Download DNMT1 as a Molecular Target in a Multimodality

DNMT1 as a Molecular Target in a Multimodality-Resistant
Phenotype in Tumor Cells
Mark V. Mishra, Kheem S. Bisht, Lunching Sun, Kristi Muldoon-Jacobs, Rania Awwad,
Aradhana Kaushal, Phuongmai Nguyen, Lei Huang, J. Daniel Pennington,
Stephanie Markovina, C. Matthew Bradbury, and David Gius
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
Abstract
Introduction
We have previously shown that hydrogen peroxide –
resistant permanent (OC-14) cells are resistant to the
cytotoxicity of several exogenous oxidative and
anticancer agents including H2O2, etoposide, and
cisplatin; and we refer to this process as an oxidative
multimodality-resistant phenotype (MMRP).
Furthermore, OC-14 cells contain increased activator
protein 1 activity, and inhibition of activator protein 1
reversed the MMRP. In this study, we show that
permanent Rat-1 cell lines genetically altered to
overexpress c-Fos also displayed a similar MMRP to
H2O2, etoposide, and cisplatin as OC-14 cells. Gene
expression analysis of the OC-14 cells and
c-Fos – overexpressing cells showed increased DNMT1
expression. Where OC-14 and c-Fos – overexpressing
cells were exposed to 5-aza-2¶-deoxycytidine,
which inhibits DNMT activity, a significant but
incomplete reversal of the MMRP was observed.
Thus, it seems logical to suggest that DNMT1 might
be at least one target in the MMRP. Rat-1 cells
genetically altered to overexpress DNMT1 were also
shown to be resistant to the cytotoxicity of H2O2,
etoposide, and cisplatin. Finally, somatic HCT116
knockout cells that do not express either DNMT1
(DNMT1 / ) or DNMT3B (DNMT3B / ) were shown to
be more sensitive to the cytotoxicity of H2O2,
etoposide, and cisplatin compared with control
HCT116 cells. This work is the first example of a role
for the epigenome in tumor cell resistance to the
cytotoxicity of exogenous oxidative (H2O2) or systemic
(etoposide and cisplatin) agents and highlights a
potential role for DNMT1 as a potential molecular target
in cancer therapy. (Mol Cancer Res 2008;6(2):243 – 9)
The resistance of tumor cells to anticancer agents remains a
major cause of failure in the treatment of patients with cancer.
The classic mechanism for the acquisition of a multidrugresistant phenotype by cancer cells was believed to involve a
single molecular mechanism, such as overexpression of
P-glycoprotein (1, 2). However, it now seems that the
multidrug-resistant phenotype represents a complex, multifactorial process, with at least two or more resistance mechanisms
(1). This may include resistance associated with decreased drug
accumulation, altered intracellular drug distribution, increased
detoxification, diminished drug-target interaction, increased
DNA repair, altered cell cycle regulation, and most recently,
changes in the levels of proteins and molecules that regulate the
cellular oxidation/reduction status (1-6).
The cytotoxicity of agents that induce oxidative stress
arises from intracellular damage caused by reactive oxygen
intermediates (ROI). Ideally, a metabolically active cell should
strike a balance between ROI production and the cellular
antioxidant defense system, resulting in a reduced cellular
environment (7-9). Relatively small amounts of ROI are
natural byproducts of intracellular electron transfer reactions
that are easily tolerated by cells and may act as signaling
molecules (10, 11). However, if ROI production exceeds the
endogenous intracellular antioxidant capacities, oxidative
stress results (12). This is a detrimental cellular environment
that can result in lipid peroxidation or DNA damage that
could ultimately end in cell death. Increased ROI levels also
result from exposure to H2O2, hyperthermia (13-15), or some
chemotherapeutic agents including cisplatin (16, 17) or
etoposide (18, 19), and it has been suggested that the resultant
oxidative damage plays a role in the cytotoxicity of these
anticancer agents (16-19).
