THE RADIOBIOLOGIC CHARACTERISTICS OF DNA POLYMERASE β IN HEPATOMAS

To investigate the effects of γ rays on DNA polymerase β properties and its DNA repair functions before or after γ rays exposure, DNA polymerase βactivity, gene expression and mRNA levels in SMMC-LTNM hepatomas horn on nude mice or the samples of the liver cancer tissues from 15 patients were measured with 3H-TTP incorporation test, immunocytochemistry and cytoplasmic dot hybridization analysis, respectively.Irradiation was carried out with 60Co-γ rays at ice bath. It was found that DNA polymerase β activity, gene expression and the amount of mRNA were much higher in hepatoma cells than those in normal hepatocytes (P<0.01). In vitro studies, the enzyme activity both in hepatoma and normal liver cells appeared unchanged within 40 Gy γ-ray exposure. Following whole-body exposure of the nude mice bearing SMMC-LTNM with 2 Gy or 4 Gy of γ rays, DNA polymerase β activity in hepatoma increased temorarily at 48 hours postirradiation, and its gene expression seemed more active.The euzyme mRNA increased to 1.76-fold of the control group. 72 hours after exposure, all of these changes returned to normal levels. DNA polymerase βparticipated in DNA repair synthesis and this effect was different between hepatoma and hepatocytes because there were some biologic differences of the enzyme between hepatoma cells and normal liver cells. These data suggested that DNA polymeraseβactivity, its gene expression and mRNA level in hepatomas could increased temporarily after γ rays exposure, which may facilitate the cells to repair DNA damages from radiation.

Expression of liver cancer associated gene HCCA3

AIM: To study and clone a novel liver cancer reisted gene,and to explore the molecular basis of liver cancer genesis.METHODS: Using mRNA differential display polymerasechain reaction (DDPCR), we investigated the difference of mRNA in human hepatocellular carcinoma (HCC) and paired surrounding liver tissues, and got a gene probe. By screening a human placenta cDNA library and genomic homologous extend, we obtained a full-length cDNA named HCCA3. We analyzed the expression of this novel gene in 42pairs of HCC and the surrounding liver tissues, and distribution in human normal tissues by means of Northern blot assay.RESULTS: A full-length cDNA of liver cancer associated gene HCCA3 has been submitted to the GeneBank nucleotide sequence databases ( Accession No. AF276707 ). The positive expression rate of this gene was 78.6% (33/42) in HCC tissues, and the clinical pathological data showed that the HCCA3 was closely associated with the invasion of tumor capsule ( P = 0.023) and adjacant small metastasis satellite nodules lesions ( P= 0.041). The HCCA3 was widely distributed in the human normal tissues, which was intensively expressed in lungs, brain and colon tissues,while lowly expressed in the liver tissues.CONCLUSION: A novel full-length cDNA was cloned and differentiated, which was highly expressed in liver cancer tissues. The high expression was closely related to the tumor invasiveness and metastasis, that may be the late heredited change in HCC genesis.

Cloning of differentially expressed genes in human hepatocellular carcinoma and nontumor liver

INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6].The new genes,especially the functional genes directly related with tumor are still worth being found.The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone.Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up. PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomicDNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgeneexpressing cells was 65 ± 10 ng / L per 106 cells,420 ± 45 ng/L per 106 cells in sense group and 485± 30 ng/L per 106 cells in the negative control group, (P＜0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S. C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells.CONCLUSION Expression of antisense VEGFRNA in SMMC-7721 cells could decrease thetumorigenicity, and antisense-VEGF genetherapy may be an adjuvant treatment forhepatoma.

人TNFα真核表达载体的构建及其在人肝癌耐药细胞株HepG2/ADM中的稳定表达

目的:构建可在真核细胞内表达人肿瘤坏死因子alpha(hTNF-α)基因的真核表达载体,建立稳定表达hTNF-α基因的细胞模型。方法:应用RT-PCR的方法从分离的正常人外周血单核细胞(LPS刺激)中,扩增出hTNF-αcDNA,连接至载体pMD18-Tvector中,经酶切鉴定与序列测定证实;以亚克隆法构建于真核表达载体pBK-CMV的相应酶切位点,转化至大肠埃希菌DH5α,最后将表达的pBK-hTNFα重组蛋白行SDS-PAGE电泳,并作考马斯亮蓝染色分析,Westernblot鉴定;经脂质体Lipo-fectamine2000转染HepG2/ADM细胞后,经G418筛选,通过ELISA、RT-PCR方法检测其在体外转染肝癌耐药细胞HepG2/ADM后hTNFα基因和蛋白的表达。结果:自正常人外周血单核细胞中克隆的hTNF-αcDNA成功连接到pMD18-Tvector,用BamHⅠ和HindⅢ双酶切、经序列测定证实,与Genbank的序列完全一致;pBK-hTNFα的克隆表达重组蛋白经Westernblot证实此蛋白为hTNFα。克隆基因正确插入载体pBK-CMV中。pBK-hTNFα经脂质体介导转染肝癌耐药细胞后,ELISA、RT-PCR方法检测到其在转染细胞中稳定表达。结论:通过DNA重组技术成功地构建了可在体外稳定表达hTNF-αcDNA全长的真核表达载体pBK-hTNFα。

人剪切修复基因XPD对p52和p21基因的影响

目的:探讨人剪切修复基因XPD转染人HepG2肝癌细胞后p52和p21基因表达的变化以及对肝癌细胞生长的影响。方法:人肝癌细胞HepG2为靶细胞,XPD基因通过Lipofectamine2000脂质体转染HepG2。将细胞分为重组质粒HepG2-pEGFP-N2-XPD组(XPD组)、空载质粒HepG2-pEGFP-N2组(N2组)和HepG2空白对照组。分别用逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Westernblot)法检测各组细胞中XPD、p52以及p21的mRNA和蛋白质的表达量,并用四甲基偶氮唑盐微量酶反应比色(MTT)法检测各组细胞的增殖能力。结果:XPD组中的p52的mRNA表达较其他2组显著下调,XPD、p21的mRNA表达较其他2组明显上调(均P<0.01)。Westernblot检测结果显示各组细胞中XPD、p52及p21的蛋白表达各组间差异与其mRNA各组间差异一致。MTT检测显示XPD转染入HepG2后细胞增殖能力减弱。结论:XPD基因可以抑制癌细胞的生长和基因p52的表达,促进p21的表达。