Studies On The Proliferation Effect And Protein Signal Pathway Of P16-Rb-HDAC1-E2F1 In Biliary Tract Cancer

Tumorigenesis is a process of multi-factor and multi-step, which involved in the aberration of chromosome structure, inactivation of antioncogene, activation of oncogene, regulation of cell cycle, activation of gene promoter, epigenetics, and so on. Many researches were carried out in these aspects. Regulation of genes became new direction when human genome program had been completed. Regulation and expression of genetic information depend on the effect of protein signals. So study on the protein becomes a hot spot.Previous studies of Shengquan Zou' group indicate that p16 and HDAC1 play important roles in molecular cytogenetics and epigenetics in extrahepatic cholangiocarcinoma. Studies have demonstrated that Rb/E2F1 pathway influence the activation of HDAC1, and function of Rb/p16 pathway was recognized gradually also. It is a hint that p16, Rb, E2F1 and HDAC1 constitute a protein signal pathway. These protein signals were involved in many aspects of tumor origin and were representative in tumorigenesis. Our research is to study the effect and protein signal pathway of p16-Rb-HDAC1-E2F1 of extrahepatic cholangiocarcinoma and gallbladder carcinoma from many aspects by plasmid transfection, immuneprecipitation, Western blot, RT-PCR, MTT, immunehistochemistry, and so on. To study the correlation between regulation of gene and function of these proteins, which offer the new evidence of the effect of these proteins on biological behaviours of biliary tract cancer. And to analysis the correlation between the expression of these proteins with cinical epidemiology, which offer the new evidence on cause of biliary tract cancer. Although level of our research is limited, we will work further in future. Part I Studies on the interaction among p16-Rb-HDAC1-E2F1 proteins in biliary tract cancerThesis 1 The expression of p16-Rb-HDAC1-E2F1 in biliary tract cancer cell linesObjective To study the expression of proteins and mRNA of p16-Rb-HDAC1-E2F1 in cholangiocarcinoma cell lines (QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line). Methods Measure the expression of proteins and mRNA of p16-Rb-HDAC1-E2F1 in cholangiocarcinoma cell lines (QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line (Mz-ChA-1 cell line) by immunehistochemistry and RT-PCR assay. Results1. There is weak expression of p16 mRNA in QBC₉₃₉ cell line, there is weak expression of Rb mRNA in KMBC cell line, there are weak expressions of HDAC1 mRNA in QBC₉₃₉, KMBC, OZ and Mz-ChA-1 cell lines respectively, there are weak expressions of E2F1 mRNA in QBC₉₃₉, KMBC and Mz-ChA-1 cell lines respectively. There is expression of p16 protein in QBC₉₃₉ cell line, there is expression of Rb protein in KMBC cell line, there are expressions of HDAC1 protein in QBC₉₃₉, KMBC, OZ and Mz-ChA-1 cell lines respectively, there are expressions of E2F1 protein in QBC₉₃₉, KMBC and Mz-ChA-1 cell lines respectively.2. There is weak expression of p16 protein in tumor tissue of QBC₉₃₉ cell line transplanted nude mice model, there is weak expression of Rb protein in cancer tissue of KMBC cell line transplanted nude mice model. There are expressions of HDAC1 protein in tumor tissue of QBC₉₃₉, KMBC, OZ and Mz-ChA-1 cell lines transplanted nude mice model respectively, but their positive rates are different. There are weak expressions of E2F1 protein in cancer tissue of QBC₉₃₉, KMBC and Mz-ChA-1 cell lines transplanted nude mice model respectively.Conclusions P16-Rb-HDAC1-E2F1 play some roles in billiary tractcancer. Biological behaviours of biliary tract cancer cell lines canbe changed by internal environment. Thesis 2 Studies on the interaction among p16-Rb-HDAC1-E2F1proteins in biliary tract cancerObjective To study the mechanism and interaction among p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer.Methods By using the method of immuneprecipitation, we studied the interaction among p16-Rb-HDAC1-E2F1 proteins of extrahepatic cholangiocarcinoma cell lines (QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) which infected with p16 plasmid and pRb plasmid respectively. Results1. There are Rb protein(QBC₉₃₉, KMBC, OZ and Mz-ChA-1 cell