title: Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines.
abstract: Alterations in DNA methylation patterns accompany the establishment of immortal cell lines. De novo methylation of CpG islands within the control regions of growth-regulatory genes may inactivate their transcription, giving cells selective growth advantages in culture. We exposed seven human tumor cell lines and two human fibroblast cell strains to the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-CdR), to determine whether the silencing of growth-regulatory genes by de novo methylation in immortalized cell lines could be reversed, possibly restoring growth control. After recovery from the immediate cytotoxic effects of 5-Aza-CdR, this agent suppressed cellular growth in all seven tumor lines but not in either fibroblast strain. Because alterations in the p16 (CDKN2/MTS1) cell cycle regulatory gene are associated with numerous cancers, we analyzed expression of this gene before and after 5-Aza-CdR treatment. The gene was reactivated by 5-Aza-CdR treatment in three of four tumor cell lines not expressing p16, whereas the fourth tumor line contained a p16 homozygous deletion. p16 was shown to be hypermethylated only in the cell lines and its up-regulation by 5-Aza-CdR was associated with demethylation of the p16 promoter. The remaining tumor lines expressed p16 at constant levels before and after 5-Aza-CdR treatment and showed minimal p16 promoter methylation, suggesting that other growth-regulatory genes may have been silenced by de novo methylation in these cells. p16 expression, cell growth inhibition, and G1 cell cycle arrest by 5-Aza-CdR in the T24 bladder tumor cell line were also heritable after prolonged passage in culture. Furthermore, a dormant p16 gene was reactivated in T24 cells growing in nu/nu rats, and 5-Aza-CdR treatment of T24 cells before inoculation into nu/nu mice decreased the rate of tumor growth. These results suggest that 5-Aza-CdR may slow the growth of tumor cells by reactivating growth-regulatory genes silenced by de novo methylation.
authors.affiliation: Urological Cancer Research Laboratory, USC/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles 90033, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Bender
authors.forename: M M
authors.initials: MM
authors.lastname: Pao
authors.forename: P A
authors.initials: PA
authors.lastname: Jones
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000187:drug effects* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D000077209:Decitabine; D016193:G1 Phase; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D011923:Rats, Nude; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D019941:Cyclin-Dependent Kinase Inhibitor p16; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine