug-free period 69
4-10 Effects of various concentrations of MGBG on viability of
v i i

MGBG-resistant L1210 cells after one month of drug-free
period 70
4-11 Accumulation of MGBG in the MGBG-resistant L1210 cells 71
4-12 Competitive inhibition of MGBG uptake by spermidine, spermine
and putrescine in MGBG-resistant L1210 cells 72
4-13 Comparison of MGBG dose-response curve of parental
drug-sensitive (AuxBi) and multidrug-resistant (CHRC5)
CHO cell lines 74
4-14 Correlation between intracellular content of MGBG and the
rate of cell proliferation in various type of L1210 cells 76
v i i i

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
THE TRANSPORT OF METHYLGLYOXAL BIS(GUANYLHYDRAZONE)
AND ITS ROLE IN THE DEVELOPMENT OF RESISTANCE
IN MURINE LEUKEMIA L1210 CELLS
By
Sukanya Kanthawatana, M.D.
December 1989
Chairman: Allen H. Neims, M.D., Ph.D.
Major Department : Pharmacology and Therapeutics
When murine leukemia L1210 cells were exposed to methylglyoxal
bisfguanylhydrazone] (MGBG), drug concentrations less than 0.3 pM had no
effect on cell proliferation, and concentrations greater than 5 pM were lethal
except in subclones of cells selected for resistance. Intermediate
concentrations decreased the rate of cell growth temporarily. L1210 cells
accumulated MGBG avidly, and this accumulation was crucial for cytotoxicity
and the development of resistance. My studies focused on the transport
process, and most were conducted with 0.3 - 5 pM MGBG. With 1 pM MGBG in
the medium, the apparent intracellular concentration of MGBG reached
approximately 2000 pM in 24 hours. Transport of MGBG seemed to be energy
dependent and carrier-mediated with saturable kinetics characterized by a Vmax
and Km in the presence of sodium, of 296 + 19 pmole/106 cells/hour and 4.72 +

0.169 pM, respectively. The rate of transport depended on temperature,
extracellular pH and cell proliferation. Inhibitors of energy metabolism,
ionophores which dissipate the plasma membrane electrochemical gradient,
and the replacement of sodium ions by other cations, all significantly decreased
the rate of accumulation of MGBG. Naturally-occurring polyamines, spermidine
and spermine, and putrescine competed with MGBG for uptake.
Within 48 hours of exposure to 1 pM MGBG, the rate of growth of L1210 cells
had decreased substantially. Despite the continued presence of MGBG in the
medium, the intracellular concentration of MGBG decreased to approximately
500 pM by day 6 and even further to 100 - 400 pM well in advance of the full
recovery of cell proliferation by 2 weeks. The development of resistance during
this time was due to a decrease in MGBG influx; the rate of efflux remained
unchanged. In addition, an MGBG-resistant subclone of cells also exhibited a
deficiency in MGBG transport. In both types of resistant cells, as well as in
previously untreated cells, the relative but not absolute affinity of the transporter
for MGBG, spermidine, spermine and putrescine were constant. In all cells, an
apparent intracellular concentration of MGBG greater than 400 pM was
associated with toxicity.
Resistance to MGBG was not associated with multiple drug resistance or
expression of P-glycoprotein.
x

CHAPTER 1
INTRODUCTION
Specific Aims
Methylglyoxal bis[guanylhydrazone] (MGBG) has been used clinically in the
treatment of cancer for several years. MGBG accumulates quite extensively in
sensitive targets like mouse leukemia L1210 cells. I have observed that even a
relatively small decrease in the extent of accumulation of MGBG results in
resistance to the antiproliferative and cytotoxic actions of the drug. My
dissertation focuses on the transport of MGBG. In general terms, I propose to
1. characterize the transport and accumulation of MGBG in sensitive
( previously untreated) L1210 cells.
2. define the mechanism(s) of resistance to MGBG that develops quickly
(days) when L1210 cells are exposed to sublethal concentrations of MGBG.
3. define the mechanism(s) of resistance to MGBG in a clone of L1210 cells
selected for resistance to MGBG (resistance persists even when the cells are
grown in drug-free medium).
Background
The structure of MGBG is depicted in Figure 1-1. The figure also includes
structures of putrescine, spermidine, spermidine, N-acetylspermidine and
alpha-difluoromethyl ornithine (DFMO), a well-known inhibitor of ornithine
decarboxylase. The bis[guanylhydrazone] represent a chemical family of
compounds in which terminal amidine groups are separated by variable
aromatic or aliphatic structures frequently containing interposed nitrogen
1

2
groups. Although a number of these compounds posses significant antitumor
activity, they differ substantially in their mode of action and in their ability to
interfere with various biochemical pathways. For example, the antileukemic
action of the aromatic bis[guanylhydrazone], of which 4,4'-diacetyldiphenylurea
bis[guanylhydrazone] is probably the best studied example, has been
correlated with the ability of these compounds to bind to calf thymus DNA and to
inhibit DNA-dependent DNA polymerase (Dave slaL, 1977). The mode of
action of the aliphatic compounds is much less understood. Unlike the aromatic
bis[guanylhydrazone], MGBG and other aliphatic analogs bind weakly to DNA
and do not inhibit the activity of DNA polymerase (Dave et a]., 1977).
Early clinical trials revealed that MGBG had very impressive antiproliferative
activity, particularly against acute myelocytic leukemia (Carbone el ai-, 1964,
Freireich, el a]., 1962, Levin ala}., 1965, Porter alai-. 1979, and Regelson and
Holland, 1963). At optimal dose levels (150 mg/m2 daily), MGBG alone
produced complete remission in about 45% of patients (Levin alai., 1965).
This drug can also be used in combination with other chemotherapeutic drugs,
such as 6-mercaptopurine, and remission rates of 35 - 45% have been reported
among leukemia patient (Boiron alai-. 1965 and Weil alai-. 1969). MGBG was
also found to be effective in the treatment of some solid tumors, including
Hodgkin's disease (46% responsive) and non-Hodgkin's lymphoma (37%
responsive). One of the interesting approaches in the use of MGBG was its
sequential administration after DFMO (alpha-difluoromethyl ornithine), an
inhibitor of polyamine biosynthesis.
Despite being introduced into clinical use as an anticancer agent about 30
years ago, the molecular basis for MGBG's activity remains obscure. Until
recently, the mechanism of action of MGBG was thought to be related to its
various relationships with the biological polyamines. MGBG has some similarity

3
to spermidine (Hamilton and La Placa, 1968), competes with spermidine and
spermine for uptake into the cell (Dave and Caballes, 1973, Porter giai., 1981
and 1982), strongly inhibits the polyamine biosynthetic enzyme, S-
adenosylmethionine decarboxylase (Holtta ei aL, 1973, Williams-Ashman and
Schenone, 1972) and inhibits the polyamine degradative enzyme, diamine
oxidase (Holtta el aL, 1973). It was the inhibition of S-adenosylmethionine
decarboxylase by MGBG that was thought initially to be responsible for the
antiproliferative activity of the drug. In cells treated with MGBG, the
concentration of putrescine increases whereas spermidine and spermine
decrease slowly as a consequence of pathway blockade. The observation that
spermidine prevented the antiproliferative effects of MGBG (Mihich, 1965), led
initially to the conclusion that this effect on the naturally-occurring polyamines
was important. The action of spermidine is now attributed more to competition
for cellular uptake than to replenishment of polyamine pools. A number of
laboratories (Holtta et aL, 1979, 1981, Newton and Abdel-Monem, 1977,
Pleshkewych §igL, 1982, Seppanen glaL, 1980) have been unable to correlate
these changes in polyamine pool size with inhibition of cell growth. Moreover,
DFMO, a highly specific inhibitor of polyamine biosynthesis, is cytostatic
(Mamont §i aL, 1978), whereas MGBG is cytotoxic, a finding that suggests
different mechanisms of action for the two drugs.
On the basis of considerable ultrastructural and biochemical evidence, there
is now sufficient cause to believe that the mitochondrion may, in fact, be the
target responsible for the antiproliferative activity of MGBG. Indeed, 4, 4'-
diacetyldiphenylurea-bis[guanylhydrazone], an aromatic bisfguanylhydrazone]
with potent antiproliferative activity, causes profound ultrastructural damage to
mitochondria (Mikles-Robertson gigL, 1981) but has no effect on polyamine
biosynthesis (Corti et gl., 1974). Ultrastructural studies in a variety of cell types

4
treated in vitro (Mikles-Robertson alai-, 1979, Pathakalal-, 1977, and
Wiseman alai-, 1980) or in vivo (Pleshkewych alai-, 1980 and Porter el ai-,
1979) with MGBG reveal that mitochondria were selectively and significantly
damaged.
In addition, the mitochondria were functionally impaired by MGBG. The
metabolic effects of MGBG include 1) decreased rates of oxidation of pyruvate
(Pleshkewych alai-, 1980) and long chain fatty acids (Nikula alai-, 1984 and
Brady alai-, 1987) ; 2) a decrease in the intracellular concentration of ATP with
concommitant increases in ADP concentration (Pine and DiPaolo, 1966 and
Regelson and Holland, 1963) and lactate production (Porter alai-, 1982) ; and
3) a decreased rate of incorporation of acetate into lipid (Pine and DiPaolo,
1966).
A study of the effects of MGBG on isolated rat liver mitochondria (Byczkowski
alai-, 1981) demonstrated that at drug concentrations comparable to those
attained intracellularly, MGBG significantly inhibits state 4 respiration, but has
less of an effect on either state 3 or uncoupled respiration. Byczkowski alai-
speculated that 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. Since MGBG is a cation under physiological conditions, it might
accumulate at or within the inner membrane because of its negative potential.
Similar selective binding characteristics have been noted for other cationic
compounds. Rhodamine dyes that are positively charged stain mitochondria
specifically, whereas uncharged rhodamines and the negatively charged dye,
fluorescein, do not. It is possible that MGBG neutralizes the net negative surface
potential of the mitochondrial inner membrane, perhaps by binding to
phospholipids. This might influence cation binding and/or transport and
secondarily affect mitochondrial bioenergetics (Toninello alai-, 1988). In any

5
case, the exact mechanism by which MGBG influences mitochondrial
metabolism is unknown. It is likewise not known whether or not these actions
play a role in the effects of MGBG on mitochondrial DNA replication described
below.
MGBG inhibits the replication of mitochondria DNA (mtDNA) at
concentrations of drug that do not seem to interfere with the replication of
nuclear DNA (Feuerstein eial, 1979). Drug-induced inhibition of mtDNA
replication is likely to occur shortly after exposure to MGBG (Nass, 1984). Our
laboratory has observed that the decrease in cellular content of mtDNA with
time can be accounted for by lack of replication coupled with dilution of the
residual genome upon repetitive cellular division (Bortell, 1987). These results
were obtained in our laboratory with use of dot-blot hybridization methods
involving whole cell lysates and 35S labelled full-length mouse mtDNA probe.
Although we know little about the mechanism(s) by which MGBG adversely
affects the ultrastructure, bioenergetics and replication of mitochondria, it does
seem likely that these actions relate to the antiproliferative and cytotoxic effects
of the drug and many of its congeners.
MGBG seems to accumulate in dividing cells by utilizing the facilitated
carrier mechanism for spermidine (Dave and Caballes, 1973, Porter el ai-.
1981). Seppanen eiai. (1981) found that MGBG uptake is critically dependent
on the growth rate of tumor cells (i.e., slowly dividing cells transport less MGBG
than rapidly dividing cells). Moreover, Mikles-Robertson el a[. (1979) found that
MGBG cytotoxicity also correlates with growth rate. Athough several
descriptions of the transport of MGBG have been published, the process is still
poorly defined. MGBG uptake is thought to involve an energy dependent
saturable carrier (Porter el al., 1982; Field el al., 1964), in that cells concentrate
the drug, and concentration gradients across the plasma membrane as high as

6
1000-fold have been reported. Polyamine and/or MGBG transport has been
studied in different cell lines and tissues including neuroblastoma (Rinehart and
Chen, 1984), fibroblast (Pohjanpelto, 1976), Ehrlich ascites carcinoma
(Seppanen glai-, 1980,1981,1982 ; Seppanen, 1981), mammary gland
explants (Kano and Oka, 1976), slices of rat lung and other tissues
(Gordonsmith alai-. 1985; Smith and Wyatt, 1981), and rat lung perfused in situ
(Reynolds et al., 1985). In general, it appears that putrescine, spermidine,
spermine, and MGBG are transported by common membrane transport systems
which are distinguishable from other defined systems (e.g., for various amino
acids). The transport is saturable and temperature-dependent, with maximal
rates evident at 37°C. MGBG transport is inhibited by uncouplers of oxidative
phosphorylation and certain respiratory poisons, dependent on the proliferative
status of the cells and, in some instances, subject to hormonal regulation.
Whether polyamine/MGBG transport actually occurs via the same mechanisms
in all types of cells in any particular organism is also unclear. The development
of more efficacious drugs in this class of compounds seems to depend on
further understanding of the mechanism of transport, and, perhaps more
importantly, how the transport process is regulated.

