摘要

大部分的子宮頸癌均顯示細胞曾被人類乳突病毒 (HPVs) 感染。其中HPV-E7 (以下簡稱 E7 ) 蛋白會與 Rb 蛋白結合，抑制 Rb 蛋白調控細胞週期的功能。在過去本實驗室的研究中，發現亞砷酸鈉和 cisplatin 藉由降低 HPV-E6蛋白表現，回復 p53 蛋白功能，而造成 SiHa 和 T15 (轉殖 HPV-16 E6 基因的 TK6 細胞) 細胞程序性細胞死亡。 SiHa 細胞內同時存在 E6 和 E7 蛋白， E7 蛋白是否也參與亞砷酸鈉和 cisplatin 誘導的程序性細胞死亡機制，目前並不清楚。本實驗希望釐清E7蛋白對亞砷酸鈉、cisplatin和X-ray誘導程序性細胞死亡的影響。利用含有HPV16-E7/Neo的載體 pLXSN分別轉入有HPV-16子宮頸癌細胞株 (SiHa) 和沒有HPV-16的人類纖維母細胞株 (HF)。從含有G418的培養液中，篩選出成功轉殖的SiHa-E7 (#4, #8和#14) 和HF-E7 (#8) 細胞株。
在SiHa-E7 #14細胞內，藉由免疫螢光染色方法得知，轉入E7基因後，細胞內之E7蛋白，大部分位在細胞核內，小部分位在細胞核周圍的細胞質，與SiHa內原本的E7蛋白位置相同。且表現的E7蛋白並不會改變SiHa-E7 #14細胞中p53蛋白的分布位置。另外，由西方墨點方法獲得：轉入E7基因後，SiHa-E7 #14細胞內的hypo-phosphorylated-Rb (pRb) 蛋白表現有增加1-3倍，而背景值的p53和p21蛋白的表現量降低。在HF-E7 #8細胞內，轉入E7基因，可獲得背景值p53表現量增加，為原來HF的3到6倍。
利用 Parental 細胞和成功轉入E7基因的細胞 SiHa-E7 #14和HF-E7 #8分別處理亞砷酸鈉、cisplatin或X-ray。以流式細胞計數器 (A) 分析細胞族群中sub-G1細胞百分比，另一方面以染SRB到細胞膜的方式 (B)，做SRB細胞毒性分析。結果顯示，處理0-8 Gy X-ray和0-5 μM cisplatin 24小時之後，在SiHa-E7 #14及HF-E7 #8細胞株，都比原來SiHa以及HF細胞所得之sub-G1百分比多1-3倍，而SRB細胞毒性分析結果也多1.5-2倍。在0-32 μM亞砷酸鈉處理24小時後，兩種SiHa無論在 (A) 與 (B) 兩種分析方式所得結果，都沒有明顯差異。此結果可支持本實驗室過去的研究：亞砷酸鈉誘導的SiHa細胞程序性細胞死亡，主要是藉由降低E6蛋白，達到回復p53蛋白的功能，E7蛋白對此機制沒有太大影響。至於在HF和HF-E7 #8兩種細胞對應亞砷酸鈉敏感度方面，後者可得較多的sub-G1細胞。
在SiHa和SiHa-E7 #14細胞中，分別處理X-ray與cisplatin後所得之pRb以及p53蛋白表現都隨處理劑量 (0-8 Gy X-ray；0-10 μM cisplatin 24小時) 增加而增多。然而，受p53調控的p21蛋白表現，反而隨處理計量增加而減少。另一方面，處理劑量增加，兩種細胞間表現的active-caspase-3蛋白也沒有明顯改變。由此推論E7一方面藉由與pRb鍵結，另一方面藉由降低細胞內p21蛋白表現，促進Rb蛋白磷酸化，破壞Rb-E2F複合體，釋放E2F進入細胞核，透過磷酸化p53蛋白，使較多SiHa細胞進行程序性細胞死亡。雖然，HF細胞中，經過X-ray或cisplatin處理後，p53蛋白的表現有隨處理劑量 (0-4 Gy X-ray；0-2 μM cisplatin 24小時) 增加而增多，但是在有轉殖E7的細胞，p53的表現差異不明顯。
本研究成功的轉殖 E7 到有 HPV 和沒有 HPV 的兩種細胞內，經分析後發現，E7蛋白並不影響亞砷酸鈉誘導的程序性細胞死亡。但另一方面，E7 的轉殖確實改變細胞對 X-ray 和 cisplatin 的敏感度。而 E7 蛋白主要是透過與pRb 鍵結及降低細胞內 p21 蛋白表現量，促進細胞的程序性細胞死亡。

Abstract

Most human cervical cancers are caused by human papilloma virus (HPVs) infection. The HPV-E7 protein of HPV can bind to retinoblastoma protein (Rb) then inhibits the cell cycle regulation activity of Rb protein. Previous study of this lab showed that sodium arsenite (SA) could restore p53 tumor suppressor pathway in human cervical carcinoma SiHa cells, but not mutant-p53-SiHa (SiHa-p53m) cells. In present study showed that cisplatin could restore p53 expression in HPV-16 E6 containing SiHa cells after 10 mM cisplatin treatment for more than 4 hours (4 h) and enhance the radiosensitivity (in sub-G1 apoptosis) in HPV16 E6 containing SiHa cells, but not SiHa-p53m cells. Whether E7 involving in SA or cisplatin restored p53 function or cisplatin enhanced radiosensitivity (X-ray) in E6/E7 containing SiHa cells was not yet known. In order to determine E7 function in SiHa cells after treat with SA, cisplatin or X-ray, SiHa and human fibroblast (HF) cells were transfected with the retrovirus-derived vector pLXSN containing both the neo and HPV-16 E7 gene. Four individual clones (SiHa-E7#4, SiHa-E7#8, SiHa-E7#14 and HF-E7#8) were selected because of their high level of E7 protein and hypo-phosphorylated Rb protein (pRb) among a total of 30 (15 SiHa-E7; 15 HF-E7) G418-resistant transfected clones.
