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Drug-induced DNA hypermethylation: A potential mediator of acquired drug resistance during cancer chemotherapy
Mutation Research 386 Ž1997. 153–161
Drug-induced DNA hypermethylation: A potential mediator of acquired drug resistance during cancer chemotherapy Jonathan W. Nyce
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Department of Molecular Pharmacology and Therapeutics, EpiGenesis Pharmaceuticals, 101–204 West Fourteenth Street, GreenÕille, NC 27858, USA Accepted 6 December 1996
1. Introduction Enhanced genetic diversity within tumor cell populations is well documented w1x. Tumor cells are known to possess higher rates of mutation than their normal counterparts w2,3x, possibly due to inactivation of critical cell cycle check point proteins w4x, DNA repair mechanisms w5x, and other cellular processes w6x. Such genetic instability in tumor cells is thought to play a major role in tumor progression, metastasis, and the development of resistance to anticancer agents. While less well documented, there is a growing literature which suggests that epigenetic diversity is also enhanced within tumor cell populations w7–28x. Epigenetic instability may also play a significant role in tumor progression and metastasis. My laboratory has studied epigenetic changes within tumor cells that are caused by exposure to cancer chemotherapy drugs, and which confer a state of drug resistance upon the cancer cells w29,30x. Specifically, we have found that: Ø Exposure of tumor cells to DNA synthesis-inhibiting concentrations of anticancer agents is associated with extensive DNA hypermethylation; Ø Such drug-induced DNA hypermethylation is very
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Tel.: q1 Ž919. 752-7036; Fax: q1 Ž919. 752-9148.
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much part of the response to drug-induced toxicity since it is observed primarily in tumor cell populations experiencing ) 95% lethality; Such drug-induced DNA hypermethylation can silence the expression of genes being synthesized during the window of drug-induced toxicity; Such drug-induced DNA hypermethylation can create drug resistance by randomly inactivating genes whose products are required to activate cancer chemotherapy agents to their cancer-killing forms; Drug-induced DNA hypermethylation can be blocked, in a dose-dependent manner, by inhibitors of DNA methylation such as 5-aza-2X-deoxycytidine; Drug-induced DNA hypermethylation is not a tissue culture phenomenon since it occurs in patients undergoing cancer chemotherapy; Cisplatin is the most potent inducer of DNA hypermethylation, possibly due to conformational changes induced by cisplatin adducts which render the affected DNA a better substrate for DNA cytosine 5-methyltransferase; Topoisomerase inhibitors generally inhibit DNA methylation, possibly by inducing conformational changes which render the affected DNA a poor substrate for DNA cytosine 5-methyltransferase; Drug-induced changes in DNA methylation, both
1383-5742r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII 1 3 8 3 - 5 7 4 2 Ž 9 6 . 0 0 0 5 1 - 6
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hyper- and hypomethylation, contribute to tumor cell epigenetic and genetic instability. A discussion of each of these points follows. 1.1. Exposure of tumor cells to DNA synthesis-inhibiting concentrations of anticancer agents is associated with extensiÕe oÕermethylation of newly replicated DNA A broad selection of anticancer drugs were found to induce DNA hypermethylation when applied at toxic concentrations in vitro ŽFig. 1.. For example, cytosine arabinoside ŽaraC., 5-fluorouracil and methotrexate each produced dose-dependent increases in the methylation content of DNA. As expected, the methylation inhibitor 5-azadeoxycytidine Ž5-azadC. decreased methylation in treated tumor cells. Unexpected was the finding that 6-thioguanine inhibited DNA methylation ŽFig. 1.. One possible explanation for this is suggested by recent work which demonstrated that both O6-methylguanine Ža DNA adduct induced by exposure to endogenous and exogenous methylating agents. and 6-thioguanine are repaired by the same short patch repair mechanism w31x. Delayed remethylation of the repair patch could account for the hypomethylation observed w32x. Alternatively, 6-thioguanine could inhibit methylation of 5X cytosines directly. Most of the drug-induced DNA hypermethylation appears within newly replicated DNA, and most Žbut
Fig. 1. Alterations in DNA methylation induced by broad classes of anticancer drugs. HTB-54 human lung epidermoid carcinoma cells were exposed to the indicated concentrations of drugs for 24 h, in the presence of w6- 3 Hxdeoxycytidine, and 5-methylcytosine content was quantitated as previously described w30x. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
not all., within the canonical methylation target dinucleotide CpG w33x. Methylation occurring in dinucleotide fractions other than CpG Že.g., CpA, CpT, CpC. appears to be gradually lost over several cell cycles, but a significant percentage remains even after five cell cycle times w34x. The simplest explanation for drug-induced DNA hypermethylation is that DNA methylase enzymes, which typically trail replication forks, ‘catch up’ to replication forks stalled as a result of drug-induced DNA synthesis inhibition. Due to their increased contact time with newly replicated cytosines in the stalled replication forks, methylase enzymes Žaccording to this view. modify a much larger percentage of cytosines than they normally would. Both cytosines within CpG dinucleotides and cytosines in other dinucleotide fractions apparently become methylated during this process w29,30,34x. 1.2. Drug-induced DNA hypermethylation is part of the response to drug-induced toxicity Although it occurs in a dose-dependent fashion, drug-induced DNA hypermethylation is only readily apparent in tumor cells responding to extremely toxic concentrations of anticancer agents. Typically, greater than 95% cell kill is required, and analytical techniques must be employed that detect methylation only during the window of toxic drug exposure w33x. One such technique is the use of radiolabelled w 14 Cxdeoxycytidine which is incorporated into DNA and the subsequent methylation of which can then be determined by HPLC or HPTLC w29x. Such assays, nevertheless, remain difficult because drug-induced DNA hypermethylation is inversely proportional to the rate of DNA synthesis, and the highest levels of hypermethylation are detected at the lowest levels of w 14 Cxdeoxycytidine incorporation. Alternatively, the drug-stalled replication forks in which hypermethylation occurs may be isolated from bulk DNA and examined directly for 5-methylcytosine content w33x. 1.3. Drug-induced DNA hypermethylation can silence the expression of genes being synthesized during the window of drug-induced toxicity We showed recently that 2X ,3X-dideoxy-3Xazidothymidine ŽAZT., although a weak inducer of
J.W. Nyce r Mutation Research 386 (1997) 153–161
Fig. 2. Correlation between the level of AZT-induced DNA hypermethylation and induction of TKy epimutants in V-79 cells. ŽReprinted with permission from Proc. Natl. Acad. Sci. USA, 90, 2960–2964, 1993..
DNA hypermethylation, was nevertheless capable of leading to a dose-dependent Žboth with respect to micromolar concentration of AZT and level of DNA hypermethylation. induction of thymidine kinase epimutants ŽFig. 2.. Such epimutants were observed to have transcriptionally inactivated thymidine kinase genes ŽTKy. that were rapidly reactivatable upon exposure to the demethylating agent 5-azadC w30x. The fact that the AZT-induced TKy clones were reactivatable to the TKq phenotype at extremely high frequency Žtypically ) 50%., rules out classical mutation as the mechanism of phenotypic reversion, and provides strong evidence that the initial gene silencing event was epigenetic rather than genetic in origin.
