Group Title: Molecular Cancer 2004, 3:3
Title: N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA
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Title: N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA
Series Title: Molecular Cancer 2004, 3:3
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Creator: Jaiswal AS
Multani AS
Pathak S
Narayan S
Publication Date: 38002
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Volume ID: VID00001
Source Institution: University of Florida
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N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like
growth arrest in colon cancer cells is associated with loss of
adenomatous polyposis coli protein, microtubule organization, and
telomeric DNA
Aruna S Jaiswal1, Asha S Multani2, Sen Pathak2 and Satya Narayan*1


Address: 1UF Shands Cancer Center and Anatomy and Cell Biology, College of Medicine, Academic Research Building, Room R4-216, PO Box
100232, University of Florida, Gainesville, FL 32610, USA and 2Department of Molecular Genetics, unit & 011, The University, the University of
Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
Email: Aruna S Jaiswal ajaiswal@ufscc.ufl.edu; Asha S Multani amultani@mail.mdanderson.org;
Sen Pathak spathak@mail.mdanderson.org; Satya Narayan* snarayan@ufscc.ufl.edu
* Corresponding author



Published: 16 January 2004 Received: 18 December 2003
Molecular Cancer 2004, 3:3 Accepted: 16 January 2004
This article is available from: http://www.molecular-cancer.com/content/3/l/3
2004 Jaiswal et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.



Abstract
Background: Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and
altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the
present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is able to induce
senescence in the HCT- 1 6 colon cancer cell line.
Results: Cells treated with lower concentrations of MNNG (0-25 microM) for 50 h showed a dose-dependent increase
in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50-100 microM)
showed a senescence-like Go/GI phase arrest which was confirmed by increased expression of P-galactosidase, a
senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of
p53 protein, but the senescence-like Go/G phase arrest was dissociated with p53 protein levels, since the p53 protein
levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a
transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in
senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after
treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-
related proteins such as p21, p27, cyclin BI, Cdc2, c-Myc and max. The expression levels of these proteins were
increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells
treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role
in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals
with cellular senescence in MNNG-treated cells. We found that protein and mRNA levels of APC were drastically
reduced in cells treated with higher concentrations of MNNG. The loss of APC expression might lead to chromosomal
instability as well as microtubular disorganization through its dissociation with tubulin. In fact, the protein level of a-
tubulin was also drastically decreased in senescence-like arrested cells treated with higher concentrations of MNNG.
The levels of telomeric DNA also decreased in cells treated with higher concentrations of MNNG.
Conclusions: These results suggest that in response to DNA alkylation damage the senescence-like arrest of HCT-1 16
cells was associated with decreased levels of APC protein, microtubular organization, and telomeric DNA.




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Background
Cellular senescence is a biological process leading to irre-
versible arrest of cell division. It was initially described in
cultures of human fibroblast cells that lost the ability to
divide indefinitely [1]. The proliferative life-span of nor-
mal human cells is limited by the replicative or cellular
senescence [2,3]. The major feature of senescent pheno-
type includes an irreversible arrest of cell division, resist-
ance to apoptotic cell death, specific changes in cellular
functions, and senescent associated secretion of a variety
of molecules such as proteases, cytokines and growth fac-
tors [4]. Phenotypically, similar processes can be achieved
by accelerating senescence using various DNA-damaging
agents such as y-radiations [5,6], oncogenic stimulations
[7,8], and genetic or pharmacological manipulations [9].
It is evident from the literature that the loss of tumor sup-
pressor function is one of the major causes of transforma-
tion and immortalization of normal cells. Inactivation of
tumor suppressor gene p53 or Adenomatous polyposis
coli (APC) are among the most common causes of colon
cancer development [10-12]. Very often, the defective
expressions of tumor suppressor genes with cancer devel-
opment are linked with genetic instability. In colorectal
cancer, genetic instability occurs in two forms micro-
satelite instability (MSI) and chromosomal instability
(CIN) [13]. In MSI instability, there is a defect in mis-
match repair machinery that consequently results in the
instability of repetitive DNA sequences [14]. In CIN,
tumors exhibit a defect in chromosomal segregation,
which results in variation of chromosome numbers
among individual cells [15]. Recently, mutations in the
APC gene have been linked with CIN [16]. Mutations in
the APC gene produce truncated proteins. Many of the
somatic mutations in the APC gene are located in the cen-
tral region of the gene which is called as mutation cluster
region (MCR) [12]. Cellular levels of APC are critical for
maintaining cytoskeletal integrity, cellular adhesion, and
Wnt signaling [17-19]. APC also binds and stabilizes
microtubules in vivo and in vitro [17] and clusters at the
ends of microtubules near the plasma membrane of inter-
phase cells [16].