Most E7 protein were located in the nucleus of SiHa and SiHa-E7#14 cells after immuno-fluorescence stain, but some E7 protein were found in cytoplasm surrounding the nucleus. Compared to parental SiHa cells, the SiHa-E7#14 cells have 1-3 fold more pRb protein, but less p53 and p21 protein. In HF-E7#8 cells, the E7 protein increased 3-6 fold of p53 expression than parental HF cells.
The E7 effects on sub-G1 apoptosis and cytotoxicity were assayed by means of Flow-Cytometry (A) and SRB assay (B) in E7-transfected cells and parental cells. SiHa-E7#14 and HF-E7#8 cells both induced 1-3 fold sub-G1 apoptosis than parental cells after 0-8 Gy X-ray treatments. They also have 1-3 fold more sub-G1 apoptosis than parental cells after 0-5 μM cisplatin treatment for 24 h. SiHa-E7 #14 and HF-E7 #8 cells both appeared to have a 1.5-2 fold SRB-cytotoxicity than parental cells after the same dose of X-ray or cisplatin treatment. After 0-32 μM SA treatment for 24 h, SiHa and SiHa-E7#14 cells showed no significant difference in cytotoxic results from protocol (A) and (B). These results confirmed the previous study of this lab: SA induced SiHa cells’ apoptosis via decreasing E6 gene expression and then restoring p53 protein function. E7 protein had no significant effects on SA-induced apoptosis. In contrast HF-E7#8 cells induced more sub-G1 apoptosis than parental HF cells after the same dose of SA for 24 h treatment.
Western blotting indicated that an increase in dose-dependent p53 and pRb protein expression (according to X-ray 0-8 Gy or cisplatin 0-10 μM for 24 h) was found in SiHa and SiHa-E7 #14 cells. In the other hand p21, p53 down stream protein, expression was a decreased in dose-dependent. The results from the above treatment showed that, active-caspase-3 protein in two kinds of SiHa cells were no different before or after treatment. P53 protein increased in both cells with agent doses in HF cells, but p53 expression level showed no difference in HF-E7#8 cells.
In this study, E7 gene was successfully transfected into SiHa and HF cells stably. Flow-cytometry and SRB assay showed that, E7 protein had no significant effect on SA-induced apoptosis, but increased the sensitivity of SiHa cells to cisplatin and X-ray. The E7 protein may through binding pRb protein and decreasing p21 protein expression to increase cells undergoing apoptosis.

References

Almasan, A., Y. Yin, R.E. Kelly, E.Y. Lee, A. Bradley, W. Li, J.R. Bertino, and G.M. Wahl. 1995. Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis. Proc Natl Acad Sci U S A. 92:5436-40.
Alonso, L.G., M.M. Garcia-Alai, A.D. Nadra, A.N. Lapena, F.L. Almeida, P. Gualfetti, and G.D. Prat-Gay. 2002. High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners. Biochemistry. 41:10510-8.
Arbeit, J.M., K. Munger, P.M. Howley, and D. Hanahan. 1994. Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice. J Virol. 68:4358-68.
Chou, R.H., and H. Huang. 2002a. Restoration of p53 tumor suppressor pathway in human cervical carcinoma cells by sodium arsenite. Biochem Biophys Res Commun. 293:298-306.
Chou, R.H., and H. Huang. 2002b. Sodium arsenite suppresses human papillomavirus-16 E6 gene and enhances apoptosis in E6-transfected human lymphoblastoid cells. J Cell Biochem. 84:615-24.
