Fiskerstrand T, Christensen B, Tysnes O B, Ueland P M, Refsum H
Department of Clinical Biology, University of Bergen, Haukeland Hospital, Norway.
Cancer Res. 1994 Sep 15;54(18):4899-906.
We investigated the biochemical changes which accompanied the development and reversion of methionine dependence in a human glioma cell line GaMg. This cell line attained a higher proliferation rate and more malignant 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 established by exposing the cells to 5-aza-2-deoxycytidine, followed by culture in a Met-Hcy+ medium. In these genetically related cell lines, we investigated homocysteine remethylation and the functional state of cobalamin-dependent methionine synthase, the enzyme responsible for remethylation of homocysteine to methionine. The methionine synthase activity in cell extracts was similar in all cell sublines. Intact cell methionine 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 addition to adenosylcobalamin, were high in P10, declined progressively in P45 and P60, and were restored in P60R. P25 cells had some unique features among the methionine-independent phenotypes because both homocysteine remethylation and the level of 5-methyltetrahydrofolate were low in Met+Hcy- medium. The maximal homocysteine export rate in the presence of nitrous oxide, which reflects the overall transmethylation rate, was high in P60 and even higher in P60R compared to the lower passages. The basis for development of methionine dependence during culture of this glioma cell line seems related to the combined effects of reduced methionine biosynthesis and an increased overall transmethylation rate. The single parameter which most closely correlated to the ability to use homocysteine for growth was methylcobalamin. These data support a model for methionine dependence, which implies impaired provision of cobalamin to methionine synthase.
我们研究了人胶质瘤细胞系GaMg中伴随蛋氨酸依赖性发展和逆转的生化变化。随着体外传代次数增加，该细胞系增殖速率更高，形态更具恶性特征。早期传代细胞（P10、P25和P45）能够在补充有同型半胱氨酸的蛋氨酸缺陷培养基（Met-Hcy+）中生长，而后期传代细胞（P60）失去了这种能力，即变得依赖蛋氨酸。通过将P60细胞暴露于5-氮杂-2'-脱氧胞苷，然后在Met-Hcy+培养基中培养，建立了一个不依赖蛋氨酸的回复突变体（P60R）。在这些遗传相关的细胞系中，我们研究了同型半胱氨酸的再甲基化以及钴胺素依赖性蛋氨酸合酶的功能状态，该酶负责将同型半胱氨酸再甲基化为蛋氨酸。所有细胞亚系中细胞提取物中的蛋氨酸合酶活性相似。完整细胞的蛋氨酸生物合成和一氧化二氮依赖性同型半胱氨酸输出分别反映了在Met-Hcy+和含蛋氨酸（Met+Hcy-）培养基中培养的细胞中的同型半胱氨酸再甲基化。这两个参数，以及底物5-甲基四氢叶酸的细胞含量、辅因子甲基钴胺素以及腺苷钴胺素，在P10中含量较高，在P45和P60中逐渐下降，在P60R中恢复。P25细胞在不依赖蛋氨酸的表型中具有一些独特特征，因为在Met+Hcy-培养基中同型半胱氨酸再甲基化和5-甲基四氢叶酸水平都较低。在一氧化二氮存在下反映总体转甲基速率的最大同型半胱氨酸输出率在P60中较高，与早期传代细胞相比，在P60R中甚至更高。该胶质瘤细胞系培养过程中蛋氨酸依赖性发展的基础似乎与蛋氨酸生物合成减少和总体转甲基速率增加的综合作用有关。与利用同型半胱氨酸生长能力最密切相关的单一参数是甲基钴胺素。这些数据支持了一个蛋氨酸依赖性模型，该模型表明向蛋氨酸合酶提供钴胺素受损。
