Reston J T, Gould-Fogerite S, Mannino R J
Department of Biomedical Sciences, School of Public Health, Albany, NY.
Biochim Biophys Acta. 1993 May 8;1177(1):49-53. doi: 10.1016/0167-4889(93)90156-j.
The efficiency of stable gene transfer and expression in NIH3T3 cells has been shown to be significantly enhanced by a brief treatment with the phorbol ester tetradecanoylphorbol 12,13-acetate (TPA) immediately following calcium-phosphate transfection. Several lines of evidence indicated that this effect was mediated through protein kinase C activation. These studies were expanded to determine whether this was a consistent and widespread phenomenon among other cell lines. The efficiency of transfection in two other established fibroblast lines, LMtk- and 2A3 3T3, was unaffected by TPA treatment, and primary human foreskin fibroblasts were similarly unaffected. Transfection was inhibited by TPA treatment in the transformed cell lines EJ and HeLa. Protein kinase C enzyme assays indicated that TPA causes a translocation of the enzyme from cytosol to membrane in both NIH3T3 and EJ cells, suggesting that the PKC translocation event does not account for the TPA effect on transfection. The TPA-mediated inhibition of transfection in EJ cells was not blocked by sphingosine, suggesting that this phenomenon is unrelated to PKC activation. The results suggest that TPA treatment may either enhance, inhibit, or have no effect on transfection, depending on the cell line.
磷酸钙转染后立即用佛波酯十四烷酰佛波醇12,13 - 乙酸酯（TPA）进行短暂处理，已证明可显著提高NIH3T3细胞中稳定基因转移和表达的效率。多项证据表明，这种效应是通过蛋白激酶C激活介导的。这些研究得以扩展，以确定这在其他细胞系中是否是一种一致且普遍的现象。另外两种已建立的成纤维细胞系LMtk-和2A3 3T3的转染效率不受TPA处理的影响，原代人包皮成纤维细胞同样不受影响。在转化细胞系EJ和HeLa中，TPA处理会抑制转染。蛋白激酶C酶活性测定表明，TPA在NIH3T3和EJ细胞中均会导致该酶从胞质溶胶转位至细胞膜，这表明蛋白激酶C转位事件并不能解释TPA对转染的影响。EJ细胞中TPA介导的转染抑制作用不受鞘氨醇的阻断，这表明该现象与蛋白激酶C激活无关。结果表明，TPA处理对转染的影响可能因细胞系而异，可能会增强、抑制或无影响。