lines) and E2F1 protein(QBC₉₃₉, KMBC and Mz-ChA-1 cell lines) lie in the precipitation of anti-HDAC1.2. There are HDAC1 protein(QBC₉₃₉, KMBC, OZ and Mz-ChA-1 cell lines) and E2F1 protein (QBC₉₃₉, KMBC and Mz-ChA-1 cell lines) lie in the precipitation of anti-Rb.Conclusions1. Rb protein, HDAC1 protein and E2F1 protein can make the specific interaction in biliary tract cancer, and there are the direct pathways among these protein signals.2. P16 protein has not direct relationship with Rb protein, HDAC1 protein, and E2F1 protein. Part II Relation between p16-Rb-HDAC1-E2F1 proteins and biological behaviours of biliary tract cancerThesis 3 Effect and interaction of p16-Rb-HDAC1-E2F1 proteins in biliary tract cancer cell lines in vivo and in vitroObjective To study the effect and interaction of p16-Rb-HDAC1-E2F1 onthe growth of cholangiocarcinoma cell lines (QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) in vivo and in vitro. Methods1. Transfect p16 plasmid and pRb plasmid into cholangiocarcinoma cell lines(QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line), measure the growth change of biliary tract cancer cell lines after transfection by MTT assay, measure the expression change of pRb mRNA after transfected with p16 plasmid and measure the expression change of p16 mRNA after transfected with pRb plasmid by RT-PCR.2. By using the same methods to observe the effect of histone deacetylase inhibitor-trichostatin A(TSA),which effect is against HDAC1, on the growth of cholangiocarcinoma cell lines(QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line).3. Successfully establish the transplanted gallbladder carcinoma nude mice models, and measure the growth change of the transplanted tumor after being transfected and treated with TSA.Results1. The expression of Rb mRNA of KMBC cell line transfected with p16 plasmid was inhibited. There are weak expressions of p16 mRNA in OZ and Mz-ChA-1 cell lines transfected with pRb plasmid. 2. The proliferation of cholangiocarcinoam cell lines(KMBC cell line) transfected with p16 plasmid was inhibited, the proliferation of cholangiocarcinoam cell lines (QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) transfected with pRb plasmid was inhibited, in which the inhibition of proliferation of QBC₉₃₉ cell line transfected with pRb plasmid was the most distinct.3. TSA can inhibit the proliferation of cholangiocarcinoma cell lines (QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line). These effects were dose-dependent and time-dependent.4. Successfully established the gallbladder carcinoma transplanted nude mice model, the growth of cancer was inhibited in the nude mice transplanted with gallbladder carcinoma cell line which transfected with pRb plasmid and TSA treatment.Conclusions1. There was negative feedback adjustment between p16 and Rb in biliary tract cancer.2. Rb and TSA can inhibit the growth of gallbladder carcinoma cell line in vivo, Rb and TSA can inhibit the growth of cholangiocarcinoma cell lines and gallbladder carcinoma cell line in vitro.3. P16 can only inhibit the growth of biliary tract cancer cell lines which express Rb, this indicates that the effect of p16 in biliary tract cancer rely on the function of Rb. Thesis 4 Mechanism of HDAC1 in immune escape of gallbladder carcinoma and correlation between HDAC1 and apoptosisObjective1. To study the role of Fas/FasL and HDAC1 in biological behaviours of gallbladder carcinoma.2. To study their correlated action and mechanism in tumor escape. Methods1. Immunohistochemistry technique was used to study the expression of Fas/FasL protein and HDAC1 protein in gallbladder carcinoma tissues, gallbladder adenoma tissues, gallbladder dysplasia tissues and chronic cholecystis tissues.2. Apoptosis of the infiltrating lymphocytes in these tissues was studied by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method.3. Expressions of both proteins and apoptosis of the tumor infiltrating lymphocytes in cancer tissues of primary foci were compared with clinicopathological features of gallbladder carcinoma.4. Study the correlation between Fas/FasL pathway and HDAC1. Results1. The positive rates of Fas were not significantly difference among carcinoma, adenoma, dysplasia and chronic cholecystis. The positive rate of FasL in carcinoma was significantly higher than that in chronic cholecystis (X²=4. 