Functional Characterization Of P53 In Nasopharyngeal Carcinoma By Proteomics And RNAi Techniques

Nasopharyngeal carcinoma (NPC) is a high-incidence malignancy in Southern China and Southeast Asia. As an etiologically multi-factorial disease, carcinogenesis of the nasopharynx may result from combined effects of Epstein-Barr viral, genetic and environmental factors. 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 factors in multistage NPC carcinogenesis.p53 gene is a very important tumor-suppressor gene, whose inactivation is implicated in a high percentage of individual cases of human cancer, p53 is primarily a sequence-specific transcriptional activator, which binds to cognate p53 responsive elements within the genome and activates the transcription of genes residing in the vicinity of these binding sites. The molecules activated by p53 induce cell cycle arrest, senescence, differentiation, DNA repair to conserve the genome and apoptosis. Mutation of p53 gene detected in more than 50% of human tumors has been found to be the most frequent molecular alteration in human malignancy. Although NPC had a relatively low frequency of p53 mutations, as compared to other types of cancer, numerous studies showed an overexpression or accumulation of p53 protein in over 60% NPC and in amost all NPC cell lines, p53 overexpression is also associated with the proliferation, micro-vessel density, craniosacral invasion and neck lymph node metastasis of NPC as well as the NPC patients' prognosis, Which suggest that overexpressed p53 protein in NPC might be dysfunction or inactivation. But till now the p53 protein function and the mechanism of accumulation in NPC have remained unclear and have not been fully elucidated yet.RNA interference (RNAi) is a sequence-specific and post- transcriptional gene silencing method initiated by double-stranded RNAs. Since RNAi represents a powerful and convenient tool for determining the functions of specific genes via analysis of loss-of-function phenotype, we used stable expression of small interfering RNAs (siRNA) of p53 gene to determine the function of p53 in NPC. Then, the effects of persistent down-regulation of p53 gene expression on the transcriptional inactivation of the p53-responsive genes and on cell phenotype including cell cycle, cell proliferation, cell radiation sensitivity and cell apoptosis rate after ionizing radiation were demonstrated. Furthermore, comparative proteomic approach was performed to high-throughout identify the differential proteins between p53 knockdown CNE2 and control cells, which may be associated with p53 function. The results presented here will no doubt provide clues to further study the functional mechanism of p53 in NPC.In our study, we successfully used RNAi technology constructing the pSUPER/sip53 and established CNE2sip53 cell lines including CNE2sip53-1 and CNE2sip53-2 cell lines, which had stable expression of p53 siRNA in CNE2 cell line and satisfactory inhibition of the target p53 protein expression. Then the mRNA expression level of p53 gene and the p53-responsive gene p21 and MDM2 were detected by real-time quantitative RT-PCR. The p53 mRNA levels of CNE2sip53-1 and CNE2sip53-2 decreased 99.9% and 97.5% respectively of that in control cells including CNE2 and CNE2/pSUPER cell lines. The mRNA levels of p53-responsive gene p21 decreased 79.5% and 74% and the MDM2 mRNA levels decreased 81.9% and 77.1%. Consistent with the down-regulation of mRNA, western blot analysis showed that the p21 and MDM2 protein levels in CNE2sip53 cell lines were significantly diminished after 6 Gy ionizing radiation (IR) when compared with the control cell lines. These results indicated that the overexpressed p53 in CNE2 cells still plays a pivotal role in regulating the transcription of genes. Their biological characteristic analysis was then performed as follows: (1) FACS analysis of CNE2sip53-1 and CNE2sip53-2 cell lines revealed a significant decrease of G1 phase with a corresponding increase of S phase populations when compared with the control cell lines, and the G1/S ratio of CNE2sip53-1 and CNE2sip53-2 decreased 33.9% and 26.4% respectively when compared with CNE2 cell line (p＜0.05). (2) The MTT assay showed that knockdown p53 expression in CNE2 cells was associated with a marked increase of cell growth. (3) The monolayer growth experiment showed that the clonogenicity of CNE2sip53-1 and CNE2sip53-2 was significantly higher than those of the controls (CNE2sip53-1: 73%, CNE2sip53-2: 