ALTERED METHIONAL HOMEOSTASIS IS ASSOCIATED WITH DECREASED APOPTOSIS IN BAF(3) BCL(2) MURINE LYMPHOID-CELLS

Methional is a potent inducer of apoptosis in an interleukin 3-depende nt murine lymphoid cell line BAF(3) b(0) when it is added to the cultu re medium. In these cells transfected with the bcl(2) gene, BAF(3) bcl (2), the apoptotic-inducing activity of methional is dramatically redu ced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenas e) in order to reduce methional oxidation brought about an increase in apoptosis in BAF(3) b(0) but not in BAF(3) bcl(2) cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BA F(3) b(0) and BAF(3) bcl(2) cells. The extent of DNA fragmentation in BAF(3) bcl(2) cells approached that in BAF(3) b(0) cells in the presen ce of quercetin and exogenous methional, suggesting a defect in methio nal biosynthesis in BAF(3) bcl(2) cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the s odium salt of [U-C-14]4-methylthio-2-oxobutanoic acid (MTOB), the prec ursor of methional. The 80% decrease in labelled methional in BAF(3) b cl(2) compared with BAF(3) b(0) cells was accompanied by a concomitant rise in the transamination of [C-14]MTOB to [C-14]methionine in BAF(3 ) bcl(2) cells. Inhibition of the transaminase, however, by a syntheti c transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF(3) b(0) but not in BAF(3) bcl(2) cells, confi rming that the defect in BAF(3) bcl(2) cells was not in the transamina se itself but rather in the oxidative decarboxylation step MTOB --> me thional. In addition, no evidence was obtained for the synthesis of [C -14]malondialdehyde from [(14)]methional in BAF(3) bcl(2) cells. as th ese cells show no deficiency in their content of reactive oxygen speci es compared with that of BAF(3) b(0) cells, they may possess some othe r defect in the beta-hydroxylase enzyme system itself.