89, P<0.05).2. The apoptotic index in carcinoma was significantly higher than that in adenoma(t'=4.19, P<0.01)and chronic cholecystis (t' =8. 06,P<0. 01). The apoptotic index was significantly lower in well-differentiated carcinoma and Nevin I -III carcinoma than that in poorly-differentiated carcinoma(t'=2. 63, P<0. 05) and Nevin IV-V carcinoma(t' =3. 33, P<0. 01).3. The confidence interval of infiltrating lymphocytes in adenoma, chronic cholecystis, well-differentiated carcinoma and Nevin I -III carcinoma was very significantly lower than that in carcinoma(t' =6. 99, P<0. 01), adenoma(t' =3.66, P<0. 01), poorly-differentiated carcinoma (t' =5.31, P<0. 01) and Nevin IV-V carcinoma(t' =3.76, P<0. 01) respectively.4. The confidence interval of apoptosis of infiltrating lymphocytes in well-differentiated carcinoma was significantly lower than that in poorly-differentiated carcinoma(t=2. 52, P<0. 05), and was not significantly lower in Nevin I-III carcinoma than in Nevin IV-V carcinoma(t=1. 42, P>0.05). Apoptosis of infiltrating lymphocytes was not discovered in adenoma and chronic cholecystis.5. The expression of FasL and HDAC1 in gallbladder carcinoma displayed some extent positive correlation(P<0. 05), the expression of Fas and HDAC1 in gallbladder carcinoma were not significant correlation. Conclusions1. FasL protein expressed in gallbladder carcinoma cells permits tumor cells to escape from immune surveillance of organism by inducing apoptosis in infiltrating lymphocytes of carcinoma tissues. Up-regulation of FasL protein expression plays an important role in invasive depth, histological classification and metastasis of gallbladder carcinoma.2. The evidence of the role of Fas protein in immune escape of gallbladder carcinoma do not be found.3. There is the correlation between HDAC1 and immune escape of gallbladder carcinoma. Part III Effect of histone deacetylase inhibitor on p16-Rb-HDAC1-E2F1 proteins signal in biliary tract cancerThesis 5 Effect of histone deacetylase inhibitor onp16-Rb-HDAC1-E2F1 proteins signal in cholangiocarcinoma and gallbladder carcinomaObjective To study the effect of histone deacetylase inhibitor-trichostatin A(TSA) on p16-Rb-HDAC1-E2F1 signal pathway in extrahepatic cholangiocarcinoma and gallbladder carcinoma.Methods Treat cholangiocarcinoma cell lines(QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line) with TSA, and measure the mRNA expression change of p16, Rb, HDAC1 and E2F1 by RT-PCR assay and the protein expression change by Western blot method. Then transplanted these cells into subcutaneous tissue of nude mice to establish the transplanted cholangiocarcinoma and gallbladder carcinoma nude mice models, and observed the effect of TSA on the expression of Rb, HDAC1, DNMT1, E2F1 of transplanted cancer tissues in vivo. Results1. TSA can down-regulate the expression of protein and mRNA of HDAC1 in cholangiocarcinoma cell lines(QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line (Mz-ChA-1 cell line), can up-regulate the expression of protein and mRNA of p16 in KMBC cell line, there are no effect in expression of Rb and E2F1.2. TSA can not down- or up-regulate the expression of p16, Rb, HDAC1 and E2F1 proteins in the transplanted biliary tract cancer nude mice models. Conclusions TSA might down-regulate the expression of HDAC1 in cholangiocarcinoma cell lines(QBC₉₃₉, KMBC and OZ cell lines) and gallbladder carcinoma cell line(Mz-ChA-1 cell line), and up-regulate the expression of p16 in Rb⁺ cell lines, consequently effect the transcription of gene. The effect of TSA reply on the length of time. Part IV Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the gallbladder carcinomaThesis 6 Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the gallbladder carcinomaObjective To study the characteristics of clinicopathology of gallbladder carcinoma and the expression and significance of p16-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma. Methods1. Analysis 65 cases of gallbladder carcinoma in Tongji hospital of Huazhong university of science and technology retrospectively (1991-2005).2. Research the expression and