72%; CNE2: 43%, CNE2/pSUPER: 42%, p＜0.01). (4) Also soft agar growth experiments revealed that the colony formation abilities of CNE2sip53-1 and CNE2sip53-2 was significantly higher than those of the controls (CNE2sip53-1: 37%, CNE2sip53-2: 34.1%; CNE2: 20.5%, CNE2/pSUPER: 22.1%, p＜0.01). (5) The result of nude mice tumor formation assay showed that tumor formation of CNE2sip53-1 was earlier than that of CNE2 and CNE2/pSUPER cells and tumor growth were significantly faster than those inoculated with CNE2 or CNE2/pSUPER cells (P＜0.01, mean size 1.22±0.25 mm~3 in the CNE2 group, n=6, and 1.09±0.29 mm~3 in the CNE2/pSUPER group, n=6 vs 2.55±0.71 mm~3 in the CNE2sip53 group, n=6). Furthermore, the effect of p53 knockdown in CNE2 cells on radiation sensitivity was examined by performing a clonogenic survival assay and radiation-induced apoptosis was also determined at 24 h following irradiation by Hoechst 33258 staining. We constructed the survival curves according to the single-hit multitarget model of cell survival, which showed that the survival fraction at 2 Gy was 0.41 for CNE2 cell line, and 0.40 for CNE2/pSUPER cell line whereas the survival fractions at 2 Gy were 0.65 and 0.66, and the vaule of SER were 0.79 and 0.78 respectively for the two CNE2sip53 cell lines. After exposed to 6 Gy irradiation, more apoptotic cells were detected in CNE2 and CNE2/pSUPER cells than in CNE2sip53 cells (CNE2sip53-1: 69.1%, CNE2sip53-2: 68.4%; CNE2: 52.1%, CNE2/pSUPER: 53.2%, p＜0.05), consistent with the data obtained from clonogenic assay that knockdown the expression of p53 in CNE2 cells resulted in increased radioresistance. Our results from above experiments demonstrated that stable knockdown the expression of p53 enhanced the ability of CNE2 cells to proliferate in vitro and in vivo and to form tumors, and decreased the radiation sensitivity.On the other hand, basing on establishing p53 knockdown human NPC CNE2 cell line using stable RNA interference, we compared the proteomic changes between CNE2sip53 (that is CNE2sip53-1) and control cell line CNE2/pSUPER using two-dimensional gel electrophoresis (2-DE). Twenty-two differentially expressed proteins between the two cell lines were identified by both matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Electrospray ionization mass spectrometry (ESI-Q-TOF-MS). Among 22 differentially expressed proteins, 14 proteins including HSP27, 14-3-3σ, GRP78, HSP70 and so on were up-regulated in CNE2sip53 cells, while 8 protein including translation initiation factor eukaryotic translation initiation factor 4B (elF4B), tumor protein translationally-controlled 1 (TPT1), heterogenous nuclear ribonuclear protein K ( hnRNP K) and so on were down-regulated. The differential expression levels of the partial proteins (HSP27, 14-3-3σ, GRP75) were confirmed by western blot analysis. Furthermore, several differential proteins including HSP27, HSP70, GRP75 and GRP78 were verified as p53 interacting proteins in NPC by immunoprecipitation and western blot analysis, and the suppression of HSP27 expression by HSP27 antisense oligonucleotides could decrease the p53 protein level. Our data suggested that these differentially expressed proteins may be associated with the function of p53 in NPC.Above all, our study employed the siRNA approach to stably knockdown the expression of p53 in CNE2 cells for assessing its biochemical and biological consequences. We demonstrate that down-regulation of p53 in CNE2 cells was associated with significantly diminished expression of p53 target genes p21 and MDM2, accelerated cell cycle progression, elevated proliferation ability in vitro and in vivo, and increased radioresistance, which indicated that the overexpressed p53 in CNE2 cells remains functionally proficient and plays a pivotal role in regulating cell cycle, differentiation and apoptosis. The 22 differentially expressed proteins that were initiated by a comparative proteomic study of CNE2sip53 and CNE2/pSUPER cell lines might be the proteins associated with the function of p53 in NPC. Partial differential proteins including HSP27, HSP70, GRP75 and GRP78 might interact with p53 protein and stabilize p53 protein by increasing the half-life of p53 protein, resulting in the overexpression or accumulation of p53 protein in NPC. Our study not only indicated that the overexpressed p53 in CNE2 cells remains functionally proficient and provided the important basis and clue to comprehensively investigate the accumulation mechanisms of p53 in NPC.