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products are required for activation of drugs to their to x ic fo rm . In th e a b o v e e x a m p le , hypermethylation-mediated TK gene inactivation creates a situation of resistance to AZT, as well as to FudR w30x, which also requires activation to its phosphorylated form in order to be toxic. Other examples, potential and realized, include the hypermethylation-mediated transcriptional inactivation of the deoxycytidine kinase gene and resistance to araC; of the hypoxanthine phosphoribosyl transferase ŽHPRT. gene and 6-thioguanine resistance w34x; of topoisomerase II gene inactivation and resistance to etoposide w35x; and of an unidentified gene and resistance to interferon a w36x. 1.5. Drug-induced DNA hypermethylation is not a tissue culture phenomenon since it occurs in patients undergoing cancer chemotherapy In two separate series of experiments we have shown that drug-induced DNA hypermethylation occurs in patients undergoing cancer chemotherapy. In the first study, a pediatric patient undergoing unsuccessful high dose araC chemotherapy for acute myelomonocytic leukemia ŽFig. 3. and an adult patient undergoing hydroxyurea chemotherapy for acute myelocytic leukemia ŽFig. 4. were observed to have
1.4. Drug-induced DNA hypermethylation can create drug resistance by randomly inactiÕating genes whose products are required to actiÕate cancer chemotherapy agents to their cancer-killing forms AZT is inactive until it is metabolized to its phosphorylated forms by thymidine kinase and thymidylate kinase. Induction of TKy, AZT-resistant epimutants correlated with large decreases in TKspecific mRNA levels in AZT-treated cells. Subsequent exposure to 5-azadC, which resensitizes such epimutants to AZT, also is associated with large scale increases in TK-specific mRNA transcripts w30x. Drug-induced DNA hypermethylation is thus capable of transcriptionally inactivating genes whose
Fig. 3. Drug-induced DNA hypermethylation in the leukemic cells of a pediatric patient undergoing unsuccessful high dose cytosine arabinoside ŽHDAC.rhydoxyurea ŽHU.rVP-16 Žetoposide. treatment for myelomonocytic leukemia. Post hydroxyurea s 36 h after administration. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
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Fig. 4. Drug-induced DNA hypermethylation in vivo in the acute myelocytic leukemia cells of an adult patient undergoing treatment with hydroxyurea. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
4- to 5-fold increases in their 5-methylcytosine content which, in the former case, had not returned to normal one month post treatment w29x. In a subsequent study, a series of patients with advanced acute lymphocytic leukemia treated with carboplatin, an isomer of cisplatin, were observed to have significant tumor-associated DNA hypermethylation up to 1 month following carboplatin exposure. This study is described in more detail below. 1.6. Drug-induced DNA hypermethylation can be blocked, in a dose-dependent manner, by inhibitors of DNA methylation such as 5-aza-2X-deoxycytidine Cisplatin-induced DNA hypermethylation in HTB-54 human lung adenocarcinoma cells was found to be preventable, in a dose-dependent fashion, by simultaneous exposure to 5-azadC at doses ranging from 0.001 to 10 mM ŽFig. 5.. AraC-induced DNA hypermethylation was similarly found to be inhibited by simultaneous exposure to 5-azadC within the same dose range w29x. In the clinical study of acute lymphocytic leukemia mentioned above, a series of patients were treated with 5-azacytidine Ž50 mgrm2rday on days 1 through 5. in addition to carboplatin Ž250 mgrm2rday on days 3 through 7.. Such exposure to 5-azacytidine was observed to transiently prevent carboplatin-induced DNA hypermethylation ŽTable 1.. The transient nature of this effect was probably due to the short half-life of 5-azacytidine as compared to carboplatin, which per-
Fig. 5. Prevention of cisplatinum-induced DNA hypermethylation in HTB-54 human lung adenocarcinoma cells pretreated with X varying concentrations of the methylation inhibitor, 5-aza-2 -deoxycytidine ŽazadC.. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
mitted carboplatin to induce hypermethylation once effective DNA methylation-inhibiting levels of 5azacytidine had diminished. 1.7. Cisplatin: A special case of drug-induced DNA hypermethylation Cisplatin is one of the most commonly used anticancer drugs. However, the development of resisTable 1 Patient
a
T0
Ž5mC.
Ž5mC.
Day 1–3
Day 4–8
Day 9–30
1 2 3 4 5 6 7 8 9 10
4.4"0.26 2.3"0.01 4.4"0.91 3.6"1.50 4.4"0.03 3.6"0.01 6.5"0.02 3.6"0.20 5.6"0.21 6.1"0.11
1.2"0.22 1.4"0.02 ) n.d. 2.8"1.0 3.3"0.02 n.d. 4.6"0.42 3.6"0.01 ) n.d. n.d.
8.5"1.4
n.d.
5.6"0.8 3.8"1.1 3.7"0.03 3.2"0.01 n.d. n.d. ) 8.4"0.03 n.d.
7.3"0.7 n.d. n.d. 5.9"0.5 5.5"0.1
Average
4.5"1.2
2.8"1.3
5.5"2.4
6.9"1.6
9.8"0.22 6.0"1.6
T0 Ž5mC., pretreatment levels of 5-methylcytosine; Ž5mC. day 1–3, levels of 5-methylcytosine on days 1, 2 or 3, depending on availability of clinical specimen; Ž5mC. day 4–8, levels of 5methylcytosine on days 4, 5, 6, 7 or 8, depending on availability of clinical specimen; Ž5mC. day 9–30, 5-methylcytosine levels obtained on clinical specimens obtained between 8 and 30 days post treatment initiation. a Patient expired within this time frame.
J.W. Nyce r Mutation Research 386 (1997) 153–161
tance to cisplatin represents a major obstacle in its effective use. The extreme level of DNA hypermethylation induced by cisplatin exposure is noteworthy w29x. It is in contrast to alkylating agents, which also directly modify DNA, but which inhibit DNA methylation w29x, possibly by inactivating the sulfhydrylrich DNA methylase enzyme w37x. Cisplatin exposure increases the 5-methylcytosine content of DNA to a greater degree than any other compound tested. This suggests that some process other than DNA synthesis inhibition might be contributing. Recent studies have shown that the major adduct induced by cisplatin in DNA is an intrastrand crosslink between two adjacent guanines on the same DNA strand w38x. The presence of such dŽG)pG). cisplatin adducts in DNA significantly alters the DNase I cleavage pattern w39,40x. It is thought that the enhanced Dnase I cleavage of DNA containing dŽG)pG). cisplatin adducts is due to distortions induced in DNA by the adducts. The dŽG)pG). adduct bends the double helix 32–348 towards the major groove, thus widening the minor groove, and unwinds the DNA by approximately 138 w41x. The crystallographic structure of the Dnase I-DNA complex indicates that a loop of the enzyme penetrates into the minor groove of the DNA and interacts asymmetrically with the backbone of both strands, contacting two phosphate groups on each side of the cleaved bond and two phosphate groups on the adjacent strand across the minor groove w42x. We suggest that the cisplatin adduct-induced distortions in DNA, in a manner similar to that for DNase I, may render DNA a better substrate for DNA methyltransferase. This could occur by virtue of the modified DNA offering better access to the five carbon atom of cytosines within the major groove, or greater affinity of the enzyme for cisplatin-kinked DNA. An alternative perspective is that methylation marks ‘suspect’ DNA for scrutiny by other enzymes, such as those involved in DNA repair processes w43x. Precedent for this perspective might be obtained from experiments demonstrating that transfected DNA is rapidly methylated and inactivated w44x, and that viral DNA is a target for DNA methylase and gene inactivation w45x, w46x. However, the recent finding that 5-methylcytosine inhibits repair of 3X nearest neighbor O6-methylguanines by O6-methylguanine DNA methyltransferase w47x suggests that drug-induced DNA hyper-
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methylation is unlikely to play a general role in marking DNA containing adducts in need of repair. 1.8. DNA topoisomerase II inhibitors: A special case of DNA hypomethylation We have shown that topoisomerase II inhibitors, including nalidixic acid, novobiocin, etoposide and teniposide, induce DNA hypomethylation w33,48x. This is interesting in view of data showing that exposure to topoisomerase II inhibitors alters gene expression and induces differentiation in a number of experimental systems w49,50x and the strong association of DNA hypomethylation with gene activation w44x. One explanation of our findings that could relate them to the effects of topoisomerase inhibitors upon gene expression is that topoisomerase II releases DNA from that topological conformation that constitutes an efficient substrate for DNA methylase. Thus, even in the presence of increased amounts of DNA methylase in some malignant cells w25x, large stretches of DNA might be rendered methylation-resistant by topoisomerase activity. ŽIt should be pointed out that topoisomerase II inhibitors generally permit topoisomerase activity Žstrand breaking, relief of torsional stress. but prevent the strand rejoining activity of the enzyme.. The ability of topoisomerase inhibitors to induce DNA hypomethylation could offer a potential explanation for their demonstrated effects upon gene expression and differentiation. Since demethylating agents have been demonstrated to be capable of activating transcriptionally repressed genes, it is reasonable to postulate that the ability of topoisomerase inhibitors to activate gene expression might be related to their ability to inhibit DNA methylation w48x. We have suggested that the enhanced topoisomerase activity observed in tumor cells w51,52x, by virtue of its potential to inhibit DNA methylation, might account for the ectopic gene expression long considered to be a hallmark of the malignant state. We and others have shown that exposure of mammalian cells to topoisomerase II inhibitors leads to the generation of polyploid DNA content, another hallmark of the malignant cell. Increased levels of topoisomerase enzymes in malignant cells could thus provide a theoretical link between the induction of polyploidy, DNA hypomethylation and ectopic gene expression,
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three markers for genetic instability in malignant cells. 1.9. Genetic effects of drug-induced DNA hypermethylation The dinucleotide CpG is strongly underrepresented in human and animal DNA, being present at only about 20–25% of its expected frequency. There is good evidence that this ‘CpG suppression’ is due to the fact that CpG is the target for DNA methylation, and that the product, 5-methylcytosine, undergoes spontaneous deamination to thymidine. Over time, then, CpG tends to be replaced by TpG and its replication product CpA. Evidence has arisen that such spontaneous deamination of 5-methylcytosine in DNA may represent one of the most important types of mutation in human disease w53–56,7,57x. To the extent that cells containing hypermethylated replication forks recover from toxic drug exposure, they would be expected to have an increased mutation rate due to the subsequent spontaneous deamination of the newly methylated cytosines. Thus, epigenetic changes occurring during the carcinogenic process or as a result of exposure to drugs during cancer chemotherapy, should increase the level of genetic instability within the tumor cell population. Changes in DNA methylation may, in fact, play a major role in promoting genetic instability within tumor cell populations. Methylation-mediated changes in genomic stability could result from gene activation in regions of hypomethylation Že.g., of genes associated with metastasis, angiogenesis, recombination, etc.., gene inactivation within regions of hypermethylation Že.g., of tumor suppresser genes or genes whose products are required to activate anticancer drugs, etc.., reduced ability to identify the parental template strand during DNA mismatch repair, or through the generation of mutation ‘hotspots’ in hypermethylated regions of the tumor cell genome. As mentioned above, it is also possible that changes in DNA methylation occurring in tumor cells might set in motion a cascade of ectopic gene expression events that might release tumor cells from normal homeostatic controls, as well as transducing epigenetic events into genetic ones by the induction of polyploidy or via mutagenic deamination of 5-methylcytosine.