7
H2N(CH2)4NH2 H2N(CH2)3NH(CH2)4NH2
putrescine spermidine
H2N(CH2)3NH(CH2)4NH(CH2)3NH2
spermine
CH3CONH(CH2)3NH(CH2)4NH2
N1-acetylspermidine
NH CH3 NH
II I II
h2ncnhn=c-ch=nnhcnh2
MGBG
chf2
I
H2N(CH2)3C-NH2
I
COOH
DFMO
FIGURE 1-1 : Structure of polyamines, related compounds and
inhibitors of polyamine synthesis.

CHAPTER 2
MATERIALS AND GENERAL METHODS
Cell Culture
Murine L1210 cells (obtained from American Type Culture Collection,
Rockville, Maryland) were allowed to grow in suspension in 25 or 75 cm2
canted-neck tissue culture flasks (Fisher Scientific) in an incubator (National
Appliance Company, Portland, Oregon) in an atmosphere of air: CO2 (95 : 5,
v/v) at 37°C. Cells were incubated in Roswell Park Memorial Institute (RPMI)
1640 medium (CellgroR, GIBCO Laboratories) supplemented with 10 % heat-
inactivated horse serum (GIBCO Laboratories), 16 mM HEPES (3-[N-
morpholino] propanesulphonic acid) and 8 mM MOPS (N-2-hydroxyethyl-
piperazine-N'-2-ethanesulphonic acid), at a final pH of 7.40. All chemicals were
obtained from Sigma Chemical Co. unless otherwise specified. Cells were
maintained in logarithmic growth by reseeding an aliquot of cells into fresh
medium every two days to a starting concentration of 7 x 104 cells per ml.
Cell Counts
Aliquots (100 or 200 pi) of cells were diluted in 10 ml of Hematall diluent
(Fisher Scientific). Cell number was determined by electronic particle analysis
(Coulter Counter, Model ZF, Coulter Electronics, Hialeah, FL). Two aliquots
were counted from each flask, and the mean was determined. Because cells
were reseeded every two days, a calculation was devised to plot cell
accumulation as a single, continuous line over the the entire period of an
experiment. Cumulative counts were calculated as:
8

9
cone, of viable cells (n+1)
Cumulative count(n+1) = Cumulative count (n) X
cone, of viable cells (n)
The population doubling time of the cells was determined as follows: N =
Noe+kt; where t = time, No Is the number of cells present at t = 0, k = In 2/
doubling time, and the doubling time Is the value of t when N = 2No.
Cell Viability
Trypan blue dye (Eastman Kodak, Rochester, NY) at a final concentration of
0.06% was added to 100 pi aliquots of cells. 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 examined by
phase contrast microscopy (Ernst Leitz, Wetzlar, Germany) at 200X
magnification. Percent viability of cells was determined as follows:
number of cells excluding trypan blue dye x 100
% viability =
total number of cells
Clonoaenic Assay
Colony-forming ability of L1210 cells was determined by growth in soft agar
(Chu and Fischer, 1968). Pre-autoclaved Agar Noble (DIFCO Laboratories,
Detroit, Ml) was admixed at 37°C with RPMI 1640 medium containing 20% heat-
inactivated horse serum to achieve 1mg agar/ml final concentration . A
4 ml aliquot of the prepared medium was transferred into each cloning tube.
L1210 cells from selected experitments were counted and resuspended into
cloning tubes over the concentration range of 100 to 10,000 cells per tube.
Cloning tubes were cooled down at 0 - 4°C for 15 minutes prior to incubation at
37°C. Colony counts were determined 2 weeks after the start of incubation.
Transport of MGBG
The assay of MGBG influx and efflux was based on the use of methylglyoxal-
bis[14C]guanylhydrazone ([14C] MGBG) (specific activity 92.8 pCi/mg or 25.6

mCi/mmole, Amersham). A stock solution of 1 mM [14C] MGBG was prepared
aseptically. Aliquots of this stock solution were added to the incubation medium
to obtain the indicated final concentration of drug.
Murine L1210 cells were exposed to various concentrations of [14C] MGBG
for the indicated times at 37°C and pH 7.40 unless otherwise indicated.
Incubations were terminated by centrifugation. One-ml aliquots of cell
suspensions were centrifuged at 15,000g for 10 seconds in Eppendorf tubes.
The supernatant was discarded, and the cell pellet was washed twice by
centrifugation with one ml of ice-cold medium containing 1 mM unlabelled
MGBG. The tip of each Eppendorf tube, which contained the cell pellet, was cut
off and transferred to a scintillation vial containing 10 ml of LiquiscintR (National
Diagnostics, Manville, NJ) for quantitation by liquid scintillation counting in
Beckman model LS 7000.
Cell counts and viability were obtained with use of a hemacytometer. The
diameter and volume of cells were determined by the method of Schwartz el ai.
(1983). Uniform polymeric microspheres (Polyscience, Warrington, PA) from
4.72 to 10.0 11 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 diameter and volume for each size of calibrated
microbead to obtain a standard curve.
Aliquots of 1 x106 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 and volume. The
diameter of drug-naive L1210 cells was 10.6 p and cell volume was 5.68 pi per
106 cells. There was no significant change in the size of L1210 cells during or
after an exposure to MGBG when the cells were observed under a light

microscope in the presence of standard beads. The apparent intracellular
concentration of [14C] MGBG (pM) in all experiments was calculated assuming
a volume of 5.68 pi per 106 cells. In studies of efflux, cells were preincubated in
[14C] MGBG as indicated in subsequent chapters.
Preliminary experiments had revealed that 1) the rate of accumulation of
MGBG by cells was constant for at least 20 minutes; and 2) although 1 mM
unlabelled MGBG was routinely included in the wash of cells, its presence did
not make much difference.
Stability of fHCI MGBG
It is generally believed that MGBG does not undergo biotransformation in
higher animals (Warrell and Burchenal, 1983), in part because no metabolites
of MGBG have been detected in urine, feces and various tissues and no
radioactive carbon dioxide was expired after in vivo administration of [14C]
MGBG. Nonetheless, we further established the stability of [14C] MGBG upon
incubation with L1210 cells by thin layer chromatography. L1210 cells were
exposed to [14C] MGBG for 24 and 48 hours. Aliquots of cell suspension were
taken, centrifuged and the supernatant was then removed. The cell pellet was
washed twice at 0 - 4°C with media containing 1 mM unlabelled MGBG, then
resuspended in deionized water to lyse the cells. Trichloroacetic acid was
added to a final concentration of 10% and the precipitated protein was removed
by centrifugation. Aliquots of supernatant were spotted on precoated thin layer
plates of Silica Gel 60 F254 (E. Merck, Darmstadt, Germany). Chromatograms
were developed with 50% n-butanol, 30% water and 20% glacial acetic acid.
Unlabelled MGBG was located by fluorescence quenching, and its Rf was found
to be 0.49 - 0.53. [14C] MGBG was located by autoradiography with use of
Kodak SB Film, and its migration was indistinguishable from that of authentic
MGBG. The only radioactivity observed after 24 and 48 hours of incubation of

[14C] MGBG with L1210 cells was that of the drug itself. This observation
confirmed that [14C] MGBG was stable and was not metabolized by the cells.
Protein Assay
Protein was measured by the method of Lowry eiaK (1951) with use of
bovine serum albumin as standard.
Statistical Analysis
The two-tailed Student t-test was used to analyze comparisons; the
significance level was set at P < 0.05. Lineweaver-Burk graphs were evaluated
by least squares regression analysis.

CHAPTER 3
TRANSPORT OF MGBG AND ITS EFFECT ON SENSITIVE L1210 CELLS
This chapter deals with 1) the effects of varying doses of MGBG on the
proliferation, viability and clonogenicity of L1210 cells; 2) the characterization
of the processes involved in the transport of MGBG into and out of these cells
upon first exposure to the drug; 3) an assessment of the intracellular
distribution of the MGBG, especially with regard to possible accumulation within
mitochondria; and 4) the relationship between transport of MGBG and the rate
of cell growth.
Effects of MGBG on Growth. Viability and Clonoaenicitv
To determine the effects of MGBG on cell growth and viability, L1210 cells in
logarithmic growth were incubated continuously in 0 to 20 pM drug at 37°C, pH
7.40. Cells were reseeded every two days in fresh medium containing MGBG.
Cell count and viability were determined daily. The cumulative count of viable
cells is depicted in Figure 3-1, and the percent of total cells that were viable is
presented in Figure 3-2. Untreated cells exhibit a doubling time of
approximately 9 hours.
MGBG at concentrations as high as 20 pM had minimal effect on cell growth
or viability for the first few days of exposure. Our laboratory (Bortell, 1987) has
reported that the cellular content of mt DNA decreases markedly during this
time. The decrease was found to reflect dilution of the mitochondrial DNA due
to lack of its replication coupled with continuing cell division. Thereafter cell
13

growth was inhibited by MBGB in a dose-dependent manner, perhaps because
of the depletion of mt DNA. At MGBG concentrations between 0.3 and 1.0 pM,
the growth rates were markedly inhibited for about 10 days after which recovery
to rates virtually identical to untreated cells was observed. At these doses,
viability was also adversely affected in a concentration-dependent manner, but
it did not fall below 60%. Viability recovered to the control value of nearly 100%
when growth rate recovered. At the intermediate dose of 5.0 pM MGBG, the
percent of viable cells decreased to a plateau of about 40%, and recovery of
growth had not occurred by day 17 of treatment. The cytotoxic affect of MGBG
was apparent when the medium contained 20 pM MGBG. Cell viability
decreased progressively, and no living cells could be detected after day eight.
Further experiments were performed to determine the effects of MGBG on
clonogenicity of murine L1210 cells in soft agar. L1210 cells were pretreated
with different concentrations of MGBG (0-10 pM) for 24 or 72 hours before
reseeding in drug-free RPMI 1640 medium containing 1 gm/ml Agar Noble.
MGBG inhibited the clonogenicity of L1210 cells in soft agar in a concentration-
dependent manner (Fig 3-3). The inhibitory effect of MGBG on the
clonogenicity of L1210 cells was evident as early as 24 hours after MGBG
exposure, whereas the cell viability was unaffected at that time.
Characterization of the Initial Transport of MGBG
Intracellular Accumulation of MGBG During the First 20 Hours of Exposure
Although we have measured the intracellular content of MGBG over a few
weeks of incubation (see below), this portion of the proposal focuses on the first
20 hours of incubation. It is during this time that the L1210 cells accumulated
large amounts of MGBG. By 20 hours, the replication of mitochondrial DNA has
been inhibited, but the rate of cell growth and cell viability have remained
unaffected.

cumulative cell count
15
time (days)
FIGURE 3-1 : Effects of various concentrations of MGBG on the proliferation of
murine L1210 cells. Cells in logarithmic growth were incubated
continuously in 0 - 20 pM MGBG and were reseeded every two days in
fresh medium containing MGBG. Cell counts were determined by
electronic particle analysis (Coulter Counter). Cumulative cell counts were
calculated and cell accumulation was plotted as a single continuous line
over the entire period of an experiment.

% viability
16
FIGURE 3-2 : Effects of various concentrations of MGBG on the viability of
murine L1210 cells over time. Cells in logarithmic growth were incubated
continuously in 0 - 20 pM MGBG and were reseeded every two days in
fresh medium containing MGBG. Cell viability was determined with use of
the trypan blue dye exclusion method.

clonogenicity (% control)
17
\iM MGBG
o— 72hr in MGBG
* 24hr in MGBG
FIGURE 3-3 : Effects of MGBG on the clonogenicity of L1210 cells. L1210 cells
were exposed to different concentrations of MGBG (0-10 pM) for 24 or 72
hours prior to reseeding in RPMI 1640 medium containing 1 mg/ml Agar
Noble. Clonogenicity was determined by an ability of cells to form colonies in
agar after 2 weeks of incubation, and values are expressed as percent control.