characteristics of p16-Rb-HDAC1-E2F1 proteins respectively existing in tissues of gallbladder carcinoma, gallbladder adenoma, and chronic cholecystitis by immunehistochemistry. Results1. The incidence of coexistence of gallstones in gallbladder carcinoma was 44.64%(25/56). The incidence of hepatitis B in gallbladder carcinoma was 26.79%(15/56). The incidence of schistosome in gallbladder carcinoma was 30.36%(17/56). The incidence of multiple pregnancies in gallbladder carcinoma was 72.98% (27/37). Gallbladder carcinoma accounted for 0.64%(56/8807) of cholecystectomy. Preoperative mis-diagnose were often the gallstone, adenoma and liver cancer. Diagnostic rates of B-ultrasound, CT and MRI were 42. 3% (22/52), 57. 1%[ (14-6) / (20-6) ]and 50%[ (5-2) / (8-2) ] respectively. The γ -GT were all positive in gallbladder carcinoma(4/4).2. The position of the primary carcinoma of gallbladder located in the collum of gallbladder was 29.73%(11/37), in body of gallbladder was 29.73%(11/37), in fundus of gallbladder was 16.22%(6/37). Pathologic histology type of gallbladder carcinoma included adenocarcinoma (83.93%, 47/56), adenoma malignant progression (7.14%, 4/56), squamous cell carcinoma(5.36%, 3/56) and undifferentiated carcinoma (3.57%, 2/56). The incidence of gallbladder carcinoma in Nevin I stage was 14.29% (8/56), IV stage was 14.29%(8/56), V stage was 10.71%(6/56). There were not significant correlation among sex,the primary position and malignant degree of the gallbladder carcinoma. Liver was often infiltrated by tumor(14/56). Nerve bundle of gallbladder wall were infiltrated by two of them.3. The positive rates of pl6 and Rb proteins in gallbladder carcinoma tissue are respectively higher than that in adenoma tissue and cholecystitis tissue.The positive rates of HDAC1 and E2F1 proteins in gallbladder carcinoma tissue are respectively higher than that in adenoma tissue and cholecystitis tissue. The positive rate of pl6-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma tissue has no relationship with the differentiation degree of gallbladder carcinoma, Nevin stage, age, gender and gallstone. The expression of pl6 in gallbladder carcinoma infected with hepatitis B virus was 27. 7%, and 55. 6% in the others, there was significant different between them(P<0. 05). It took on some extent positive correlation between the expression of pl6 and the expression of Rb in gallbladder carcinoma( γ=0. 6091, t =4. 48, P<0. 001), it took on some extent negative correlation between the expression of pl6 and the expression of HDAC1 in gallbladder carcinoma( γ=-0. 5215, t=3.56, P<0. 005), it took on some extent negative correlation between the expression of Rb and the expression of HDAC1 in gallbladder carcinoma ( γ=-0.3953, t=2.51, P<0. 05). Conclusions1. Gallstones, hepatitis B, schistosome and multiple pregnancies are risk factors of gallbladder carcinoma. The γ -GT may be potential tumor marker.2. Gallbladder carcinoma often locates in collum and body of gallbladder, pathologic histology type of gallbladder carcinoma is often adenocarcinoma, tumor can infiltrate along nerve bundle of gallbladder wall. There are not significant correlation among sex, the primary position and malignant degree of the gallbladder carcinoma.3. The expression of p16-Rb-HDAC1-E2F1 proteins in gallbladder carcinoma tissue has no relationship with the differentiation degree of gallbladder carcinoma, Nevin stage, age, and gender.4. The expression of p16 protein in gallbladder carcinoma tissue is significantly correlative with hepatitis B.5. The down-regulating expression of p16 protein and Rb protein and down-regulating expression of HDAC1 protein and E2F1 protein is relative with the gallbladder carcinoma pathological characteristics, there are interaction among p16 protein, Rb protein and HDAC1 protein of the gallbladder carcinoma. Thesis 7 Analysis of the correlation between the p16-Rb-HDAC1-E2F1 proteins and clinicopathological factor in the unsuspected gallbladder carcinomaObjective To study the characteristics of clinicopathology in unsuspected gallbladder carcinoma and the expression and significance of p16-Rb-HDAC1-E2F1 proteins in unsuspected gallbladder carcinoma. Methods Analysis 23 cases of unsuspected gallbladder carcinoma in Tongji hospital retrospectively, characteristics of clinicopathology were compared with preoperative diagnosed gallbladder carcinoma in same period.Research the expression and characteristics of p16-Rb-HDAC1-E2F1 proteins respectively existing in tissues of unsuspected gallbladder carcinoma by immunehistochemistry.Results The incidence of coexistence of gallstones in unsuspected gallbladder carcinoma was significantly higher than in preoperative diagnosed gallbladder carcinoma(P<0. 