Cordon-Cardo, C. 1995. Mutations of cell cycle regulators. Biological and clinical implications for human neoplasia. Am J Pathol. 147:545-60.
Debbas, M., and E. White. 1993. Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev. 7:546-54.
DeGregori, J., T. Kowalik, and J.R. Nevins. 1995. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol. 15:4215-24.
Demers, G.W., S.A. Foster, C.L. Halbert, and D.A. Galloway. 1994a. Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7. Proc Natl Acad Sci U S A. 91:4382-6.
Demers, G.W., C.L. Halbert, and D.A. Galloway. 1994b. Elevated wild-type p53 protein levels in human epithelial cell lines immortalized by the human papillomavirus type 16 E7 gene. Virology. 198:169-74.
Dimri, G.P., M. Nakanishi, P.Y. Desprez, J.R. Smith, and J. Campisi. 1996. Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein. Mol Cell Biol. 16:2987-97.
Du, J., G.G. Chen, A.C. Vlantis, P.K. Chan, R.K. Tsang, and C.A. van Hasselt. 2004. Resistance to apoptosis of HPV 16-infected laryngeal cancer cells is associated with decreased Bak and increased Bcl-2 expression. Cancer Lett. 205:81-8.
Eck-Enriquez, K., T.L. Kiefer, L.L. Spriggs, and S.M. Hill. 2000. Pathways through which a regimen of melatonin and retinoic acid induces apoptosis in MCF-7 human breast cancer cells. Breast Cancer Res Treat. 61:229-39.
Eichten, A., M. Westfall, J.A. Pietenpol, and K. Munger. 2002. Stabilization and functional impairment of the tumor suppressor p53 by the human papillomavirus type 16 E7 oncoprotein. Virology. 295:74-85.
Germolec, D.R., J. Spalding, H.S. Yu, G.S. Chen, P.P. Simeonova, M.C. Humble, A. Bruccoleri, G.A. Boorman, J.F. Foley, T. Yoshida, and M.I. Luster. 1998. Arsenic enhancement of skin neoplasia by chronic stimulation of growth factors. Am J Pathol. 153:1775-85.
Gorospe, M., C. Cirielli, X. Wang, P. Seth, M.C. Capogrossi, and N.J. Holbrook. 1997. p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene. 14:929-35.
Harper, J.W., G.R. Adami, N. Wei, K. Keyomarsi, and S.J. Elledge. 1993. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 75:805-16.
Hatakeyama, M., and R.A. Weinberg. 1995. The role of RB in cell cycle control. Prog Cell Cycle Res. 1:9-19.
Helt, A.M., J.O. Funk, and D.A. Galloway. 2002. Inactivation of both the retinoblastoma tumor suppressor and p21 by the human papillomavirus type 16 E7 oncoprotein is necessary to inhibit cell cycle arrest in human epithelial cells. J Virol. 76:10559-68.
Huang, H., S.Y. Huang, T.T. Chen, J.C. Chen, C.L. Chiou, and T.M. Huang. 2004. Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells. J Cell Biochem. 91:756-65.
Huang, H., C.Y. Li, and J.B. Little. 1996. Abrogation of P53 function by transfection of HPV16 E6 gene does not enhance resistance of human tumour cells to ionizing radiation. Int J Radiat Biol. 70:151-60.
Hwang, S.G., D. Lee, J. Kim, T. Seo, and J. Choe. 2002. Human papillomavirus type 16 E7 binds to E2F1 and activates E2F1-driven transcription in a retinoblastoma protein-independent manner. J Biol Chem. 277:2923-30.
Jaattela, M., H. Mouritzen, F. Elling, and L. Bastholm. 1996. A20 zinc finger protein inhibits TNF and IL-1 signaling. J Immunol. 156:1166-73.
Jones, D.L., R.M. Alani, and K. Munger. 1997a. The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2. Genes Dev. 11:2101-11.
Jones, D.L., and K. Munger. 1997. Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein. J Virol. 71:2905-12.
Jones, D.L., D.A. Thompson, and K. Munger. 1997b. Destabilization of the RB tumor suppressor protein and stabilization of p53 contribute to HPV type 16 E7-induced apoptosis. Virology. 239:97-107.
Kaznelson, D.W., S. Bruun, A. Monrad, S. Gjerlov, J. Birk, C. Ropke, and B. Norrild. 2004. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation. Virology. 320:301-12.
Lambert, P.F., H. Pan, H.C. Pitot, A. Liem, M. Jackson, and A.E. Griep. 1993. Epidermal cancer associated with expression of human papillomavirus type 16 E6 and E7 oncogenes in the skin of transgenic mice. Proc Natl Acad Sci U S A. 90:5583-7.