Cells in logarithmic growth were incubated in various concentrations of
[14C] MGBG at 37° C, pH 7.40. At various times, aliquots of cells were
harvested and washed at 0 °C by centrifugation. Although the routine washing
procedure involves the use of unlabelled 1 mM MGBG, initial experiments
revealed that little added radiolabel was displaced from the cells by the carrier.
In addition, the rate of efflux of radiolabel from the cells is sufficiently slow even
at 37°C to permit this approach to separation of cells from incubation medium.
The results are presented in Figure 3-4. MGBG accumulated within cells at
reasonably constant rates dependending on the initial extracellular MGBG
concentration. It is apparent in Figure 3-4 that the apparent intracellular
concentration of MGBG exceeded that of the medium by about 1000-fold within
12 hours.
Kinetic Characterization of the Initial Uptake Process
The initial uptake of MGBG occurs by a saturable process. To determine Km
and Vmax, L1210 cells in logarithmic growth were reseeded at a density of 3 x
105 cells per ml in prewarmed (37°C) media containing different concentrations
of [14C] MGBG at pH 7.40. "Intracellular" radioactivity was determined 20
minutes later (see Chapter 2 for details of centrifugation and washing of the
cells). As noted above, all of the radioactivity, even 48 hours after the start of
incubation, remained stable as [14C] MGBG. The amount of drug in cells was
expressed as pmole per 106 cells. Four separate experiments were conducted.
Lineweaver-Burk plots were constructed (Figure 3-5), and Km and Vmax were
determined by linear regression. The Km and Vmax for uptake of MGBG at 37°C
were 4.72 ± 0.17 pM and 296 ± 19 pmole/106 cells/hour, respectively.
Interestingly, the amount of intracellular [14C] MGBG over the first 20 hours of
exposure to the drug (Figure 3-4) could be predicted with use of the kinetic
parameters determined over 20 minutes (Table 3-1). Given the volumes of cells

|iM pellet [MGBG]
19
time (hrs.)
FIGURE 3-4 : Accumulation of MGBG in L1210 cells during the first 20 hours of
exposure. Cells in logarithmic growth were incubated in 0.1 - 5 pM [14C]
MGBG. At various times, aliquots of cells were harvested and cell pellet was
washed twice by centrifugation with use of medium containing 1 mM MGBG.
Cell counts and viability were obtained with use of a hemacytometer and the
results were expressed as apparent intracellular concentrations of [14C]
MGBG (pM).

1/pmole MGBG/milion cells/hr.
20
1/jiM [MGBG]
FIGURE 3-5 : Lineweaver-Burk plot of MGBG uptake in control L1210 cells. L1210
cells in logarithmic growth were reseeded at a density of 3 x 105 cells per ml
in media containing different concentrations of [14C] MGBG. Intracellular
radioactivity was determined 20 minutes later and the amount of drug in cells
was expressed as pmole [14C] MGBG per 106 cells. The Km and Vmax were
determined by linear regression of the Lineweaver-Burk Plot.

TABLE 3-1 : Comparison between the calculated and observed intracellular concentrations of MGBG during
the first 20 hours of exposure.
hrs. after Intracellular concentration of [14-C] MGBG (pMI
incubation 0.1 pM 0.5 uM 0.93 uM 4.8 pM
calculated observed calculated observed calculated observed calculated observed
5.7
60
38.9 (1.5)
271.0
289.8 (11.3)
487.8
561.5 (34.5)
1488.9
1420.5
(49.1)
13.3
141.3
131.7 (9.6)
637.6
735.0 (32.9)
1147.6
1374.9 (79.5)
3503.1
3423.5
(224)
20.1
169.5
164.8 (10.1)
960.8
1110 (120)
1729.2
1946.5 (136)
5074.5
3944.5
(263)
Note : The intracellular concentration of MGBG at various times was calculated with use of Michaelis-Menten Equation
(v = Vmax[S]/Km + [S]), using the predetermined values for the Vmax and Km of 296 ± 19 pmole/106 cells/hour and 4.72
±_0.169 pM, respectively. Efflux was ignored in the calculations. Each observed value represents the mean + SEM.

22
and of the incubation media, the change in extracellular concentrations of
MGBG over 20 hours did not exceed 25% of the initial concentration. Indeed,
except for the lowest initial concentration of drug, the decrease in extracellular
[MGBG] was considerably less than 25%.
Experiments were also conducted at 0 - 4°C. Rates of uptake of 10 pM
MGBG at 0 - 4°C was less than 10% of that seen at 37°C. This result would be
anticipated if the uptake of MGBG depended on active transport.
Inhibition of MGBG Uptake bv Spermidine. Spermine and Putrescine
L1210 cells were incubated in medium containing different concentrations of
[14C] MGBG (1.2 - 58.6 pM) with or without 10 pM spermidine, spermine or
putrescine. The incubation was carried out for 20 minutes at 37 °C. Uptake of
MGBG was inhibited competitively by spermidine, spermine and putrescine
(Figure 3-6). This result implies that MGBG and the naturally occurring
polyamines, spermidine, spermine and putrescine, share a common transport
mechanism. The K¡'s of spermidine, spermine and putrescine for the inhibition
of MGBG uptake were 0.524 ± 0.03, 0.582 ± 0.029 and 3.74 ± 0.029 pM,
respectively.
Effect of Extracellular pH on the Initial Uptake of MGBG
Logarithmically-growing L1210 cells were exposed to [14C] MGBG (2.0 pM)
for 0 to 10 minutes at 37°C over the range of extracellular pH between 7.0 and
7.7. The rates of uptake of MGBG were strongly influenced by pH . An increase
in extracellular hydrogen ion concentration enhanced the rate of uptake of [14C]
MGBG. Similar results were observed when the experiments were conducted
for 0 to 75 minutes with 0.8 and 8.5 pM [14C] MGBG (Figure 3-7). MGBG is a
weak base with pKa's of about 7.5 and 9.2 at 25°C (Williams-Ashman and
Seidenfeld, 1986). At physiological pH, MGBG exists primarily as a mixture of
monovalent and divalent cations. The results depicted in Figure 3-7 suggest

23
that it is the divalent form of MGBG (MGBG 2+) that is transported into L1210
cells. Some evidence for this idea derived from consideration of the competitive
inhibition of MGBG transport by the naturally occurring polyamines. For
example, spermidine, the extended molecular conformation of which is quite
similar to that of MGBG, has pKa values of about 8.4, 9.8 and 10.8 at 25°C
(Williams-Ashman and Seidenfeld, 1986), and exists almost exclusively as a
mixture of divalent and trivalent cations at physiological pH. The hypothesis that
MGBG2+ is the substrate for transport is supported further by the good
agreement between the observed and calculated values of MGBG uptake
presented in Table 3-2. The calculations were based on Henderson-
Hasselbach computations of the concentrations of MGBG2+ at different pH's
and on the assumption that only MGBG2+ is transported.
Efflux of MGBG
The following experiments were designed to study the characteristics of
efflux of MGBG from L1210 cells. Logarithmically-growing L1210 cells were
exposed to 1.5 pM [14C] MGBG for four hours before centrifugation and
resuspension in drug-free medium. The apparent intracellular concentration of
MGBG was about 400 pM at the time of resuspension. The efflux of radioactivity
with time is depicted in Figure 3-8. Efflux occurs by a process with
approximately first-order characteristics with a half-life of about three hours.
Experiments were also conducted at 0 - 4°C. The results reveal that the efflux
of MGBG at this low temperature was minimal (Figure 3-9), which would be
anticipated if the efflux of MGBG depended on active transport.
Countertransport of Unlabelled MGBG
To explore the possibility that MGBG transport is mediated by an antiport
carrier, the effect of preloading the cells with unlabelled MGBG on the
subsequent rate of uptake of [14C] MGBG was assessed. L1210 cells were

1/pmole MGBG/10e6 cells/hr.
24
â–¡ control
♦ 10 nM spd
d lO^Mspm
« 10|iMputres
FIGURE 3-6 : Competitive inhibition of MGBG uptake by spermidine, spermine and
putrescine in control L1210 cells. Control L1210 cells in logarithmic growth
were reseeded at a density of 3 x 105 cells per ml in media containing
different concentrations of [14C] MGBG (1.7 - 80.6 pM) in the presence or
absence of 10 pM spermidine (spd), spermine (spm) or putrescine (putres).
The uptake of [14C] MGBG at 37°C was assessed after 20 minutes
incubation. The K¡'s of spermidine, spermine and putrescine for the uptake
of MGBG were 0.524 ± 0.03, 0.582 ± 0.029 and 3.74 ± 0.029 pM,
respectively.

|iM pellet [MGBG]
25
medium
pH
□—
6.90
•—
7.10
□—
7.36
e—
7.50
■—
7.75
□—
8.35
FIGURE 3-7 : Effect of extracellular pH on the initial uptake of MGBG.
Logarithmically-growing L1210 cells were exposed to [14C] MGBG (0.8
pM) for 0 - 75 minutes over the range of extracellular pH between 6.90
and 8.35. Intracellular radioactivity was determined at various times (0 -
75 minutes) after the exposure.

26
TABLE 3-2 : Comparison between the observed rates of MGBG uptake at
different extracellular pH's with rates calculated on the basis of transport
of MGBG2+ only.
Extracellular pH [MGBG1 +]/[MGBG2+]
m/m
calculated v
(pM/min.)
observed v
(pM/min.)
At 0.8 pM[14C] MGBG
6.90
0.16/0.64
1.04
1.07
7.10
0.23/0.57
0.92
1.08
7.36
0.34/0.46
0.77
0.72
7.50
0.40/0.40
0.67
0.34
7.75
0.64/0.34
0.58
0.28
8.35
0.70/0.10
0.18
0.10
At 8.5 pM [14C] MGBG
6.90
1.70/6.79
5.12
5.06
7.10
2.42/6.08
4.89
5.65
7.36
3.57/4.93
4.43
4.22
7.50
4.25/4.25
4.11
2.52
7.75
5.44/3.06
3.41
1.48
8.35
7.45/1.05
1.58
0.96
Note : L1210 cells were exposed to 0.8 and 8.5 pM [14C] MGBG in RPMI 1640 medium
which was adjusted to different pH's with use of concentrated HCI or NaOH. Cells were
incubated at 37 °C and aliquots of cell suspension were taken intermittently during the
course of exposure to determine pellet radioactivity. Observed values of the uptake
rate of [14C] MGBG were then compared to the values calculated with an assumption
that the Km and Vmax of MGBG uptake (4.72 pM and 8.68 pM per min., respectively)
were unchanged at different pH's but apply only to MGBG2+. The Henderson-
Hassenbach equation (pH = pKa + log [MGBG1+]/[MGBG2+] was used to calculate an
availability of divalent form of MGBG and the Michaelis-Menten equation (v = Vmax
[S]/Km + [S]), to predict the intracellular accumulation of [14C] MGBG if only the
divalent form of MGBG was transported into L1210 cells.

(iM pellet [MGBG]
27
time after crossover (hrs.)
FIGURE 3-8 : Efflux of MGBG. Logarithmically-growing L1210 cells were exposed to
1.5 (iM [14C] MGBG for 4 hours on the first day (day 0) of incubation before
centrifugation and resuspension in drug-free medium. Intracellular
radioactivity was determined at various times (0 - 48 hours) after
resuspension.

|iM pellet [MGBG]
28
FIGURE 3-9 : Effect of temperature on the efflux of MGBG. L1210 cells at 3 x 105
cells per ml were incubated in medium containing 5 pM [14C] MGBG for
1.5 hours at 37 °C. Cell pellet was washed once with drug-free medium at
0 - 4 °C before subsequent resuspension in drug-free medium, and the
cell suspension then was incubated at 0 - 4 °C or 37 °C. Intracellular
radioactivity was determined for 0 - 12 hours after the resuspension.

29
exposed to 5 pM MGBG for two hours before centrifugation and resuspension
into medium containing 1 pM [14C] MGBG. The apparent intracellular
concentration of nonradioactive MGBG would be expected to be about 400 pM
at the time of resuspension. The results shown in Figure 3-10 revealed that
preloading the cells with MGBG did enhance modestly the uptake of [14C]
MGBG.
We also explored the effects of various extracellular concentrations of MGBG
on the rate of efflux of [14C] MGBG from preloaded L1210 cells (Figure 3-11).
No stimulation of efflux was noted; when the extracellular concentration of
MGBG was 1 mM, some inhibition of efflux was observed.
In summary, the presence of unlabelled MGBG inside cells stimulated the
uptake of [14C] MGBG, but the presence of unlabelled MGBG in the medium did
not stimulate the efflux of [14C] MGBG. Interpretation of this observation is quite
complex because of the presence of naturally occurring polyamines in these
L1210 cells.
Effects of Various Cations on the Uptake of MGBG
Sodium-dependency of MGBG uptake
The accumulation of [14C] MGBG (4.0 pM) by L1210 cells was determined,
at 0, 5, 15 and 30 minutes in an Earle's balanced salt solution (EBSS) which
contains NaCI (116 mM). This rate of accumulation was compared to
incubations in which NaCI was replaced iso-osmotically by choline choride
(Figure 3-12). The results suggest that the uptake of MGBG by L1210 cells is
sodium dependent. Other experiments were done to further establish this
conclusion. L1210 cells were exposed to different concentrations of [14C]
MGBG (0.5 - 30 pM) in 10 mM HEPES-TRIS buffer, pH 7.40, which contains
either 100 mM NaCI or choline chloride. The results shown in figure 3-13
revealed an MGBG concentration dependent enhancement of [14C] MGBG in

HM pellet [MGBG]
30
preloaded MGBG (|j.M)
Q— none
•— 5.0
o— 10.0
o— 20.0
FIGURE 3-10 : Effect of MGBG preloading on the uptake of labelled MGBG. L1210
cells were exposed to 0 - 20 pM MGBG for two hours before centrifugation
and resuspension into a medium containing 10 pM [14C] MGBG. Intracellular
radioactivity was determined for 0-120 minutes after resuspension.

|iM pellet [MGBG]
31
medium [MGBG]
□—
â–  none
â–  10 \xM
• 100 |iM
o
1 mM
FIGURE 3-11 : Effect of medium concentration of MGBG on the efflux of labelled
MGBG. L1210 cells were exposed to 1.0 pM [14C] MGBG for one hour
before centrifugation, and the cell pellet was resuspended into medium
containing MGBG (10 pM - 1 mM). Intracellular radioactivity was
determined for 0 - 90 minutes after resuspension.

