Wesierska-Gadek J, Schmid G
Institute of Cancer Research, University of Vienna, Austria.
Cell Mol Biol Lett. 2001;6(2):117-40.
We investigated the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53 using two different approaches. In the first approach, we used primary and immortalized cells derived from wt and PARP-1 -/- mice. We examined whether PARP-1 deficiency would affect the expression of the wild-type (wt) p53 protein. The inactivation of the PARP-1 gene markedly affected the constitutive expression of the wt p53 protein. Interestingly, only the regularly spliced form of wt p53 was reduced to a barely detectable level in consequence to an approximately 8-fold shortening of its half-life, whereas the level of alternatively spliced p53 remained unchanged. Moreover, reconstitution of cells lacking the PARP-1 gene with the human counterpart restored the normal stability of the regularly spliced p53 protein. In the second approach, we performed experiments with c-Ha-ras transformed primary rat cells overexpressing the p53135val mutant alone or in combination with PARP-1. The advantage of this temperature sensitive p53135val mutant is its oncogenic character at 37 degrees C, connected with cytoplasmic localization of p53, and its tumor suppressor activity at 32 degrees C, accompanied by p53 translocation into the nucleus. No noticeable differences in proliferation and G1 accumulationwere observed between cells expressing p53135val with or without PARP-1. On the other hand, a comparison of the recovery of G1 arrested cells after a shift up to 37 degrees C for both cell lines showed dramatic differences in the kinetics. While cells expressing p53135val rapidly reached the characteristic S-phase level after a shift up to basal temperature, cells additionally expressing PARP-1 rested in G1 despite the temperature elevation. This coincided with exclusively cytoplasmic p53 protein in cells expressing p53135val and predominantly nuclear localization of p53 in p53135val +PARP-1 cells, as evidenced by immunostaining. Determination of the p53 level during the maintenance of cells at 32 degrees C revealed a marked decrease in the level of p53 in cells expressing p53135val alone, whereas in cells coexpressing PARP-1, the level of p53 remained largely unaffected. This indicates that the stability of wild-type p53 greatly differed between both cell lines. Furthermore, the inhibition of PARP-1 activity in G1 arrested cells by 3-aminobenzamide abolished its stabilizing effect on the wild-type p53 protein. Taken together, our results indicate that PARP-1 regulates the stability of the wt p53 protein and that its enzymatic activity is necessary for this stabilizing action.
我们采用两种不同方法研究了聚（ADP - 核糖）聚合酶 - 1（PARP - 1）与肿瘤抑制基因p53产物之间的相互作用。在第一种方法中，我们使用了源自野生型（wt）和PARP - 1基因敲除（-/-）小鼠的原代细胞和永生化细胞。我们研究了PARP - 1缺陷是否会影响野生型（wt）p53蛋白的表达。PARP - 1基因的失活显著影响了wt p53蛋白的组成性表达。有趣的是，由于其半衰期缩短了约8倍，wt p53仅正常剪接形式降低到几乎检测不到的水平，而可变剪接的p53水平保持不变。此外，用人源PARP - 1基因重建缺乏PARP - 1基因的细胞，恢复了正常剪接的p53蛋白的正常稳定性。在第二种方法中，我们对单独过表达p53135val突变体或与PARP - 1共同过表达的c - Ha - ras转化的原代大鼠细胞进行了实验。这种温度敏感的p53135val突变体的优点在于其在37℃时具有致癌特性，与p53的细胞质定位相关，而在32℃时具有肿瘤抑制活性，伴随着p53易位到细胞核。在表达p53135val的细胞中，无论有无PARP - 1，在增殖和G1期积累方面均未观察到明显差异。另一方面，对两种细胞系在温度升至37℃后G1期停滞细胞恢复情况的比较显示，动力学上存在显著差异。当表达p53135val的细胞温度升至基础温度后迅速达到特征性的S期水平时，额外表达PARP - 1的细胞尽管温度升高仍停滞在G1期。免疫染色证明，这与表达p53135val细胞中p53蛋白仅存在于细胞质中以及p53135val + PARP - 1细胞中p53主要定位于细胞核一致。在32℃维持细胞期间对p53水平的测定显示，单独表达p53135val的细胞中p53水平显著降低，但在共表达PARP - 1的细胞中，p53水平基本未受影响。这表明野生型p53在两种细胞系中的稳定性存在很大差异。此外，3 - 氨基苯甲酰胺对G1期停滞细胞中PARP - 1活性的抑制消除了其对野生型p53蛋白的稳定作用。综上所述，我们的结果表明PARP - 1调节wt p53蛋白的稳定性，并且其酶活性对于这种稳定作用是必需的。