1.10. Implications for cancer chemotherapy Drug-induced DNA hypermethylation represents one potential mechanism of drug resistance for those agents requiring enzymatic activation to achieve cytotoxicity. Since not all drugs induce DNA hypermethylation, but in fact induce DNA hypomethylation Že.g., alkylating agents, 6-thioguanine, 5-azadeoxycytidine, etc.., it may be possible to reduce that portion of drug resistance that results from drug-induced DNA hypermethylation by appropriately combining DNA methylation inhibitors with hypermethylating drugs. In our limited experience in vivo, however, it has not been possible to achieve long lasting blockade of DNA hypermethylation in patients undergoing sequential treatment with 5azacytidine and carboplatin. This was probably due to the differences in half lives of the two drugs. Alternative regimens employing longer lasting methylation inhibitors, or methylation inhibitors applied by continuous infusion, might achieve durable inhibition of drug-induced DNA hypermethylation. However, precise titration of the hypermethylationrhypomethylation phenomenon to achieve steady state levels of DNA methylation during cytotoxic chemotherapy may be difficult or impossible to achieve. An additional problem is that hypomethylation itself might contribute to the problem of acquired drug resistance. Perry et al. have shown, for example, that 5-aza-2X-deoxycytidine exposure significantly enhances both the frequency and the rate of gene amplification in vitro w58x. Gene amplification is thought to underly the development of resistance to a wide array of anticancer agents w59,60x. Attempts to moderate drug-induced DNA hypermethylationmediated drug resistance by addition of hypomethylating agents to chemotherapy protocols could result in the amplification of genes associated with drug resistance, e.g., the MDR1 gene associated with multi drug resistance. The principal paradigm for anticancer drug design for the past 50 years has been cytotoxicity – the search for ever more potent toxins – and an increasing clinical focus on applying anticancer drugs at their maximum tolerable dosage. Our results suggest that such a treatment strategy may be problematic in the absence of 100% tumor cell kill since the surviving cancer cell population, because of its enhanced
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epigeneticrgenetic diversity, may be substantially superior to normal cell populations with respect to their ability to withstand further selective pressure, i.e., drug exposure. At present, significant effort is being spent by many laboratories on designing new paradigms for anticancer therapy, including therapies targeting tumor cell differentiation and apoptosis. These approaches are based upon the premises that differentiated cells do not divide, and that the normal program of cell death, apoptosis, is aberrantly inactivated in malignant tumor cells. Some of these therapeutic strategies are based on setting in motion epigenetic cascades in order to reprogram aberrant patterns of gene expression in the tumor cell. In exploring these alternative therapeutic strategies, one should consider the dual possibilities that epigenetic instability may contribute to tumor progression in a manner similar to that of genetic instability; and that these two processes intersect in their cause and effect relationships.
Acknowledgements The technical efforts of Ms. Sherry Leonard, Ms. So Wong, and Ms. Dawn Mylott are gratefully acknowledged. Patient specimens were kindly provided by Drs. Spencer Raab Ždeceased., C. Tate Holbrook, and Alan D. Kritz. This work was performed with support from grant CA RO1 47217 from the National Cancer Institute.
References w1x Usmani, B.A. Ž1993. Genomic instability and metastatic progression, Pathobiology 61Ž2., 109–116. w2x Sweezy, M.A. and Fishel, R. Ž1994. Multiple pathways leading to genomic instability and tumorigenesis, Ann. NY Acad. Sci. 726, 165–177. w3x Cheng, K.C. and Loeb, L. Ž1993. Genomic instability and tumor progression: mechanistic considerations, Adv. Cancer Res., 60, 121–156. w4x Hartwell, L. Ž1992. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells, Cell, 71Ž4., 543–546. w5x Honchel, R., Halling, K.C. and Thibodeau, S.N. Ž1995. Genomic instability in neoplasia, Sem. Cell Biol., 6Ž1., 45– 52.
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w6x Russo, C.A., Weber, T.K., Volpe, C.M., Stoler, D.L., Petrelli, N.J., Rodriguez-Bigas, M., Burhans, W.C. and Anderson, G.R. Ž1995. An anoxia inducible endonuclease and enhanced DNA breakage as contributors to genomic instability in cancer, Cancer Res., 55Ž5., 1122–1128. w7x Nyce, J., Weinhouse, S. and Magee, P.N. Ž1983. 5-Methylcytosine depletion during tumor development: an extension of the miscoding concept, Br. J. Cancer, 48Ž4., 463–475. w8x Yamasaki, H., Mesnil, M. and Nakazawa, H. Ž1992. Interaction and distinction of genotoxic and non-genotoxic events in carcinogenesis, Toxicol. Lett., 64–65, 597–604. w9x Nyce, J., Leonard, S., Canupp, D., Schulz, S. and Wong, S. Ž1993. Epigenetic mechanisms of drug resistance: drug-induced DNA hypermethylation and drug resistance, Proc. Natl. Acad. Sci. USA, 90Ž7., 2960–2964. w10x Ribieras, S., Song-Wang, X.G., Martin, V., Lointier, P., Frappart, L. and Dante, R. Ž1994. Human breast and colon cancers exhibit alterations of DNA methylation patterns at several DNA segments on chromosomes pp. 11 and pp. 17. J. Cell. Biochem., 56Ž1., 86–96. w11x Rideout, W.M. 3rd, Eversole-Cire, P., Spruck, C.H. 3rd, Hustad, C.M., Coetzee, G.A., Gonzales, F.A. and Jones, P.A. Ž1994. Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation, Mol. Cell. Biol., 14Ž9., 6143–6152. w12x Herman, J.G., Latif, F., Weng, Y., Lerman, M.I., Zbar, B., Liu, S., Samid, D., Duan, D.S., Gnarra, J.R. and Linehan, W.M. et al. Ž1994. Silencing of the VHL tumor-suppresser gene by DNA methylation in renal carcinoma, Proc. Natl. Acad. Sci. USA, 91Ž21., 9700–9704. w13x Lee, Y.W., Klein, C.B., Kargacin, B., Salnikow, K., Kitahara, J., Dowjat, K., Zhitkovich, A., Christie, N.T. and Costa, M. Ž1995. Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens, Mol. Cell. Biol., 15Ž5., 2547–2557. w14x Greger, V., Debus, N., Lohmann, D., Hopping, W., Passarge, E. and Horsthemke, B. Ž1994. Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma, Human Genet., 94Ž5., 491–496. w15x Costa, M. Ž1995. Model for the epigenetic mechanism of action of nongenotoxic carcinogens, Am. J. Clin. Nutrition 61Ž3 Suppl.., 666S–669S. w16x Ferguson, A.T., Lapidus, R.G., Baylin, S.B. and Davidson, N.E. Ž1995. Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression, Cancer Res., 55Ž11., 2279–2283. w17x Yoshiura, K., Kanai, Y., Ochiai, A., Shimoyama, Y., Sugimura, T. and Hirohashi, S. Ž1995. Silencing of the E-cadherin invasion-suppresser gene by CpG methylation in human carcinomas, Proc. Natl. Acad. Sci. USA, 92Ž16., 7416–7419. w18x Pieretti, M., Powell, D.E., Gallion, H.H., Conway, P.S., Case, E.A. and Turker, M.S. Ž1995. Hypermethylation at a chromosome 17 ‘hot spot’ is a common event in ovarian cancer, Hum. Pathol., 26Ž4., 398–401. w19x Moulton, T., Crenshaw, T., Hao, Y., Moosikasuwan, J., Lin,
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w23x
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w25x w26x