32
the presence of NaCI. To determine kinetics of the sodium-dependent process,
uptake in choline chloride was subtracted from uptake in the presence of NaCI.
A Lineweaver-Burk plot of the sodium-dependent uptake as a function of [14C]
MGBG concentration was linear (Figure 3-14). These results indicated that the
sodium-dependent component of uptake of MGBG was a single transport
system with a Km and Vmax of 2.10 ± 0.08 pM and 226.8 ±18.0 pmole/106
cells/hour, respectively. Michaelis-Menten calculations revealed that at an
extracellular MGBG concentration of 1 pM, which is cytotoxic, the sodium
dependent transport of MGBG accounts for more than 90 % of total uptake.
Results of experiments in which both sodium and choline are replaced by
mannitol are interesting. Uptake of [14C] MGBG in10 mM HEPES-TRIS buffer,
pH 7.40 containing 200 mM mannitol was surprisingly high (Figure 3-15).
However, Lineweaver-Burk plots of MGBG uptake as a function of MGBG
concentration in the presence of mannitol, like choline chloride but unlike
sodium chloride, revealed a low affinity process (high Km).
Ability of various cations to substitute for sodium in uptake of MGBG
To determine the cation specificity of MGBG uptake, sodium chloride was
replaced by a variety of monovalent cation chloride salts in incubations of
L1210 cells with [14C] MGBG (Figure 3-17 and Table 3-3). Choline and lithium
maintained rates of uptake of MGBG about 2/3 that of sodium. Cesium and
potassium were less effective in that the observed uptakes ranged from only 7
to 24 % of sodium controls. Table 3-4 depicts experiments in which MGBG
uptake was assessed in the presence of varying mixtures of sodium, potassium
and choline chloride. The detrimental effect of potassium, even in comparison
to choline, is again apparent.
The specificity toward chloride was also examined. Substitution of sodium
chloride with sodium gluconate had no effect on MGBG uptake. Replacement of

% control uptake rate
33
FIGURE 3-12: Sodium dependency of MGBG uptake in L1210 cells. The uptake of
[14C] MGBG ( 4.0 (iM) in logarithmic-growing L1210 cells was measured in an
Earle's balanced salt solution in which NaCI was iso-osmotically replaced by
varying amount (0, 25, 50, 75 and 100%) of choline chloride. In each case,
the uptake of MGBG was assessed at 0, 5,15 and 30 minutes. The rate of
uptake at 100% (116 mM) NaCI was set at 100% and the other rates were
plotted against the fractional content of sodium. Each point represents the
average of three separate experiments, with triplicate measurements in each
experiment.

MGBG in pellet (|i.M/30 minutes)
34
FIGURE 3-13 : Relationship between MGBG concentration and the rate of uptake
of MGBG in the presence or absence of sodium. L1210 cells were
exposed to different concentrations of [14C] MGBG (0.5 - 30 pM) for 30
minutes in 10 mM HEPES-TRIS buffer, containing either 100 mM NaCI or
100 mM choline chloride.

1/d uptake NaCI-Choline Cl (pM/30 minutes)
35
â–¡ 1/d uptake
FIGURE 3-14 : Lineweaver-Burk plot of the sodium-dependent MGBG uptake
in L1210 cells. Sodium-dependent uptake of MGBG refers to the
diference between uptake in the presence of sodium chloride and
choline chloride as indicated in Figure 3-13.

MGBG in pellet (pM/30 minutes)
36
600
500
400
300
200
100
0
0 1 0 20 30 40
MGBG concentration (p.M)
NaCI
Choline Cl
Mannitol
FIGURE 3-15 : The relationship between MGBG concentration and the rate of
MGBG uptake in sodium chloride, choline chloride and mannitol. Cells
were incubated for 30 minutes in [14C] MGBG in 10 mM HEPES-TRIS
buffer, pH 7.40, containing 100 mM NaCI, 100 mM choline chloride or
200 mM mannitol.

1/pmole/milMon cells/hr.
37
â–¡ NaCI
♦ Choline Cl
â–  Mannitol
FIGURE 3-16 : Lineweaver-Burk plot of MGBG uptake in sodium chloride,
choline chloride and mannitol. The data are presented in Figure 3-15.
Vmax of MGBG uptake in NaCI, choline chloride and mannitol were
362.4 ± 19.7, 450.6 ± 31.6, 2372.8 ± 103 pmole/106 cells/hour with Km
of 9.34 + 1.04, 61.5 ± 2.52 and 50.7 + 2.10 pM, respectively.

38
chloride ion with the relatively impermeable anion, sulfate, did not affect the
initial (up to 10 minutes) uptake of MGBG, but did decrease uptake thereafter.
Effect of lonoohores on the Uptake of MGBG
Two classes of ionophores, mobile ion carriers and channel formers, with
different cation specificities, were evaluated with regard to their effect on MGBG
uptake. L1210 cells were preincubated with the ionophore (5 pM final
concentration) for 20 minutes in Earle's balanced salt solution before [14C]
MGBG was added to achieve the final concentration of 6 pM. The results are
shown in table 3-5. Among the ionophores tested, gramicidin A was the most
effective inhibitor of MGBG uptake. It is a channel-forming ionophore and does
not discriminate between sodium and potassium ions. Therefore, it can
dissipate both sodium and potassium electrochemical potential differences
across the plasma membrane. In contrast, valinomycin, a neutral ionophore
which is specific for the potassium ion did not significantly affect the uptake of
MGBG. Effects of monensin, an ionophore specific for sodium ion, and A23187,
which is specific for calcium ion (but also allows an entry of sodium and
potassium ions), were intermediate. These results support the notion that a
sodium electrochemical gradient across the plasma membrane is important in
attaining maximal uptake of MGBG.
Effect of Inhibitors of Energy Metabolism on the Uptake of MGBG
The effect of metabolic inhibitors on the uptake of [14C] MGBG was assessed
in order to gain some insight into the energetics of the transport process. The
inhibitors included antimycin A (an inhibitor of cytochrome reductase), 2, 4-
dinitrophenol (2, 4-DNP, an uncoupler of oxidative phosphorylation), and
potassium cyanide (KCN, an inhibitor of cytochrome oxidase). The effects of
these inhibitors of mitochondrial energetics (0.01 mM antimycin A, 0.1 mM 2,4-
DNP and 0.1 mM KCN) on drug uptake were studied by incubating L1210 cells

pM pellet [MGBG]
39
â–  NaCI
0 KCI
â–¡ Choline Cl
0 LICI
â–¡ CsCI
â–  Sod. gluconate
Ü Sod. sulfate
time (minutes)
FIGURE 3-17 : Effects of various ions on MGBG uptake in L1210 cells.
Incubations were performed in 10 mM HEPES-TRIS buffer, pH 7.40,
containing 100 mM NaCI, KCI, choline Cl, LiCI, CsCI, Na2SC>4 or Na
gluconate. Mannitol was also added in the buffer to achieve 100 mM
concentration.

40
TABLE 3-3 : Effects of various ions on MGBG uptake in L1210 cells.
salt
10 min. uotake
30 min, uptake
pM
%
pM
%
NaCI
39.2 ±1.2
100 a
124.7 ±5.2
100 a
KCI
7.0 ±1.2**
17.8
9.26 ±0.7**
7.4
Choline Cl
26.2 ± 1.5*
66.8
71.5 ± 1.3*
57.3
LiCI
26.7 ± 1.9*
68.1
74.0 ±4.4
59.3
CsCI
9.3 ±0.3**
23.7
29.3 ±1.2*
23.5
Na gluconate
35.8 ± 1.8
91.3
126.3 ±3.5
101.3
Na2SÜ4
34.7 ±0.9
88.5
67.9 ±1.61*
54.4
Note : All salts were included at a concentration of 100 mM (except Na2SC>4, 50 pM) in
the medium containing 100 mM mannitol and 10 mM HEPES-TRIS solution, pH
7.40. Results are mean + SE of four determinations of three separate experiments.
The final concentration of [14C] MGBG in the incubating medium was 3.0 pM. a The
uptake in NaCI was defined as 100 % (** p < 0.01, * p < 0.05).

41
TABLE 3-4 : Effects of potassium and choline chloride on the sodium-
dependent MGBG uptake in L1210 cells.
EBSS treatment
percent
100 % NaCI
100a
50 % NaCI : 50 % choline Cl
81.0
100 % choline chloride
57.3
50 % NaCI : 50 % KCI
58.6
100 % KCI
7.4
50 % KCI : 50 % choline Cl
42.6
Note : The transport rates of [14C] MGBG (3-4 |xM) in logarithmically-growing L1210
cells were measured, respectively, for 30 - 35 minutes in an Earle's balanced salt
solution where NaCI was displaced iso-osmotically by choline chloride and/or KCI (100
% NaCI corresponds to 116 mM). At the end of incubation period, cells were washed
and cell-associated radioactivity was determined as described in " Materials and
General Methods The data represents the average of two separated experiments,
with quadruplicate measurements in each experiment. a The uptake in 100 % NaCI
was defined as 100 %.

42
TABLE 3-5 : Effects of ionophores on MGBG uptake in L1210 cells.
Addition
Cation Specificity
MGBG Uptake
(|iM/20mlnutes)
% Inhibition
None
24.1 ±1.45
0
Valinomycin
K+
23.5 ± 1.05
2.5
Monensln
Na+
19.0 ± 0.65*
21.0
Gramicidin A
Na+, K+
10.0± 0.45**
58.3
A23187
Ca++
16.4 ± 0.4*
32.1
Note : L1210 cells were preincubated with the individual ionophores (5 jxM) for 20
minutes in Earle's balanced salt solution prior to an addition of [14C] MGBG (6
(xM final concentration). MGBG uptake was measured at 0, 20 and 40 minutes
after adding MGBG. Pellet radioactivity was determined as described earlier.
Each value represents the mean ± SEM, of three separate experiments, each
conducted in triplicate (* p < 0.05, ** p < 0.01).

pellet [MGBG]
43
â–  control
E3 antimycln A
B 2,4-DNP
â–¡ KCN
time (min.)
FIGURE 3-18 : Effects of inhibitors of energy metabolism on MGBG uptake.
L1210 cells were pretreated with 0.01 mM Antimycin A, 0.1 mM 2,4-
DNP or 0.1 mM KCN in PBS solution for 20 minutes, prior to addition
of [14C] MGBG (5 pM final concentration). The cells were incubated in
[14C] MGBG for the indicated times. Values are expressed as
intracellular concentrations of MGBG (pM).

% control uptake
44
â–  Antimycln A
0 2,4-DNP
El KCN
time (min.)
FIGURE 3-19 : Effects of inhibitors of energy metabolism on MGBG uptake.
L1210 cells were pretreated with 0.01 mM Antimycin A, 0.1 mM 2,4-
DNP or 0.1 mM KCN in PBS solution for 20 minutes, prior to addition
of [14C] MGBG (5 pM final concentration). The cells were incubated in
[14C] MGBG for the indicated times. Values are expressed as percent
of control (no inhibitor).

