Expression And Functional Study Of P300/CBP-associated Factor In Intestinal-type Gastric Carcinoma

PCAF (P300/CBP associated factor), a recently identified protein, also known as the first mammalian histone acetyltransferases which have been found.The present studies demonstrated that PCAF functions as a negative regulator of cell transformation and tumor cell growth through the regulation of histones and other cancer-related non-histones.PCAF might be broadly involved in diverse type of activated signaling pathways and tumorigenesis. The identification of PCAF supplies a new aim for molecular mechanism of tumor. Consequentially, it is a new target gene of much promise for gene therapy, but the study on PCAF gene in cancer just now began.To our knowledge, this is the first report about the expression of PCAF in gastric carcinoma.According to the mentioned above,we performed the following experiments. Firstly,we investigated the expression of PCAF in intestinal type gastric cancer gastric cancer tissue samples and explored its correlation with clinical and pathological characters.Then we established and identified gastric carcinoma cell lines(SGC-7901) transfected by exogenous gene PCAF.Based on above work, we investigated whether the exogenous gene PCAF transfer could inhibit the growth of gastric carcinoma cell lines in vitro and in vivo, and explored the possible machanism of this effect.Purpose a theoretical basis of genic diagnosis and therapy for intestinal-type gastric carcinoma. Part I Expression of PCAF in intestinal-type gastric carcinoma tissues and gastric adenocarcinoma cell lines.Objective:To determine the expression of PCAF in gastric cancer and to investigate the association of PCAF with gastric cancer.Methods:Wesern blot analysis were used to detect the expression level of protein of PCAF in gastric carcinoma cell lines (SGC-7901, MKN-45, AGS) and immortalized gastric mucosa epithelial cell line GES-1. Immunohistochemistry was 1performed to evaluate the expression of PCAF in a large subset containing 406 intestinal type gastric cancer samples. Correlationship between the expression of PCAF and clinical pathological characters was investigated.Results:1. Western blot analysis revealed that all gastric cancer cell lines including AGS, MKN-45 and SGC-7901 exhibited significantly lower levels of PCAF expression compared to those in immortalized gastric mucosa epithelial cell line GES-1 (P<0.05).2. Statistical analysis displayed a significant correlation in PCAF expression with the gastric wall invasion, tumor size, TNM stage, p21, pRb (P<0.001) and PCNA (P<0.01) in intestinal type gastric cancer specimens.3. A reduced PCAF protein expression correlated significantly with a mutant type p53 protein expression (P<0.01).4. Univariate analysis indicated that the patients demonstrating the high-PCAF/wild type p53 expression have a significantly (P<0.0001) better overall survival (OS).5. Multivariate analysis indicated that the location, lymph node metastasis, PCAF/p53 (P<0.0001), gastric wall invasion (P=0.001) and PCNA (P=0.018) are independently significant prognostic factors for OS.Conclusions:Reduced expression of PCAF plays an important role in the development of intestinal type gastric cancer and correlates with a poor clinical outcome. PCAF/p53 phenotype may be an independent bio-marker for prognosis in gastric carcinoma Part II Biological effect of recombinant vector in stably transfected gastric cacinoma cell lines PCAF-7901Objective:To construct a eukaryotic expression plasmid of PCAF gene:pcDNA3.1-PCAF, in order to study its effect on the biological behavior of gastric carcinoma cell line SGC-7901 in vitro and in vivo.Methods:1.(1) PCAF clone (ATCC Catalog No.10435572) was obtained from the Mammalian Genome Collection.(2) The full-length PCAF cDNA insert was isolated from pBluescriptR by double digestion of EcoRI and Acc65I, and then subcloned into the mammalian expression vector pcDNA3.1 (-) with the sites of EcoRI and Acc65I. The eukaryotic expression vector of PCAF was constructed by double restriction endonucleases cleavage directional clone method.(3) Double restriction endonucleases cleavage and sequencing methods were used to identify the eukaryotic expression vector of PCAF gene.(4) DNA sequencing was performed to confirm the construction.2.Two different plasmids including a recombinate pcDNA3.1-PCAF and an empty pcDNA3.1 vector were prepared by transformation of bacterium, amplification and purification of plasmids.Then two kinds of plasmids were reseparately transferred into gastric carcinoma cell line SGC-7901 cultured in vitro by using lipofectamine 2000. After transfection, positive clones were screened with G418 and expanded by culture.The expression of the Neo'tag-gene was detected by RT-PCR to confirm whether the combinant vector DNA integrated with the genomic DNA of SGC-7901. Western blot methods were used to analysis expression of PCAF protein in SGC-7901 cells before and after transfection.3.MTT growth test, flow cytometry analysis were used in vitro to study the effects of PCAF expression on transfected cells proliferation. Tumor growth in nude mice was used to access the tumorigenicity of gastric cancer cells. Apoptosis cells were detected by TUNEL staining. Results:1.(1) Vector pcDNA3.1 was introduced into coli DH5a with Lipo 2000.(2) Vector pcDNA3.1 can be successful cleavage by double restriction endonucleases EcoRl and Acc65I.(3) Double restriction endonucleases cleavage identification indicated the expected straps.(4) DNA sequencing was performed to confirm the construction.2.All of the two cell lines transfected by cooresponding plasmid acquired resistance to neomycin. The Neo'-tag gene was detected by RT-PCR in cell lines un-transfected due to lack of Neo'-tag. Western-blot showed that the PCAF were expressed higher in cell lines transfected by pcDNA3.1-PCAF than in cell lines un-transfected and transfected by an empty vector on protein level.3.Transfected with PCAF eukaryotic expression vector could significantly inhibit growth compared with the untransfected cells in the MTT assay. Flow cytometry analysis showed the proportion of G0/G1 phase cells in PCAF-7901 group was significantly higher than the proportion in the control group. The apoptotic indexes among PCAF-7901, Vec-7901 and SGC-7901 were determined by TUNEL. There was no obvious difference among the three groups.Conclusions:1. The eukaryotic expression vector of PCAF gene was successfully constructed by techniques of gene engineering.2. The eukaryotic vector was transfected successfully to SGC-7901 cells with liposome2000, which integrated with SGC-7901 cell genome and markedly enhanced the expression of PCAF protein in SGC-7901 cells.3. PCAF was able to suppress tumorigenicity of gastric cancer cells both in vitro and in vivo, including colony formation in soft agar and tumor formation in nude mice. PCAF could also inhibit intestinal type gastric cancer cells entering S phase from G1 phase. PCAF cannot induce apoptosis...