Another important aspect of APC is its transcriptional acti-
vation by p53 in response to DNA-damaging agents
[20,21]. The activation of p53 by DNA-damaging agents
induces cell cycle arrest, apoptotic cell death [22], or
senescence [23]. The role of p53 in cell cycle arrest in G,
phase is mediated by transcriptional activation of cyclin
dependent kinase (CDK) inhibitor p21(Waf-1/Cipl),
whereas in apoptosis it is mediated by transcriptional acti-
vation of mediators including p53 upregulated modulator
of apoptosis (PUMA) and p53-induced gene 3 (PIG3)
[24]. p53 also plays a role in cell cycle arrest in G2 phase
by transcriptionally activating the expression of 14-3-3 0
gene [25]. Biochemical and functional analysis of p53 has


also demonstrated its participation in the regulation of
DNA damage-induced senescence and DNA repair, which
can suppress tumorogenesis [22]. During cellular senes-
cence, the length of telomeric DNA also decreases, possi-
bly due to the absence of telomerase activity [26].
Senescent cells remain viable indefinitely and express spe-
cific phenotype markers, such as senescence-associated 3-
galactosidase [27]. In the present study, we investigated
whether DNA-damaging agent MNNG can induce senes-
cence-like cell cycle arrest in colon cancer cells. Data is
presented to determine whether in these cells the expres-
sion levels of p53, APC, a-tubulin and telomeric DNA are
associated with senescence-like cell cycle arrest.

Results
G2IM phase arrest, apoptosis, and senescence in HCT-I 16
cells treated with different concentrations of MNNG
Cellular senescence is a process of irreversible arrest of cell
division that can be induced by DNA-damaging agents
[6,28]. DNA-damaging agents can also activate p53-
dependent and -independent pathways leading to cell
cycle arrest and apoptosis. Since HCT-116 cells contain a
wild-type p53 and p21 genes, their role in G2/M phase
arrest and apoptosis is highly likely [29].

In order to asses the effect of DNA-damaging agent
MNNG on cell cycle arrest and apoptosis of HCT-116
cells, we treated the synchronized cells with varying con-
centrations of MNNG (0 to 100 iM) for 50 h. Cells were
harvested and analyzed for their distribution into differ-
ent phases of cell cycle by FACS analysis. We found an
increased G2/M phase arrest in cells treated with lower
concentrations of MNNG (0-25 iM), which dropped to
the control level in cells treated with higher concentra-
tions of MNNG (50 and 100 iM) (Figure 1). The number
of cells in the GQ/G1 phase decreased in a dose-dependent
manner as concentrations of MNNG treatment increased
to 25 iM, but beyond this concentration of MNNG treat-
ment, an increased GG/G1 phase arrest in HCT-116 cells
was evident. Interestingly, at 100 iM of MNNG treatment,
the HCT-116 cells showed a major change in their cell
cycle arrest profile in which the G,/G, phase arrest was sig-
nificantly increased, and the G2/M phase arrest was
decreased as compared to control cells. Furthermore, the
sub-G, cells, which represent the apoptotic cells, increas-
ingly accumulated until 50 iM of MNNG treatment and
then drastically decreased to the control level at 100 iM
of MNNG treatment (Figure 1). These results suggest that
HCT-116 cells exhibit a dual response, i.e., a G2/M phase
arrest and apoptosis and GQ/G1 phase arrest, respectively,
after treatment with lower or higher concentrations of
MNNG.






