Sanchez-Prieto R, Lleonart M, Ramón y Cajal S
Department of Pathology, Clínica Puerta de Hierro, Madrid, Spain.
Oncogene. 1995 Aug 17;11(4):675-82.
p53 tumor suppressor protein is required for efficient execution of apoptosis after DNA-damage in many cell systems. Since the oncogene E1a confers susceptibility to DNA-damaging agents and stabilizes p53 protein, we investigate whether the sensitivity to anticancer drugs of E1a-expressing cells was mediated by binding to a specific set of cellular proteins (p60, p105, p107 and p300) and related to the induction of apoptosis and the level of p53 protein. We studied the effect of cisplatin (CDDP), doxorubicin (DOX) and ionizing radiation (RX) on murine keratinocytes (PAM 212) transfected by the wild type E1a oncogene or several E1a mutants which bind to different subsets of cellular proteins. Keratinocytes transfected with the mutant d1787N (which binds to p60, p105, p107 and p300) showed a lethality in response to CDDP (10 micrograms ml-1) fourfold higher than controls and threefold higher in response to DOX and radiation (5 grays). The sensitivity of keratinocytes carrying the mutant NTd1598 (binding to p105, p107 and p60) to DNA-damaging agents was similar to that of control keratinocytes, while mutant d1922/947 (binding only to p300) were resistant to CDDP and RX but sensitive to DOX. Apoptosis (after 24 h) studied by DNA fragmentation and flow cytometry was only observed in cells carrying the wild type E1a or the mutant d1787N. After treatment with DNA-damaging agents, p53 protein expression increased in all the cell lines and no rE1ation to sensitivity to anticancer agents or induction of apoptosis was observed. From these results, we conclude that cell sensitivity to cisplatin and ionizing radiation induced by the E1a oncogene requires binding to p105, p107 and p300 cellular proteins, while sensitivity to Doxorubicin requires binding only to p300. Interestingly, p53 protein levels were related to the binding to the p300 protein. The high levels of p53 after CDDP and DOX in the mutant d1922/947, which are only sensitive to DOX, suggest that E1a oncogene products may induce sensitivity to DNA-damaging agents by p53-related and unrelated pathways.
在许多细胞系统中，p53肿瘤抑制蛋白是DNA损伤后有效执行细胞凋亡所必需的。由于癌基因E1a使细胞对DNA损伤剂敏感并稳定p53蛋白，我们研究了表达E1a的细胞对抗癌药物的敏感性是否通过与一组特定的细胞蛋白（p60、p105、p107和p300）结合介导，以及是否与细胞凋亡的诱导和p53蛋白水平相关。我们研究了顺铂（CDDP）、阿霉素（DOX）和电离辐射（RX）对转染了野生型E1a癌基因或几种与不同细胞蛋白亚群结合的E1a突变体的小鼠角质形成细胞（PAM 212）的影响。用突变体d1787N（与p60、p105、p107和p300结合）转染的角质形成细胞对CDDP（10微克/毫升）的致死率比对照高四倍，对DOX和辐射（5戈瑞）的致死率高两倍。携带突变体NTd1598（与p105、p107和p60结合）的角质形成细胞对DNA损伤剂的敏感性与对照角质形成细胞相似，而突变体d1922/947（仅与p300结合）对CDDP和RX耐药，但对DOX敏感。通过DNA片段化和流式细胞术研究的细胞凋亡（24小时后）仅在携带野生型E1a或突变体d1787N的细胞中观察到。在用DNA损伤剂处理后，所有细胞系中p53蛋白表达均增加，未观察到与对抗癌剂的敏感性或细胞凋亡诱导的相关性。从这些结果中，我们得出结论，E1a癌基因诱导的细胞对顺铂和电离辐射的敏感性需要与p105、p107和p300细胞蛋白结合，而对阿霉素的敏感性仅需要与p300结合。有趣的是，p53蛋白水平与与p300蛋白的结合有关。仅对DOX敏感的突变体d1922/947在CDDP和DOX处理后p53水平较高，这表明E1a癌基因产物可能通过p53相关和不相关途径诱导对DNA损伤剂的敏感性。
