Yan-Fang Zheng1, Zhi-Guo Rao, Ji-Ren Zhang. 1. Oncology Center, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China. zyfcn@yahoo.com
Abstract
OBJECTIVE: To study the characterization of anti-HPV16E6-ribozyme transfected into cultured human cervical cancer cell line, and to investigate the effect of ribozyme on the proliferation and apoptosis of the cells. METHODS: By way of lipofectin transfection, anti-HPV16E6-ribozyme, a ribozyme designed to specifically cleave the HPV16E6 gene, and empty vector expression plasmids were respectively transfected into CaSKi cells which were subsequently designated as CaSKi-R and CaSKi-P accordingly. The expression of ribozyme in the transfected cells was observed by RNA dot blot analysis, and E6 mRNA expression was detected by Northern blotting in the 3 kinds of cells whose growth curves and clone-forming ability on soft agar were studied, with their tumorgenicity observed by percutaneous inoculation of the cells in nude mice. Flow cytometry was employed to assess the apoptosis rates and the expression of the genes including c-myc, bcl-2, p53, and fas etc. RESULTS: Stable expression of anti-HPV16E6-ribozyme was observed in CaSKi-R cells that had less E6 mRNA expression than CaSKi had as shown by Northern blotting. When compared with those of CaSKi cells, the growth rate and clone-forming ability on soft agar of CaSKi-R were reduced, while those of CaSKi-P evinced no significant changes. The tumorgenicity of CaSKi-R in nude mice was also comparatively decreased, with markedly elevated apoptosis rate. Anti-HPV16E6-ribozyme reduced the expressions of E6, c-myc, bcl-2, PCNA and C-erbB2 genes but increased p53 expression in CaSKi-R cells, which, however, did not happen in CaSKi-P cells. The expression of Fas underwent no significant changes in response to the transfection. CONCLUSION: Anti-HPVE6-ribozyme inhibits proliferation and induces apoptosis in CaSKi cells, possibly due to the decrease of E6 gene expression that triggers a series of changes in the gene expressions of the cells.
OBJECTIVE: To study the characterization of anti-HPV16E6-ribozyme transfected into cultured human cervical cancer cell line, and to investigate the effect of ribozyme on the proliferation and apoptosis of the cells. METHODS: By way of lipofectin transfection, anti-HPV16E6-ribozyme, a ribozyme designed to specifically cleave the HPV16E6 gene, and empty vector expression plasmids were respectively transfected into CaSKi cells which were subsequently designated as CaSKi-R and CaSKi-P accordingly. The expression of ribozyme in the transfected cells was observed by RNA dot blot analysis, and E6 mRNA expression was detected by Northern blotting in the 3 kinds of cells whose growth curves and clone-forming ability on soft agar were studied, with their tumorgenicity observed by percutaneous inoculation of the cells in nude mice. Flow cytometry was employed to assess the apoptosis rates and the expression of the genes including c-myc, bcl-2, p53, and fas etc. RESULTS: Stable expression of anti-HPV16E6-ribozyme was observed in CaSKi-R cells that had less E6 mRNA expression than CaSKi had as shown by Northern blotting. When compared with those of CaSKi cells, the growth rate and clone-forming ability on soft agar of CaSKi-R were reduced, while those of CaSKi-P evinced no significant changes. The tumorgenicity of CaSKi-R in nude mice was also comparatively decreased, with markedly elevated apoptosis rate. Anti-HPV16E6-ribozyme reduced the expressions of E6, c-myc, bcl-2, PCNA and C-erbB2 genes but increased p53 expression in CaSKi-R cells, which, however, did not happen in CaSKi-P cells. The expression of Fas underwent no significant changes in response to the transfection. CONCLUSION: Anti-HPVE6-ribozyme inhibits proliferation and induces apoptosis in CaSKi cells, possibly due to the decrease of E6 gene expression that triggers a series of changes in the gene expressions of the cells.