Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells

Objective. In cervical cancer, high-risk human papillomavirus (HPV) genes a re expressed solely in cancerous cells and have been proposed to be the mos t important etiological factors for cervical cancer, thus making them suita ble targets for gene therapy. In this study, we aim to inactivate the HPV16 E7 in CaSki cells and test the possibility of reducing the tumorigenicity of these cells. Methods. The full-length HPV16 E7 cDNA was cloned in the pBabe-puro or pWZL -Hygro retrovirus vector in reverse orientation and was stably transfected into CaSki cells by replication-defective retrovirus infection giving rise to CaSki-E7AS and CaSki-E7AS2X cells. Immunoprecipitation/Western analysis and real-time RT-PCR were performed to document the levels of HPV16 E7 gene product. Flow cytometry was performed to study changes in the cell cycle i n response to reduced E7 protein. The expression of bcl-2, RE, and E2F-1 wa s studied using Western blot analysis. Tumorigenicity of CaSki, CaSki-E7AS, and CaSki-E7AS2X cells was assayed with subepidermal tumor growth in nude mice. Results. We have documented that the delivery of the antisense gene constru ct resulted in the reduction of HPV16 E7 protein expression and cell prolif eration in CaSki cells. Furthermore, we demonstrated that these changes wer e accompanied by cell cycle arrest, up-regulation of RE, and down-regulatio n of E2F-1 and bcl-2 proteins. More importantly, dose-dependent transductio n of the antisense HPV16E7 construct was able to inhibit and/or retard the tumorigenicity of CaSki cells in vivo. Conclusions. Down-regulation of HPV16 E7 with antisense RNA is beneficial i n reducing the tumorigenicity of CaSki cells and can potentially be useful for HPV-associated malignancy gene therapy. (C) 2000 Academic Press.