CONTRIBUTION OF 4-METHYLTHIO-2-OXOBUTANOATE AND ITS TRANSAMINASE TO THE GROWTH OF METHIONINE-DEPENDENT CELLS IN CULTURE - EFFECT OF TRANSAMINASE INHIBITORS

The growth in culture of methionine-dependent transformed cells of hum an, rat and mouse origin was arrested in the absence Of L-methionine ( Met) but took place in the presence of 4-methylthio-2-oxobutanoic acid (MTOB), the keto acid of Met. From 24 hr after seeding, cells grew in 0.1 mM MTOB medium at a rate comparable to that in 0.1 mM Met medium. Using [S-35]MTOB, it was found that the Met synthesized was used in n ormal MRC-5 cells and in transformed HeLa cells to the same extent for protein, adenosylmethionine and adenosylhomocysteine syntheses. Howev er, when the free Met content was examined, it was found to be 3-fold greater in HeLa than in MRC-5 cells. To examine the importance of this free Met for the growth of transformed cells, the transaminase respon sible for converting MTOB to Met was chosen as a target enzyme for the synthesis of compounds with potential inhibitory activity. Since this is a multisubstrate enzyme, reduced Schiff bases were prepared contai ning both pyridoxal or other aromatic groups, as one constituent, and L-Met or other amino-acids in the free acid or ester or amide form, as the other constituent. Only esters containing the pyridoxal moiety an d Met or certain of its structural analogues exhibited good selective growth inhibitory activity in that there was little (20%) or no effect on the growth of normal MRC-5 and derm cells, respectively, while tha t of transformed HeLa, HEp-2 and L1210 cells was strongly inhibited (8 0%). This inhibition was accompanied by a concomitant decrease in the activity of the MTOB transaminase in both HeLa and MRC-5 cells treated with 3c the most potent inhibitor. However, using [S-35]MTOB it was f ound that MTOB itself accumulated 48% in HeLa but only 12% in MRC-5 ce lls treated with 3c. On the contrary [S-35]Met formed from [S-35]MTOB increased 3.7-fold in MRC-5 inhibitor-treated cells showing 20% growth inhibition whereas it decreased 38% in HeLa-treated cells showing 80% growth inhibition. This decrease in cellular Met in HeLa is not respo nsible for growth arrest. Indeed the growth of HeLa cells could not be restored by adding a 10-fold excess of Met. Since MTOB can alleviate Met-dependence, the intracellular homeostasis of this metabolite may p lay a hitherto unsuspected role in controlling cell growth.