GFP/HPV-16E6 fusion protein induces apoptosis in MCF-7 and 293T cells using a transient expression system

Zhang,Ge; Liu,Yan; Yu,Liwei; Sun,Lina

doi:10.3892/or.2012.1976

GFP/HPV-16E6 fusion protein induces apoptosis in MCF-7 and 293T cells using a transient expression system

Authors:
- Ge Zhang
- Yan Liu
- Liwei Yu
- Lina Sun
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Affiliations: Beijing Haidian Hospital, Beijing 100080, P.R. China, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, P.R. China, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P.R. China
Published online on: August 22, 2012 https://doi.org/10.3892/or.2012.1976
Pages: 1673-1680

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Abstract

Since mucosal high-risk human papillomavirus (HPV) E6 can target and degrade the tumor suppressor p53, it is recognized as a major causative agent of cervical cancer. However, to date the distribution of high-risk HPV-E6 protein remains elusive. Thus, in the present study we used a mammalian green fluorescent protein (GFP) expression system to express a GFP/HPV-16E6 fusion protein (GFP-16E6) in wild-type (wt) p53 cells, such as MCF-7 and 293T cells to investigate the trafficking and localization of E6 and p53. Following transfection, we observed that the overexpressed GFP-16E6 was a nuclear protein, and that endogenous wt p53 localized to the nucleus together with GFP-16E6. Strikingly, p53 levels were not decreased but increased in 24 h transfected with pGFP-16E6. Furthermore, we observed significant apoptosis induced by GFP-16E6, which proved to be dependent on p53 expression.