A class of proto-oncogenes referred to as immediate early
response genes is activated as a consequence of a wide variety
of environmental agents that induce oxidative stress (13, 20,
21). These genes encode nuclear transcription factors, including
the activator protein 1 (AP-1) complex, which play central roles
in the transmission of intracellular information through multiple
downstream signaling pathways (22-25). One possible role for
the induction of these transcription factors is to modulate the
expression of specific target genes involved in a protective
and/or reparative cellular response to the damaging effects of
oxidative stress induced by exogenous cytotoxic agents
(26-29). As such, knowledge of these signaling pathways
provides fundamental insight into how tumor cells respond to
cytotoxic agents.
Received 8/8/07; revised 10/2/07; accepted 10/4/07.
Grant support: Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research, and the Radiation Oncology Branch. M.
Mishra was supported by the NIH Clinical Research Training Program as a
Medical Student Research Fellow.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: M.V. Mishra and K.S. Bisht contributed equally to this manuscript.
Requests for reprints: David Gius, Radiation Oncology Branch, National Cancer
Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-496-5457.
E-mail: [email protected]
Copyright D 2008 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-07-0373
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FIGURE 1. Fos and OC-14 – overexpressing
cell lines exhibit MMRP and increased expression of DNMT1 . Clonogenic cell survival curves
with control Rat-1 cells or Rat-cells genetically
altered to overexpress Fos (CMV-c-Fos ). These
cells were treated with either (A) H2O2 or (B)
cisplatin. CMV-c-Fos cells were also pretreated
with 5.0 Amol/L of 5-aza-CdR for 48 h prior to
exposure to either H 2O2 or cisplatin. After
exposure, cells were trypsinized and plated at
various densities from 200 to 20,000 cells per
60 mm dish. After roughly 7 to 10 d, colonies
were fixed, stained, counted, and the surviving
fraction was plotted versus the concentration
of cisplatin or moles per cell of H2O2. Curves are
normalized to account for agent-induced
cytotoxicity alone. Each separate assay was
done in duplicate with three dilutions of cells per
condition, and then repeated twice in their
entirety for a total of three independent experiments. Points, mean; bars, 1 SD. Statistical
significance was established by Student’s t test
(P < 0.05). C. Immunoreactive DMNT levels in
Rat-1 or CMV-c-Fos cell lines. Total cellular
protein was isolated and 30 mg of cellular protein
were separated by SDS-PAGE, transferred onto
nitrocellulose, and processed for immunoblotting
with anti-DNMT antibody (Oncogene Products
Research). Equal protein loading was determined
using a Bradford protein assay. D. Northern
analysis of HA-1 and OC-14 cells. Total RNA was
isolated from HA-1 and OC-14 cells (Qiagen)
separated by electrophoresis, blotted, and
probed with a complementary sequence to the
DNMT1 gene. The DNMT1 and h-actin bands
and fluorogram sections were obtained using a
TYPHOON Phosphorimager. E. OC-14 cells
overexpress DNMT1 and exposure to 5-azaCdR reverses their MMRP. OC-14 hydrogen –
resistant hamster cells were exposed to 1.0 or
5.0 mmol/L of 5-aza-CdR for 24 or 48 h, and
exposed to two different concentrations of
cisplatin. Clonogenic survival assays were done
as described above. Points, mean; bars, 1 SD.
We have previously shown that H2O2 stress – resistant
OC-14 cells have increased AP-1 DNA-binding activity and
are resistant to the damaging effects of agents that induce
oxidative stress (30). In addition, the inhibition of the AP-1
complex reverses the multimodality resistance phenotype
(MMRP), suggesting potential downstream targets. This form
of tumor cell resistance is referred to as a MMRP. These
observations are consistent with the idea that immediate early
genes respond to oxidative stress, resulting in the induction of
downstream genes that detoxify and protect against the
accumulation of intracellular ROI. In this work, we expand
our previous results and suggest that one downstream target of
Fos in the process of a MMRP may be the methyltransferase-1
(DNMT1) gene. The totality of these results suggests that the
epigenome may play a role in tumor cell resistance to oxidative
stress and highlights a potential role for DNMT1 as a potential
molecular target in cancer therapy.
