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. 1993 Apr 1;90(7):2960–2964. doi: 10.1073/pnas.90.7.2960

Epigenetic mechanisms of drug resistance: drug-induced DNA hypermethylation and drug resistance.

J Nyce 1, S Leonard 1, D Canupp 1, S Schulz 1, S Wong 1
PMCID: PMC46216  PMID: 8464912

Abstract

In a model system employing Chinese hamster V-79 cells, the DNA synthesis inhibitor 3'-azido-3'-deoxythymidine (BW A509U, AZT) was shown to induce genome-wide DNA hypermethylation, low-frequency silencing of thymidine kinase (TK; EC 2.7.1.21) gene expression, and resistance to AZT. Twenty-four hours of exposure of V-79 cells to 150 microM AZT led to > 2-fold enhancement of genomic 5-methylcytosine levels and produced TK- epimutants at a rate approximately 43-fold above background. Such AZT-induced TK- epimutants were shown to be severely reduced in their capacity to activate AZT to its proximate antiviral form, AZT 5'-monophosphate, as compared with the TK+ parental cell line from which they were derived. TK- clones isolated under these conditions were shown to be 9- to 24-fold more resistant to the cytotoxic effects of AZT than the parental TK+ cell line and showed collateral resistance to 5-fluoro-2'-deoxyuridine. Three of four TK- epimutants could be reactivated at very high frequency (8-73%) to the TK+ AZT-sensitive phenotype by 24 hr of exposure to the demethylating agent 5-azadeoxycytidine (5-azadC), implying that drug-induced DNA hypermethylation, rather than classical mutation, was involved in the original gene-silencing event in these three clones. These 5-azadC-induced TK+ revertants concomitantly regained the ability to metabolize AZT to its 5'-monophosphate. RNA slot blot analyses indicated that the four AZT-induced TK- clones expressed 8.9%, 15.6%, 17.8%, and 11.1% of the parental level of TK mRNA. The three clones that were reactivatable by 5-azadC showed reexpression of TK mRNA to levels 84.4%, 51.1%, and 80.0% that of the TK+ parental cell line. These experiments show that one potential mechanism of drug resistance involves drug-induced DNA hypermethylation and resulting transcriptional inactivation of cellular genes whose products are required for drug activation.

