Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells.

Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF 3 b 0 when it is added to the culture medium. In these cells transfected with the bcl 2 gene, BAF 3 bcl 2 , the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) 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 BAF 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 presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF 3 bcl 2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium salt of [U- 14 C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF 3 bcl 2 compared with BAF 3 b 0 cells was accompanied by a concomitant rise in the transamination of [ 14 C]MTOB to [ 14 C]methionine in BAF 3 bcl 2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF 3 b 0 but not in BAF 3 bcl 2 cells, confirming that the defect in BAF 3 bcl 2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB →methional. In addition, no evidence was obtained for the synthesis of [ 14 C]malondialdehyde from [ 14 C]methional in BAF 3 bcl 2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF 3 b 0 cells, they may possess some other defect in the β-hydroxylase enzyme system itself.