Results
Fos Overexpressers Exhibit MMRP and Increased
Expression of DNMT1
We have previously shown that H2O2-resistant OC-14 cells
have an MMRP phenotype characterized by increased resistance to H2O2, cisplatin, and etoposide (30). It was also shown
that OC-14 cells constitutively overexpress c-Fos and have
increased AP-1 activity, and that the chemical inhibition of
AP-1 complex reversed the MMRP (30). To determine if c-Fos
might be one target in the process, permanent rat fibroblast cell
lines (208F) genetically altered to overexpress c-Fos (CMV-cFos) were used (31). Increased c-Fos levels were confirmed
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DNMT as a Target in MMRP
through Western blot analysis (data not shown). Clonogenic cell
survival experiments showed that CMV-c-Fos cells were more
resistant to the cytotoxicity of H2O2 (Fig. 1A, o versus .),
cisplatin (Fig. 1B, o versus .), and etoposide (data not shown)
when compared with the parental 208F cells. In these
experiments, 30 10 13 and 60 10 13 mol/cell roughly
corresponded with 45 and 90 Amol/L of H2O2, respectively.
The results of these experiments suggest that overexpression
of Fos results in a similar MMRP as that observed for
OC-14 cells.
It has been previously shown that DNMT1 is one target in
the process of how overexpression of Fos transforms NIH3T3
cells (31). In addition, it is well established that the AP-1
signaling pathway up-regulates the expression of DNMT1 (32).
Thus, it seemed logical to determine if CMV-c-Fos and/or
H2O2-resistant OC-14 cells, both of which have a MMRP,
might overexpress DNMT1. Western blot analysis showed a
significant increase in DNMT1 protein level in the Fos
overexpressing CMV-c-Fos cells (Fig. 1C). In addition,
Northern analysis showed an increase in DNMT1 expression
in OC-14 cells (Fig. 1D). Northern analysis was done because
DNMT1 antibody does not cross-react in hamster cells (data not
shown). The results of these experiments suggest that DMNT1
may be at least one target in the MMRP observed in OC-14 and
Fos-overexpressing cells.
Inhibition of DNMT1 in CMV-c-Fos and OC-14 Cells by
5-Aza-2¶-Deoxycytidine Reverses Their MMRP
To further investigate a possible role of DNMT1 in MMRP,
H2O2-resistant OC-14 cells that overexpress DNTM1 (Fig. 1D)
were treated with 1 or 5 Amol/L of 5-aza-2¶-deoxycytidine
(5-aza-CdR), an inhibitor of DNMT activity. Clonogenic cell
survival experiments in OC-14 cells pretreated with 5-aza-CdR
for 24 (Fig. 1E, left) and 48 h (right) followed by exposure to
cisplatin showed that exposure to 5-aza-CdR resulted in a dosedependent loss in their previously observed MMRP. Similar
results were observed when cells were exposed to 5-aza-CdR
and H2O2 (data not shown). These experiments were repeated
in Rat-1 cells overexpressing Fos. Pretreatment of CMV-c-Fos
cells with 5-aza-CdR significantly, but not completely, reversed
their resistance to H2O2 (Fig. 1A, o versus E) and cisplatin
(Fig. 1B, o versus E).
Overexpression of DNMT1 Is Associated with MMRP
Because OC-14 and CMV-c-Fos cells overexpress DNMT,
contain a MMRP, and exposure to 5-aza-CdR reverses this
phenotype, it seemed logical to determine if cells overexpressing DNMT1 would have a similar MMRP. Clonogenic
cell survival experiments using Rat-1 cells genetically altered
to overexpress DNMT1 were also found to be resistant to the
cytotoxicity of H2O2 (Fig. 2A), cisplatin (Fig. 2B), and
etoposide (Fig. 2C). The results of these experiments suggest
that DNMT1 may be at least one target downstream of Fos
that plays a role in the observed MMRP seen in CMV-c-Fos
cells.
