Ubiquitin And P53 Protein Expression In Bile Duct And Gallbladder Carcinomas And Clinical Significance

Background:Ubiquitin protein (Ub) is a highly conserved protein of 8 kDa that becomes covalently attached to lysine (Lys) residues of target proteins. Ubiquitin was discovered as a destruction tag to cope with fault foldback protein and useless protein degradation.〔1,3〕This occurs through a three-step process involving Ub-activating (E1), Ub-conjugating (E2) and Ub-ligating (E3) enzymes [1]. Recently ,some data show that DNA damage and DNA repair are signaling responses that are tightly linked to ubiquitylation.(2 Ubiquitylation and cell signaling Kaisa Haglund and Ivan Dikic). While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. Natalia Lukashchuk and Karen H. Vousden find that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways (3).The p53 tumor suppressor gene encodes a protein that accumulates in the cells in response to a variety of stresses,activating a number of responses that include cell cycle arrest, apoptosis, and DNA repair. p53 functions as a transcription factor, activating the expression of various target genes that mediate the p53 responses(4). Nearly half of tumor patients were found wild-type p53 tumor suppressor mutated. p53 is mutated in over half of all human tumors and, among almost all of the remaining tumors, the pathway of p53-induced cell cycle arrest and apoptosis is deficient(5). Unlike other solid tumors, melanomas typically lack p53 mutations and retain expression of the wild-type (WT) protein, often at high levels (6–9). This is surprising, given the highly malignant nature of melanoma and its great resistance to therapeutic intervention.MDM2 maybe the critical factor.MDM2 is the target gene in the p53 downstream. P53 can inhibit the function of MDM2, while MDM2 also can inhibit the function of p53. They are reverse feedback. .Objective:To study the relationship of the expression of ubiquitin protein and p53 protein in human bile duct, gallbladder Carcinomas with pathology and its clinical significance. Then we try to find how cholangiocarcinoma and gallbladder carcinomas development and its usage for treatment.Methods:1) tissue blocks of bile duct carcinomas, gallbladder Carcinomas and cholecystitis were collected from the second affiliate hospital of forth military medical university. Then we fixed with 4% paraform in 100mmol/L phosphate buffer saline(pbs), 4℃, 24hours.We use immunohistochemical method to detect the expression of ubiquitin protein and in bile duct and gallbladder carcinomas, normal and inflammatory tissues. 2) Tissue blocks of bile duct carcinomas, gallbladder Carcinomas and cholecystitis collection were same to method 1. Specimen were lysed in RIPA(50mmol·L-1 Tris (PH8.0), 150mmol·L-1 NaCl, 1mmol·L-1 EDTA,1%NP-40, 0.1% SDS), plus protease inhibitors (Complete, Sigma)1μl per 100μl lysate in order to get total protein. Then we use western blotting method to detect the expression of ubiquitin and p53 proteins.3) Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), we detected the expressions of Ubiquitin mRNA in the tissues of bile duct and gallbladder carcinomas, and paraneoplastic tumor samples and from the patients. Certainly ,we also test the ubiquitin mRNA in QBC-939 cell lines first.Results:1)Ubiquitin protein expression levels and positive rates in human bile duct and gallbladder carcinomas were significantly higher than that in normal and inflammatory tissues(p<0.05). ubiquitin protein expression was significantly correlated with the clinical pathologic degrees(p<0.05). 2)In western blotting detection, ubiquitin protein expression in human bile duct ,gallbladder carcinomas and inflammatory tissues were respectively 1.80±0.29 and 1.25±0.30. Then we use independent t-test to detect the significance of the 2 groups. They were significantly different (p=0.005, T=3.394) .The result was same to the immunohistochemical detection. P53 protein expression levels in human bile duct and gallbladder carcinomas were not statistics difference with that in inflammatory tissues(p=0.394). 3). Ubiquitin mRNA were upgrade in tumor tissues, compare to the paraneoplastic tumor tissues(P<0.05).Conclusion:ubiquitin protein is upregulated in human bile duct and gallbladder Carcinomas tissues. The higher expression of ubiquitin protein was closely related to the degree of malignancy and disease progression of human bile duct and gallbladder Carcinomas. P53 protein expression in human bile duct ,gallbladder carcinomas and inflammatory tissues are not significant. This may be the reason of MDM2 affect the p53 function. We also use semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) to demonstrate the upgrading of tumor ubiquitin mRNA.