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1	Antonsson A, Spurr TP, Chen AC, et al: High prevalence of human papillomaviruses in fresh frozen breast cancer samples. J Med Virol. 83:2157–2163. 2011. View Article : Google Scholar : PubMed/NCBI
2	Jones DL, Thompson DA and Munger K: Destabilization of the RB tumor suppressor protein and stabilization of p53 contribute to HPV type 16 E7-induced apoptosis. Virology. 239:97–107. 1997. View Article : Google Scholar : PubMed/NCBI
3	Honda R and Yasuda H: Activity of MDM2, a ubiquitin Ligase, toward p53 or itself is dependent on the RING finger domain of the ligase. Oncogene. 19:1473–1476. 2000. View Article : Google Scholar : PubMed/NCBI
4	DiMaio D: The role of human papillomaviruses in cancer. J Neurovirol. 14:50. 2005.
5	Liu YM, McKalip A and Herman B: Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: Roles of p53 and caspase activation. J Cell Biochem. 78:334–349. 2000. View Article : Google Scholar : PubMed/NCBI
6	Meek DW: The p53 response to DNA damage. DNA Repair. 3:1049–1056. 2004. View Article : Google Scholar : PubMed/NCBI
7	Chang YC, Liao CB, Hsieh PY, Liou ML and Liu YC: Expression of tumor suppressor p53 facilitates DNA repair but not UV-induced G2/M arrest or apoptosis in Chinese hamster ovary CHO-K1 cells. J Cell Biochem. 103:528–537. 2008. View Article : Google Scholar : PubMed/NCBI
8	Wei J, O’Brien D, Vilgelm A, et al: Interaction of Helicobacter pylori with gastric epithelial cells is mediated by the p53 protein family. Gastroenterology. 134:1412–1423. 2008.
9	Mayer C and Grummt I: Cellular stress and nucleolar function. Cell Cycle. 4:1036–1038. 2005. View Article : Google Scholar : PubMed/NCBI
10	Qian H, Wang T, Naumovski L, Lopez CD and Brachmann RK: Groups of p53 target genes involved in specific p53 downstream effects cluster into different classes of DNA binding sites. Oncogene. 21:7901–7911. 2002. View Article : Google Scholar : PubMed/NCBI
11	Sherman L and Schlegel R: Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16. J Virol. 70:3269–3279. 1996.PubMed/NCBI
12	Liang XH, Volkmann M, Klein R, Herman B and Lockett SJ: Co-localization of the tumor-suppressor protein p53 and human papillomavirus E6 protein in human cervical carcinoma cell lines. Oncogene. 8:2645–2652. 1993.PubMed/NCBI
13	Kelley ML, Keiger KE, Lee CJ and Huibregtse JM: The global transcriptional effects of the human papillomavirus E6 protein in cervical carcinoma cell lines are mediated by the E6AP ubiquitin ligase. J Virol. 79:3737–3747. 2005. View Article : Google Scholar : PubMed/NCBI
14	Tao M, Kruhlak M, Xia S, Androphy E and Zheng ZM: Signals that dictate nuclear localization of human papillomavirus type 16 oncoprotein E6 in living cells. J Virol. 77:13232–13247. 2003. View Article : Google Scholar : PubMed/NCBI
15	Li X and Coffino P: High-risk human papillomavirus E6 protein has two distinct binding sites within p53, of which only one determines degradation. J Virol. 70:4509–4516. 1996.PubMed/NCBI
16	Momand J and Zambetti GP: Mdm-2: ‘big brother’ of p53. J Cell Biochem. 64:343–352. 1997.
17	Butz K, Shahabeddin L, Geisen C, Spitkovsky D, Ullmann A and Hoppe-Seyler F: Functional p53 protein in human papillomavirus-positive cancer cells. Oncogene. 10:927–936. 1995.PubMed/NCBI
18	Chalfie M, Tu Y, Euskirchen G, Ward WW and Prasher DC: Green fluorescent protein as a marker for gene expression. Science. 263:802–805. 1994. View Article : Google Scholar : PubMed/NCBI
19	Zheng Y, Zhang J and Rao Z: Ribozyme targeting HPV16 E6E7 transcripts in cervical cancer cells suppresses cell growth and sensitizes cells to chemotherapy and radiotherapy. Cancer Biol Ther. 3:1129–1135. 2004. View Article : Google Scholar : PubMed/NCBI
20	Gross-Mesilaty S, Reinstein E, Bercovich B, et al: Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway. Proc Natl Acad Sci USA. 95:8058–8063. 1998. View Article : Google Scholar : PubMed/NCBI
21	Ronco LV, Karpova AY, Vidal M and Howley PM: Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev. 12:2061–2072. 1998. View Article : Google Scholar : PubMed/NCBI
22	Kumar A, Zhao Y, Meng G, et al: Human papillomavirus oncoprotein E6 inactivates the transcriptional coactivator human ADA3. Mol Cell Biol. 22:5801–5812. 2002. View Article : Google Scholar : PubMed/NCBI
23	Ferrigno P and Silver PA: Regulated nuclear localization of stress-responsive factors: how the nuclear trafficking of protein kinases and transcription factors contributes to cell survival. Oncogene. 18:6129–6134. 1999. View Article : Google Scholar : PubMed/NCBI
24	Hollstein M, Rice K, Greenblatt MS, et al: Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res. 22:3551–3555. 1994.PubMed/NCBI
25	Diaz D, Santander MA and Chavez JA: HPV-16 E6 and E7 oncogene expression is downregulated as a result of Mdm2 knockdown. Int J Oncol. 41:141–146. 2012.PubMed/NCBI
26	Hwang SJ, Suh MJ, Yoon JH, et al: Identification of characteristic molecular signature of Mullerian inhibiting substance in human HPV-related cervical cancer cells. Int J Oncol. 39:811–820. 2011.PubMed/NCBI
27	Kawamata Y, Mitsuhashi A, Unno Y, et al: HPV 16-E6-mediated degradation of intrinsic p53 is compensated by upregulation of p53 gene expression in normal cervical keratinocytes. Int J Oncol. 21:561–567. 2002.PubMed/NCBI
28	Fouret P, Dabit D, Sibony M, et al: Expression of p53 protein related to the presence of human papillomavirus infection in precancer lesions of the larynx. Am J Pathol. 146:599–604. 1995.PubMed/NCBI
29	Isaacs JS, Chen P, Garza A, Hansen MF, Barrett JC and Weissman BE: Failure of HPV E6 to rapidly degrade p53 in human HeLa x PNET cell hybrids. Oncogene. 14:1669–1678. 1997. View Article : Google Scholar : PubMed/NCBI
30	Shin YC, Nakamura H, Liang X, et al: Inhibition of the ATM/p53 signal transduction pathway by Kaposi’s sarcoma-associated herpesvirus interferon regulatory factor 1. J Virol. 80:2257–2266. 2006.PubMed/NCBI
31	Accardi R, Dong W, Smet A, et al: Skin human papillomavirus type 38 alters p53 functions by accumulation of deltaNp73. EMBO Rep. 7:334–340. 2006. View Article : Google Scholar : PubMed/NCBI
32	Li L, Guo L, Tao Y, et al: Latent membrane protein 1 of Epstein-Barr virus regulates p53 phosphorylation through MAP kinases. Cancer Lett. 255:219–231. 2007. View Article : Google Scholar : PubMed/NCBI
33	Granja AG, Nogal ML, Hurtado C, et al: Modulation of p53 cellular function and cell death by African swine fever virus. J Virol. 78:7165–7174. 2004. View Article : Google Scholar : PubMed/NCBI