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S%
Go/G1 82
S 12
G2/M 7
sub-G1 13
-^ >-^---.-- --- -



S%
Go/G1 74
SS 17
G2/M 9
sub-G1 20





GoG1 70
S: S 17
G2/M 13
Ssub-G1 23




S%
GolG1 57
(1 1S 9
G2/M 35
sub-G1 28





Go/G1 72
S 16
G2/M 11
sub-G1 49




%
0 GO/G1 82
S 12
G2/M 6
sub-G1 9




Figure I
Cell cycle analysis in HCT-I 16 cells treated with MNNG. HCT- 116 cells were treated with different concentrations of
MNNG for 50 h. Cells were harvested and analyzed for cell cycle profile by FACScan analysis. The ranges of Go/G S, G2/M,
and sub-G, phase cells were established on the basis of the corresponding DNA content of the histograms. The data is repre-
sentative of three different experiments carried out independently. The Go/G I, S, G2/M and sub-GI cells are given as a percent-
age of the total counted cells.



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0 50


100


MNNG (gM)

Figure 2
Senescence associated B-Galactosidase staining in HCT-I 16 cells treated with MNNG. HCT- 116 cells were fixed
after treatment either with 50 or 100 jM MNNG and processed for P-galactosidase staining as described in Materials and
Methods. Stained cells were observed under the microscope and images were captured.


Senescence-like growth arrest in HCT- 116 cells treated
with higher concentrations ofMNNG
To examine whether increased G0/G, phase arrest of HCT-
116 cells treated with higher concentrations of MNNG
was due to increased senescence, we analyzed senescent-
associated B-galactosidase expression in untreated- and
MNNG-treated HCT-116 cells. Our result showed that
senescence-associated B-galactosidase staining increased
in cells treated with 100 jiM MNNG as compared to
untreated cells (Figure 2). We found no change in B-galac-
tosidase staining in HCT-116 cells either untreated or
treated with lower concentrations of MNNG (Figure 2).
These results suggest that treatment of HCT- 116 cells with
higher concentration of MNNG caused senescence-like
GQ/G1 phase arrest.

G2IM phase arrest and apoptosis, but not senescence-like
GolGI phase arrest, is associated with increased levels of
p53, p21 (waf- Icip- ), Cdc2/cyclin BI, and c-Myc proteins
in HCT-116 cells treated with higher concentrations of
MNNG
Since p53 is one of the important mediators of senescence
response, we analyzed the level of p53 protein in HCT-
116 cells treated for 50 h with varying concentrations of
MNNG. The increased p53 protein level was seen and cor-
related with G2/M phase arrest and apoptosis in HCT-116
cells treated with lower concentrations of MNNG. How-
ever, at higher concentrations ofMNNG treatment, a dras-


tically reduced level of p53 protein was observed. Since an
increased level of p53 protein is often associated with
senescence, the decreased level of p53 protein and
increased level of senescence in HCT-116 cells treated
with higher concentrations of MNNG suggests that the
role of p53 is probably not involved in MNNG-induced
senescence-like growth arrest in HCT-116 cells. We further
tested whether the decrease in the p53 protein level at 100
ItM MNNG treatment was a transcriptional or a post-
translational phenomenon. We carried out the Northern
blot analysis for p53 mRNA levels in HCT-116 cells treated
with different concentrations of MNNG. Results showed
an increased level of p53 mRNA up to 10 tiM MNNG
treatment. However, at higher concentrations of MNNG
treatment, a reduced level of p53 mRNA level was
observed (Figure 3). The decreased level of p53 mRNA at
25 tiM or higher concentrations of MNNG treatment did
not correlate with the increased level of p53 protein.
These results suggest that the treatment of HCT-116 cells
with 0-10 tiM MNNG is a transcriptional phenomenon,
while the treatment with 25-50 jiM MNNG is a post-
translational protein stabilization effect of p53. However,
treatment with 100 jiM MNNG caused a very low level of
both p53 mRNA and protein. We expected a post-transla-
tional stabilization of p53 at 100 jiM MNNG treatment, as
it was found after lower concentrations of MNNG treat-
ment, but this was not the case. It appears that both tran-
scription-mediated down-regulation of p53 gene


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p53 mRNA -



18 S RNA -



MNNG (VM)
for 15 h


A. Northern blot


*OO *


0 5 10 25 50 100


B. Western blot


p53 ->


MNNG (VM)
for 15 h


5 10 25 50 100


C. Quantitative analysis
400

300

SS 200

S100t
arn


0 5 10 25 50 100


MNNG (VtM) for 15 h

Figure 3
p53 mRNA and protein levels in HCT-I 16 cells treated with MNNG. HCT-1 16 cells were treated with different con-
centrations of MNNG (0-100 riM) for 15 h, and total RNA was isolated for Northern blot analysis as described in Materials
and Methods. After MNNG, treatment cells were also harvested for the preparation of whole cell lysate. Panel A shows a
representative autoradiogram of the p53 mRNA and 18 S RNA. Panel B shows the p53 protein level in HCT- 116 cells after
treatment with 50 iM MNNG treatment for different periods. Panel C is a quantitative analysis of the p53 protein level. The
data are mean SE of three different experiments. = significantly different than untreated cells.