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w35x
w36x
J.W. Nyce r Mutation Research 386 (1997) 153–161 N., Dembitzer, F., Hensle, T., Weiss, L., McMorrow, L. and Loew, T. et al. Ž1994. Epigenetic lesions at the H19 locus in Wilms’ tumor patients, Nature Genet., 7Ž3., 440–447. Laird, P.W. and Jaenisch, R. Ž1994. DNA methylation and cancer, Hum. Mol. Genet. 3 Spec. No. 1487–1495. Matsuo, K., Tang, S.H., Zeki, K., Gutman, R.A. and Fagin, J.A. Ž1993. Aberrant deoxyribonucleic acid methylation in human thyroid tumors, J. Clin. Endocrinol. Metab., 77Ž4., 991–995. Mills, K.I., Sproul, A.M. and Burnett, A.K. Ž1993. Methylation of the major breakpoint cluster region ŽM-bcr. in Philadelphia-positive CML, Leukemia, 17Ž5., 707–711. Issa, J.P., Vertino, P.M., Wu, J., Sazawal, S., Celano, P., Nelkin, B.D., Hamilton, S.R. and Baylin, S.B. Ž1993. Increased cytosine DNA-methyltransferase activity during colon cancer progression, J. Natl. Cancer Inst., 85Ž15., 1235–1240. Ohtani-Fujita, N., Fujita, T., Aoike, A., Osifchin, N.E., Robbins, P.D. and Sakai, T. Ž1993. CpG methylation inactivates the promoter activity of the human retinoblastoma tumorsuppresser gene, Oncogene, 8Ž4., 1063–1067. Baylin, S. Ž1992., Abnormal regional hypermethylation in cancer cells, AIDS Res. Hum. Retroviruses, 8Ž5., 811–820. Makos, M., Nelkin, B.D., Chazin, V.R., Cavenee, W.K., Brodeur, G.M. and Baylin, S.B. Ž1993. DNA hypermethylation is associated with 17p allelic loss in neural tumors, Cancer Res., 53Ž12., 2715–2718. Sakai, T., Toguchida, J., Ohtani, N., Yandell, D.W., Rapaport, J.M. and Dryja, T.P. Ž1991. Allele-specific hypermethylation of the retinoblastoma tumor-suppresser gene, Am. J. Human Genet., 48Ž5., 880–888. Peltomaki, P. Ž1991. DNA methylation changes in human testicular cancer, Biochim. Biophys. Acta, 1096Ž3., 187–196 Nyce, J., Mylott, D., Leonard, S., Willis, L. and Kataria, A. Ž1989. Detection of drug-induced DNA hypermethylation in human cells exposed to cancer chemotherapy agents, J. Liq. Chrom., 12Ž8., 1313–1321. Nyce, J.W. Ž1989. Drug-induced DNA hypermethylation and drug resistance in human tumors, Cancer Res., 49Ž21., 5829– 5836. Griffin, S., Branch, P., Xu, Y.Z. and Karran, P. Ž1994. DNA mismatch binding and incision at modified guanine bases by extracts of mammalian cells: implications for tolerance to DNA methylation damage, Biochemistry, 33Ž16., 4787–4793. Kastan, M.B., Gowans, B.J. and Lieberman, M.W. Ž1982. Methylation of deoxycytidine incorporated by excision repair synthesis of DNA, Cell, 30Ž2., 509–516. Nyce, J., Liu, L. and Jones, P.A. Ž1986. Variable effects of DNA synthesis inhibitors upon DNA methylation in mammalian cells, Nucleic Acids Res., 14Ž10., 4353–4367. Nyce, J. Ž1991. Gene silencing in mammalian cells by direct incorporation of electroporated 5-methyl-2X deoxycytidine 5’triphosphate, Somatic Cell Mol. Genet., 17, 543–550. Leonard, S.A., Wong, S.C. and Nyce, J.W. Ž1993. Quantitation of 5-methylcytosine by one-dimensional high-performance thin-layer chromatography, J. Chrom., 645, 189–192. Reid, T.R., Merigan, T.C. and Basham, T.Y. Ž1992. Resis-
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tance to interferon a in a mouse B-cell lymphoma involves DNA methylation, J. Interferon Res., 12Ž2., 131–137. Wilson, V. and Jones, P. Ž1983. Inhibition of DNA methylation by chemical carcinogens in vitro, Cell, 32, 239–246. Lepre, C.A. and Lippard, S.J. Ž1990. Interaction of platinum antitumor compounds with DNA, in: F. Eckstein and D.M. Lilley ŽEds.., Nucleic Acids and Molecular Biology, Vol. 4, pp. 3–9, Springer-Verlag, Berlin. Visse, R. and deRuijter, M., Brouwer, J.A. and van de Putte, P. Ž1991. Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA, J. Biol. Chem., 266, 7609–7617. Schwartz, A. and Leng, M. Ž1994. Dnase I footprinting of cis- and trans-diamminedichloroplatinumŽII.-modified DNA, J. Mol. Biol., 236, 969–974. Bellon, S.F., Coleman, J.H. and Lippard, S.J. Ž1991. DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum ŽII., Biochemistry, 30Ž32., 8026–8035. Suck, D., Lahm, A. and Oefner, C. Ž1988. Structure refined to 2 Angstrom of a nicked DNA octanucleotide complex with DNase I, Nature, 332, 464–468. Hare, J.T. and Taylor, J.H. Ž1985. One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair, Proc. Natl. Acad. Sci. USA, 82Ž21., 7350–7354. Razin, A. and Cedar, H. Ž1991. DNA methylation and gene expression, Microbiol. Rev., 55Ž3., 451–458. Bednarik, D.P., Duckett, C., Kim, S.U., Perez, V.L., Griffis, K., Guenthner, P.C. and Folks, T.M. Ž1991. DNA CpG methylation inhibits binding of NF-kappa B proteins to the HIV-1 long terminal repeat cognate DNA motifs, New Biologist, 3Ž10., 969–976. Gutekunst, K.A., Kashanchi, F., Brady, J.N. and Bednarik, D.P. Ž1993. Transcription of the HIV-1 LTR is regulated by the density of DNA CpG methylation. J. Acq. Immune Defic. Syndromes, 6Ž6., 541–549. Bentivegna, S.S. and Bresnick, E. Ž1994. Inhibition of human O6 -methylguanine DNA methyltransferase by 5-methylcytosine, Cancer Res., 54Ž2., 327–329. Schonfeld, S., Schulz, S. and Nyce, J.W. Ž1992. Effects of inhibitors of topoisomerases I and II on DNA methylation and DNA synthesis in human colonic adenocarcinoma cells in vitro, Intern. J. Oncology, 1, 807–814. Nakaya, K., Chou, S., Kaneko, M., and Nakamura, Y. Ž1991. Topoisomerase inhibitors have potent differentiation-inducing activity for human and mouse myeloid leukemia cells. Jpn. J. Cancer Res., 82, 184–191. Rappa, G., Lorico, A. and Sartorelli, A.C. Ž1990. Induction of the differentiation of WEHI-3B Dqmonomyelocytic leukemia cells by inhibitors of topoisomerase II, Cancer Res., 50, 6723–6730. Holden, J.A., Rolfson, D.H. and Wittwer Ž1990. Human DNA topoisomerase II: evaluation of enzyme activity in normal and neoplastic tissues, Biochemistry, 29Ž8., 2127– 2134. Hsiang, Y.H., Wu, H.Y. and Liu, L.F. Ž1988. Proliferation-
J.W. Nyce r Mutation Research 386 (1997) 153–161
w53x
w54x
w55x w56x
dependent regulation of DNA topoisomerase II in cultured cells, Cancer Res., 48, 3230–3235. Shapiro, R. and Klein, R.S. Ž1966. The deamination of cytidine and cytosine by acidic buffer solutions. Mutagenic implications, Biochemistry, 5Ž7., 2358–2362. Coulondre, C., Miller, J.H., Farabaugh, P.J. and Gilbert, W. Ž1978. Molecular basis of base substitution hotspots in Escherichia coli, Nature, 274Ž5673., 775–780. Duncan, B.K. and Miller, J.H. Ž1980. Mutagenic deamination of cytosine residues in DNA, Nature, 287Ž5782., 560–561. Wang, R.Y., Kuo, K.C., Gehrke, C.W., Huanh, L.H. and Ehrlich, M. Ž1982. Heat and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA, Biochim. Biophys. Acta, 697Ž3., 371–377.
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w57x Rideout, W.M. 3d, Coetzee, G.A., Olumi, A.F. and Jones, P.A. Ž1990. 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes, Science, 249Ž4974., 1288–1290. w58x Perry, M.E., Rolfe, M., McIntyre, P., Commane, M. and Stark, G.R. Ž1992. Induction of gene amplification by 5-azaX 2 -deoxycytidine, Mutation Res., 276Ž3., 189–197. w59x Roninson, I.B. Ž1992. From amplification to function: the case of the MDR1 gene, Mutation Res., 276Ž3., 151–161. w60x Harnevo. L.E. and Agur, Z. Ž1992. Drug resistance as a dynamic process in a model for multistep gene amplification under various levels of selection stringency, Cancer Chemother. Pharmacol., 30Ž6., 469–476.
