Liteplo R G, Hipwell S E, Rosenblatt D S, Sillaots S, Lue-Shing H
Department of Experimental Oncology, Ottawa Regional Cancer Centre, Ontario, Canada.
J Cell Physiol. 1991 Nov;149(2):332-8. doi: 10.1002/jcp.1041490222.
Our aim was to identify the biochemical defect responsible for the inability of highly growth autonomous human tumor cells to proliferate in culture medium devoid of methionine, but containing homocysteine and 5-methyletrahydrofolic acid. We have adopted the terms "homocysteine-responsive" and "homocysteine-nonresponsive" to describe cells which can or cannot proliferate in methionine-free homocysteine-supplemented medium. Using a panel of genetically related homocysteine-responsive and -nonresponsive human melanoma cell lines, the results from a number of experiments indicate that acquisition of the "homocysteine-nonresponsive phenotype" is associated with the reduced intracellular accumulation of methyl-cobalamin, a critical cofactor of the methionine synthase enzyme. When in vitro methionine synthase assays were performed in the presence of exogenously added methyl-cobalamin, specific methionine synthase activity in extracts obtained from homocysteine-responsive cells was only twofold greater than that observed with extracts prepared from homocysteine-nonresponsive cells. However, when exogenous methyl-cobalamin was omitted from the enzyme assays, methionine synthase activity in extracts derived from homocysteine-nonresponsive cells was dramatically reduced, compared with the small decrease observed with homocysteine-responsive cell extracts. Compared with their homocysteine-responsive counterparts, homocysteine-nonresponsive cells exhibited increased levels of cobalamin efflux and decreased intracellular accumulation of methyl-cobalamin. There was a clear relationship between the abilities of these related melanoma cell lines to proliferate in methionine-free homocysteine-supplemented medium, and the extent of cobalamin loss and capacity of exogenously added methyl-cobalamin to stimulate in vitro methionine synthase activity. These results indicate a link between alterations in the intracellular trafficking and/or metabolism of cobalamin and the increased growth autonomy of human melanoma cells.
我们的目标是确定导致高度生长自主性的人类肿瘤细胞无法在不含蛋氨酸但含有同型半胱氨酸和5-甲基四氢叶酸的培养基中增殖的生化缺陷。我们采用了“同型半胱氨酸反应性”和“同型半胱氨酸无反应性”这两个术语来描述在补充了同型半胱氨酸的无蛋氨酸培养基中能够或不能增殖的细胞。使用一组遗传相关的同型半胱氨酸反应性和无反应性人类黑色素瘤细胞系，多项实验结果表明，“同型半胱氨酸无反应性表型”的获得与甲基钴胺素（蛋氨酸合酶的关键辅因子）细胞内积累减少有关。当在体外进行蛋氨酸合酶测定时，在外源添加甲基钴胺素的情况下，从同型半胱氨酸反应性细胞中获得的提取物中的特异性蛋氨酸合酶活性仅比从同型半胱氨酸无反应性细胞中制备的提取物中观察到的活性高两倍。然而，当酶测定中省略外源甲基钴胺素时，与同型半胱氨酸反应性细胞提取物中观察到的小幅下降相比，同型半胱氨酸无反应性细胞提取物中的蛋氨酸合酶活性显著降低。与它们的同型半胱氨酸反应性对应物相比，同型半胱氨酸无反应性细胞表现出钴胺素外排水平增加和甲基钴胺素细胞内积累减少。这些相关黑色素瘤细胞系在补充了同型半胱氨酸的无蛋氨酸培养基中增殖的能力与钴胺素损失程度以及外源添加甲基钴胺素刺激体外蛋氨酸合酶活性的能力之间存在明显关系。这些结果表明钴胺素细胞内运输和/或代谢的改变与人类黑色素瘤细胞生长自主性增加之间存在联系。
