Bae I, Fan S, Bhatia K, Kohn K W, Fornace A J, O'Connor P M
Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Cancer Res. 1995 Jun 1;55(11):2387-93.
We investigated temporal relationships between ionizing radiation-induced G1 arrest and induction of the p53-regulated genes GADD45, CIP1/WAF1, and MDM2 in a series of Burkitt's lymphoma and lymphoblastoid cell lines that differed in p53 gene status. Emphasis was placed on characterization of the EW36 cell line, which despite expressing wild-type p53 genes, is defective in G1 arrest following gamma-irradiation (P. M. O'Connor et al., Cancer Res., 53: 4776-4780, 1993). Induction of CIP1/WAF1, GADD45, and to a lesser extent MDM2 mRNA was observed in all wild-type p53 lines that arrested in G1. Cell lines that contained only mutant p53 genes or were heterozygous for p53 mutations failed to induce appreciable levels of these p53-regulated transcripts and did not arrest in G1. G1 arrest in the wild-type p53 cell line WMN was more prolonged than elevation of CIP1/WAF1, GADD45, or MDM2 transcripts, suggesting that G1 arrest duration must be dependent upon stability of these newly synthesized proteins. In agreement, we found that p21Cip1/Waf1, a potent inhibitor of G1-S phase cyclin-dependent kinases, was maintained at elevated levels throughout the period that WMN cells remained arrested in G1. EW36 cells exhibited normal induction of CIP1/WAF1, GADD45, and MDM2 mRNA following gamma-irradiation, suggesting that the defect in G1 arrest must reside downstream of p53 transactivation. Investigations into the stability of p53 and p21Cip1/Waf1 revealed that EW36 cells failed to maintain elevated levels of these proteins following irradiation. p53 levels decreased within 4 h of irradiation, and p21Cip1/Waf1 levels decreased shortly after the normal decline of CIP1/WAF1 mRNA levels. Degradation of p21Cip1/Waf1 coincided with the escape of EW36 cells from G1 arrest. Our studies suggest that p21Cip1/Waf1 stability may determine G1 arrest duration and that premature degradation of this protein could provide an alternative route to subversion of the G1 checkpoint in cancer cells.
我们研究了一系列在p53基因状态上存在差异的伯基特淋巴瘤和淋巴母细胞系中，电离辐射诱导的G1期阻滞与p53调控基因GADD45、CIP1/WAF1和MDM2诱导之间的时间关系。重点是对EW36细胞系进行特征描述，尽管该细胞系表达野生型p53基因，但在γ射线照射后G1期阻滞存在缺陷（P.M.奥康纳等人，《癌症研究》，53:4776 - 4780，1993）。在所有阻滞于G1期的野生型p53细胞系中均观察到CIP1/WAF1、GADD45以及程度较轻的MDM2 mRNA的诱导。仅含有突变型p53基因或为p53突变杂合子的细胞系未能诱导出这些p53调控转录本的显著水平，且未阻滞于G1期。野生型p53细胞系WMN中的G1期阻滞比CIP1/WAF1、GADD45或MDM2转录本的升高持续时间更长，这表明G1期阻滞的持续时间必定取决于这些新合成蛋白质的稳定性。与此一致的是，我们发现p21Cip1/Waf1，一种G1 - S期细胞周期蛋白依赖性激酶的有效抑制剂，在WMN细胞停留在G1期的整个期间都维持在升高水平。EW36细胞在γ射线照射后表现出CIP1/WAF1、GADD45和MDM2 mRNA的正常诱导，这表明G1期阻滞缺陷必定存在于p53反式激活的下游。对p53和p21Cip1/Waf1稳定性的研究表明，EW36细胞在照射后未能维持这些蛋白质的升高水平。p53水平在照射后4小时内下降，p21Cip1/Waf1水平在CIP1/WAF1 mRNA水平正常下降后不久也下降。p21Cip1/Waf1的降解与EW36细胞从G1期阻滞中逃逸同时发生。我们的研究表明，p21Cip1/Waf1的稳定性可能决定G1期阻滞的持续时间，并且该蛋白质的过早降解可能为癌细胞中G1期检查点的颠覆提供另一条途径。