45
with the inhibitors for 20 minutes at 37°C prior to the addition of [14C] MGBG.
These uptake studies were performed in the phosphate-buffered saline without
glucose to minimizing energy derived from glycolysis. The uptake of drug at 15,
30 and 60 minutes is expressed as an intracellular concentration of MGBG (pM)
in Figure 3-18 and as a percentage of control uptake in Figure 3-19. All are
corrected for rapid binding. The results reveal a significant decrease in the
accumulation of [14C] MGBG in the cells pretreated with the metabolic inhibitors
(antimycin » 2,4-DNP > KCN). The data suggest a requirement of energy for
the drug uptake.
Intracellular Distribution of MGBG
I was particularly interested in exploring whether or not the striking
intracellular accumulation of MGBG reflected accumulation of the drug into
mitochondria. Two factors prompted this interest: 1) the cationic nature of
MGBG; and 2) the effects of MGBG on mitochondrial structure, function and
replication. Attempts to preload the cells with [14C] MGBG and then isolate
mitochondria were not pursued vigorously because the procedure involved in
the preparation of a mitochondrial fraction from these cells would predispose to
redistribution of radiolabel. Rather, two other experimental approaches were
taken: 1) uptake of [14C] MGBG by isolated L1210 mitochondria; and 2) digitonin
permeabilization of the plasma membrane of cells preloaded with [14C] MGBG.
Uptake of MGBG bv Isolated Mitochondria
Exponentially-growing L1210 cells were incubated with 0.1% w/v digitonin
in dimethylsulfoxide (DMSO) until approximately 70% of the cells no longer
excluded trypan blue. Cells were then washed once with a solution containing
250 mM sucrose, 2 mM EDTA and bovine serum albumin (1 mg/ml) at pH 7.40.
The cell pellet was immediately homogenized on ice with a 2 ml tapered glass
tissue grinder until more than 90% of the cells were ruptured. The homogenate

46
was centrifuged at 1000g for 10 minutes. The resulting supernatant fraction
was centrifuged at 16,000g for 15 minutes to obtain the mitochondrial fraction.
The respiratory control ratio (RCR) or the state 3/state 4 respiratory ratio of
the mitochondrial fraction was 2.0 to 2.5; with intact cells the RCR was 3.0 to
3.5. Oxygen consumption was measured with an oxygen electrode with
succinate as substrate in an incubation medium containing 150 mM sucrose, 25
mM glycylglycine, 40 mM KCI, and bovine serum albumin (1 mg/ml).
Mitochrondrial pellets were resuspended in 2.5 pM [14C] MGBG in RPMI
1640 medium at 37°C, pH 7.40. Radioactivity in the mitochondrial pellet was
measured after 0, 0.5, 1,2, and 3 hours of incubation. The mitochrondrial pellet
was collected by centrifugation and washed once in medium containing 1 mM
unlabelled MGBG before recentrifugation. The uptake of [14C] MGBG by
mitochondria (in pmoles per mg protein) is presented in Table 3-4. The
concentration of [14C] MGBG in the mitochondrial pellet even after three hours of
exposure to 2.5 pM drug is less than 2.5 pM (for cells, about 15 pmoles per mg
protein is equivalent to 2.5 pM MGBG). Furthermore, mitochondrial uptake is
not likely to be the driving force for sequestration of MGBG within the cell
because the observed rate of uptake is only about 1% of that predicted by the
kinetic constants determined by experiments with intact cells.
Selective Release of Cytosolic Constituents by Diqitonin
Because of its low content of cholesterol, the inner membrane of
mitochondria is more resistant than the plasma membrane to disruption by
digitonin. I used this difference to assess whether or not the MGBG that had
accumulated within L1210 cells was predominately localized in mitochondria;
rhodamine 123 was used as a positive control since it is well established that
this cationic dye is highly concentrated within mitochondria (Johnson £t al-.
1981).

L1210 cells were exposed to 0.5 pM [14C] MGBG for 2 to 8 days. In addition
to determination of the apparent intracellular concentration of MGBG, one-ml
aliquots of cells were admixed gently in an ice bath with enough 0.1% (w/v)
digitonin in DMSO to cause 90 to 95% of the cells to become permeable to
trypan blue. The [14C] MGBG remaining in the pellet of the intact and
permeabilized cells was compared (Figure 3-20). Less than 6% of the drug in
the intact cells was recovered in the pellet of cells with disrupted plasma
membranes.
Concurrent experiments with rhodamine 123 provided evidence that a
compound that is accumulated by mitochondria remained within the cell after
this treatment with digitonin. One million 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 at the
final concentration of 1 pg/ml. The samples were incubated at 37°C for 10
minutes and washed once by centrifugation. The stained cells were observed
under a Zeiss epifluorescence microscope and were virtually indistinguishable
(in terms of mitochondrial fluorescence at an excitation wavelength of 485 nm)
from cells not disrupted by digitonin.
The experiments with both isolated mitochondria and digitonin lead me to
conclude that the large accumulation of MGBG within the cells involved active
transport of MGBG across the plasma membrane, some degree of intracellular
binding, and passive equilibration with the various intramitochondrial
compartments.
Effect of Growth Rate of L1210 cells on MGBG Transport
L1210 cells were seeded at an initial concentration of 3 x 104 cells/ml and
incubated at 37°C for five days without reseeding or adding fresh medium. The
cells divided exponentially until their concentration reached 1.8 x 106 cells/ml

48
TABLE 3-6 : Uptake of MGBG by isolated mitochondria a.
Time of incubation in
MGBG (hrs)
Mitochondrial MGBG
(pmole/mg protein)
% of expected intracellular
MGBG b
0.5
0.50 ±0.02*
0.36
1
3.83 ±0.04
1.35
2
1.94 ±0.07
0.35
3
2.68 ±0.06
0.32
Note : a The mitochondrial fraction obtained from L1210 cells was incubated in 2.5 p.M
[14C] MGBG for 0 - 3 hrs. The radioactivity in the mitochondrial pellet was determined
after two washes with ice-cold 1 mM MGBG. The results were expressed as pmole
mitochondrial [14C] MGBG/mg protein (* mean ± SEM ).
b I assumed for this calculation that the uptake of MGBG by L1210 cells was due
totally to accumulation of the drug in mitochondria. The kinetic constants obtained from
experiments with intact cells were used to predict rates of uptake by mitochondria. It
can be seen that the actual rates of uptake were only about 1 % of the prediction.

pellet cpm/million cells
49
gf control
gg digitonized
* p < 0.0001
Time (days)
FIGURE 3-20 : MGBG remaining in the cell pellet after permeabilization of the
plasma membrane by digitonin. L1210 cells were exposed to 0.5 pM [14C]
MGBG for 2 to 8 days. Prior to determination of the apparent intracellular
concentration of MGBG, one-ml aliquots of cells were admixed gently in an
ice bath with enough 0.1% (w/v) digitonin in dimethylsulfoxide until
approximately 95% of the cells became permeable to trypan blue dye. The
[14C] MGBG remaining in the pellet of the intact and permeabilized cells
was determined and compared.

50
(Figure 3-21). Plateau growth ensued when the concentration of cells reached
2 x106 cells/ml. Cell viability exceeded 95% when the cells were in logarithmic
growth, but it declined progressively to 40% by the fifth day of incubation when
the cells were at plateau (Figure 3-22).
Cells in logarithmic growth were compared to those in early plateau with
regard to transport of MGBG. L1210 cells at the two growth rates were
reseeded in fresh media containing 0.5 pM [14C] MGBG at starting
concentrations of 1 x 106 and 2 x 106 cells/ml, respectively. The intracellular
content of radiolabel was determined at 0, 24 and 48 hours after the start of
incubation at 37°C, pH 7.40. The accumulation of MGBG was higher in the
logarithmically-growing cells (Figure 3-22). Kinetic experiments with cells just
entering plateau phase revealed that the decrease in uptake of MGBG in these
cells reflected more on an increase in Km (9.04 ± 0.34 pM, P < 0.01) than a
decrease in Vmax (0.271 ± 0.019 nmoles/106 cells/hour) relative to cells in
exponential growth.

cumulative cell count
51
FIGURE 3-21 : Growth curve of L1210 cells. L1210 cells were seeded at an initial
concentration of 3 x 104 cells/ml and incubated for 5 days without reseeding
or adding fresh medium. Cell counts were determined by electronic particle
analysis (Coulter Counter).

% cell viability
52
FIGURE 3-22 : Viability of L1210 cells as a function of growth phase. L1210 cells
were seeded at an initial concentration of 3 x 104 cells/ml and incubated for 5
days without reseeding or adding fresh medium. Cell viability was
determined with use of Trypan Blue dye exclusion method.

|j.M pellet [MGBG]
53
FIGURE 3-23 : Uptake of MGBG as a function of growth rate. Exponentially
growing L1210 cells were seeded at initial concentrations of 0.1 - 2.0 x 106
cells/ml to attain different subsequent rate of growth. In addition, cells
already in the plateau phase were seeded at 2.0 x 106 cells/ml. All cells
were exposed to fresh medium containing 0.5 pM [14C] MGBG at starting
concentrations of 1 x 10^, 3 x 10^ and 2 x 10^ cells per ml. The intracellular
content of radiolabel was determined at 0, 24, and 48 hours after the start of
incubation.

CHAPTER 4
TRANSPORT OF MGBG AND DRUG RESISTANCE
When L1210 cells were treated with MGBG at concentrations that were low
enough (< 5 pM) to allow survival of some cells, the residual cells reacquired
the control rate of proliferation within one to three weeks (Figures 3-1 and 3-2)
despite the continued presence of MGBG in the medium. As will be
documented below, this resistance seems to reflect a decreased
intracellular/extracellular concentration ratio of MGBG. This Chapter describes
the changes that account for this decrease in the intracellular accumulation of
MGBG. As discussed in Chapter 3, MGBG had accumulated in cells for the first
20 hours of exposure to drug in accord with a saturable process with a Vmax of
296 ± 19 pmole/106 cells/hour and a Km of 4.72 ± 0.17 pM. Our focus now
turns to subsequent 19 days of exposure.
Concentration of MGBG in L1210 Cells Purina 20 Davs of Drug Exposure
This experiment resembles that depicted in Figure 3-1 in which the effects of
varying concentrations of MGBG on L1210 cell growth were ascertained except
that the current experiment incorporated the use of [14C] MGBG to allow
intermittent measurement of radiolabel in the cell pellet. Logarithmically-
growing L1210 cells were reseeded every two days at a density of 8 x 104
cells/ml in media at 37°C, pH 7.40 containing 0.1 to 5.0 pM [14C] MGBG.
MGBG, 0.1 pM, has minimal, if any, effect on growth or viability. MGBG, 5.0 pM,
is occasionally compatible with long-term cell survival. The intracellular content
54

55
of [14C] MGBG was determined intermittently (Figure 4-1). The apparent
intracellular concentration of MGBG depended both on the concentration of
drug added to the medium and on the duration of exposure. At 1.0 pM MGBG in
the medium, the intracellular concentration of drug reached 2000 pM about 24
hours after the start of incubation (as described in Chapter 3). Despite
continued presence of the drug in the medium (including reseeding the cells
into fresh drug every 48 hours), the intracellular concentration of MGBG
decreased to 500 pM by day 6 and 100 pM by day 20. The same pattern
applied to all doses of MGBG as long as they were high enough to affect cell
growth; peak intracellular concentrations occurred between 24 and 72 hours
after the start of the incubation and decreased thereafter. When the
concentration of MGBG was too low to have a detectable effect on the growth
rate or viability of L1210 cells, no such maximum was seen.
Although it is tempting to attribute, or at least relate, this decrease in
intracellular content of MGBG to the decrease in growth rate that occurs a few
days after the start of exposure to MGBG, the rate of cell proliferation returned to
the control value within two weeks of exposure to MGBG (Figure 3-1), a time
when the intracellular/extracellular ratio of MGBG is still decreasing.
Intracellular content of putrescine, spermidine and spermine in L1210 cells
was determined with use of high performance liquid chromatography (HPLC) in
our laboratory by M. Kelly (Table 4-1). By 48 hours of exposure in 0.5 pM
MGBG, the intracellular content of putrescine increased while the levels of
spermidine and spermine had decreased. This result was anticipated because
MGBG is an effective inhibitor of S-adenosylmethionine decarboxylase. By day
20, when rate of cell proliferation had recovered, the intracellular
concentrations of polyamines had also returned to control.

|xM pellet [MGBG]
56
FIGURE 4-1 : Intracellular content of MGBG upon prolonged exposure to the drug.
Logarithmically-growing L1210 cells were reseeded every two days at a
density of 8 x 104 cells/ml in media containing 0.1 to 5.0 pM [14C] MGBG.
The intracellular content of [14C] MGBG was determined periodically.

