Kawamata Hitoshi, Omotehara Fumie, Nakashiro Koh-Ichi, Uchida Daisuke, Shinagawa Yasuhiro, Tachibana Masatsugu, Imai Yutaka, Fujimori Takahiro
Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Mibu, Shimo-Tsuga, Tochigi, Japan.
Int J Oncol. 2007 May;30(5):1089-97.
A p53 functional analysis system, which can identify the types of abnormality of p53, such as loss of function, dominant negative function, or gain of oncogenic function, is now required. In this study, we examined the functional diversity of several mutations of p53 derived from human head and neck cancer cells. The entire open reading frame of p53 cDNA was subcloned into a mammalian expression vector, pEGFP-C3, and genetic mutations were determined. Then, intracellular localization and transcriptional activity of the tumor-derived p53 proteins were examined in Saos-2 cells. A mutant-p53 (Glu17Lys, His193Leu) or a truncated p53 (Delta121) did not activate the reporters containing p53 responsive elements from p21waf1, BAX, MDM2, p53AIP1, and PUMA genes at all. However, a mutant-p53 (Asn30Ser) showed the transcriptional activity on all of the reporters as wild-type p53 did. On the other hand, a mutant-p53 (Asp281His) activated the p21waf1 promoter strongly and the MDM2 promoter faintly, but did not activate the BAX promoter. Interestingly, this mutant-p53 prevented Saos-2 cells from undergoing apoptosis after treatment with a DNA damaging agent, adriamycin. This mutant-p53 induced cell cycle arrest but not apoptosis. Furthermore, another mutant-p53 (Glu17Lys, His193Leu) also prevented the cells from undergoing apoptosis after DNA damage probably in a transcription-independent manner. These results suggest that some cancer cells may contain the oncogenic mutation of the p53 gene, and the oncogenic p53 protein prevents cancer cells from undergoing apoptosis after DNA damage. Detailed information for mutated p53 gene in cancer cells might provide useful suggestions for the therapeutic strategy.
现在需要一种能够识别p53异常类型的功能分析系统，例如功能丧失、显性负功能或致癌功能获得。在本研究中，我们检测了源自人头颈部癌细胞的几种p53突变的功能多样性。将p53 cDNA的整个开放阅读框亚克隆到哺乳动物表达载体pEGFP-C3中，并确定基因突变。然后，在Saos-2细胞中检测肿瘤来源的p53蛋白的细胞内定位和转录活性。突变型p53（Glu17Lys，His193Leu）或截短型p53（Delta121）根本不激活含有来自p21waf1、BAX、MDM2、p53AIP1和PUMA基因的p53反应元件的报告基因。然而，突变型p53（Asn30Ser）与野生型p53一样，对所有报告基因都显示出转录活性。另一方面，突变型p53（Asp281His）强烈激活p21waf1启动子，微弱激活MDM2启动子，但不激活BAX启动子。有趣的是，这种突变型p53在用DNA损伤剂阿霉素处理后可防止Saos-2细胞发生凋亡。这种突变型p53诱导细胞周期停滞但不诱导凋亡。此外，另一种突变型p53（Glu17Lys，His193Leu）可能以转录非依赖的方式在DNA损伤后也阻止细胞发生凋亡。这些结果表明，一些癌细胞可能含有p53基因的致癌突变，致癌性p53蛋白可防止癌细胞在DNA损伤后发生凋亡。癌细胞中p53基因突变的详细信息可能为治疗策略提供有用的建议。