DNMT1 Expression Is Induced by Oxidative Stress
Agents
If DNMT1 is one target in the MMRP observed in H2O2resistant OC-14 cells, then it seemed logical to determine
whether expression of DNMT1 could be increased by specific
exogenous agents that induce intracellular oxidative stress.
As such, HCT116 cells were exposed to three agents that
induce oxidative stress and levels of DNMT1 expression
were subsequently determined. Western blot analysis showed
a dose-dependent increase in DNMT1 protein concentration
following exposure to hypoxia (Fig. 3A, left), H2O2 (right),
and ionizing radiation (data not shown). The induction in
intracellular DNMT1 protein after exposure to oxidative
stress was also confirmed by immunofluorescence in
HCT116 cells (data not shown). In addition, global DNA
methylation was also increased following exposure to
hypoxia (Fig. 3B) and hydrogen peroxide (Fig. 3C). The
results of these experiments would suggest that oxidative
stress induces methyltransferase activity as well as an
intracellular protective response.
FIGURE 2. Overexpression of DNMT1 is associated with MMRP. Clonogenic cell survival curves with control Rat-1 cells or Rat-cells genetically altered to
overexpress DNMT1 (CMV-c-DNMT1). Cells were treated with either (A) H2O2, (B) cisplatin, or (C) etoposide. After exposure, cells were trypsinized and
plated at various densities from 200 to 20,000 cells per 60 mm dish. After roughly 7 to 10 d, colonies were fixed, stained, counted, and the surviving fraction
was plotted versus the concentration of cisplatin or etoposide or moles per cell of H2O2. Points, mean; bars, 1 SD. Statistical significance was established by
Student’s t test (P < 0.05).
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FIGURE 3. DNMT1 expression induced by
oxidative stress agents. A. Western blot analysis
of HCT116 colon cancer cells treated with hypoxia
(0.1% for 6 h; left ) or H2O2 (right ). Cells were
harvested and 30 Ag of cellular protein was
separated by SDS-PAGE, transferred onto
nitrocellulose, and processed for immunoblotting
with immunoblotted anti-DNMT1 antibody
(Oncogene Products Research). Equal protein
loading was determined using a Bradford protein
assay. HCT116 colon cancer cells were exposed
to either (B) hypoxia (0.1% for 6 h) or (C) H2O2,
and cells were harvested to determine global DNA
methylation.
DNMT1 ( / ) and DNMT3B ( / ) Somatic Knockout
HCT116 Cell Lines Show Increased Sensitivity to H2O2
and Etoposide
If overexpression of DNMT1 results in resistance to H2O2,
cisplatin, and etoposide then it would seem reasonable to
hypothesize that cells lacking methyltransferase genes would
display increased sensitivity to these agents. To address this
idea, clonogenic cell survival assays were done with somatic
HCT116 knockout cells that did not express either DNMT1
or DNMT3B. Somatic knockout cell lines for DNMT1
(DNMT1 / ) and DNMT3B (DNMT3B / ) have been
previously constructed and characterized (32), and represent
an ideal model system to investigate the epigenetic
prosurvival response. As shown, tumor cells lacking expression of the DNMT1 (DNMT1 / ) or DNMT3B (DNMT3B / )
gene are more sensitive to the cytotoxicity of H2O2 (Fig. 4A),
etoposide (Fig. 4B), or cisplatin (data not shown) as compared
with parental HCT116 cells. These results further suggest that
DNMT may play a role in tumor cell resistance as well as act
as a potential molecular target.
Discussion
A relatively new theme in cancer research involves the idea
that the same genes implicated in the process of cellular
transformation, such as the proto-oncogene c-Fos, are subsequently used by tumor cells to evade the damaging and
cytotoxic effects of anticancer agents. An understanding of the
potential relationship of these intracellular factors, which are
altered as a result of malignant progression, may be critical to
predicting how tumor cells respond to therapeutic intervention.
