Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line

To investigate the effects of anti HPV16E6 ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line Methods HRz was designed by computer programs HRz’s activity was identified by cleavage experiments in vitro HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi R and CaSKi P, respectively The expression of ribozyme in transfected cells was observed by RNA dot blot The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot Cell growth curves and soft agar forming ability were studied The ability of each cell line to form tumors was assessed in nude mice Apoptosis rates and expression of c myc, bcl 2, p53 and Fas were detected by flow cytometry (FCM) Antigens of tumor cells, HLA 1, HLA 2, B7 1 and B7 2 were also detected NK, LAK, and CD 3AK cells were induced Their cytotoxicities were detected in CaSKi R, CaSKi P, and CaSKi cells Results In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site specific manner HRz could be expressed stably in transfected CaSKi cells Northern blot analysis showed that E6 mRNA levels were lower in CaSKi R than in CaSKi The growth rate of CaSKi R was slower than those of CaSKi and CaSKi P The soft agar forming rate of CaSKi R was lower compared with those of CaSKi and CaSKi P cells The ability of CaSKi R to form tumors in nude mice was also poor The apoptosis rate of CaSKi R cells was much higher than those of CaSKi and CaSKi P HRz could reduce the expression of E6, c myc and bcl 2 proteins, and increase the expression of p53 as well HRz could increase the expression of HLA 2, B7 1 and B7 2 antigens The cytotoxicity of NK, LAK and CD 3AK cells was much higher in CaSKi R than in CaSKi P and CaSKi cells Conclusion HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells

Effects of blocking androgen receptor expression with specific hammerhead ribozyme on in vitro growth of prostate cancer cell line

Objective To study the possibility of gene therapy for prostate cancer by blocking androgen receptor (AR) gene expression using a specific hammerhead ribozyme (RZ). Methods The hammerhead ribozyme expression vector pcDNA-hAR-RZ,specific to AR mRNA,was constructed and transfected into the prostate cancer cell line LNCaP by using lipofectamine. Androgen receptor expression was measured by RT-PCR and immunohistochemical methods. Cellular proliferation activities were assayed using the tetrazolium bromide colorimetry method; cell cycle changes were observed by flow cytometry; and cell apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling method. Results One to seven days after transfection with the ribozyme expression vector,AR mRNA expression at molecular and protein levels in LNCaP cells decreased by 32.6%-40.7% ( P <0.05) and 21.0%-87.64% ( P <0.05) respectively,and cell proliferation was inhibited by 18.28%-35.34% ( P <0.05). Meanwhile,the cell cycle was arrested at the G 2/M stage,and apoptotic morphological changes occurred with an apoptosis rate of 25.17% ( P <0.01).Conclusion Ribozyme specific against AR mRNA is capable of inhibiting the expression AR and inducing the apoptosis in prostate cancer cells.