TEMOZOLOMIDE INDUCED DIFFERENTIATION OF K562 LEUKEMIA CELLS IS NOT MEDIATED BY GENE HYPOMETHYLATION M. ZUCCHE~I,C. V. CATAPANO,~. FILIPPESCHI, E. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR ERBA and M. D'INCALCI* Istituto di Recerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, 20157 Milan, Italy (Receiue~ 2 March 19&8;nccepred 10 December 1988) Abetraet-Temozolomide (8-earbamoyl-lmethylimidazo[5,Id]-l,2,3,5-tetrazin-4-(3H)-one), an exper- imental antitumor agent which spontaneously decomposes to 5-(3,3-methyl-l-triazeno) imidazoie-4- carboxamide, the active metabolite of the antineoplastic drug DTIC, causes erythroid differentiation of K562 leukemia cells. The increase in E and y globin gene expression after temozolomide treatment does not appear to be due to drug-induced hypomethylation of the genes. In other genes containing many methylated sequences such as the proto-oncogenes c-myc and C-Ha-ras, temozolomide caused no detectable change in methylation. In contrast, in the same genes S-aza-2’-deoxycytidine induced hypomethylation. Temozolomide caused DNA alkali-labile sites and an arrest of the cell cycle in G2 phase. Ethazolastone (its 3-ethyhmidazo analogue) which does not cause differentiation of K562 produced no significant DNA damage and G2 phase blockade. DNA damage rather than hypomethyl- ation may be responsible for induction of differentiation. c N\ CH3 Fig. 1. Chemical structure of temozolomide. 5 -(3,3 - Dimethyl - zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1 - tri~no)i~d~le - 4 - carbox- amide (DTIC) is an ~tin~plastic agent used for the therapy of several human rnali~~n~es [I, 2] and is the drug of choice against melanoma [3]. DTIC undergoes oxidative Ndemethylation, forming of 5- (3,3-methyl-1-triazeno) imidazole+carboxamide (MTIC) an alkylating agent probably responsible for DTIC’s activity [4,5]. Temozolomide (8-carbamoyl-3-methylimidazo- [5,1-d]-1,2,3,.5-tetrazin-4-(3H)-one) (Fig. l), pre- viously called methazolastone, is a recently syn- thesized compound which decomposes spontaneously to MTIC [6] without requiring meta- bolic activation. Temozolomide may overcome the problem of the variable and/or insufficient activation of DTIC and for this reason is under clinical inves- tigation , In addition it is useful for in vitro studies to eluci- date the mode of action of methyltriazenes, which is still unclear. These monofunctional alkylators not only have cytotoxic activity, probably due to alky- lation of cellular macromolecules, but other inter- esting effects related to modulation of the expression of some genes have been reported. Methyltriazenes * To whom requests for reprints should be addressed at the Istituto di Ricerche Farmacologiche ‘Mario Negri’, via Eritrea 62, 20157 Milan, Italy. cause increased expression of some tumoral cell- associated antigens 171,and their antitumoral activity might thus be partially due to an increased sus- ceptibility to immunological mechanisms. Recently Tisdale [8] reported that temozolomide but not ethazolastone (its 3-ethylimidazo analogue), induced differentiation in K562 erythroleukemic cells. He found that after temozolomide treatment the content of 5-methylcytosine fell from 3.5 to 2.2% in the total genome and proposed that differentiation was due to gene hypomethylation (81, as previously described for azacytidine [9? lo]. The data on the total presence of 5-methyIcytosine can only be taken as indicative at this stage and more detailed studies on the methylation status of single genes are required to confirm this hypothesis. In the present study we investigated whether temo- zolomide induces changes in the methylation status of the E globin gene, y globin gene, c-myc oncogene and ras oncogene. The data do not support the hypothesis that temozolomide-induced differenti- ation of KS62 erythroleukemia cells is mediated by gene hypomethylation. MATERIALS AND METHODS Drugs. Temozolomide and 8-carbamoyi-3~thyl- imid~o[5,1~]-1,2,3,5-tetr~n~-(3H)~ne (ethazo- lastone) were kindly provided by Dr C. G. Newton, May & Baker Ltd., Dagenham, Essex (RMlO7XS). The drugs were dissolved in 0.5% DMSO immedi- ately before use. S-Aza-2’-deoxycytidine was pro- vided by the Drug Synthesis and Chemistry Branch, National Cancer Institute, MD. It was dissolved in RPM1 1640 medium before administration. Cell culture. KS62 erythroleukemia cells [ 1 l] were grown at 37” in suspension culture in RPMI 1640 medium (Gibco Europe, Glasgow, Scotland) sup- plemented with 10% heat-inactivated (56”, 30 min)