We have previously shown that the AP-1 transcription factor
complex displays increased activity in hydrogen peroxide –
resistant tumor cells that exhibit a MMRP to several
commonly used oxidative and anticancer agents (30). The
MMRP observed in these cells was reversed following
treatment with an agent that inhibits Fos, clearly implicating
Fos in this process. In this work, we expand on these
observations and show that downstream targets of Fos, such
as DNMT1, which are up-regulated as a result of transformation may play a role in resistance to exogenous agents. In
addition, when these results are combined with our previous
work (30), it suggests one model whereby oxidative stress
increases methyltransferase activity by redox-sensitive transcription factors such as Fos.
DNMT1, which catalyzes the transfer of methyl groups from
S-adenosyl methionine to the C-5 position of cytosines, has
been previously shown to be a target gene of Fos (31) during
cellular transformation. Furthermore, overexpression of DNMT
and increased methylation of the promoters of tumor suppressor
genes and their associated silencing have been found in
retinoblastoma (33-35) as well as many other tumors types
(36). As a result, it has been suggested that silencing of tumor
suppressor genes through hypermethylation by DNMT1 results
in a cellular environment permissive to the development of
chromosomal instability or genomic instability (35), ultimately
resulting in cellular transformation (37).
Although the DNMT genes have been clearly implicated
in the process of cellular transformation, no studies to date
have examined the role of the DNMT genes in the process of
oxidative tumor cell resistance to exogenous or anticancer
agents. In the present model system, DNMT1 overexpression
was confirmed to be linked to c-Fos expression (30). It is
also shown for the first time that DNMT expression and
activity are up-regulated following treatment with agents such
as H2O2 and hypoxia which induce oxidative stress.
Furthermore, increased DNMT1 expression was also associated with increased resistance to two commonly used
systemic chemotherapeutics. However, we cannot rule out
that the tumor cell resistance observed in these agents might
be due, at least in part, to a more generalized transformed
cellular phenotype induced by DNMT1 overexpression.
However, the results observed in the HCT116 somatic
knockout cells would suggest at least some direct effect on
tumor cell resistance. Taken together, these results indicate
that the DNMT genes may play a role in how tumor cells
evade the damaging and cytotoxic effects of anticancer
agents.
Previous studies have shown that methylation of specific
genes might be potential molecular markers in thyroid (38),
breast (39), prostate (40), gastric (41), and colon carcinomas
(42). Thus, based on this study and previous observations, it
seems plausible that the functional status of DNMT genes may
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DNMT as a Target in MMRP
be potential molecular markers that can be profiled in tumors to
either idealize cancer therapies or determine potential treatment
outcome as the technology to study both the genome and
epigenome increases and improves.
Even more intriguing is the idea that the epigenome may be
a potential molecular target for the treatment of tumors that are
resistant to oxidative stress and/or chemotherapeutics known to
induce oxidative stress. In the present study, the MMRP
exhibited by DNMT1-overexpressing OC-14 cells was reversed
following treatment with the nucleoside analogue 5-aza-CdR,
which inhibits methyltransferase activity. Furthermore, somatic
colon cancer cells genetically manipulated to knock out the
DNMT1 and DNMT3B showed increased sensitivity to a
variety of anticancer agents. These results suggest that DNA
methylation – targeting drugs such as 5-aza-CdR may be used as
an adjunct in cancer therapy to abolish the MMRP displayed by
potential tumor types.
Theoretically, an ideal molecular target should (a) be
overexpressed or constitutively active in tumor cells, (b)
enhance tumor proliferation, (c) inhibit watchdog or fidelity
genes, (d) incite a prosurvival effect, and (e) enhance
resistance to therapeutic modalities (e.g., IR and chemotherapy). The DNMT genes clearly meet several of these
criteria. Alterations in methylation patterns and activity have
been observed in countless tumors and tumor cell lines, and
agents such as 5-aza-CdR, which inhibits DNMT activity,
have been shown to inhibit tumor cell proliferation, induce
cell death (32, 42), and in the present study, to reverse
MMRP seen in several tumor cells. Thus, based on the
results from this work, combined with previous findings, it
seems logical to assume that the DNMT genes might be
effective molecular targets in cancer therapy.