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p21 -41



p27 0 .



Cdc2 4 ql



Cyclin B1 -


c-Myc ->



Max -4


0 10 25 50 100


MNNG (pM)


Figure 4
Western blot analysis of cell cycle-related proteins.
HCT-1 16 cells were treated with different concentrations of
MNNG and then processed for Western blot analysis of cell
cycle related proteins such as p21(Waf-I/Cipl), p27, Cdc2,
Cyclin BI, c-Myc and Max. Arrows indicate the position of
the bands. The molecular sizes of p21 (Waf- I/Cip I), p27,
Cdc2, cyclin B I, c-Myc and Max proteins on the Western
blots were identified as 21, 27, 34, 51, 67, and 41 kDa,
respectively.




expression and de-stabilization of p53 protein are playing
a role to exhibit reduced levels of p53 protein at 100 tiM
MNNG treatment (Figure 3).

We further examined the involvement of other cell cycle
related proteins in the senescence-like G0/G1 phase arrest
in HCT-116 cells treated with 100 jiM MNNG. In these
experiments, we analyzed the protein levels of cyclin-
dependent kinase inhibitors p21(Wafl/Cipl) and p27 in
MNNG-treated HCT-116 cells. Our results showed that
the protein levels of p21(Wafl/Cipl) and p27 were
increased up to 25 jiM MNNG treatment and then
decreased after higher concentrations of MNNG treatment


(Figure 4). Since Cdc2 and cyclin B1 are associated with
G2/M phase arrest [30], we also determined their protein
levels in HCT-116 cells treated with MNNG. The result
showed an increased Cdc2 and cyclin B1 levels in cells
treated with up to 25 jiM MNNG and then decreased after
higher concentrations of MNNG treatment as compared
to control cells (Figure 4). The increased Cdc2/cyclin B1
levels correlated with G2/M phase arrest in HCT-116 cells
treated with lower concentrations of MNNG. However, in
senescence-like arrested cells it appeared that Cdc2/cyclin
B 1 does not play any role, since the expression of these
proteins was drastically decreased in these cells. We next
determined whether the c-Myc and Max protein levels
were altered after MNNG treatment. c-Myc transcription
factor heterodimerizes with Max and regulates the expres-
sion of genes involved in cellular proliferation, differenti-
ation and apoptosis [31]. It also regulates cell cycle
progression and expression of other genes, which control
cellular senescence [32]. In our previous studies, we found
an increased level of c-Myc in HCT-116 cells treated with
25 and 50 jiM MNNG as compared to control cells, which
decreased after 50 jiM MNNG treatment. On the other
hand, the Max protein level increased at higher concentra-
tions of MNNG treatment (Figure 3). These results suggest
that Max, but not c-Myc, might be playing some role in
MNNG-induced senescence-like growth arrest in HCT-
116 cells, however, the mechanism is not yet clear.

Loss ofAPC protein level is associated with senescence-like
GolGI phase arrest in HCT-116 cells treated with higher
concentrations of MNNG
There are reports showing that the loss of APC function
can result in chromosomal instability [12,16,33]. How-
ever, there are no reports available to suggest a clear
involvement of APC in senescence. In our previous stud-
ies, we have indicated that treatment with lower concen-
trations of MNNG induces APC gene expression in HCT-
116 cells [20]. The HCT-116 cells express a wild-type APC
gene and translate a full-length APC protein [20]. In the
present investigation, we tested whether the APC gene
expression is affected at higher concentrations of MNNG
treatment and whether the APC protein levels are associ-
ated with senescence-like growth arrest in HCT-116 cells.
The results showed an increased APC protein level in
HCT-116 cells treated with up to 25 jiM MNNG and a
decreased level ofAPC protein after treatment with higher
concentration of MNNG (Figure 5). To further determine
whether the decrease in the APC protein level after treat-
ment with higher concentrations of MNNG was due to a
transcriptional or a post-transcriptional effect, we meas-
ured the APC mRNA level in HCT-116 cells. The results
showed an increased level of APC mRNA in a dose-
dependent manner up to 50 jiM MNNG treatment, while
at the 100 jiM MNNG treatment, the APC mRNA level was
drastically decreased. In these experiments, the cells were