Liang, P., and A.B. Pardee. 2003. Analysing differential gene expression in cancer. Nat Rev Cancer. 3:869-76.
Liang, Y., C. Yan, and N.F. Schor. 2001. Apoptosis in the absence of caspase 3. Oncogene. 20:6570-8.
Liu, Y., A. McKalip, and B. Herman. 2000. 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. 78:334-49.
Lowe, S.W., and H.E. Ruley. 1993. Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev. 7:535-45.
Lowe, S.W., H.E. Ruley, T. Jacks, and D.E. Housman. 1993. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell. 74:957-67.
Mantovani, F., and L. Banks. 2001. The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene. 20:7874-87.
Mantovani, F., P. Massimi, and L. Banks. 2001. Proteasome-mediated regulation of the hDlg tumour suppressor protein. J Cell Sci. 114:4285-92.
Morgenbesser, S.D., B.O. Williams, T. Jacks, and R.A. DePinho. 1994. p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature. 371:72-4.
Ohtani, K., J. DeGregori, and J.R. Nevins. 1995. Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci U S A. 92:12146-50.
Pan, H., and A.E. Griep. 1994. Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development. Genes Dev. 8:1285-99.
Polyak, K., T. Waldman, T.C. He, K.W. Kinzler, and B. Vogelstein. 1996. Genetic determinants of p53-induced apoptosis and growth arrest. Genes Dev. 10:1945-52.
Pomerantz, J., N. Schreiber-Agus, N.J. Liegeois, A. Silverman, L. Alland, L. Chin, J. Potes, K. Chen, I. Orlow, H.W. Lee, C. Cordon-Cardo, and R.A. DePinho. 1998. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell. 92:713-23.
Powers, J.T., S. Hong, C.N. Mayhew, P.M. Rogers, E.S. Knudsen, and D.G. Johnson. 2004. E2F1 uses the ATM signaling pathway to induce p53 and Chk2 phosphorylation and apoptosis. Mol Cancer Res. 2:203-14.
Qin, X.Q., D.M. Livingston, W.G. Kaelin, Jr., and P.D. Adams. 1994. Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proc Natl Acad Sci U S A. 91:10918-22.
Rogoff, H.A., M.T. Pickering, M.E. Debatis, S. Jones, and T.F. Kowalik. 2002. E2F1 induces phosphorylation of p53 that is coincident with p53 accumulation and apoptosis. Mol Cell Biol. 22:5308-18.
Rogoff, H.A., M.T. Pickering, F.M. Frame, M.E. Debatis, Y. Sanchez, S. Jones, and T.F. Kowalik. 2004. Apoptosis associated with deregulated E2F activity is dependent on E2F1 and Atm/Nbs1/Chk2. Mol Cell Biol. 24:2968-77.
Shiyanov, P., S. Bagchi, G. Adami, J. Kokontis, N. Hay, M. Arroyo, A. Morozov, and P. Raychaudhuri. 1996. p21 Disrupts the interaction between cdk2 and the E2F-p130 complex. Mol Cell Biol. 16:737-44.
Slebos, R.J., M.H. Lee, B.S. Plunkett, T.D. Kessis, B.O. Williams, T. Jacks, L. Hedrick, M.B. Kastan, and K.R. Cho. 1994. p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein. Proc Natl Acad Sci U S A. 91:5320-4.
Stoppler, H., M.C. Stoppler, E. Johnson, C.M. Simbulan-Rosenthal, M.E. Smulson, S. Iyer, D.S. Rosenthal, and R. Schlegel. 1998. The E7 protein of human papillomavirus type 16 sensitizes primary human keratinocytes to apoptosis. Oncogene. 17:1207-14.
Wang, Y., I. Okan, K. Pokrovskaja, and K.G. Wiman. 1996. Abrogation of p53-induced G1 arrest by the HPV 16 E7 protein does not inhibit p53-induced apoptosis. Oncogene. 12:2731-5.
Weinberg, R.A. 1995. The retinoblastoma protein and cell cycle control. Cell. 81:323-30.
Wu, E.W., K.E. Clemens, D.V. Heck, and K. Munger. 1993. The human papillomavirus E7 oncoprotein and the cellular transcription factor E2F bind to separate sites on the retinoblastoma tumor suppressor protein. J Virol. 67:2402-7.
Wu, X., and A.J. Levine. 1994. p53 and E2F-1 cooperate to mediate apoptosis. Proc Natl Acad Sci U S A. 91:3602-6.
zur Hausen, H. 1996. Papillomavirus infections--a major cause of human cancers. Biochim Biophys Acta. 1288:F55-78.
zur Hausen, H. 2000. Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis. J Natl Cancer Inst. 92:690-8.