This work also suggests that agents which induce oxidative
stress, hypoxia, H2O2, and ionizing radiation also increase both
DNMT1 protein levels as well as overall global DNA
methylation. The methods used for the experiments presented
here only measured total methylation and it is possible, and
even likely, that the chromatin methylation changes are more
complex, and specific promoters may be hypomethylated
whereas others, in a greater amount, are methylated. However,
the effects produced by agents which induce oxidative stress
suggest that the epigenome may be an example of another
cellular preprogrammed stress response pathway to defend cells
against potentially damaging and/or cytotoxic exogenous
genotoxic agents.
The epigenome is an especially intriguing target in cancer
therapy because epigenetic changes observed in tumor cells,
such as hypermethylation, unlike genomic changes, can be
reversed with therapeutic intervention. In this regard, several
clinical studies including phase II trials of DNMT inhibitors
have been completed (43). However, the results of these studies
are unclear, and it has been suggested that this is due to a lack
of specificity and targeting of these agents to specific
potentially responsive tumor subtypes.
Thus, it is becoming increasingly clear that changes in the
epigenome may play a critical role in both cellular
transformation and carcinogenesis as well as how tumor cells
defend themselves against the damaging and cytotoxic effects
of therapeutic modalities. However, molecular profiling as
well as the identification of specific tumors or tumor subtypes
will be necessary to move these agents forward in preclinical
and early clinical studies. Taken together, these observations
would suggest a role for testing epigenetic alterations in
tumors to determine potential clinical indications. Similar to
many of the new agents currently in clinical studies, a
rigorous set of translational work must also be done to
establish and validate molecular markers or targets with
clinically significant end points such as clinically complete
FIGURE 4. DNMT1( / ) and DNMT3B( / ) cell lines showed increased
sensitivity to anticancer agents. HCT116 methyltransferase somatic
knockout cells that do not express either DNMT1 (DNMT1 / ) or
DNMT3B (DNMT3B / ) were used in clonogenic cell survival experiments. HCT116 or knockout cells were exposed to either hydrogen
peroxide or etoposide at various concentrations. Cells were then trypsinized and plated at various densities from 200 to 20,000 cells per 60 mm
dish. After roughly 7 to 10 d, colonies were fixed, stained, counted, and the
surviving fraction was plotted versus the concentration. Points, mean;
bars, 1 SD; statistical significance was established by Student’s t test
(P < 0.05).
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response, local and distant control, and disease-free and
overall survival.
Materials and Methods
Rat fibroblast cells (208F, CMV-c-Fos, and CMV-DNMT1)
which overexpress either c-Fos and DNMT1 (a kind gift from
Tom Curran, Department of Pathology and Laboratory
Medicine, University of Pennsylvania, Philadelphia, PA). HA-1
and the selected H2O2-resistant OC-14 (4, 5) were grown in
Eagle’s MEM supplemented with Earle’s basic salt solution,
10% heat-inactivated FCS (+penicillin; 100 units/mL), and
streptomycin (100 Ag/mL) in a humidified 5% CO2/95% air
atmosphere at 37jC. The DNMT1 somatic knockout cells
(DNMT1 / and DNMT3B / ) and parental colon cancer cell
line (HCT116) have been previously described (44) and were
grown in McCoy’s 5A medium (Invitrogen). Cells were seeded
at 2 105 cells/100 mm culture dish and grown to 75% to 85%
confluence (4-5 106 cells/100 mm dish) prior to experimental
treatment unless otherwise stated. H2O2 stock solutions were
made in sterile PBS, and concentrations were determined by a
spectrophotometric method previously described (4). Doses of
H2O2 were delivered directly to the growth medium. Likewise,
cisplatin (Sigma) stock solutions (0.25% w/v) were made in
sterile water immediately prior to treatment of cells to prevent
chemical hydration and potency degradation. Cells were treated
with cisplatin added directly to the growth medium. Doses of
H2O2 and cisplatin were chosen to allow for the calculation of a
dose modifying factor (DMF) at 10% or 50% iso-survival (4, 6),
where DMF(10, 50) = [dose to reach 10% (or 50%) survival in
OC-14] / [dose to reach 10% (or 50%) survival in HA-1]. Cells
were treated with designated doses of 5-aza-CdR (Sigma).