Drug-induced DNA hypermethylation: A potential mediator of acquired drug resistance during cancer chemotherapy Jonathan W. Nyce
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Department of Molecular Pharmacology and Therapeutics, EpiGenesis Pharmaceuticals, 101–204 West Fourteenth Street, GreenÕille, NC 27858, USA Accepted 6 December 1996
1. Introduction Enhanced genetic diversity within tumor cell populations is well documented w1x. Tumor cells are known to possess higher rates of mutation than their normal counterparts w2,3x, possibly due to inactivation of critical cell cycle check point proteins w4x, DNA repair mechanisms w5x, and other cellular processes w6x. Such genetic instability in tumor cells is thought to play a major role in tumor progression, metastasis, and the development of resistance to anticancer agents. While less well documented, there is a growing literature which suggests that epigenetic diversity is also enhanced within tumor cell populations w7–28x. Epigenetic instability may also play a significant role in tumor progression and metastasis. My laboratory has studied epigenetic changes within tumor cells that are caused by exposure to cancer chemotherapy drugs, and which confer a state of drug resistance upon the cancer cells w29,30x. Specifically, we have found that: Ø Exposure of tumor cells to DNA synthesis-inhibiting concentrations of anticancer agents is associated with extensive DNA hypermethylation; Ø Such drug-induced DNA hypermethylation is very
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Tel.: q1 Ž919. 752-7036; Fax: q1 Ž919. 752-9148.
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much part of the response to drug-induced toxicity since it is observed primarily in tumor cell populations experiencing ) 95% lethality; Such drug-induced DNA hypermethylation can silence the expression of genes being synthesized during the window of drug-induced toxicity; Such drug-induced DNA hypermethylation can create drug resistance by randomly inactivating genes whose products are required to activate cancer chemotherapy agents to their cancer-killing forms; Drug-induced DNA hypermethylation can be blocked, in a dose-dependent manner, by inhibitors of DNA methylation such as 5-aza-2X-deoxycytidine; Drug-induced DNA hypermethylation is not a tissue culture phenomenon since it occurs in patients undergoing cancer chemotherapy; Cisplatin is the most potent inducer of DNA hypermethylation, possibly due to conformational changes induced by cisplatin adducts which render the affected DNA a better substrate for DNA cytosine 5-methyltransferase; Topoisomerase inhibitors generally inhibit DNA methylation, possibly by inducing conformational changes which render the affected DNA a poor substrate for DNA cytosine 5-methyltransferase; Drug-induced changes in DNA methylation, both
1383-5742r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII 1 3 8 3 - 5 7 4 2 Ž 9 6 . 0 0 0 5 1 - 6
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hyper- and hypomethylation, contribute to tumor cell epigenetic and genetic instability. A discussion of each of these points follows. 1.1. Exposure of tumor cells to DNA synthesis-inhibiting concentrations of anticancer agents is associated with extensiÕe oÕermethylation of newly replicated DNA A broad selection of anticancer drugs were found to induce DNA hypermethylation when applied at toxic concentrations in vitro ŽFig. 1.. For example, cytosine arabinoside ŽaraC., 5-fluorouracil and methotrexate each produced dose-dependent increases in the methylation content of DNA. As expected, the methylation inhibitor 5-azadeoxycytidine Ž5-azadC. decreased methylation in treated tumor cells. Unexpected was the finding that 6-thioguanine inhibited DNA methylation ŽFig. 1.. One possible explanation for this is suggested by recent work which demonstrated that both O6-methylguanine Ža DNA adduct induced by exposure to endogenous and exogenous methylating agents. and 6-thioguanine are repaired by the same short patch repair mechanism w31x. Delayed remethylation of the repair patch could account for the hypomethylation observed w32x. Alternatively, 6-thioguanine could inhibit methylation of 5X cytosines directly. Most of the drug-induced DNA hypermethylation appears within newly replicated DNA, and most Žbut
Fig. 1. Alterations in DNA methylation induced by broad classes of anticancer drugs. HTB-54 human lung epidermoid carcinoma cells were exposed to the indicated concentrations of drugs for 24 h, in the presence of w6- 3 Hxdeoxycytidine, and 5-methylcytosine content was quantitated as previously described w30x. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
not all., within the canonical methylation target dinucleotide CpG w33x. Methylation occurring in dinucleotide fractions other than CpG Že.g., CpA, CpT, CpC. appears to be gradually lost over several cell cycles, but a significant percentage remains even after five cell cycle times w34x. The simplest explanation for drug-induced DNA hypermethylation is that DNA methylase enzymes, which typically trail replication forks, ‘catch up’ to replication forks stalled as a result of drug-induced DNA synthesis inhibition. Due to their increased contact time with newly replicated cytosines in the stalled replication forks, methylase enzymes Žaccording to this view. modify a much larger percentage of cytosines than they normally would. Both cytosines within CpG dinucleotides and cytosines in other dinucleotide fractions apparently become methylated during this process w29,30,34x. 1.2. Drug-induced DNA hypermethylation is part of the response to drug-induced toxicity Although it occurs in a dose-dependent fashion, drug-induced DNA hypermethylation is only readily apparent in tumor cells responding to extremely toxic concentrations of anticancer agents. Typically, greater than 95% cell kill is required, and analytical techniques must be employed that detect methylation only during the window of toxic drug exposure w33x. One such technique is the use of radiolabelled w 14 Cxdeoxycytidine which is incorporated into DNA and the subsequent methylation of which can then be determined by HPLC or HPTLC w29x. Such assays, nevertheless, remain difficult because drug-induced DNA hypermethylation is inversely proportional to the rate of DNA synthesis, and the highest levels of hypermethylation are detected at the lowest levels of w 14 Cxdeoxycytidine incorporation. Alternatively, the drug-stalled replication forks in which hypermethylation occurs may be isolated from bulk DNA and examined directly for 5-methylcytosine content w33x. 1.3. Drug-induced DNA hypermethylation can silence the expression of genes being synthesized during the window of drug-induced toxicity We showed recently that 2X ,3X-dideoxy-3Xazidothymidine ŽAZT., although a weak inducer of
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Fig. 2. Correlation between the level of AZT-induced DNA hypermethylation and induction of TKy epimutants in V-79 cells. ŽReprinted with permission from Proc. Natl. Acad. Sci. USA, 90, 2960–2964, 1993..
DNA hypermethylation, was nevertheless capable of leading to a dose-dependent Žboth with respect to micromolar concentration of AZT and level of DNA hypermethylation. induction of thymidine kinase epimutants ŽFig. 2.. Such epimutants were observed to have transcriptionally inactivated thymidine kinase genes ŽTKy. that were rapidly reactivatable upon exposure to the demethylating agent 5-azadC w30x. The fact that the AZT-induced TKy clones were reactivatable to the TKq phenotype at extremely high frequency Žtypically ) 50%., rules out classical mutation as the mechanism of phenotypic reversion, and provides strong evidence that the initial gene silencing event was epigenetic rather than genetic in origin.
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products are required for activation of drugs to their to x ic fo rm . In th e a b o v e e x a m p le , hypermethylation-mediated TK gene inactivation creates a situation of resistance to AZT, as well as to FudR w30x, which also requires activation to its phosphorylated form in order to be toxic. Other examples, potential and realized, include the hypermethylation-mediated transcriptional inactivation of the deoxycytidine kinase gene and resistance to araC; of the hypoxanthine phosphoribosyl transferase ŽHPRT. gene and 6-thioguanine resistance w34x; of topoisomerase II gene inactivation and resistance to etoposide w35x; and of an unidentified gene and resistance to interferon a w36x. 1.5. Drug-induced DNA hypermethylation is not a tissue culture phenomenon since it occurs in patients undergoing cancer chemotherapy In two separate series of experiments we have shown that drug-induced DNA hypermethylation occurs in patients undergoing cancer chemotherapy. In the first study, a pediatric patient undergoing unsuccessful high dose araC chemotherapy for acute myelomonocytic leukemia ŽFig. 3. and an adult patient undergoing hydroxyurea chemotherapy for acute myelocytic leukemia ŽFig. 4. were observed to have
1.4. Drug-induced DNA hypermethylation can create drug resistance by randomly inactiÕating genes whose products are required to actiÕate cancer chemotherapy agents to their cancer-killing forms AZT is inactive until it is metabolized to its phosphorylated forms by thymidine kinase and thymidylate kinase. Induction of TKy, AZT-resistant epimutants correlated with large decreases in TKspecific mRNA levels in AZT-treated cells. Subsequent exposure to 5-azadC, which resensitizes such epimutants to AZT, also is associated with large scale increases in TK-specific mRNA transcripts w30x. Drug-induced DNA hypermethylation is thus capable of transcriptionally inactivating genes whose
Fig. 3. Drug-induced DNA hypermethylation in the leukemic cells of a pediatric patient undergoing unsuccessful high dose cytosine arabinoside ŽHDAC.rhydoxyurea ŽHU.rVP-16 Žetoposide. treatment for myelomonocytic leukemia. Post hydroxyurea s 36 h after administration. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
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Fig. 4. Drug-induced DNA hypermethylation in vivo in the acute myelocytic leukemia cells of an adult patient undergoing treatment with hydroxyurea. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
4- to 5-fold increases in their 5-methylcytosine content which, in the former case, had not returned to normal one month post treatment w29x. In a subsequent study, a series of patients with advanced acute lymphocytic leukemia treated with carboplatin, an isomer of cisplatin, were observed to have significant tumor-associated DNA hypermethylation up to 1 month following carboplatin exposure. This study is described in more detail below. 1.6. Drug-induced DNA hypermethylation can be blocked, in a dose-dependent manner, by inhibitors of DNA methylation such as 5-aza-2X-deoxycytidine Cisplatin-induced DNA hypermethylation in HTB-54 human lung adenocarcinoma cells was found to be preventable, in a dose-dependent fashion, by simultaneous exposure to 5-azadC at doses ranging from 0.001 to 10 mM ŽFig. 5.. AraC-induced DNA hypermethylation was similarly found to be inhibited by simultaneous exposure to 5-azadC within the same dose range w29x. In the clinical study of acute lymphocytic leukemia mentioned above, a series of patients were treated with 5-azacytidine Ž50 mgrm2rday on days 1 through 5. in addition to carboplatin Ž250 mgrm2rday on days 3 through 7.. Such exposure to 5-azacytidine was observed to transiently prevent carboplatin-induced DNA hypermethylation ŽTable 1.. The transient nature of this effect was probably due to the short half-life of 5-azacytidine as compared to carboplatin, which per-
Fig. 5. Prevention of cisplatinum-induced DNA hypermethylation in HTB-54 human lung adenocarcinoma cells pretreated with X varying concentrations of the methylation inhibitor, 5-aza-2 -deoxycytidine ŽazadC.. ŽReprinted with permission from Cancer Res., 49, 5829–5836, 1989..
