S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), which catalyzes the synthesis of AdoMet from methionine and ATP, is the major methyl donor for transmethylation reactions and propylamino donor for the biosynthesis of polyamines in biological systems. We have reported previously that wild-type C-1300 murine neuroblastoma (wMNB) cells, made resistant to the nucleoside analogue (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (MDL 28,842), an irreversible inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), express increased AdoMet synthetase activity (M. R. Hamre et al., Oncol. Res., 7: 487-492, 1995). In the present study, immunoblot analyses of AdoMet Synthetase with isoform-specific (MATII) antibodies demonstrated an elevation in the AdoMet synthetase immunoprotein in nucleoside analogue-resistant MNB cells (rMNB-MDL) when compared to wild-type, nonresistant MNB cells. An increase of 2.1-fold was observed in the alpha2/alpha2' catalytic subunit, which differed significantly from the much smaller increment in the noncatalytic beta-subunit of AdoMet synthetase. Densitometric analyses revealed that an increased expression of AdoMet synthetase in rMNB-MDL cells was due to overexpression of the alpha2 (Mr 53,000; 2.6-fold) and alpha2' (Mr 51,000; 1.8-fold) subunits. AdoMet synthetase mRNA expression in rMNB-MDL cells was remarkably greater than wMNB cells, as determined by quantitative competitive reverse transcription-PCR (QC-PCR) analysis. DNA (cytosine) methyl transferase expression, measured by reverse transcription-PCR analysis, was also elevated significantly in rMNB-MDL cells. In contrast, Western blot analyses demonstrated down-regulation (1.6-fold) of AdoMet synthetase in doxorubicin-resistant human leukemia cells (HL-60-R) expressing multidrug resistance protein when compared with wild-type, nonresistant HL-60 cells. The resistance of rMNB-MDL cells to nucleoside analogue inhibitors of S-adenosylhomocysteine hydrolase correlates directly with overexpression of the alpha2/alpha2' subunits of AdoMet synthetase. Cellular adaptation allows sufficient AdoMet to be synthesized, so that viability of the MNB cells can be maintained even in the presence of high AdoHcy concentrations. This novel mechanism of drug resistance does not appear to require multidrug resistance protein (P-glycoprotein) overexpression.