Gronostajski R M, Goldberg A L, Pardee A B
J Cell Physiol. 1984 Oct;121(1):189-98. doi: 10.1002/jcp.1041210124.
When cultured fibroblasts are deprived of serum, the degradation of long-lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum-deficient medium (0.5% serum), the rate of degradation of long-lived proteins increased about twofold within 30 min. This increase was reduced by 50-70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24-48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two- to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24-48 hours had about twice the rate of protein synthesis and two- to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475-treated cells were also about 30% larger than the untreated cells. Thus, the protease-inhibitor prevented much of the atrophy induced by serum deprivation. The serum-deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum-deprived cells. Other agents which inhibited protein breakdown in serum-deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475-treated cells began DNA synthesis 1-2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.
当培养的成纤维细胞缺乏血清时，长寿蛋白和RNA的降解增加，细胞停止增殖，并且体积减小。为了确定蛋白质分解代谢增加在这些反应中的作用，我们研究了溶酶体内蛋白水解抑制剂对Balb/c 3T3细胞的影响。当将这些细胞置于血清缺乏培养基（0.5%血清）中时，长寿蛋白的降解速率在30分钟内增加了约两倍。溶酶体硫醇蛋白酶抑制剂（Ep475和亮抑蛋白酶肽）或提高溶酶体内pH值的试剂（氯喹和氯化铵）可使这种增加降低50 - 70%。相比之下，这些化合物对在10%血清中生长的细胞中的蛋白质降解几乎没有影响。因此，与先前的研究一致，溶酶体似乎是血清剥夺后蛋白水解增加的部位。当3T3细胞被剥夺血清24 - 48小时时，蛋白质合成速率、蛋白质和RNA含量以及细胞体积降低了两到四倍。蛋白酶抑制剂Ep475减少了蛋白质合成速率的降低以及细胞蛋白质和RNA的损失。被剥夺血清并用Ep475处理24 - 48小时的细胞，其蛋白质合成速率约为对照血清剥夺细胞的两倍，蛋白质和RNA水平高两到四倍。经Ep475处理的细胞也比未处理的细胞大30%左右。因此，蛋白酶抑制剂防止了血清剥夺诱导的大部分萎缩。血清剥夺的成纤维细胞也停止增殖并积聚在细胞周期的G1期。用Ep475处理的细胞以与未处理的血清剥夺细胞相同的方式积聚在G1期。其他抑制血清剥夺细胞中蛋白质分解的试剂也不能阻止细胞增殖的停滞。因此，血清剥夺期间蛋白水解的增强似乎是细胞大小和蛋白质合成减少所必需的，但可能不是细胞增殖停止所必需的。当在有或没有Ep475的情况下被剥夺血清的细胞通过重新添加血清刺激增殖时，较大的经Ep475处理的细胞比未处理的较小细胞晚1 - 2小时开始DNA合成。因此，用Ep475处理后，血清刺激后的细胞周期进程速率与细胞大小、蛋白质或RNA含量或蛋白质合成速率不成比例。
