No abstract

Human papillomavirus (HPV) E6 and E7 viral oncogenes are very well known to cause cervical cancer, because E6 degrades TP53 tumor suppressor protein, and E7 inactivates the tumor suppressor retinoblastoma (pRb) protein. Thus E6 and E7 oncogenes of HPV are supposed to be promising targets of gene therapy against HPV mediated cervical cancer. Here, we attempted to study the regulation of TP53/pRb proteins dynamic behaviour after HPV E6/E7 small interfering RNA (siRNA) transfection in cervical cancer cells. HPV positive (HeLa and Caski) cell lines were selected for these experiments. Herein, we also validated the dynamics of TP53 in response to antiviral siRNA targeting the E6 and E7 oncogenes. At first, we analyzed the effect of HPV E6/E7 siRNA on TP53/pRb reporter gene activity by using pathway profiling luciferase system. Our result clearly indicates that when compared with E6/E7 siRNA pool alone, siRNA pool in combination cisplatin dramatically increases the TP53/pRb and decreases E2F response elements regulating luciferase reporter gene activity. TP53 protein and its target gene expression were also confirmed by western blot and qRT-PCR analysis in a time dependent manner. In our TP53 reporter gene (pGreenFire1 GFP reporter vector with EF1-puro) assay by using live imaging analysis shows that combination of HPVE6/E7 siRNA pool with cisplatin, induces the TP53 expression within 10h and sustained that leads to cell death. Our imaging results also suggested that combining cisplatin with inhibition of HPV E6/E7 oncogene would synergize to stimulate a sustained GFP-TP53 expression can trigger either cell cycle arrest or apoptosis in response to DNA damage. In addition, we generated GFP-TP53 reporter stable HeLa cell line (pGreenFire1 GFP reporter vector with EF1-puro), in order to find the specificity of HPV E6/E7 siRNA induced TP53 expression in in vitro and in vivo. Similar GFP-TP53 dynamic pattern was observed in E6/E7 siRNA transfected GFP-TP53 reporter stable HeLa cell line. Furthermore to analyze the dynamic behaviour of pRb/E2F in live cells, reporter vector containing luciferase gene was recombinantly engineered to replace with RFP gene. Taken together, our results suggest that increase of TP53/Rb expression by silencing E6/E7 is associated with its chemo-sensitizing effect in cervical cancer cells. Overall, stabilization and maximal activation of the TP53/pRb signaling network can facilitate determination of the optimal therapeutic strategy for human cervical carcinoma. . Citation Format: Nirmal Rajasekaran, Hun Soon Jung, Young Deug Kim, Deuk Ae Kim, Tae Kyung Ha, Yoo Ha Na, Young kee Shin. Effect of small interfering RNA targeting HPV E6/E7 gene on the regulation of TP53/Rb dynamic behaviour in cervical cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2115. doi:10.1158/1538-7445.AM2015-2115