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A. Western blot


B. Quantitative analysis


250


200


150
00
S100


1 2 3 4 5 6

0 5 10 25 50 100
MNNG (pM)


0 5 10 25
MNNG (pM)


Figure 5
APC protein levels in HCT-I 16 cells treated with MNNG. HCT- 116 cells were treated with different concentrations
of MNNG (0-100 rIM) for 15 h, and then APC protein was analyzed as described in Materials and Methods. Panel A shows a
representative autoradiogram of the APC protein.Panel B is a quantitative analysis of the APC protein level in MNNG-treated
HCT- 116 cells. The data are mean SE of three different experiments. = significantly different than untreated cells.


treated for 15 h with MNNG (Figure 6, Panel A). However,
after 50 h of treatment with MNNG, the APC mRNA levels
decreased (Figure 6, Panel B), which paralleled with the
decreased levels of APC protein (Figure 5). These results
suggest that the decreased level of APC protein is due to
reduced APC gene expression in HCT-116 cells treated
with higher concentrations of MNNG.

Loss of microtubule organization is associated with
senescence-like Go/GI arrest in HCT- 116 cells treated with
higher concentrations of MNNG
The sub-cellular distribution of APC, its interaction with
microtubules [34] and its role in chromosomal segrega-
tion [16,33] has been reported. Microtubules are impor-
tant components to keep kinetochores intact. It has been
shown that a microtubule-depolymerizing agent can dis-
sociate APC from kinetochores resulting in impaired seg-
regation of chromosomes [16,33]. Based upon these
findings, we suspected that the loss of APC in HCT-116
cells treated with higher concentrations of MNNG may
lead to microtubule disorganization. For these


experiments, the HCT-116 cells were grown on cover slips
and treated with different concentrations of MNNG for 50
h. Cells were fixed and analyzed for APC and microtubule
structural protein, a-tubulin, organization in HCT-116
cells by immunohistochemistry. After treatment with
MNNG, cells were also examined for their morphological
changes followed by immunostaining. Morphological
observations suggested that cells were still viable, but their
growth was drastically reduced at higher concentrations of
MNNG treatment as compared to untreated cells. Immu-
nostaining showed a reduced level of APC protein in
HCT-116 cells treated with 100 |iM MNNG. The analysis
of a-tubulin after immunostaining also showed a loss of
structural integrity of cells treated with 100 |iM MNNG. A
disrupted association of a-tubulin and APC can be seen in
the cells treated at this concentration (Figure 7, see
merged staining). These results indicate that the structural
organization of APC and a-tubulin proteins is disrupted
in MNNG-induced senescence-like G0/G1 phase arrested
HCT-116 cells.



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APC-+ *w-4 I 4 4 fp M


50 100


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A. 15 h treatment


APC mRNA ->




18 S RNA -









APC mRNA -





18 S RNA -



!tM MNNG


1 2 3 4 5 6
0 5 10 25 50 100


Figure 6
APC mRNA levels in HCT-I 16 cells treated with
MNNG. HCT- 1 6 cells were treated with different concen-
trations of MNNG (0-100 -iM) either for 15 h or for 50 h.
After the treatment, the total RNA was isolated for North-
ern blot analysis as described in Materials and Methods.
Panel A shows a representative autoradiogram of APC
mRNA and corresponding 18 S RNA after 15 h of MNNG
(0-100 jIM) treatment. Panel B shows a representative
autoradiogram of APC mRNA and corresponding 18 S RNA
after 50 h of MNNG (0-100 -iM) treatment. The level of 18 S
mRNA was used for normalizing loading errors and transfer
efficiency.