mitted carboplatin to induce hypermethylation once effective DNA methylation-inhibiting levels of 5azacytidine had diminished. 1.7. Cisplatin: A special case of drug-induced DNA hypermethylation Cisplatin is one of the most commonly used anticancer drugs. However, the development of resisTable 1 Patient
a
T0
Ž5mC.
Ž5mC.
Day 1–3
Day 4–8
Day 9–30
1 2 3 4 5 6 7 8 9 10
4.4"0.26 2.3"0.01 4.4"0.91 3.6"1.50 4.4"0.03 3.6"0.01 6.5"0.02 3.6"0.20 5.6"0.21 6.1"0.11
1.2"0.22 1.4"0.02 ) n.d. 2.8"1.0 3.3"0.02 n.d. 4.6"0.42 3.6"0.01 ) n.d. n.d.
8.5"1.4
n.d.
5.6"0.8 3.8"1.1 3.7"0.03 3.2"0.01 n.d. n.d. ) 8.4"0.03 n.d.
7.3"0.7 n.d. n.d. 5.9"0.5 5.5"0.1
Average
4.5"1.2
2.8"1.3
5.5"2.4
6.9"1.6
9.8"0.22 6.0"1.6
T0 Ž5mC., pretreatment levels of 5-methylcytosine; Ž5mC. day 1–3, levels of 5-methylcytosine on days 1, 2 or 3, depending on availability of clinical specimen; Ž5mC. day 4–8, levels of 5methylcytosine on days 4, 5, 6, 7 or 8, depending on availability of clinical specimen; Ž5mC. day 9–30, 5-methylcytosine levels obtained on clinical specimens obtained between 8 and 30 days post treatment initiation. a Patient expired within this time frame.
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tance to cisplatin represents a major obstacle in its effective use. The extreme level of DNA hypermethylation induced by cisplatin exposure is noteworthy w29x. It is in contrast to alkylating agents, which also directly modify DNA, but which inhibit DNA methylation w29x, possibly by inactivating the sulfhydrylrich DNA methylase enzyme w37x. Cisplatin exposure increases the 5-methylcytosine content of DNA to a greater degree than any other compound tested. This suggests that some process other than DNA synthesis inhibition might be contributing. Recent studies have shown that the major adduct induced by cisplatin in DNA is an intrastrand crosslink between two adjacent guanines on the same DNA strand w38x. The presence of such dŽG)pG). cisplatin adducts in DNA significantly alters the DNase I cleavage pattern w39,40x. It is thought that the enhanced Dnase I cleavage of DNA containing dŽG)pG). cisplatin adducts is due to distortions induced in DNA by the adducts. The dŽG)pG). adduct bends the double helix 32–348 towards the major groove, thus widening the minor groove, and unwinds the DNA by approximately 138 w41x. The crystallographic structure of the Dnase I-DNA complex indicates that a loop of the enzyme penetrates into the minor groove of the DNA and interacts asymmetrically with the backbone of both strands, contacting two phosphate groups on each side of the cleaved bond and two phosphate groups on the adjacent strand across the minor groove w42x. We suggest that the cisplatin adduct-induced distortions in DNA, in a manner similar to that for DNase I, may render DNA a better substrate for DNA methyltransferase. This could occur by virtue of the modified DNA offering better access to the five carbon atom of cytosines within the major groove, or greater affinity of the enzyme for cisplatin-kinked DNA. An alternative perspective is that methylation marks ‘suspect’ DNA for scrutiny by other enzymes, such as those involved in DNA repair processes w43x. Precedent for this perspective might be obtained from experiments demonstrating that transfected DNA is rapidly methylated and inactivated w44x, and that viral DNA is a target for DNA methylase and gene inactivation w45x, w46x. However, the recent finding that 5-methylcytosine inhibits repair of 3X nearest neighbor O6-methylguanines by O6-methylguanine DNA methyltransferase w47x suggests that drug-induced DNA hyper-
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methylation is unlikely to play a general role in marking DNA containing adducts in need of repair. 1.8. DNA topoisomerase II inhibitors: A special case of DNA hypomethylation We have shown that topoisomerase II inhibitors, including nalidixic acid, novobiocin, etoposide and teniposide, induce DNA hypomethylation w33,48x. This is interesting in view of data showing that exposure to topoisomerase II inhibitors alters gene expression and induces differentiation in a number of experimental systems w49,50x and the strong association of DNA hypomethylation with gene activation w44x. One explanation of our findings that could relate them to the effects of topoisomerase inhibitors upon gene expression is that topoisomerase II releases DNA from that topological conformation that constitutes an efficient substrate for DNA methylase. Thus, even in the presence of increased amounts of DNA methylase in some malignant cells w25x, large stretches of DNA might be rendered methylation-resistant by topoisomerase activity. ŽIt should be pointed out that topoisomerase II inhibitors generally permit topoisomerase activity Žstrand breaking, relief of torsional stress. but prevent the strand rejoining activity of the enzyme.. The ability of topoisomerase inhibitors to induce DNA hypomethylation could offer a potential explanation for their demonstrated effects upon gene expression and differentiation. Since demethylating agents have been demonstrated to be capable of activating transcriptionally repressed genes, it is reasonable to postulate that the ability of topoisomerase inhibitors to activate gene expression might be related to their ability to inhibit DNA methylation w48x. We have suggested that the enhanced topoisomerase activity observed in tumor cells w51,52x, by virtue of its potential to inhibit DNA methylation, might account for the ectopic gene expression long considered to be a hallmark of the malignant state. We and others have shown that exposure of mammalian cells to topoisomerase II inhibitors leads to the generation of polyploid DNA content, another hallmark of the malignant cell. Increased levels of topoisomerase enzymes in malignant cells could thus provide a theoretical link between the induction of polyploidy, DNA hypomethylation and ectopic gene expression,
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three markers for genetic instability in malignant cells. 1.9. Genetic effects of drug-induced DNA hypermethylation The dinucleotide CpG is strongly underrepresented in human and animal DNA, being present at only about 20–25% of its expected frequency. There is good evidence that this ‘CpG suppression’ is due to the fact that CpG is the target for DNA methylation, and that the product, 5-methylcytosine, undergoes spontaneous deamination to thymidine. Over time, then, CpG tends to be replaced by TpG and its replication product CpA. Evidence has arisen that such spontaneous deamination of 5-methylcytosine in DNA may represent one of the most important types of mutation in human disease w53–56,7,57x. To the extent that cells containing hypermethylated replication forks recover from toxic drug exposure, they would be expected to have an increased mutation rate due to the subsequent spontaneous deamination of the newly methylated cytosines. Thus, epigenetic changes occurring during the carcinogenic process or as a result of exposure to drugs during cancer chemotherapy, should increase the level of genetic instability within the tumor cell population. Changes in DNA methylation may, in fact, play a major role in promoting genetic instability within tumor cell populations. Methylation-mediated changes in genomic stability could result from gene activation in regions of hypomethylation Že.g., of genes associated with metastasis, angiogenesis, recombination, etc.., gene inactivation within regions of hypermethylation Že.g., of tumor suppresser genes or genes whose products are required to activate anticancer drugs, etc.., reduced ability to identify the parental template strand during DNA mismatch repair, or through the generation of mutation ‘hotspots’ in hypermethylated regions of the tumor cell genome. As mentioned above, it is also possible that changes in DNA methylation occurring in tumor cells might set in motion a cascade of ectopic gene expression events that might release tumor cells from normal homeostatic controls, as well as transducing epigenetic events into genetic ones by the induction of polyploidy or via mutagenic deamination of 5-methylcytosine.
