科创中国

AIM: To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence. METHODS: A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments. RESULTS: Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783. In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues. CONCLUSION: The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.

You Han;Xiao Bing;Cui Da-Xiang;Shi Yong-Quan;Fan Dai-Ming

World Journal of Gastroenterology

1998

AIM: To compare the expression level of Fas gene and Bcl-2 gene in gastric cancer cells SGC7901 and gastric cancer multidrug resistant cells (MDR) SGC7901/ VCR, to transduce Fas cDNA and Bcl-2 antisense nucleic acid into SGC7901/ VCR cells respectively, and to observe the expression of two genes in transfectants and non-transfectants as well as their drug sensitivity. METHODS: Eukaryotic expression vector pBK-Fas cDNA and pDOR anti-Bcl2 were constructed and transfected into SGC7901/ VCR cells by lipofectamine, respectively. Northern blot and Western blot were used to detect the expression of mRNA and protein in SGC7901/ VCR and SGC7901 cells and transfectants, and drug sensitivity of transfectants for VCR, CDDP and 5-FU was analyzed with MTT assay. RESULTS: After gene transfection, 80 for Fas and 120 for antisense Bcl-2 drug-resistant clones were selected from 2 × 10 ⁵ cells, transfection rate being 0.04% and 0.06%. Two clones of SGC7901 Fas/ VCR cells and SGC7901 anti Bcl-2/ VCR cells were randomly selected for further incubation. Hybridization results showed that the expression level of Fas mRNA and protein in SGC7901/ VCR cells was much lower, but that of Bcl-2 mRNA and protein was higher than that in SGC7901 cells. The expression of Fas mRNA and protein in SGC7901 Fas/ VCR cells was higher, and of Bcl-2 mRNA and protein was lower in SGC7901 anti Bcl-2/ VCR cells than that in non-transfectants. MTT assay showed that transfectants were more sensitive to VCR, CDDP, 5-FU than non-transfectants. CONCLUSION: Bcl-1 gene displayed high expression while Fas gene had low expression in drug resistant gastric cancer cells. Expression of Bcl-2 protein was effectively blocked in SGC7901 anti Bcl-2/ VCR cells by gene transfection. In contrast, the expression of Fas mRNA and protein in SGC7901 Fas/ VCR cells increased. Fas gene and Bcl-2 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to chemotherapeutic drugs. These results suggest cell apoptosis plays an important role in the mechanism of MDR, and enhancing apoptosis might reverse MDR.

Xiao Bing;Shi Yong-Quan;Zhao Yan-Qiu;You Han;Wang Zuo-You;Liu Xian-Ling;Yin Fang;Qiao Tai-Dong;Fan Dai-Ming

World Journal of Gastroenterology

1998

AIM: To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs. METHODS: A 1.03kb cDNA sequence of Hsp90β was obtained from the primary plasmid phHsp90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90β expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with Western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry. RESULTS: In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90β remained what had been designed and the gene constructs were named pcDNA-Hsp90. AH-SGC7901, AH-SGC7901/ VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/ VCR, AH-HCC7402 and AH-EC109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/ VCR and AH-Ec109 cells, G 1 phase cells were increased; S phase and G 2 phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G 1 phase cells were decreased, G 2 phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G 1 , S and G 2 phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/ VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/ VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P < 0.05). CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells. Copyright©1999 by the WJG Press.

Liu Xian-Ling;Xiao Bing;Yu Zhao-Cai;Guo Jian-Cheng;Zhao Qing-Chuan;Xu Li;Shi Yong-Quan;Fan Dai-Ming

World Journal of Gastroenterology

1999

The gastric cancer associated antigen McAb MG7-Ag was detected by means of a newly established method, termed immuno-PCR. A McAb recombinant DNA chimeric molecule was made which possesses bispecific binding affinity for antigen that had been immobilized on microtiter wells and the segment of the attached DNA was amplified by PCR. The antigen of gastric cancer cell line KATO III was monitored by this method. Analysis of PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 20 cells to be detected readily and reproducibly. Immuno-PCR showed a 10⁴ enhancement in detection sensitivity compared with ELISA assay. When the same numbers of cells (2 x 10⁶/ml) were immobilized and then the serial diluted chimeric molecule was added, 3.8 x 10^-14 moles and 3.0 x 10^-11 moles were needed to give positive results with the immuno-PCR and ELISA assay, respectively. Therefore, immuno-PCR could give an enormous amplification capability with good specificity, and has a sensitivity much higher than any existing techniques for antigen detection.

Ren-jun ;Fan D.;Zhou S.