01). The incidence of hepatitis B in unsuspected gallbladder carcinoma was 21. 74%(5/23), in preoperative diagnosed gallbladder carcinoma was 30. 30%(10/33). The incidence of schistosome in unsuspected gallbladder carcinoma was 39. 13%(9/23), in preoperative diagnosed gallbladder carcinoma was 24.24%(8/33). The incidence of multiple pregnancies in unsuspected gallbladder carcinoma was 56. 52%(13/23), in preoperative diagnosed gallbladder carcinoma was 42. 42%(14/33). The γ-GT were all positive in unsuspected gallbladder carcinoma(2/2) and preoperative diagnosed gallbladder carcinoma(2/2). The position of the primary carcinoma of unsuspected gallbladder carcinoma ofen located in the collum and body of the gallbladder, the position of the primary carcinoma of preoperative diagnosed gallbladder carcinoma ofen located in the body and fundus of the gallbladder. The incidence of Nevin I stage and II stage in unsuspected gallbladder carcinoma was significantly higher than that in preoperative diagnosed gallbladder carcinoma(P<0. 05). The incidence of Nevin IV stage in unsuspected gallbladder carcinoma was significantly lower than that in preoperative diagnosed gallbladder carcinoma(P<0. 05). The incidence of Nevin V stage in unsuspected gallbladder carcinoma was significantly lower than that in preoperative diagnosed gallbladder carcinoma(P<0. 01). There was not significant difference between expression of p16-Rb-HDAC1-E2F1 proteins of unsuspected gallbladder carcinoma and expression of p16-Rb-HDAC1-E2F1 proteins of preoperative diagnosed gallbladder carcinoma.Conclusions Unsuspected gallbladder carcinoma is different from preoperative diagnosed gallbladder carcinoma in the position of the primary carcinoma, the malignant degree and the incidence of coexistence of gallstones. Gallstones, hepatitis B , schistosome and multiple pregnancies are risk factors of unsuspected gallbladder carcinoma. Unsuspected gallbladder carcinoma has similar molecular biological characteristics with preoperative diagnosed gallbladder carcinoma. Ingenuity in this study:1. The protein signals of p16-Rb-HDAC1-E2F1 in biliary tract cancer involved in the inactivation of antioncogene, regulation of cell cycle, histone acetylation activation of gene promoter, epigenetics, and so on, which was representative in tumorigenesis. The research methods we used have combined these aspects, which apply a available pattern in studying the pathogenesis of cancer.2. Our study displayed that Rb protein, HDAC1 protein and E2F1 protein can make the specific interaction in biliary tract cancer, and there are the indirect pathways among p16 protein signals and the others, the effect of pl6 in biliary tract cancer rely on the function of Rb, which indicates that p16, Rb, E2F1 and HDAC1 constitute a protein signal pathway.3. Our study connects the protein signal conduction with the epigenetics, which may clarify the regulation of gene even more. Our study display that the epigenetics can influence cell differentiation by regulation of gene, and the change of the epigenetics connects with protein signals.4. To extrahepatic cholangiocarcinoma and gallbladder carcinoma, the rate of excision is low and the sensitivity to chemotherapy and actinotherapy is poor. We study the effect of histone deacetylase inhibitor on the growth of biliary tract cancer in vivo and in vitro based on the previous datas of Shengquan Zou' group, which was valuable experiment in biological treatment of biliary tract cancer.5. The previous datas of Shengquan Zou' group indicate that hepatitisvirus may influence the expression of gene in cholangiocarcinoma. Our research indicates that the expression of pl6 protein in gallbladder carcinoma is significantly correlative with hepatitis B.