Schwartz M A, Both G, Lechene C
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, MA.
Proc Natl Acad Sci U S A. 1989 Jun;86(12):4525-9. doi: 10.1073/pnas.86.12.4525.
Growth of normal fibroblasts requires an adhesive substratum on which to spread, whereas transformed cells can grow in suspension. Since an alkaline cytoplasm has been shown to be required for growth, we measured cytoplasmic pH in individual cells as a function of spreading. The degree of spreading was controlled by coating tissue culture plastic with varying amounts of the nonadhesive polymer polyHEMA. Completely round BALB/c 3T3 cells were 0.15 pH unit more acidic than spread cells. In short-term experiments, cells were treated with the peptide Gly-Arg-Gly-Asp-Ser-Pro to induce rounding or by plating on fibronectin to induce spreading. When cells were induced to change shape, pH changed rapidly and reversibly. All of the anchorage-dependent cell lines tested behaved similarly, but 3T3 cells transformed by the plasma membrane oncogene src or ras were able to maintain a relatively alkaline pH even when completely round. Cells transformed by the nuclear oncogene myc behaved like normal cells. Our results suggest that the requirement for spreading may in part be mediated by cytoplasmic pH. Anchorage-independent growth due to oncogenes that localize to the plasma membrane is associated with loss of this control mechanism.
正常成纤维细胞的生长需要一个可供其铺展的黏附基质，而转化细胞能够在悬浮状态下生长。由于已表明生长需要碱性细胞质，我们测量了单个细胞的细胞质pH值随铺展情况的变化。通过用不同量的非黏附性聚合物聚甲基丙烯酸羟乙酯（polyHEMA）包被组织培养塑料来控制铺展程度。完全呈圆形的BALB/c 3T3细胞比铺展的细胞酸性强0.15个pH单位。在短期实验中，用肽Gly-Arg-Gly-Asp-Ser-Pro处理细胞以诱导变圆，或通过接种在纤连蛋白上以诱导铺展。当细胞被诱导改变形状时，pH值迅速且可逆地变化。所有测试的锚定依赖性细胞系表现相似，但由质膜癌基因src或ras转化的3T3细胞即使完全呈圆形也能够维持相对碱性的pH值。由核癌基因myc转化的细胞表现得与正常细胞一样。我们的结果表明，对铺展的需求可能部分由细胞质pH介导。由于定位于质膜的癌基因导致的非锚定依赖性生长与这种控制机制的丧失有关。
