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.