DEVELOPMENT AND REVERSION OF METHIONINE DEPENDENCE IN A HUMAN GLIOMA CELL-LINE - RELATION TO HOMOCYSTEINE REMETHYLATION AND COBALAMIN STATUS

We investigated the biochemical changes which accompanied the developm ent and reversion of methionine dependence in a human glioma cell line GaMg. This cell line attained a higher proliferation rate and more ma lignant morphology with increasing passages in vitro. Early passages ( P10, P25, and P45) were able to grow in a methionine-deficient medium supplemented with homocysteine (Met(-)Hcy(+)), while a later passage ( P60) had lost this ability, i.e., it had become methionine-dependent. From P60 cells, a methionine-independent revertant (P60R) was establis hed by exposing the cells to 5-aza-2-deoxycytidine, followed by cultur e in a Met(-)Hcy(+) medium. In these genetically related cell lines, w e investigated homocysteine remethylation and the functional state of cobalamin-dependent methionine synthase, the enzyme responsible for re methylation of homocysteine to methionine. The methionine synthase act ivity in cell extracts was similar in all cell sublines. Intact cell m ethionine biosynthesis and nitrous oxide-dependent homocysteine export reflect homocysteine remethylation in cells cultured in a Met(-)Hcy() and methionine-containing (Met(+)Hcy(-)) medium, respectively. Both of these parameters, as well as the cellular content of the substrate 5-methyltetrahydrofolate, and the cofactor methylcobalamin, in additio n to adenosylcobalamin, were high in P10, declined progressively in P4 5 and P60, and were restored in P60R. P25 cells had some unique featur es among the methionine-independent phenotypes because both homocystei ne remethylation and the level of 5-methyltetrahydrofolate were low in Met(+)Hcy(-) medium. The maximal homocysteine export rate in the pres ence of nitrous oxide, which reflects the overall transmethylation rat e, was high in P60 and even higher in P60R compared to the lower passa ges. The basis for development of methionine dependence during culture of this glioma cell line seems related to the combined effects of red uced methionine biosynthesis and an increased overall transmethylation rate. The single parameter which most closely correlated to the abili ty to use homocysteine for growth was methylcobalamin. These data supp ort a model for methionine dependence, which implies impaired provisio n of cobalamin to methionine synthase.