Chinese Journal of Oncology

1994

Aim: To observe the drug sensitizing effect and related mechanisms of fas gene transduction on human drug-resistant gastric cancer cell SGC7901/VCR (resistant to Vincristine). Methods: The cell cycle alteration was observed by FACS. The sensitivity of gastric cancer cells to apoptosis was determined by in vitro apoptosis assay. The drug sensitization of cells to several anti-tumor drugs was observed by MTT assay. Immunochemical method was used to show expression of P-gp and Topo II in gastric cancer cells. Results: Comparing to SGC7901 and pBK-SGC7901/VCR, fas-SGC7901/VCR showed decreasing G2 cells and increasing S cells, the G2 phase fraction of pBK-SGC7901/VCR was about 3.0 times that of fas-SGC7901/VCR, but S phase fraction of fas-SGC7901/VCR was about 1.9 times that of pBK-SGC7901/VCR, indicating S phase arrest of fas-SGC7901/VCR. FACS also suggested apoptosis of fas-SGC7901/VCR. fas-SGC7901/VCR was more sensitive to apoptosis inducing agent VM-26 than pBK-SGC7901/VCR. MTT assay showed increased sensitization of fas-SGC7901/VCR to DDP, MMC and 5-FU, but same sensitization to VCR according to pBK-SGC7901/VCR. SGC7901, pBK-SGC7901/VCR and fas-SGC7901/VCR had positively stained Topo II equally. P-gp staining in pBK-SGC7901/VCR was stronger than in SGC7901, but there was little staining of P-gp in fas-SGC7901/VCR. Conclusion: fas gene transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree, possibly because of higher sensitization to apoptosis and decreased expression of P-gp.

Yin F.;Shi Y. Q.;Zhao W. P.;Xiao B.;Miao J. Y.;Fan D. M.

World Journal of Gastroenterology

2000

AIM: To construct and express in E. coli a fusion gene of gastric cancer MG7 mimic epitope with heat shock protein 70 (hsp70). METHODS: A cDNA fragment encoding MG7 mimic epitope (GC23) was added to 3′ terminus of hsp70 gene by PCR amplification. The PCR products were cloned into pUCm-T vector and were sequenced. The fusion gene was subcloned into vector PET-21a (+) and expressed in E. coli. SDS-PAGE and Western blot were employed to identify the expression of the fusion gene. RESULTS: A fragment about 2.0kb was amplified by the PCR. Sequence analysis revealed that the sequence of GC23 was connected successfully to 3′ terminus of hsp70. The fusion gene was cloned into PET-21a(+) identified by enzyme digestion and PCR. SDS-PAGE and Western blot showed that a Mr72 000 fusion protein was expressed that could be recognized by anti-hsp70 antibody. CONCLUSION: The fusion gene of GC23/hsp70 has been successfully constructed and expressed in E. coli.

Ning X. X.;Wu K. C.;Shi Y. Q.;Wang X.;Zhao Y. Q.;Fan D. M.

World Chinese Journal of Digestology

2001

Cao X. Y.;Liu J.;Lian Z. R.;Clayton M.;Hu J. L.;Zhu M. H.;Fan D. M.;Feitelson M.

World Journal of Gastroenterology

2001

Cao X. Y.;Liu J.;Lian Z. R.;Clayton M.;Hu J. L.;Zhu M. H.;Fan D. M.;Feitelson M.

World Journal of Gastroenterology

2001

Aim: To develop the single chain variable fragment of MG 7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. Methods: mRNA was isolated from MG 7 -producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG 7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOIII of highly expressing MG 7 -binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG 7 ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG 7 ScFv. Finally, the relative molecular mass of soluble MG 7 ScFv was measured by SDS-PAGE. Results: The V H , V L and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 10 ⁶ and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG 7 antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG 7 ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG 7 ScFv was 32. Conclusion: The MG 7 ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Yu Z. C.;Ding J.;Nie Y. Z.;Fan D. M.;Zhang X. Y.

World Journal of Gastroenterology

2001

Insulin-like growth factor I (IGF-1) is a well-established mitogen to many different cell types and is implicated in progression of a number of human cancers, notably breast cancer. The prolyl isomerase Pin1 plays an important role in cell cycle regulation through its specific interaction with proteins that are phosphorylated at Ser/Thr-Pro motifs. Pin1 knockout mice appear to have relatively normal development yet the Pin1^-/- mouse embryo fibroblast (MEF) cells are defective in re-entering cell cycle in response to serum stimulation after G0 arrest. Here, we report that Pin1^-/- MEF cells display a delayed cell cycle S-phase entry in response to IGF stimulation and that IGF-1 induces Pin1 protein expression which correlates with the induction of cyclin D1 and RB phosphorylation in human breast cancer cells. The induction of Pin1 by IGF-1 is mediated via the phosphatidylinositol 3-kinase as well as the MAP kinase pathways. Treatment of PI3K inhibitor LY294002 and the MAP kinase inhibitor PD098059, but not p38 inhibitor SB203580, effectively blocks IGF-1-induced upregulation of Pin1, cyclin D1 and RB phosphorylation. Furthermore, we found that Cyclin D1 expression and RB phosphorylation are dramatically decreased in Pin1^-/- MEF cells. Reintroducing a recombinant adenovirus encoding Pin1 into Pin1^-/- MEF cells restores the expression of cyclin D1 and RB phosphorylation. Thus, these data suggest that the mitogenic function of IGF-1 is at least partially linked to the induction of Pin1, which in turn stimulates cyclin D1 expression and RB phosphorylation, therefore contributing to G0/G1-S transition. © 2001 Wiley-Liss, Inc.

You Han;Uchida Takafumi;Xiao Zhi-Xiong Jim;Zheng Hongwu;Fan Daiming;Murray Steven A.;Yu Qiang

Journal of Cellular Biochemistry

2001