Published online Jun 15, 1999. doi: 10.3748/wjg.v5.i3.199
Revised: March 19, 1999
Accepted: April 10, 1999
Published online: June 15, 1999
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 1.03 kb cDNA sequence of Hsp90β was obtained from the primary plasmid phHSP90 by EcoRI and BamHI nuclease diges tion and was cloned to the EcoRI and BamHI 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 protecti on 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 EcoRI and BamHI 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-SGC790, 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, G1 phase cells were increased; S phase and G2 phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G1 phase cells were decreased, G2 phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G1, S and G2 phase cells remai ned 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.