Wesierska-Gadek J, Schmid G
Institute of Cancer Research, University of Vienna, Borschkegasse 8 a, A-1090 Vienna, Austria.
J Cell Biochem. 2000 Sep 18;80(1):85-103.
We have previously reported that in cells ectopically expressing temperature-sensitive p53(135val) mutant, p53 formed tight complexes with poly(ADP-ribose) polymerase (PARP). At elevated temperatures, p53(135val) protein, adopting the mutant phenotype, was localized in the cytoplasm and sequestered the endogenous PARP. To prove whether an excess of p53(135val) protein led to this unusual intracellular distribution of PARP, we have established cell lines overexpressing p53(135val) + c-Ha-ras alone or in combination with PARP. Interestingly, immunostaining revealed that PARP is sequestered in the cytoplasm by mutant p53 in cells overexpressing both proteins. Simultaneous overexpression of PARP had no effect on temperature-dependent cell proliferation and only negligibly affected the kinetics of p53-mediated G(1) arrest. However, if the cells were completely growth arrested at 32 degrees C and then shifted up to 37 degrees C, coexpressed PARP dramatically delayed the reentry of transformed cells into the cell cycle. Even after 72 h at 37 degrees C the proportion of S-phase cells was reduced to 20% compared to those expressing only p53(135val) + c-Ha-ras. The coexpressed PARP stabilized wt p53 protein and its enzymatic activity was necessary for stabilization.
我们之前报道过，在异位表达温度敏感型p53(135val)突变体的细胞中，p53与聚（ADP - 核糖）聚合酶（PARP）形成紧密复合物。在升高的温度下，呈现突变表型的p53(135val)蛋白定位于细胞质中，并隔离内源性PARP。为了证明过量的p53(135val)蛋白是否导致PARP这种异常的细胞内分布，我们建立了单独过表达p53(135val) + c - Ha - ras或与PARP联合过表达的细胞系。有趣的是，免疫染色显示，在同时过表达这两种蛋白的细胞中，PARP被突变型p53隔离在细胞质中。PARP的同时过表达对温度依赖性细胞增殖没有影响，并且对p53介导的G(1)期阻滞动力学的影响可以忽略不计。然而，如果细胞在32℃完全生长停滞，然后转移到37℃，共表达的PARP会显著延迟转化细胞重新进入细胞周期。即使在37℃培养72小时后，与仅表达p53(135val) + c - Ha - ras的细胞相比，S期细胞的比例也降至20%。共表达的PARP稳定了野生型p53蛋白，其酶活性对于这种稳定是必需的。