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Selected References

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  1. Adams R. L. DNA methylation. The effect of minor bases on DNA-protein interactions. Biochem J. 1990 Jan 15;265(2):309–320. doi: 10.1042/bj2650309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alizon M., Wain-Hobson S., Montagnier L., Sonigo P. Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell. 1986 Jul 4;46(1):63–74. doi: 10.1016/0092-8674(86)90860-3. [DOI] [PubMed] [Google Scholar]
  3. Avramis V. I., Mecum R. A., Nyce J., Steele D. A., Holcenberg J. S. Pharmacodynamic and DNA methylation studies of high-dose 1-beta-D-arabinofuranosyl cytosine before and after in vivo 5-azacytidine treatment in pediatric patients with refractory acute lymphocytic leukemia. Cancer Chemother Pharmacol. 1989;24(4):203–210. doi: 10.1007/BF00257619. [DOI] [PubMed] [Google Scholar]
  4. Balzarini J., Pauwels R., Baba M., Herdewijn P., de Clercq E., Broder S., Johns D. G. The in vitro and in vivo anti-retrovirus activity, and intracellular metabolism of 3'-azido-2',3'-dideoxythymidine and 2',3'-dideoxycytidine are highly dependent on the cell species. Biochem Pharmacol. 1988 Mar 1;37(5):897–903. doi: 10.1016/0006-2952(88)90178-5. [DOI] [PubMed] [Google Scholar]
  5. Blum M. R., Liao S. H., Good S. S., de Miranda P. Pharmacokinetics and bioavailability of zidovudine in humans. Am J Med. 1988 Aug 29;85(2A):189–194. [PubMed] [Google Scholar]
  6. Broder S. Clinical applications of 3'-azido-2',3'-dideoxythymidine (AZT) and related dideoxynucleosides. Med Res Rev. 1990 Oct-Dec;10(4):419–439. doi: 10.1002/med.2610100403. [DOI] [PubMed] [Google Scholar]
  7. Chandler L. A., Jones P. A. Hypomethylation of DNA in the regulation of gene expression. Dev Biol (N Y 1985) 1988;5:335–349. doi: 10.1007/978-1-4615-6817-9_12. [DOI] [PubMed] [Google Scholar]
  8. Coffin J. M. Genetic variation in AIDS viruses. Cell. 1986 Jul 4;46(1):1–4. doi: 10.1016/0092-8674(86)90851-2. [DOI] [PubMed] [Google Scholar]
  9. Devare S. G., Srinivasan A., Bohan C. A., Spira T. J., Curran J. W., Kalyanaraman V. S. Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5718–5722. doi: 10.1073/pnas.83.15.5718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Furman P. A., Fyfe J. A., St Clair M. H., Weinhold K., Rideout J. L., Freeman G. A., Lehrman S. N., Bolognesi D. P., Broder S., Mitsuya H. Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8333–8337. doi: 10.1073/pnas.83.21.8333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hahn B. H., Shaw G. M., Taylor M. E., Redfield R. R., Markham P. D., Salahuddin S. Z., Wong-Staal F., Gallo R. C., Parks E. S., Parks W. P. Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS. Science. 1986 Jun 20;232(4757):1548–1553. doi: 10.1126/science.3012778. [DOI] [PubMed] [Google Scholar]
  12. Holliday R. DNA methylation and epigenetic mechanisms. Cell Biophys. 1989 Aug-Oct;15(1-2):15–20. doi: 10.1007/BF02991575. [DOI] [PubMed] [Google Scholar]
  13. Howard J., Hudspeth A. J. Mechanical relaxation of the hair bundle mediates adaptation in mechanoelectrical transduction by the bullfrog's saccular hair cell. Proc Natl Acad Sci U S A. 1987 May;84(9):3064–3068. doi: 10.1073/pnas.84.9.3064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jeggo P. A., Holliday R. Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells. Mol Cell Biol. 1986 Aug;6(8):2944–2949. doi: 10.1128/mcb.6.8.2944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Larder B. A., Darby G., Richman D. D. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science. 1989 Mar 31;243(4899):1731–1734. doi: 10.1126/science.2467383. [DOI] [PubMed] [Google Scholar]
  16. Larder B. A., Kemp S. D. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. doi: 10.1126/science.2479983. [DOI] [PubMed] [Google Scholar]
  17. Nyce J. Drug-induced DNA hypermethylation and drug resistance in human tumors. Cancer Res. 1989 Nov 1;49(21):5829–5836. [PubMed] [Google Scholar]
  18. Nyce J. Gene silencing in mammalian cells by direct incorporation of electroporated 5-methyl-2'-deoxycytidine 5'-triphosphate. Somat Cell Mol Genet. 1991 Nov;17(6):543–550. doi: 10.1007/BF01233619. [DOI] [PubMed] [Google Scholar]
  19. Nyce J., Liu L., Jones P. A. Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells. Nucleic Acids Res. 1986 May 27;14(10):4353–4367. doi: 10.1093/nar/14.10.4353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Scanlon K. J., Funato T., Pezeshki B., Tone T., Sowers L. C. Potentiation of azidothymidine cytotoxicity in cisplatin-resistant human ovarian carcinoma cells. Cancer Commun. 1990;2(10):339–343. doi: 10.3727/095535490820874128. [DOI] [PubMed] [Google Scholar]
  21. Scanlon K. J., Kashani-Sabet M., Sowers L. C. Overexpression of DNA replication and repair enzymes in cisplatin-resistant human colon carcinoma HCT8 cells and circumvention by azidothymidine. Cancer Commun. 1989;1(4):269–275. [PubMed] [Google Scholar]
  22. Vazquez-Padua M. A., Starnes M. C., Cheng Y. C. Incorporation of 3'-azido-3'-deoxythymidine into cellular DNA and its removal in a human leukemic cell line. Cancer Commun. 1990;2(1):55–62. doi: 10.3727/095535490820874740. [DOI] [PubMed] [Google Scholar]
  23. Yoshiyama H., Kobayashi N., Matsui T., Nakashima H., Kajii T., Yamato K., Kotani S., Miyoshi I., Yamamoto N. Transmission and genetic shift of human immunodeficiency virus (HIV) in vivo. Mol Biol Med. 1987 Dec;4(6):385–396. [PubMed] [Google Scholar]

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