Loss of telomeric DNA is associated with senescence-like
Gol/G phase arrest in HCT-116 cells treated with higher
concentrations of MNNG
It has been reported that telomere shortening triggers rep-
licative senescence in human cells [3]. The exact relation-
ship between DNA damage-induced telomere loss and
cellular senescence has not been clearly defined. In the
present study, we examined whether senescence-like G0/
G, phase arrest of HCT-116 cells after treatment with


higher concentrations of MNNG was associated with the
loss oftelomeric DNA. HCT-116 cells were treated with 50
and 100 jiM MNNG for 50 h and processed for Q-FISH
analysis. Results showed a dose-dependent decrease in tel-
omeric DNA signals after treatment with MNNG as com-
pared to untreated cells (Figure 8). These results suggest
that the decreased level of telomeric DNA is associated
with MNNG-induced senescence in HCT-116 cells.

Discussion
Mutations in the Adenomatous polyposis coli (APC) gene
are believed to be an early event in the tumorogenesis and
results in the production of truncated APC protein
[12,35]. Previously, it has been shown that the primary
effect of the loss of expression of the APC gene in polyps
is the accumulation and stabilization of B-catenin protein
[19,36]. The stabilized B-catenin then translocates to the
nucleus and binds to T-cell factor (Tcf)/lymphoid
enhancer factor (Lef), a nuclear transcription factor, and
induces target genes such as cyclin Dl and the oncogene c-
myc [37,38]. In the present study, we determined the
involvement of the APC protein in DNA damage-induced
senescence-like G0/G1 phase arrest of HCT-116 cells. Our
results showed that the treatment of HCT-116 cells with
lower concentrations of MNNG (0-50 jiM) for 50 h
resulted in a dose-dependent increase of the G2/M phase
arrest and apoptosis. The G2/M phase arrest and apoptosis
was associated with an increased level of cellular p53 pro-
tein, which was consistent with previous observations
[22,29]. The p53 responds to DNA damage by activating
transcription-dependent and -independent pathways
leading to cell cycle arrest and/or apoptosis and prevent-
ing proliferation of cells with damaged genome [39]. The
increased or stabilized level of p53 and p21(Waf-1/Cipl)
has been suggested to play a critical role in the G2/M phase
arrest and apoptosis [24,40], which is consistent with our
previous studies in HCT-116 cells treated with lower con-
centrations of MNNG [41]. However, HCT-116 cells
treated with higher concentrations of MNNG showed a
senescence-like Go/G1 phase arrest but did not show any
increase in p53 protein level. These results suggest that the
p53-mediated pathway was not involved in the senes-
cence-like Go/G1 phase arrest of HCT-116 cells. It has been
suggested that increased transcriptional activity of p53
protein also plays an important role in the senescence-like
GQ/G1 phase arrest via transactivation of p21 (Waf-1/Cipl)
gene [42]. Since p21(Waf-1/Cipl) protein level was
decreased in HCT-116 cells treated with higher concentra-
tions of MNNG, our findings further suggest that the p53/
p21(Waf-1/cipl) pathway is not involved in senescence-
like Go/G1 phase arrest of HCT-116 cells. Further, we
found that other cell cycle related proteins such as p27,
cdc2, Cyclin B1, c-Myc and Max were also not involved in
senescence-like Go/G1 phase arrest in HCT-116 cells;
although, their role in senescence-like Go/G1 arrest have


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B. 50 h treatment


.09-9


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MNNG (jM)


100


Figure 7
Immunohistochemical analysis of APC and a-tubulin proteins in HCT-1 16 cells after treatment with MNNG.
Cells were grown in 0.5% FBS containing medium on cover slips and treated with different concentrations of MNNG for 50 h.
Cells were fixed and processed for immunofluorescence staining as described in the Materials and Methods. Images were cap-
tured on the Zeiss upright microscope. Morphological changes were also recorded on an inverted Zeiss Microscope.



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A. FISH


0.6


0.4


0.2


0.0


B. Q-FISH



_ *,


MNNG (iM) 0


50 100


Figure 8
Determination of telomeric DNA levels by Q-FISH analysis in HCT-I 16 cells treated with MNNG. HCT-1 16
cells were treated with 50 and 100 IM of MNNG and then processed for Q-FISH analysis as described in Materials and Meth-
ods. Panel A shows the data of a FISH analysis. Panel B shows the quantitative analysis of the FISH data of the control and
MNNG-treated cells. The percentage telomeric area of the interphase nuclei in FISH preparations was quantified using a soft-
ware package (Metaview Imaging system-3.6a; Universal Imaging Co., Westchester, PA). From each sample at least 50 inter-
phase nuclei were quantified, and mean values of percent telomeric area compared to the nuclear area were calculated. The
values for the treated cells were compared to the control cells.