1.10. Implications for cancer chemotherapy Drug-induced DNA hypermethylation represents one potential mechanism of drug resistance for those agents requiring enzymatic activation to achieve cytotoxicity. Since not all drugs induce DNA hypermethylation, but in fact induce DNA hypomethylation Že.g., alkylating agents, 6-thioguanine, 5-azadeoxycytidine, etc.., it may be possible to reduce that portion of drug resistance that results from drug-induced DNA hypermethylation by appropriately combining DNA methylation inhibitors with hypermethylating drugs. In our limited experience in vivo, however, it has not been possible to achieve long lasting blockade of DNA hypermethylation in patients undergoing sequential treatment with 5azacytidine and carboplatin. This was probably due to the differences in half lives of the two drugs. Alternative regimens employing longer lasting methylation inhibitors, or methylation inhibitors applied by continuous infusion, might achieve durable inhibition of drug-induced DNA hypermethylation. However, precise titration of the hypermethylationrhypomethylation phenomenon to achieve steady state levels of DNA methylation during cytotoxic chemotherapy may be difficult or impossible to achieve. An additional problem is that hypomethylation itself might contribute to the problem of acquired drug resistance. Perry et al. have shown, for example, that 5-aza-2X-deoxycytidine exposure significantly enhances both the frequency and the rate of gene amplification in vitro w58x. Gene amplification is thought to underly the development of resistance to a wide array of anticancer agents w59,60x. Attempts to moderate drug-induced DNA hypermethylationmediated drug resistance by addition of hypomethylating agents to chemotherapy protocols could result in the amplification of genes associated with drug resistance, e.g., the MDR1 gene associated with multi drug resistance. The principal paradigm for anticancer drug design for the past 50 years has been cytotoxicity – the search for ever more potent toxins – and an increasing clinical focus on applying anticancer drugs at their maximum tolerable dosage. Our results suggest that such a treatment strategy may be problematic in the absence of 100% tumor cell kill since the surviving cancer cell population, because of its enhanced
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epigeneticrgenetic diversity, may be substantially superior to normal cell populations with respect to their ability to withstand further selective pressure, i.e., drug exposure. At present, significant effort is being spent by many laboratories on designing new paradigms for anticancer therapy, including therapies targeting tumor cell differentiation and apoptosis. These approaches are based upon the premises that differentiated cells do not divide, and that the normal program of cell death, apoptosis, is aberrantly inactivated in malignant tumor cells. Some of these therapeutic strategies are based on setting in motion epigenetic cascades in order to reprogram aberrant patterns of gene expression in the tumor cell. In exploring these alternative therapeutic strategies, one should consider the dual possibilities that epigenetic instability may contribute to tumor progression in a manner similar to that of genetic instability; and that these two processes intersect in their cause and effect relationships.
Acknowledgements The technical efforts of Ms. Sherry Leonard, Ms. So Wong, and Ms. Dawn Mylott are gratefully acknowledged. Patient specimens were kindly provided by Drs. Spencer Raab Ždeceased., C. Tate Holbrook, and Alan D. Kritz. This work was performed with support from grant CA RO1 47217 from the National Cancer Institute.
References w1x Usmani, B.A. Ž1993. Genomic instability and metastatic progression, Pathobiology 61Ž2., 109–116. w2x Sweezy, M.A. and Fishel, R. Ž1994. Multiple pathways leading to genomic instability and tumorigenesis, Ann. NY Acad. Sci. 726, 165–177. w3x Cheng, K.C. and Loeb, L. Ž1993. Genomic instability and tumor progression: mechanistic considerations, Adv. Cancer Res., 60, 121–156. w4x Hartwell, L. Ž1992. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells, Cell, 71Ž4., 543–546. w5x Honchel, R., Halling, K.C. and Thibodeau, S.N. Ž1995. Genomic instability in neoplasia, Sem. Cell Biol., 6Ž1., 45– 52.
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w6x Russo, C.A., Weber, T.K., Volpe, C.M., Stoler, D.L., Petrelli, N.J., Rodriguez-Bigas, M., Burhans, W.C. and Anderson, G.R. Ž1995. An anoxia inducible endonuclease and enhanced DNA breakage as contributors to genomic instability in cancer, Cancer Res., 55Ž5., 1122–1128. w7x Nyce, J., Weinhouse, S. and Magee, P.N. Ž1983. 5-Methylcytosine depletion during tumor development: an extension of the miscoding concept, Br. J. Cancer, 48Ž4., 463–475. w8x Yamasaki, H., Mesnil, M. and Nakazawa, H. Ž1992. Interaction and distinction of genotoxic and non-genotoxic events in carcinogenesis, Toxicol. Lett., 64–65, 597–604. w9x Nyce, J., Leonard, S., Canupp, D., Schulz, S. and Wong, S. Ž1993. Epigenetic mechanisms of drug resistance: drug-induced DNA hypermethylation and drug resistance, Proc. Natl. Acad. Sci. USA, 90Ž7., 2960–2964. w10x Ribieras, S., Song-Wang, X.G., Martin, V., Lointier, P., Frappart, L. and Dante, R. Ž1994. Human breast and colon cancers exhibit alterations of DNA methylation patterns at several DNA segments on chromosomes pp. 11 and pp. 17. J. Cell. Biochem., 56Ž1., 86–96. w11x Rideout, W.M. 3rd, Eversole-Cire, P., Spruck, C.H. 3rd, Hustad, C.M., Coetzee, G.A., Gonzales, F.A. and Jones, P.A. Ž1994. Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation, Mol. Cell. Biol., 14Ž9., 6143–6152. w12x Herman, J.G., Latif, F., Weng, Y., Lerman, M.I., Zbar, B., Liu, S., Samid, D., Duan, D.S., Gnarra, J.R. and Linehan, W.M. et al. Ž1994. Silencing of the VHL tumor-suppresser gene by DNA methylation in renal carcinoma, Proc. Natl. Acad. Sci. USA, 91Ž21., 9700–9704. w13x Lee, Y.W., Klein, C.B., Kargacin, B., Salnikow, K., Kitahara, J., Dowjat, K., Zhitkovich, A., Christie, N.T. and Costa, M. Ž1995. Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens, Mol. Cell. Biol., 15Ž5., 2547–2557. w14x Greger, V., Debus, N., Lohmann, D., Hopping, W., Passarge, E. and Horsthemke, B. Ž1994. Frequency and parental origin of hypermethylated RB1 alleles in retinoblastoma, Human Genet., 94Ž5., 491–496. w15x Costa, M. Ž1995. Model for the epigenetic mechanism of action of nongenotoxic carcinogens, Am. J. Clin. Nutrition 61Ž3 Suppl.., 666S–669S. w16x Ferguson, A.T., Lapidus, R.G., Baylin, S.B. and Davidson, N.E. Ž1995. Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression, Cancer Res., 55Ž11., 2279–2283. w17x Yoshiura, K., Kanai, Y., Ochiai, A., Shimoyama, Y., Sugimura, T. and Hirohashi, S. Ž1995. Silencing of the E-cadherin invasion-suppresser gene by CpG methylation in human carcinomas, Proc. Natl. Acad. Sci. USA, 92Ž16., 7416–7419. w18x Pieretti, M., Powell, D.E., Gallion, H.H., Conway, P.S., Case, E.A. and Turker, M.S. Ž1995. Hypermethylation at a chromosome 17 ‘hot spot’ is a common event in ovarian cancer, Hum. Pathol., 26Ž4., 398–401. w19x Moulton, T., Crenshaw, T., Hao, Y., Moosikasuwan, J., Lin,
160
w20x w21x
w22x
w23x
w24x
w25x w26x
w27x
w28x w29x
w30x
w31x
w32x
