ABSTRACT
Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.
Subject(s)
Humans , Angiogenesis Inhibitors , Metabolism , Autoantigens , Genetics , Metabolism , Cell Proliferation , Cells, Cultured , Collagen Type IV , Genetics , Metabolism , DNA, Complementary , Genetics , Electroporation , Endothelial Cells , Cell Biology , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , Metabolism , Umbilical Cord , Cell BiologyABSTRACT
OBJECTIVE: To study the role of anti-HPV16E6-ribozyme in modulating the sensitivity of cervical carcinoma cell line to chemotherapy. METHODS: By way of lipofectin transfection, anti-HPV16E6-ribozyme antibody and empty eukaryotic expression plasmids were respectively transfected into CaSKi cells that were designated as CaSKi-R and CaSKi-P cells accordingly. Ribozyme expression in the transfected cells was subsequently observed with RNA dot blot, and the amount of E6 mRNA expression detected by Northern blotting. Cell count was performed for determining the growth rate of the non-transfected and transfected CaSKi cells and colony formation test was employed to examine the sensitivity of the cells to chemotherapy. PI/Annexin V staining was conducted to determine the apoptosis rates of each cell line in response to chemotherapy. RESULTS: As shown by dot blot analysis, stable expression of anti-HPV16E6-ribozyme was achieved in CaSKi-R cells, in which lower E6 mRNA expression level was observed by means of Northern blotting in comparison with those in CaSKi-P and CaSKi cells. Significant slow-down of the growth rate occurred in CaSKi-R cells as compared with the growth rate of the other 2 cell lines, but no differences in relative cloning efficiency and apoptosis rates of the 3 cell lines were observed in response to taxol treatment (P>0.05), while cis-platinum treatment produced comparatively deceased relative cloning efficiency and increased apoptosis rate in CaSKi-R (P<0.01). CONCLUSION: Transfection by anti-HPVE6-ribozyme may inhibit the growth of CaSKi cells and increase their sensitivity to cis-platinum but not to taxol.
