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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 Mol Cancer Res 2008;6(2). February 2008 Downloaded from mcr.aacrjournals.org on August 11, 2017. © 2008 American Association for Cancer Research. 243 244 Mishra et al. 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 Mol Cancer Res 2008;6(2). February 2008 Downloaded from mcr.aacrjournals.org on August 11, 2017. © 2008 American Association for Cancer Research. 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). Mol Cancer Res 2008;6(2). February 2008 Downloaded from mcr.aacrjournals.org on August 11, 2017. © 2008 American Association for Cancer Research. 245 246 Mishra et al. 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 Mol Cancer Res 2008;6(2). February 2008 Downloaded from mcr.aacrjournals.org on August 11, 2017. © 2008 American Association for Cancer Research. 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). Mol Cancer Res 2008;6(2). February 2008 Downloaded from mcr.aacrjournals.org on August 11, 2017. © 2008 American Association for Cancer Research. 247 248 Mishra et al. 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. Polyacrylamide-SDS Gel Electrophoresis and Western Blot Assays Equal amounts of protein, ranging from 10 to 30 Ag/sample, were mixed with Laemmli lysis buffer and boiled for 5 min. 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 1. Larsen AK, Escargueil AE, Skladanowski A. 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Balaghi M, Wagner C. DNA methylation in folate deficiency: use of CpG methylase. Biochem Biophys Res Commun 1993;193:1184 – 90. Mol Cancer Res 2008;6(2). February 2008 Downloaded from mcr.aacrjournals.org on August 11, 2017. © 2008 American Association for Cancer Research. 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. Updated version Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://mcr.aacrjournals.org/content/6/2/243 This article cites 44 articles, 16 of which you can access for free at: http://mcr.aacrjournals.org/content/6/2/243.full.html#ref-list-1 This article has been cited by 5 HighWire-hosted articles. Access the articles at: /content/6/2/243.full.html#related-urls Sign up to receive free email-alerts related to this article or journal. 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