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been shown in previous studies [32,43-45]. The difference
in these and previous studies could have been due to dif-
ferences in cell type and DNA damaging agents.

Once we determined that the p53/p21(Waf-1/Cipl) path-
way was not involved in MNNG-induced senescence-like
GQ/G1 phase arrest of HCT-116 cells, then we looked at
other parameters which are suggested to be associated
with chromosomal abnormalities and likely with
senescence. For this part of the study, we determined the
expression level of APC, a-tubulin, and telomeric DNA.
The loss of APC protein can cause chromosomal instabil-
ity (CIN) and microtubule disorganization that can lead
to aneuploidy [16]. The aneuploid cells can choose cell
death, senescence, or cell survival pathways, depending
upon the genetic pressure exerted upon these cells
[46,47]. In the present study, we found that cells exhibit-
ing senescence-like Go/G1 phase arrest after treatment
with higher concentrations of MNNG showed a drastically
reduced level of APC and a-tubulin proteins, which sug-
gest their role in MNNG-induced chromosomal instabil-
ity and perhaps senescence-like Go/G1 phase arrest of
HCT-116 cells. This statement needs to be further verified
by using the APC overexpression system to block DNA
damage-induced senescence in these cells. In fact, the
association ofAPC with microtubules plays an important
role in chromosomal segregation in which the APC is spe-
cifically detected at the kinetochores, and the binding of
APC at kinetochores requires intact microtubules [16,33].

Next, we examined whether the loss of MNNG-induced
telomeric DNA was also associated with senescence-like
GQ/G1 phase arrest of HCT-116 cells. In previous studies,
a DNA damage checkpoint response in telomere-initiated
senescence has been described in human diploid fibrob-
last (HDF) cell lines [48]. Although we have not deter-
mined the activity of checkpoint responsive CHK1 and
CHK2 kinase activities, the MNNG-induced level of telo-
meric DNA was significantly decreased in HCT-116 cells.
In earlier studies, we have shown that HCT-116 cells
treated with lower concentrations of MNNG showed a
dose-dependent loss of telomeric DNA in a p53-inde-
pendent manner [41]. In these studies, the loss in the
amount of telomeric DNA at 50 gtM MNNG treatment was
approximately two-fold. However, in the present study,
the loss in the amount of telomeric DNA at 100 itM
MNNG treatment was more than two-fold. From these
results, it appears that approximately two-fold loss of tel-
omeric DNA favors G2/M phase arrest and apoptosis, and
more than two-fold loss of telomeric DNA after treatment
with MNNG is linked with senescence-like growth.

Conclusions
Our results suggest that MNNG-induced senescence-like
growth arrest of HCT-116 cells is associated with


decreased levels of APC, a-tubulin, and telomeric DNA.
Thus, DNA damage-induced senescence-like growth arrest
can protect cells from abnormal growth and
carcinogenesis.

Materials and Method
Maintenance and treatment of cells
Human colon cancer cell line HCT-116 was grown in
McCoys 5a medium supplemented with 10% fetal bovine
serum (FBS; Cell Grow, Mediatech, VA), 100 units/ml
penicillin and 100 gig/ml streptomycin at 370C in a
humidified atmosphere of 5% CO2. After cells reached
60% confluence, fresh medium containing 0.5% FBS and
antibiotics were added to each plate and then further incu-
bated for an additional 18 h. Treatment regimen with
MNNG (Aldrich Chemical Co., Milwaukee, WI) is given
in the figure legends.

FACS analysis
A detergent and proteolytic enzyme-based technique was
used for nuclear isolation and DNA content analysis of
cells in different phases of cell cycle. After treatment with
different concentrations of MNNG for 50 h, cells were har-
vested and processed for staining of nuclei with propid-
ium iodide [49]. The cellular DNA content was analyzed
by the Becton-Dickinson FACScan flow cytometer (BD
Biosciences, San Jose, CA). At least 10,000 cells per sample
were considered in each gated region for calculations. The
ranges for Go/G,, S, G2/M and sub-G, phase cells were
established based upon their corresponding DNA con-
tents of histograms. Results were analyzed and expressed
as a percentage of the total gated cells using the ModfitLT-
V2.0 program.

