Functional characterization of p53 in nasopharyngeal carcinoma by stable shRNA expression

Abstract

Abstract. Nasopharyngeal carcinoma (NPC) is a highincidence malignancy in Southern China and Southeast Asia. Although mutation of p53 tumor-suppressor gene is a rare event in NPC, NPC has a high frequency of over-expressed/ accumulated p53 protein, which was reported to be dysfunction or inactivation in most of NPC. We report here a functional characterization of p53 in an undifferentiated NPC cell line CNE2. To elucidate the biological function of p53, we employed the RNA interference (RNAi) approach to knockdown the endogenously expressed p53 in CNE2 cells. Interestingly, suppression of p53 expression in CNE2 cells was associated with significant down-regulation of p21 WAF1/CIP1 expression and decreased HDM2 protein level in both steady state and genotoxic stress induced by ionizing radiation (IR). Consistent with these biochemical data were the accelerated cell cycle progression and the increased proliferation rate, suggesting that p53 retained growth inhibitory activity in CNE2 cells. Indeed, down-regulation of p53 in CNE2 enhanced the ability of CNE2 cells to grow anchorage-independently in vitro and to develop tumors in vivo. Together with the radioresistance acquired by CNE2sip53 cells, our data indicate that in contrast to a previous study, p53 in this NPC cell line remains functional, which may have an important therapeutical implication. IntroductionNasopharyngeal carcinoma (NPC) is featured by a remarkable racial and geographic distribution, and is a high-incidence malignancy in Southern China, Southeast Asia, Northern Africa, and Alaska, where the observed incidence rates range from 15 to 50 per 100,000 persons (1-3). NPC can occur in all age groups, but has a bimodal age distribution. The incidence peaks at 50 to 60 years of age; and a small peak is observed during late childhood (3). As an etiologically multi-factorial disease, carcinogenesis of the nasopharynx may result from combined effects of Epstein-Barr viral (EBV), genetic and environmental factors (4-7). Available information on the origin of NPC suggests that genetic alterations of tumor suppressor genes and proto-oncogenes in multiple cellular pathways may be important in multistage NPC carcinogenesis.p53 tumor-suppressor protein plays a pivotal role in regulating cell cycle, differentiation and apoptosis (8). As a transcription factor, p53 can induce the expression of p21 WAF1/CIP1 , which inhibits cyclin-dependent kinases, thereby preventing the phosphorylation of Rb and subsequent cell cycle arrest (9). Mutation of p53 tumor-suppressor gene has been found to be the most frequent genetic alteration in human malignancy. p53 mutations often result in accumulation of the mutant p53 protein, which either loses tumor suppressor function or gains oncogenic activity (10). p53 can also be functionally inactivated by mechanisms other than gene mutation (10). Screen for p53 mutations in the hot-spots for mutation revealed only a low frequency of p53 mutations ranging from 0 to 10%, which might explain the initial high sensitivity observed with ci...