w33x
w34x
w35x
w36x
J.W. Nyce r Mutation Research 386 (1997) 153–161 N., Dembitzer, F., Hensle, T., Weiss, L., McMorrow, L. and Loew, T. et al. Ž1994. Epigenetic lesions at the H19 locus in Wilms’ tumor patients, Nature Genet., 7Ž3., 440–447. Laird, P.W. and Jaenisch, R. Ž1994. DNA methylation and cancer, Hum. Mol. Genet. 3 Spec. No. 1487–1495. Matsuo, K., Tang, S.H., Zeki, K., Gutman, R.A. and Fagin, J.A. Ž1993. Aberrant deoxyribonucleic acid methylation in human thyroid tumors, J. Clin. Endocrinol. Metab., 77Ž4., 991–995. Mills, K.I., Sproul, A.M. and Burnett, A.K. Ž1993. Methylation of the major breakpoint cluster region ŽM-bcr. in Philadelphia-positive CML, Leukemia, 17Ž5., 707–711. Issa, J.P., Vertino, P.M., Wu, J., Sazawal, S., Celano, P., Nelkin, B.D., Hamilton, S.R. and Baylin, S.B. Ž1993. Increased cytosine DNA-methyltransferase activity during colon cancer progression, J. Natl. Cancer Inst., 85Ž15., 1235–1240. Ohtani-Fujita, N., Fujita, T., Aoike, A., Osifchin, N.E., Robbins, P.D. and Sakai, T. Ž1993. CpG methylation inactivates the promoter activity of the human retinoblastoma tumorsuppresser gene, Oncogene, 8Ž4., 1063–1067. Baylin, S. Ž1992., Abnormal regional hypermethylation in cancer cells, AIDS Res. Hum. Retroviruses, 8Ž5., 811–820. Makos, M., Nelkin, B.D., Chazin, V.R., Cavenee, W.K., Brodeur, G.M. and Baylin, S.B. Ž1993. DNA hypermethylation is associated with 17p allelic loss in neural tumors, Cancer Res., 53Ž12., 2715–2718. Sakai, T., Toguchida, J., Ohtani, N., Yandell, D.W., Rapaport, J.M. and Dryja, T.P. Ž1991. Allele-specific hypermethylation of the retinoblastoma tumor-suppresser gene, Am. J. Human Genet., 48Ž5., 880–888. Peltomaki, P. Ž1991. DNA methylation changes in human testicular cancer, Biochim. Biophys. Acta, 1096Ž3., 187–196 Nyce, J., Mylott, D., Leonard, S., Willis, L. and Kataria, A. Ž1989. Detection of drug-induced DNA hypermethylation in human cells exposed to cancer chemotherapy agents, J. Liq. Chrom., 12Ž8., 1313–1321. Nyce, J.W. Ž1989. Drug-induced DNA hypermethylation and drug resistance in human tumors, Cancer Res., 49Ž21., 5829– 5836. Griffin, S., Branch, P., Xu, Y.Z. and Karran, P. Ž1994. DNA mismatch binding and incision at modified guanine bases by extracts of mammalian cells: implications for tolerance to DNA methylation damage, Biochemistry, 33Ž16., 4787–4793. Kastan, M.B., Gowans, B.J. and Lieberman, M.W. Ž1982. Methylation of deoxycytidine incorporated by excision repair synthesis of DNA, Cell, 30Ž2., 509–516. Nyce, J., Liu, L. and Jones, P.A. Ž1986. Variable effects of DNA synthesis inhibitors upon DNA methylation in mammalian cells, Nucleic Acids Res., 14Ž10., 4353–4367. Nyce, J. Ž1991. Gene silencing in mammalian cells by direct incorporation of electroporated 5-methyl-2X deoxycytidine 5’triphosphate, Somatic Cell Mol. Genet., 17, 543–550. Leonard, S.A., Wong, S.C. and Nyce, J.W. Ž1993. Quantitation of 5-methylcytosine by one-dimensional high-performance thin-layer chromatography, J. Chrom., 645, 189–192. Reid, T.R., Merigan, T.C. and Basham, T.Y. Ž1992. Resis-
w37x w38x
w39x
w40x
w41x
w42x
w43x
w44x w45x
w46x
w47x
w48x
w49x
w50x
w51x
w52x
tance to interferon a in a mouse B-cell lymphoma involves DNA methylation, J. Interferon Res., 12Ž2., 131–137. Wilson, V. and Jones, P. Ž1983. Inhibition of DNA methylation by chemical carcinogens in vitro, Cell, 32, 239–246. Lepre, C.A. and Lippard, S.J. Ž1990. Interaction of platinum antitumor compounds with DNA, in: F. Eckstein and D.M. Lilley ŽEds.., Nucleic Acids and Molecular Biology, Vol. 4, pp. 3–9, Springer-Verlag, Berlin. Visse, R. and deRuijter, M., Brouwer, J.A. and van de Putte, P. Ž1991. Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA, J. Biol. Chem., 266, 7609–7617. Schwartz, A. and Leng, M. Ž1994. Dnase I footprinting of cis- and trans-diamminedichloroplatinumŽII.-modified DNA, J. Mol. Biol., 236, 969–974. Bellon, S.F., Coleman, J.H. and Lippard, S.J. Ž1991. DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum ŽII., Biochemistry, 30Ž32., 8026–8035. Suck, D., Lahm, A. and Oefner, C. Ž1988. Structure refined to 2 Angstrom of a nicked DNA octanucleotide complex with DNase I, Nature, 332, 464–468. Hare, J.T. and Taylor, J.H. Ž1985. One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair, Proc. Natl. Acad. Sci. USA, 82Ž21., 7350–7354. Razin, A. and Cedar, H. Ž1991. DNA methylation and gene expression, Microbiol. Rev., 55Ž3., 451–458. Bednarik, D.P., Duckett, C., Kim, S.U., Perez, V.L., Griffis, K., Guenthner, P.C. and Folks, T.M. Ž1991. DNA CpG methylation inhibits binding of NF-kappa B proteins to the HIV-1 long terminal repeat cognate DNA motifs, New Biologist, 3Ž10., 969–976. Gutekunst, K.A., Kashanchi, F., Brady, J.N. and Bednarik, D.P. Ž1993. Transcription of the HIV-1 LTR is regulated by the density of DNA CpG methylation. J. Acq. Immune Defic. Syndromes, 6Ž6., 541–549. Bentivegna, S.S. and Bresnick, E. Ž1994. Inhibition of human O6 -methylguanine DNA methyltransferase by 5-methylcytosine, Cancer Res., 54Ž2., 327–329. Schonfeld, S., Schulz, S. and Nyce, J.W. Ž1992. Effects of inhibitors of topoisomerases I and II on DNA methylation and DNA synthesis in human colonic adenocarcinoma cells in vitro, Intern. J. Oncology, 1, 807–814. Nakaya, K., Chou, S., Kaneko, M., and Nakamura, Y. Ž1991. Topoisomerase inhibitors have potent differentiation-inducing activity for human and mouse myeloid leukemia cells. Jpn. J. Cancer Res., 82, 184–191. Rappa, G., Lorico, A. and Sartorelli, A.C. Ž1990. Induction of the differentiation of WEHI-3B Dqmonomyelocytic leukemia cells by inhibitors of topoisomerase II, Cancer Res., 50, 6723–6730. Holden, J.A., Rolfson, D.H. and Wittwer Ž1990. Human DNA topoisomerase II: evaluation of enzyme activity in normal and neoplastic tissues, Biochemistry, 29Ž8., 2127– 2134. Hsiang, Y.H., Wu, H.Y. and Liu, L.F. Ž1988. Proliferation-
J.W. Nyce r Mutation Research 386 (1997) 153–161
w53x
w54x
w55x w56x
dependent regulation of DNA topoisomerase II in cultured cells, Cancer Res., 48, 3230–3235. Shapiro, R. and Klein, R.S. Ž1966. The deamination of cytidine and cytosine by acidic buffer solutions. Mutagenic implications, Biochemistry, 5Ž7., 2358–2362. Coulondre, C., Miller, J.H., Farabaugh, P.J. and Gilbert, W. Ž1978. Molecular basis of base substitution hotspots in Escherichia coli, Nature, 274Ž5673., 775–780. Duncan, B.K. and Miller, J.H. Ž1980. Mutagenic deamination of cytosine residues in DNA, Nature, 287Ž5782., 560–561. Wang, R.Y., Kuo, K.C., Gehrke, C.W., Huanh, L.H. and Ehrlich, M. Ž1982. Heat and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA, Biochim. Biophys. Acta, 697Ž3., 371–377.
161
w57x Rideout, W.M. 3d, Coetzee, G.A., Olumi, A.F. and Jones, P.A. Ž1990. 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes, Science, 249Ž4974., 1288–1290. w58x Perry, M.E., Rolfe, M., McIntyre, P., Commane, M. and Stark, G.R. Ž1992. Induction of gene amplification by 5-azaX 2 -deoxycytidine, Mutation Res., 276Ž3., 189–197. w59x Roninson, I.B. Ž1992. From amplification to function: the case of the MDR1 gene, Mutation Res., 276Ž3., 151–161. w60x Harnevo. L.E. and Agur, Z. Ž1992. Drug resistance as a dynamic process in a model for multistep gene amplification under various levels of selection stringency, Cancer Chemother. Pharmacol., 30Ž6., 469–476.