57
TABLE 4-1 :
: Effects of MGBG on the intracellular content of polyamines and
putrescine in L1210 cells.
Treatment
Intracellular content (pmole/million cells, mean ± SEMI
Putrescine Spermidine Spermine Total
Control
282 ±6 2577 ±81 612 ± 17 3483 ±120
Cells treated with 0.5 pM MGBG for 48 hours
1838 ± 49** 1334 ±39* 344 ± 35* 3574 ± 82
Cells treated with 0.5 pM MGBG for 20 days (after recovery)
244 ±27 2494 ±201 537 ± 26 3275 ± 249
Note : Intracellular polyamine and putrescine content was determined with use of high
performance liquid chromatography ; ** p < 0.01, * p < 0.05 when compared to the
control.

58
Kinetic Characterization of MGBG Transport as Resistance Develops
An experiment was designed to compare rates of influx and efflux of MGBG
before and after the peak intracellular concentration had been attained. The
cells were studied 4 hours and 156 hours (6.5 days) after the start of incubation
in 1.5 pM MGBG. The experiment was designed in this way in order to compare
transport in cells with an intracellular concentration of MGBG of about 400 pM
on the increase (4 hours) and on the decrease (156 hours). To be able to study
both influx and efflux in the same sets of cells, the L1210 cells were incubated
in either radioactive or nonradioactive MGBG and crossed over at 4 hours or
156 hours. For example, to study efflux after exposure to MGBG for 156 hours,
cells were incubated in 1.5 pM [14C] MGBG for 156 hours at which time the
intracellular concentration of [14C] MGBG is about 400 pM. The cells were then
collected, washed quicky, and reseeded in fresh medium containing 1.5 pM
nonradioactive MGBG , and release of label into the medium was followed.
The results depicted in Figures 4-2 and 4-3 reveal that the rate of influx of
MGBG had decreased more than two fold between 4 and 156 hours whereas
the rate of efflux remained unchanged. Within 156 hours of MGBG exposure,
the rate of influx was approximately the same as the rate of efflux (Figure 4-4),
i.e. steady-state had nearly been obtained. This observation explains the
relative stability of intracellular concentrations of MGBG thereafter.
A more detailed kinetic analysis was obtained with cells that had been
exposed to 0.5 pM MGBG for one month. After one month of such treatment, the
Km for MGBG uptake at 37°C, pH 7.40 had increased from 4.7 ± 0.2 to 24.9 ±
1.4 pM (P < 0.01) and the Vmax had decreased from 296 ± 19 to 142 ± 13
pmole/106 cells/hour (P < 0.01). If the cells were incubated in 1.0 pM MGBG,
the calculated rate of influx of drug would be less than 10% of what it had been
during the first 20 hours of exposure. These changes in the kinetic properties of

pmole MGBG/0.1 million cells
59
hrs. after crossover
— Day 0 influx
*— Day 6 influx
FIGURE 4-2 : Influx of MGBG before and after the peak intracellular concentration of
drug had been attained. L1210 cells were incubated in 1.5 pM MGBG for 4 or
156 hours before centrifugation, washing, and resuspension in fresh medium
containing 1.5 pM [14C] MGBG. The intracellular content of [14C] MGBG was
determined periodically from 0 - 26 hours after crossover.

pmole MGBG/0.1 million cells
60
hrs. after crossover
■a— Day 0 efflux
-«— Day 6 efflux
FIGURE 4-3 : Efflux of MGBG before and after the peak intracellular concentration of
drug had been attained. L1210 cells were incubated in 1.5 pM [14C] MGBG
for 4 or 156 hours before centrifugation, washing, and resuspension in fresh
medium containing 1.5 pM MGBG. The intracellular content of [14C] MGBG
was determined intermittently for 0 - 26 hours after crossover.

(iM pellet [MGBG]
61
D6 influx
D6 efflux
«— D6 Total
time (hours) after crossover
Figure 4-4 : Influx, efflux and apparent steady-state intracellular content of MGBG on
day 6 of exposure to drug. L1210 cells were incubated in 1.5 pM MGBG or
[14C] MGBG for 156 hours before centrifugation, washing and resuspension
in fresh medium containing 1.5 pM [14C] MGBG or MGBG, respectively. The
intracellular (pellet) content of t14C] MGBG was determined intermittently for
0 - 26 hours after crossover. The "D6 Total" curve (p) represents the sum of
intracellular radioactive MGBG obtained from the influx and efflux
experiments.

62
the influx process can explain the gradual decrease in the intracellular content
of MGBG that precedes the recovery of control rates of proliferation.
In Chapter 3, data were presented concerning the relative capacities of
putrescine, spermidine and spermine to compete with MGBG for uptake. Since
the kinetic characteristics of MGBG uptake had changed upon emergence of
resistance, I explored whether or not the K¡'s for putrescine, spermidine and
spermine had changed also, in order to gain some insight into the nature of the
altered transport system. The results are presented in Figure 4-5. The Kfs for
each compound had increased in rough proportion to the increase in Km for
MGBG. This finding suggests that the apparent substrate specificity of the
transport system in resistant cells had not changed.
When these cells, namely, those exposed to 0.5 pM MGBG for one month,
were transferred to drug-free medium, they did not reacquire the capacity of
untreated cells to accumulate MGBG even after incubation in drug-free medium
for 20 days (Figure 4-6). Kinetic studies were conducted with cells that had
been grown in drug-free medium for one month (Table 4-2). By this time Vmax
had returned to control value (321 ± 19 pmole/106 cells/hour) but Km had
remained increased (11.57 pM; P < 0.01 compared to control cells.
Selection of L1210 Cells Highly Resistant to MGBG
Another approach that I have taken to explore the mechanism of resistance
to MGBG involved the selection of L1210 cells presumably resistant on a
genetic basis. L1210 cells were incubated in 5 pM MGBG for two weeks and
then reseeded in medium containing 10 pM MGBG. A clone of cells retained
striking resistance to MGBG even when grown in drug-free medium for months.
When grown in drug-free medium, these resistant cells divided slowly, with a
doubling time nearly twice that of untreated cells (16 to 20 vs. 8 to 10 hours).
The dose-response curves for the resistant line of cells with MGBG are

1/pmole MGBG/10e6 cells/hr.
63
â–¡ control
• 25 p.M spd
â–  25 p.M spm
o 25 (iM putres
FIGURE 4-5 : Competitive uptake inhibition of MGBG uptake by spermidine,
spermine and putrescine in L1210 cells exposed MGBG for 3 weeks.
L1210 cells which were exposed to 0.5 pM MGBG for 3 weeks, were kept in
drug-free media for 1 week prior to this study. The cells were incubated in
the media containing different concentrations of [14C] MGBG (4.0 -117.5
pM) with or without 25 pM spermidine, spermine or putrescine. The Kfs of
spermidine, spermine and putrescine for inhibition of the uptake of MGBG
were 7.9 + 0.3, 12.0 + 0.4 and 20.9 + 0.7 pM, respectively.

|iM pellet [MGBG]
64
drug-free period
o— 20 days
•— 12 days
o— 3 days
—o— control
drug-naive
Time (min.)
FIGURE 4-6 : Uptake of MGBG in the " recovered " L1210 cells. L1210 cells that
had been exposed to 0.5 pM MGBG for one month, were transferred to
drug-free medium for 3 - 20 days. These cells were subsequently reseeded
in a medium containing [14C] MGBG (20 pM). and the uptake of drug was
followed for 75 minutes.

65
TABLE 4-2 : Kinetics of the uptake of MGBG in untreated L1210 cells, cells that
had "recovered" after exposure to MGBG, and a resistant subclone.
Cell Type and Treatment
Km (pM)
(mean ± SEM)
Vmax (pmole/106 cells/hr.
(mean ± SEM)
Control
4.72 ± 0.17
296 ± 19
Cells exposed to 0.5 pM
MGBG for 30 days and then
- 2 days drug-free
24.87 ± 1.36*
142 ± 13*
-1 month drug-free
11.57 ±1.63*
321 ± 19
MGBG-resistant subclone
- 3 days drug-free
16.18 ± 0.52*
134 ± 15*
- 2 month drug-free
5.14 ±0.48
145 ± 14*
Note : * p < 0.01 (vs control)

66
presented in Figure 4-7 (growth) and Figure 4-8 (viability). Logarithmically-
growing resistant cells were seeded at a starting concentration of 1.05 x 105
cells/ml in media containing 0 to 30 pM MGBG. The cells were reseeded into
fresh medium containing MGBG 2 days later. In comparison to the curves
depicted in Figures 3-1 and 3-2 with untreated L1210 cells, these cells exhibit
striking resistance to MGBG. Even after growth in drug-free medium for six
weeks, resistance to MGBG was retained (Figures. 4-9 and 4-10).
Transport Properties of the Resistant Cells
When these resistant cells were exposed to 2.0 to 6.7 pM [14C] MGBG at
37°C, pH 7.40, they accumulated significantly less MGBG than did control
L1210 cells (Figure 4-11). This decreased accumulation correlated with an
increased Km (16.2 ± 0.5 compared to 4.7 ± 0.2 pM; P < 0.01) and a decreased
Vmax (134 ±15 compared to 296 ± 19 pmole/106 cells/hour; P < 0.01). After six-
weeks of growth in drug-free medium, Vmax remained low (145 ±14 pmole/106
cells/hour), the cell doubling time remained long, but Km had returned to the
control value (5.1 ± 0.4 pM).
Once again, the relative affinities toward putrescine, spermidine and
spermine of the transport system in resistant cells, as assessed by K¡'s for
inhibition of MGBG uptake, remained unchanged (Figure 4-12).
Response of multiple drug resistant (MDFO cells to MGBG
Two Chinese hamster ovary (CHO) cell lines were generously provided by
Dr. Gurmit Singh (McMaster University, Ontario, Canada). The parental line
(Aux Bi) is drug sensitive. The derived line (CHRC5) exhibits multiple drug
resistant and contains P-glycoprotein. The selection of CHRC5 is described by
Ling and Thompson in1974. Briefly, cross-resistance to a variety of antitumor
antibiotics in these CHO cell line was induced by exposure to increasing
concentrations of colchicine. This pleotropic or multiple drug, resistance (MDR)

cumulative cell count
67
Time (hrs.)
FIGURE 4-7 : Effects of various concentrations of MGBG on growth of MGBG-
resistant L1210 cells. Logarithmically-growing resistant cells (see text)
were transferee! to drug-free medium for 3 days prior to the study. The
cells were reseeded at a starting concentration of 1.05 x 105 cells/ml in
media containing 0 - 30 pM MGBG. The cells were maintained in
logarithmic growth by reseeding into fresh medium containing MGBG at 2
days.

% cell viability
68
Time (hrs.)
MGBG (p.M)
—□— none
—* 1-0
—a— 10.0
—o 20.0
—m— 30.0
FIGURE 4-8 : Effects of various concentrations of MGBG on viability of MGBG-
resistant L1210 cells. See Figure 4-7: viability was assessed by the
Trypan Blue dye exclusion method.

cumulative cell count
69
Time (days)
FIGURE 4-9 : Effects of various concentrations of MGBG on growth of the MGBG-
resistant L1210 cells after one month of growth in drug-free medium.
MGBG-resistant cells (see text) were maintained in drug-free medium for
one month prior to the study. Cells in logarithmic growth were seeded in
media containing 0 - 50 (iM MGBG. Cell counts were determined daily
with use of a Coulter Counter and the cells were reseeded into fresh
media with or without drug at 2 days.

% cell viability
70
Time (days)
FIGURE 4-10 : Effects of various concentrations of MGBG on viability of the
MGBG-resistant L1210 cells after one month of drug-free period. Cell
viability was determined daily with use of the Trypan Blue dye exclusion
method (see Figure 4-9).

|iM pellet [MGBG]
71
Time (hrs.)
FIGURE 4-11 : Accumulation of MGBG in the MGBG-resistant L1210 cells. The
resistant cells (see text) were maintained in a drug-free medium for 3
weeks prior to the study. The logarithmically-growing cells were seeded in
various concentrations of [14C] MGBG for 0 - 48 hours and the intracellular
content of [14C] MGBG was determined at the times indicated. For
comparison, a previously untreated L1210 cell exposed to 1 pM MGBG
would be expected to exhibit an intracellular concentration of MGBG of
about 2000 pM by 24 hours.

1/pmole MGBG/10e6 cells/hr.
72
n control
• 25 |iM spd
â–  25 |iM spm
• 25 (iM putres
FIGURE 4-12 : Competitive inhibition of MGBG uptake by spermidine, spermine
and putrescine in MGBG-resistant L1210 cells. MGBG-resistant L1210 cells
were kept in drug-free medium for 1 week prior to this study. The cells were
incubated in the media containing different concentrations of [14C] MGBG
(4.0 - 117.5 pM) with or without 25 pM spermidine, spermine or putrescine.
The K¡'s of spermidine, spermine and putrescine for inhibition of the uptake
of MGBG were 2.68 ± 0.09, 2.51 ± 0.08 and 9.33 + 0.10 pM, respectively.