polyclonal antibody against c-Fos or DNMT1 (Oncogene
Products Research), each diluted 1:1,000 in 2.5% milk in
PBS-T. The membrane was washed thrice for 15 min each in
PBS-T, and then incubated for 1 h at room temperature with
an anti-rabbit IgG horseradish peroxidase secondary antibody
(Santa Cruz Biotechnology) diluted 1:2,000 in 2.5% milk in
PBS-T. The membrane was again washed thrice for 15 min each
in PBS-T and then analyzed via an enhanced chemiluminescence method (Amersham Pharmacia Biotech) according to the
instructions of the manufacturer.
Global DNA Methylation Assay
Global DNA methylation was determined by methyl
acceptance assay as described by Balaghi and Wagner (45).
Briefly, genomic DNA was isolated from cells using the
DNeasy kit (Qiagen, Inc.) according to the protocol of the
manufacturer. Purified genomic DNA (1 Ag) was incubated
with 3 units of SssI methylase (New England Biolabs, Inc.) and
2 ACi of 3H-labeled S-adenosyl-L-methionine (Perkin-Elmer) in
an incubation buffer [10 mmol/L EDTA, 5 mmol/L DTT, and
100 mmol/L Tris-HCl (pH 8.0)] for 1 h at 37jC. The reaction
was stopped by chilling on ice for 15 min and 15 mL of reaction
mixture was transferred onto a Whatman DE 81 filter paper.
The filters were washed twice by suction with 0.5 mol/L of
sodium phosphate buffer and rinsed with 70% ethanol and
100% ethanol successively. The air-dried filters were transferred into 10 mL of scintillation fluid vial, and radioactivity
was measured using a Beckman LS 9800 Liquid scintillation
system. An increase in methyl-3H incorporation indicates
hypomethylation of endogenous DNA.
References
Clonogenic Cell Survival Assays
Cell lines were plated at densities of 3.0 105 and 3.5 105
cells, respectively, per 100 mm tissue culture dish, grown
exponentially and then treated with chemical stressors (e.g.,
H2O2, cisplatin, or etoposide). One hour after exposure, cells were
trypsinized and counted using a Coulter Counter (Beckman
Coulter). Dilutions of the treated cells were prepared, and duplicate
60-mm tissue culture dishes were seeded with 200 to 20,000 cells
each, depending on the severity of the challenge treatment.
Colonies were allowed to form in an undisturbed, humidified,
37jC/5% CO2 environment for 7 to 10 days, fixed with 70% ethanol, stained with Coomassie blue, and counted under a dissection
microscope. Only those plates containing 25 to 250 colonies were
computed as statistically relevant. Surviving fractions from the
treated test cultures were normalized to sham-treated controls and
plotted as a function of dose on a log/linear plot.
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Equal amounts of protein, ranging from 10 to 30 Ag/sample,
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Protein samples were then separated on a denaturing polyacrylamide-SDS gel and transferred to a nitrocellulose
membrane using a semidry transfer apparatus (Owl Scientific,
Inc.). The membrane was blocked for 1 h in a 5% milk/PBS-T
and was hybridized overnight at room temperature with a
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249
DNMT1 as a Molecular Target in a Multimodality-Resistant
Phenotype in Tumor Cells
Mark V. Mishra, Kheem S. Bisht, Lunching Sun, et al.
Mol Cancer Res 2008;6:243-249.
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