Senescence-associated /?-galactosidase staining
HCT-116 cells were treated with MNNG for 50 h and then
washed three times with phosphate-buffered saline (PBS).
Cells were fixed in 4% paraformaldehyde and again
washed three times with PBS. Cells were incubated with
freshly made B-galactosidase staining solution (1 mg/ml
of 5-bromo-4-chloro-3-indolyl P-D-galactoside (X-Gal), 5
mM potassium ferricyanide, 5 mM potassium ferrocya-
nide and 2 mM magnesium chloride in PBS at 37C
(without CO2) as described earlier by Dimri et al. [27].
Staining of cells was observed under the microscope
(Leica, Manheim, Germany), and images were captured
using the magnifier program (Optelec US Inc., MA).

Western blot analysis
Changes in protein levels, subsequent to MNNG treat-
ment, were determined using Western blot analysis of
whole cell extracts as described previously [20]. In this
study, the following antibodies were used to detect vari-
ous cell cycle-related proteins: anti-APC (Ab-1) mouse
monoclonal antibody from Oncogene Research Products


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(Cambridge, MA), and anti-APC (N-15), anti-Cdc-2 p34
(17), anti-Cyclin B1 (GNS-1), anti-p21 (F-5), anti-p27,
anti-c-Myc and anti-Max were from Santa Cruz Biotech-
nology (Santa Cruz, CA).

Northern blot analysis
For northern blot analysis, the total RNA from untreated-
and MNNG-treated cells was isolated by TRIzol' reagent
as described by the manufacturer (Invitrogen Life Tech-
nologies, CA). Then 50 |ig of total RNA were separated on
1% formaldehyde-agarose gel and transferred onto a
Hybond-N+ membrane (Amersham Biosciences Corp.,
NJ). The membrane was prehybridized for 6 h at 65 C in
0.5 M sodium phosphate buffer (pH 7.2), 7% (w/v) SDS,
1 mM EDTA, and 1% (w/v) bovine serum albumin (BSA)
and then hybridized with 32P-labeled APC probe (EcoRI
fragment of APC-HFBCI43; ATCC, Manassas, VA). Later
the same membrane was reprobed with 32P-labeled EcoRI
fragment of 18 S RNA probe for normalization of RNA
loading and transfer efficiency. The membranes were
exposed to x-ray films for detection of specific mRNA
signals.

Immunohistochemical analysis
Immunohistochemical analysis was performed to exam-
ine the localization of APC and a-tubulin proteins in
untreated- and MNNG-treated cells. Briefly, 5 x 105 cells
were grown on cover slips. Once cells reached 60% con-
fluence, fresh medium containing 0.5% FBS and antibiot-
ics were added to each dish and then further incubated for
18 h. Treatment regimen with MNNG is given in figure
legends. After treatment with MNNG, cells were washed
with PBS and fixed with 4% paraformaldehyde solution
for 30 min at 220C. After fixing cells, cover slips were
washed again with PBS and incubated for 30 min with 50
mM NH4C1 in PBS containing 0.2% triton X-100. After
washing with PBS, cells were further incubated for 2 h at
22C with either anti-APC (N-15) rabbit polyclonal or
anti-a-tubulin antibody (dilution 1:100) in 5% goat
serum containing 0.2% triton X-100. Unbound antibod-
ies were washed with PBS buffer and antibodies were
stained for 1 h at 220C with anti-rabbit secondary anti-
body conjugated to FITC or rhodamine (dilution 1:200)
in 5% goat serum, 0.2% triton X-100 in PBS. After wash-
ing, a drop of DAPI (in mounting solution) was added,
and cover slips were sealed from sides using nail polish.
Slides were viewed under Zeiss Axioplan-2 imaging
upright microscope (Zeiss, Thornwood, NY) systems
using different filters, and images were captured using the
open lab program.


Quantitative fluorescence in situ hybridization (Q-FISH)
analysis
The control and MNNG-treated HCT-116 cells were har-
vested and processed for cytological preparations for Q-
FISH analysis as described previously [50].

Authors' contributions
ASJ and SN drafted the paper. AM and SP did the Q-FISH
analysis. All authors read and approved the final draft of
the manuscript.

Acknowledgements
This work was supported in part to SN by NCI-NIH grants (CA-77721,
CA-09703 I). We thank Mary Wall for proof reading this manuscript.

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