73
was associated with the presence of a 170 KDa membrane glycoprotein
(Juliano and Ling, 1976). This protein is termed the P-glyco prote in because of
its association with the plasma membrane. It behaves as an efflux pump and
hence, decreases intracellular accumulation of antineoplastic agents in
resistant cells. Both CHO cell lines were grown in alpha-modified essential
medium (alpha-MEM) with L-glutamine, ribonucleosides and
deoxyribonucleosides (GIBCO Laboratory). The medium was supplemented
with 10% fetal bovine serum (FBS), streptomycin and penicillin. The cells were
grown in Petri dishes and incubated at 37°C in 5% CO2 : 95% air. The sensitive
and MDR CHO cell lines were exposed to different concentrations of MGBG (0 -
10 |iM) for 24 to 72 hours. The initial cell number plated was such that the
control cells would not reach confluence before day 3, when the inhibitory effect
of the drug was evident. After 46 or 72 hours of MGBG exposure, the medium
was removed and the cells were trypsinized. Trypsinization was stopped by
adding two volumes of medium containing 10% FBS. The dishes were gently
shaken to dislodge the cells and repipetted to break up any clumps before
counting the cells by a Coulter Counter. Cell viability was determined with use
of trypan blue dye exclusion method.
Due to the fact that the doubling time of the Aux B1 cells is shorter than the
CHrC5 cells, the sensitivities to MGBG of these two cell lines were compared
after exposure for approximately 3 doubling times (46 and 72 hours for Aux B1
and CHrC5 cells, respectively). The results depicted in Figure 4-13 reveal that
there is no difference in the sensitivity of the CHRCs and the Aux B1 to the
antiproliferative activity of MGBG. This finding suggests that different
mechanisms of resistance exist in the MDR and the MGBG-resistant cells.
Correlation between Transport and Resistance
It is important to ask whether or not "all" of the resistance to MGBG that I

% control growth
74
* CHR C5 day3
MGBG (|iM)
FIGURE 4-13: Comparison of MGBG dose-response curve of parental drug-
sensitive (Aux B1) and multidrug resistant (CHRC5) cell lines. The two CHO
cell lines were exposed to different concentrations of MGBG (0-10 pM) for
0 - 3 days (2 days for Aux Bi cell line and 3 days for CHRC5 cell line). Cell
counts were determined by electronic particle analysis (Coulter Counter)
and cell viability was determined with use of the Trypan Blue exclusion
method. The results are expressed as percent control growth (no drug).

75
observed in various experiments, can be attributed to changes in transport of
MGBG (i.e., in the accumulation of drug within cells). I have attempted to
approach this question by comparing untreated cells, "adapted" cells (0.5 pM
MGBG for one month) and selected cells (as defined above) with regard to
intracellular concentration of MGBG and rates of growth. There was a linear
relationship (r = 0.91) between intracellular concentration of MGBG at 24 hours
and growth rate over the following day, regardless of the type of cell involved
(Figure 4-14). in other words, the data from all cells fit the same line. Growth
rates between 0 and 48 hours have also been analyzed with similar results.
These results indicate that virtually all of the resistance that develops can be
explained by changes in transport and not by some intrinsic change in
sensitivity to MGBG within cell.

% control growth (24-48 hrs.)
76
100
80
60
40
20
0
|iM pellet [MGBG] by 24 hrs.
L1210 cells
â–¡ control
o 0.5 pM exp
h selected resist
FIGURE 4-14 : Correlation between intracellular content of MGBG and the rate of
cell proliferation in various type of L1210 cells. Untreated L1210 cells,
L1210 cells that "recovered" after exposure to 0.5 pM MGBG, and MGBG-
resistant subclone (see text), were exposed to various concentrations of
[14C] MGBG for 0 - 48 hours. The intracellular content of [14C] MGBG was
determined at 24 hours. Cell counts were determined at 0, 1 and 2 days
with use of a hemacytometer, and cell viability was determined by Trypan
Blue dye exclusion method.

CHAPTER 5
DISCUSSION
Discussion will focus on: 1) the mechanism of MGBG transport, and 2)
resistance of L1210 cells to MGBG.
Mechanism of MGBG Transport
As reported earlier (Field £lal-, 1964, Dave and Caballes, 1973, and Porter
filal-, 1981), MGBG was found to accumulate extensively in the cell pellet. The
extensive accumulation of MGBG we observed, was not due to surface binding
since the radiolabelled MGBG in the cell pellet could not be displaced by 1 mM
MGBG. The fact that nongrowing L1210 cells do not accumulate MGBG also
makes significant adsorption of MGBG to the cell surface most improbable as an
important quantitative factor in accumulation. All of our experiments suggested
that the major gradient in MGBG concentration was across the plasma
membrane. Accumulation of MGBG inside the cells was time-dependent, and
the rate of accumulation was linear with time for more than 20 hours.
Interestingly, accumulation of the drug over 20 hours could be predicted by
kinetic constants from 20-minute experiment (Table 3-1).
Reported effects of MGBG on mitochondrial structure and bioenergetic
functions were described in Chapter 1. Even though mitochondria seem to be
an important site of MGBG’s action, studies with use of isolated mitochondria
and digitonin revealed that mitochondria were not the driving force for extensive
77

78
accumulation of MGBG inside the cells. In other words, there was no
suggestion of an appreciable gradient of MGBG between cytosol and
mitochondrial compartments. Most of the intracellular drug was likely to be in
the cytosol. The actual free or unbound concentration of MGBG in the cytosol or
mitochondria remains unknown. MGBG is a weak base with pKa's of about 7.5
and 9.2 at 25 °C (Wiliams-Ashman and Seidenfeld, 1986). At physiological
pH, MGBG exists primarily as a mixture of monovalent and divalent cations, and
a large number and wide variety of binding sites likely exist intracellularly. As
noted above, the accumulation of MGBG was linear for at least 20 hours; this
result suggests the binding of MGBG to intracellular binding sites. Our
experiments with digitonin to permeabilize the cell membrane, however,
revealed an immediate release of the labelled drug with cytosolic components,
a result which suggests that MGBG was loosely bound to the intracellular
binding sites at least those associated with membranes or organells. Efflux of
MGBG was close to first order (Figure 3-8), another finding which implied that
most of the intracellular MGBG was either loosely bound or dissociated from
binding sites at an appreciable rate. In addition, because MGBG is relatively
poorly protonated at physiological pH compared to the natural polyamines,
MGBG would not likely bind as tightly as spermidine or spermine to anionic
polynucleotides and phospholipids or other intracellular molecules. The
extremely low abundance of S-adenosylmethionine decarboxylase
(AdoMetDC), diamine oxidase and spermidine/spermine N'-acetyltransferase
which are high affinity binding sites for MGBG, would preclude these enzyme
from binding more than a negligible fraction of the MGBG that is found within the
cells (Williams-Ashman and Seidenfeld et a!., 1986).
As mentioned earlier, MGBG is positively charged under physiological
conditions. It is unlikely that MGBG enters the cells by simple diffusion. Indeed,

79
the influx of MGBG exhibits saturation kinetics with apparent Km's of 2.10 + 0.08
and 9.34 + 1.04 pM, for the sodium-dependent and total processes,
respectively. Thus, it seems that the uptake of MGBG involves a carrier with
reasonably high affinity for MGBG. This carrier is likely also to be involved in
the transport of the naturally occurring polyamines, spermidine and spermine,
as well as putrescine, in that each of these compounds competed with MGBG
for uptake. This competition had been recognized previously (Dave and
Caballes, 1973, Janne §t aL, 1978, Porter el a]., 1981, Williams-Ashman and
Pegg, 1981, and Heby, 1981). Studies of countertransport also support the
notion of a carrier molecule. Interpretation of this experiment, however, is
complex because of the existence of naturally occurring polyamines within the
cells. The rates of uptake of MGBG were strongly influenced by pH (Figure 3-6).
An increase in extracellular hydrogen ion concentration enhanced the rate of
uptake of MGBG. These results suggested that the divalent form of MGBG
(MGBG2+) was the actual substrate for transport into the cells. This hypothesis
was supported further by the good agreement between the observed rate of
uptake and a rate predicted using values of MGBG2+ concentration based on
Henderson-Hasselbach computations (Table 3-2).
Even though normal and malignant cells in active growth could accumulate
MGBG (Field el al., 1964, Kramer el ai-, 1985), an intracellular concentration
2000 fold higher than that in the medium concentration was observed in our
studies (Figures 3-4 and 4-1). This result does not distinguish active transport
from facilitated diffusion coupled with intracellular binding. To pursue that
issue, studies were conducted to determine whether energy was required for
the accumulation of MGBG. The uptake of MGBG at low temperature (0 - 4°C)
was only about 10% of that of uptake at 37°C. Preincubation of cells with
inhibitors of energy metabolism (antimycin A, KCN and 2,4-DNP) decreased

80
and 2,4-DNP) decreased intracellular accumulation of MGBG. These results
would be anticipated if energy was required for the accumulation of the drug. In
addition, efflux of MGBG was also affected by temperature. At 0 - 4 °C, the
efflux of MGBG was barely detectable. It is often difficult to define the energy
requirement for active transport because of the potential nonspecific effects of
inhibitors of ATP synthesis.
The transport of sugars, amino acids, and numerous other solutes in many
cell types is observed to be driven against a chemical potential gradient by
cotransport with sodium ion down its electrochemical potential gradient. The
free energy dissipated by sodium ion movement down this gradient is coupled
with the increase in free energy associated with concentration of the substrate
within the cell. In various systems of this type, the carrier is proposed to be a
two-site molecule; one site accommodating sodium ion and the other
simultaneously accomodating the transported substrate (Schafer and Barfuss,
1986). We found that transport of MGBG in L1210 cells was sodium-dependent.
The sodium-dependent uptake process was consistent with a single transport
system in that the Lineweaver-Burk plot for sodium-dependent uptake was
linear. The dependency of MGBG uptake on sodium was rather specific
because the uptake of MGBG decreased when sodium was replaced by
choline, lithium, or especially, potassium or cesium. Even though there was a
decrease in the uptake of MGBG when sodium was replaced by choline, there
was a substantial amount of the intracellular accumulation of the drug. A
comparison of kinetics in these circumstances revealed that the presence of
sodium ion did not affect Vmax but did decrease Km or increase affinity toward
MGBG. The effect of sodium was therefore particularly important at low (pM)
concentrations of MGBG.
If the sodium electrochemical gradient is involved in MGBG transport,

81
dissipation of this gradient with specific antibiotic ionophores should inhibit
uptake of MGBG. The results summarized in Table 3-5 suggested that this was
indeed the case. The ion selectivity of ionophores is a combined function of the
energy required for desolution of the ion and the liganding energy obtained on
complexation (Simon and Morf, 1973 and Eisenman el a!., 1968). Valinomycin
which exhibits 10,000 : 1 preference for K+ (radius, r = 1.33 °A) over Na+ (r =
0.95 °A) in both biological and model system (Moore and Pressman, 1964,
Pressman, 1965, 1968, 1976), has no effect on the intracellular accumulation of
MGBG in L1210 cells. Monensin, which is more specific for Na+ than K+,
exhibited a moderate inhibition of MGBG accumulation whereas Gramicidin A, a
channel-forming ionophore which does not discriminate between Na+ and K+,
was the most potent inhibitor of MGBG uptake. The inhibitory effect of monensin
and gramicidin A implied the requirement of an electrochemical potential
gradient and the possible co-transport of Na+ with MGBG to enter the cells.
Although there was no direct evidence to support the cotransport of sodium ion
with MGBG in our studies, the fact that MGBG accumulated more than 2000-fold
within the cell, and the inhibitory effects of sodium ionophores, were strongly
suggestive. A23187 also caused a decrease in intracellular MGBG
accumulation. This compound is a carboxylic ionophore which possesses a
high selectivity for divalent over monovalent ions (Pfeiffer el a)., 1974), allows
Ca++ to enter cells (Reed and Lardy, 1972, Pressman, 1973, Pressman and
deGuzman, 1974,1975) and is capable of stimulating various Ca++-dependent
reactions without disturbing preexisting balances of Na+ and K+. The inhibitory
effect of A23187 on MGBG uptake might be due to the biological effects of high
intracellular level of Ca++. A23187 also transports Mg++, but gradients of this
ion across biological membranes seldom participate in biological control
(Pressman, 1976). Interpretation of the specific effects of ionophores is very

82
complex.
Resistance of L1210 Cells to MGBG
Studies in drug resistant tumor cell lines have identified many different
mechanisms for the development of resistance. In cell culture studies, three
major mechanisms of genetic resistance have been delineated and
characterized. These mechanisms include overproduction of the drug target
(e.g., gene amplification, Schimke el al, 1983, Rice el al., 1987, Somfai-Relle et
aL, 1984, and Jones, 1984), reduced drug permeability ( Mandel and Flintoff,
1978, Sirca et ai-, 1987, Rodrigues eta}.,1987, Waud, 1987, and Kraker and
Moore, 1988 ) and altered target interaction of the drug (Herman et a}., 1979,
Schabel ai-> 1982, Seebergl aL, 1982, Hunt el ai-. 1983, and Horns el ai-,
1983). Murine leukemia L1210 cells in our studies acquired a drug resistant
phenotype to MGBG during exposure to sublethal concentrations of drug. Our
data suggest that a decrease in drug uptake was responsible for this resistance.
We found that the influx rate of MGBG in L1210 cells started to decrease
significantly within a few days of exposure whereas the rate of efflux remained
unchanged. A separate subclone of genetically stable MGBG-resistant L1210
cells also exhibited a deficiency in drug transport which was characterized by a
significant decrease in Vmax and an increase in Km for MGBG transport. In both
"adapted" cells and the MGBG-resistant subclone, as well as in previously
untreated cells, an apparent intracellular concentration of MGBG less than 400
pM was required for toxicity. A similar quantitative relationship between the
intracellular concentration of MGBG 24 hours after exposure and the growth
rate over the following day was observed for all types of L1210 cells. It is
therefore unlikely that an intracellular response to a given concentration of
MGBG plays a significant role in the development of resistance. MGBG had
been reported to be metabolically inert (Warrel and Burchenal, 1983), and we

83
have confirmed that finding.
Two sites of MGBG action have received most attention with regard to its
antiproliferative activity: mitochondria and polyamine metabolism (inhibition of
AdoMetDC). Of these, as mentioned earlier, it appears that drug effects related
to mitochondrial integrity correlate best with the antiproliferative action of MGBG
in various cell lines (Pleshkewych et a|., 1980 , Mikles-Robertson et a|., 1979
and 1981, Corti £la|., 1974, Pathak ei£Í-, 1977, Wiseman gl ah, 1980 and 1983,
and Porter el al., 1979). In general, drug effects on parameters related to
polyamine metabolism fail to correlate with the antiproliferative action of MGBG.
In addition, there is no simple correlation between direct inhibition of AdoMetDC
and antileukemic activity in vivo for a series of MGBG analogs (Mihich, 1975,
Corti nial-, 1974, and Porter el a!-. 1981). Prolonged exposure to MGBG
actually increased activity of AdoMetDC. This might be related to an increase in
the enzyme synthesis (Pegg alai-. 1973, Pegg, 1984, and Fillingame and
Morris, 1973) or its stabilization (Williams-Ashman and Seidenfield, 1986).
I explored the relationship between multiple drug (MDR) resistance and
resistance to MGBG. Studies of Chinese hamster ovary (CHO) cells that were
resistant to the drug, colchicine (Ling and Thompson, 1974 and Ling, 1975),
revealed a specific alteration of the plasma membrane in multidrug resistance.
Drug-resistant cells contained a unique glycoprotein that was absent in the
drug-sensitive cells (Juliano and Ling, 1976). This glycoprotein was large in
size (molecular weight, about 170,000) and was named P-glycoprotein for its
association with the apparent permeability barrier at the plasma membrane to
drugs that were included in multidrug resistance. Further evidence suggested
that P-glycoprotein functions as a drug efflux pump (Gros el ai-, 1986 and Chen
£ia!-. 1986). Interestingly, resistance of MDR cells to colchicine, adriamycin
and other agents can be reversed by exposure to calcium channel-blocking

84
agents, a procedure which also reverses the defect in intracellular drug
accumulation (Tsuruo si ai-, 1982, Rogan gt ai-, 1984, and Willingham el ai-,
1986). Due to the fact that the efflux rate of MGBG in L1210 cells in our studies
remained unchanged after adaptation to MGBG, it is unlikely that resistance to
MGBG would be an MDR-type resistance. When parental and MDR CHO cell
lines were exposed to different concentrations of MGBG, both cell lines were
equally sensitive to the antiproliferative activity of the drug.
Our results were discussed earlier in terms of "adaptation" for the L1210
cells which had recovered within two weeks after exposure to 0.3 - 5 pM MGBG.
Several lines of evidence from our studies suggest that this resistance to MGBG
was due to adaptation of the cells to the drug, not selection of preexisting
mutant. In recent years, it has become increasingly evident that neoplasms
contain complex subpopulations of cells, each of which differs in expression of
biological properties. A concept now receiving increased attention is that drug
resistant neoplastic stem cells often arise from mutations, and that the drug-
resistant phenotype is inherited and propagated. The concept of the
development by spontaneous mutation of cancer cells that have resistance to
drugs they not yet encountered is called Goldie-Coldman hypothesis (Goldie
and Coldman, 1979). According to the Goldie-Coldman model, rates of
mutation give rise to resistance to a variety of antineoplastic agents in a variety
of mammalian tumor cells at a prevalence of 1 per 104 to 107 cells. The fact that
MGBG uptake by cells "adapted" to the presence of 0.5 pM had not returned to
the control transport rate even after removal of cells from drug for six weeks
raised the possibility that we had selected a mutant cells rather than caused an
"adaptation" of most cells. If we assume that only mutant cells can divide during
the course of MGBG exposure, the Y-intercept obtained from an extrapolation of
the MGBG dose-response curve of L1210 cells would reveal the frequency of

85
preexisting MGBG-resistant cells in overall cell population. At 0.5 pM MGBG,
this frequency was observed to be 1 in 104 cells (Figure 3-1), a value within the
estimated range denoted above. However, clonogenic experiments (Figure 3-
3) revealed that a large fraction of cells (> 60 %) was clonogenic even 72 hours
after exposure to 0.5 pM MGBG. In addition, the intracellular content of MGBG
had decreased significantly within a few days of initial exposure to the drug.
These results suggest strongly that most cells were adapting to the presence of
MGBG as opposed to, or at least in addition to, the selection of preexisting
mutants of L1210 cells that were constitutively deficient in MGBG transport. In
other words, the concept of preexisting mutant cells is unable by itself to explain
the sudden and significant decrease in the intracellular content of MGBG
within a few days after exposure to the drug. The importance of adaptive
regulation of transport is also supported by the kinetic results obtained midway
through the change in transport (after 6 days of incubation with drug). Had two
types of cells been present at this time (one with a Km of 4.72 pM and the other
with a Km of 24.87 pM for MGBG influx), I would have anticipated a nonlinear
Lineweaver-Burk plot; no hint of nonlinearlity was observed. Furthermore, a
comparison of the cells "adapted" to MGBG and those selected for resistance to
MGBG suggests that both types of cells exhibit an adaptive increase in a Km,
but a decrease in Vmax is characteristic only of the genetically stable subclone
and not of the "adapted" cells.

CHAPTER 6
SUMMARY AND FUTURE DIRECTIONS
Summary
We have studied the effect of MGBG on cell proliferation in murine leukemia
L1210 cells. Because these L1210 cells developed resistance to MGBG during
exposure, our experiments were designed to define mechanism of resistance to
MGBG in these cells and also in a clone of L1210 cells selected for resistance
to a lethal concentration of MGBG.
The important role of transport in the development of resistance to MGBG,
was first implied by a study of the apparent intracellular concentration of MGBG
for 12 days after exposure of previously untreated L1210 cells to drug. We
confirmed that MGBG accumulated quite extensively in these cells. The
apparent intracellular concentration of MGBG depended on both the
concentration of drug added to the medium and the duration of exposure. At 1
pM MGBG in the medium, the intracelllular concentration of drug reached
approximately 2000 pM about 24 hours after the addition of MGBG. Despite
continued presence of the drug in the medium, the intracellular concentration of
MGBG decreased to approximately 500 pM by day 6 and further to 100 - 400
pM thereafter. The same pattern applied to all doses of MGBG as long as they
were high enough to affect cell growth; peak intracellular concentrations
occurred 24 - 48 hours after the start of incubation and invariably decreased by
72 hours. Decrease in the intracellular accumulation of MGBG was due to a
86

87
decrease in the influx, not an increase in the efflux of MGBG. The rate of cell
proliferation returned to the control value within two weeks of exposure, a time
when the intracellular concentration of MGBG was < 400 pM. The dose-
response (cell proliferation) curve of these L1210 cells was shifted to the right
more than 10 fold. The lethal concentration of MGBG increased from 5 to about
50 pM in these "adapted" L1210 cells. Similar results were obtained in studies
with a subclone of genetically stable MGBG-resistant L1210 cells. Higher
extracellular concentrations of MGBG were required for the L1210 resistant
subclone to achieve a given intracellular concentration of MGBG. "Adapted"
L1210 cells (0.5 pM MGBG for 1 month) and MGBG-resistant subclone, as well
as untreated L1210 cells, were compared with regard to intracellular
concentration of MGBG and rate of cell growth. There was a linear relationship
between intracellular concentration of MGBG at 24 hours and rate of cell
proliferation over the following day, regardless of the type of cell involved. At
apparent intracellular concentration in excess of 400 pM, the growth rate of all
cells decreased.
Indirect methods were used to determine the intracellular distribution of
MGBG in L1210 cells. Mitochondria were unlikely to be the driving force for an
extensive intracellular accumulation of MGBG even though the effect of MGBG
was specific for mitochondria.
Transport mechanism of MGBG was also examined. The transport of MGBG
seemed to be energy dependent and carrier-mediated. It exhibited saturable
kinetics with a Vmax and Km of 296 ± 19 pmole/106 cells/hour and 4.72 + 0.169
pM, respectively. The rate of transport depended on temperature, pH and rate
of cell proliferation. Inhibitors of energy metabolism, ionophores, and the
replacement of sodium ion by other cations, all significantly decreased the rate
of accumulation of MGBG. The experiments with ionophores suggested that

88
uptake of MGBG depended on a plasma membrane electrochemical gradient.
The facilitation of uptake by sodium ions was compatible with a process in
which sodium increased the affinity of the carrier for MGBG, but two separate
processes, sodium-dependent and sodium-independent remain possible.
Resistance to MGBG was not associated with MDR-type resistance because
the sensitivity of the parental and MDR (containing P-glycoprotein) CHO cell
lines to MGBG, was indistinguishable.
Future Directions
Since transport of MGBG is the major determinant of sensitivity, and since
the MGBG gradient occurs at the plasma membrane, future directions focus use
of membrane vesicle technique to further characterize the transport of MGBG.
There are several interesting analogies with other known transport system, e.g.
for amino acid ( Rinehart and Chen, 1984 and Karl et aL, 1989) and/or some
organic cations which was resently reported by Sobol (1989), that should be
explored. It would also be of interest to examine the sodium dependency of
MGBG transport in the two types of resistant cells, "adapted" and MGBG-
resistant subclones. Understanding of the mechanism of regulation of the
MGBG transporter may lead to a discovery of new strategies for cancer
chemotherapy, either with MGBG or with novel analogs.

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BIBIOGRAPHICAL SKETCH
Sukanya Kanthawatana was born in 1959 in Saraburi, Thailand. She
received a Bachelor of Medical Science in 1981, and Doctor of Medicine in
1983 from Chiang Mai University Faculty of Medicine, Thailand. After her
internship training at Rajvithi Hospital in Bangkok in 1984, she worked as an
instructor at the Department of Pharmacology, Chiang Mai University Faculty of
Medicine. In 1985, she was selected through a vigorous and highly competitive
examination given annually by the Royal Thai Government Civil Service
Commission as a RTG scholarship recipient to further her knowledge in
pharmacology by enrolling in a Ph.D. program in the United States. Currently
she is also enrolled in the Pediatrics Residency Training Program at University
of Florida Shands Teaching Hospital. Upon returning to Thailand, she will
resume her work at the Chiang Mai University Faculty of Medicine and will be
responsible for setting up and running the Clinical Pharmacology Research Unit
and Center for Monitoring of Plasma Drug Levels.
Miss Kanthawatana is a member of Medical Council of Thailand and she is
licensed to practice medicine in Thailand. She is also a member of the Honor
Society for International Scholars, Phi Beta Delta.
94

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, Chair
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.
Edwin M. Meyer, Cocf$iir
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 of Doctor of Philosophy.
I c < G-& v- tt
Lai C. Garg
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.
Michael s. Kiiberg
Associate Professor of Biochemistry and
Molecular Biology

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.
us*'
*â–  Raymond J. Bergefpn
Professor of Medicinal Chemistry
â– >
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.
ns:
"Thomas C. Rowe
Assistant 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.
Bruce R. Stevens
Assistant Professor of Physiology
This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfilment of
the requirements for the degree of Doctor of Philosophy.
December 1989
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA
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UNIVERSITY OF FLORIDA
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