大部分的子宮頸癌細胞，均受到高危險性人類乳突病毒 (HPV) 的感染。而表現HPV type16 病毒的兩個基因E6和E7，是造成子宮頸癌最主要的原因。近來研究發現，其中的E7致癌蛋白會抑制細胞進行細胞計畫性死亡(apoptosis; programmed cell death)。但在其他文獻指出，E7在細胞裡扮演著相反的角色(促進細胞計畫性死亡)。因此，本研究主要欲釐清E7致癌蛋白在SiHa及HF細胞中所扮演的角色，SiHa為子宮頸癌細胞，此細胞受到人類乳突病毒的感染，因此能同時表現E6和E7此兩個致癌蛋白，而HF為人類纖維母細胞，是未被人類乳突病毒感染的細胞。除了了解E7蛋白是否會造成細胞計畫性死亡外，E7蛋白會誘導或抑制哪些蛋白質的表現也是本研究的目的之一。而這些一連串的蛋白質表現，即可能會造成細胞計畫性死亡。
利用pLXSN-E7/neo載體將E7基因轉入未受HPV16感染的HF細胞株中。從含有G418的培養液中，篩選出約16個成功轉殖的HF-E7細胞株。而其中的HF-E7#8細胞株能大量表現E7及磷酸化的Rb(Retinoblastoma)蛋白，此表示轉入的E7蛋白質能將Rb蛋白磷酸化且是有功能的。
透過SRB survival fraction分析發現，轉入的E7蛋白質皆會使得HF和SiHa細胞的存活率下降。而且，經由細胞中提高的sub-G1 apoptosis推測：細胞存活率下降，是因為轉入的E7蛋白，會造成細胞自發性地進行計畫性死亡。在處理0-8 Gy X-ray 或 0-1uM cisplatin後，無論是細胞進行計畫性死亡的比例，或細胞存活率下降的程度，HF-E7/SiHa-E7細胞皆高於HF/SiHa。此證明了，將E7蛋白轉入HF/SiHa細胞內，不只會造成細胞自發性地進行計畫性死亡，還會使得細胞對於DNA damage agents(X-ray及cisplatin)更為敏感。
HF-E7和SiHa-E7細胞能表現E7蛋白質，此E7蛋白質會磷酸化Rb而造成磷酸化的Rb表現量增加。在HF-E7中，其p53、ser15-p53、 p21和 caspase 3 蛋白的表現量皆高於未轉入E7蛋白的HF細胞。這顯示E7蛋白是透過p53/ ser15p53/p21/ caspase 3的路徑，增加了HF-E7細胞計畫性死亡的比例，並且此細胞計畫性死亡是藉由活化caspase 3所造成的。另外，符合SRB及Sub-G1 apoptosis的結果，在處理0-8 Gy X-ray 和 0-1 uM cisplatin後， 轉入E7蛋白的細胞其蛋白質(p53、ser15-phospho p53、 p21和 caspase 3)表現量皆會增加，最後造成細胞計畫性死亡。
雖然在HF細胞中觀察E7蛋白的功能，能較清楚的了解E7蛋白在未受到E6蛋白的影響下，所造成HF的改變。但是，E7蛋白在子宮頸癌細胞中，並非是單獨存在的，其可能在子宮頸癌細胞中與E6致癌蛋白有非常複雜的關係，最後導致細胞進行計畫性死亡的路徑更為複雜。而經由西方點墨法的結果即印證這一觀點。轉入的E7蛋白不但沒有造成與細胞計畫性死亡的p53、ser15-phospho p53、 p21蛋白表現量增加，反而使之減少。而將細胞處理0-8 Gy X-ray 和 0-1 uM cisplatin後，p21蛋白表現量也不如預期地減少了。這些結果皆與E7對HF細胞的影響不同。但是在此二轉入E7的細胞中，其活化態的caspase 3表現量會增加，，並且細胞計畫性死亡的比例也增高了。這結果顯示，雖然E7蛋白質在HF及SiHa細胞中，誘導了不同的蛋白質表現，但最終卻同樣地增加了細胞計畫性死亡的比例。

The high risk human papillomavirus type 16 (HPV-16) have been found to be strongly associated with the carcinoma of the uterine cervix. E6 and E7 proteins of HPV-16 viruses are thought to underlie the development of cervical cancer. Recent reports indicated that E7 would suppress apoptosis (Lee et al., 1998; Thompson et al., 2001). Interestingly, E7 have also been reported to mediate pro-apoptotic effects (Basile et al., 2001; Hickman et al., 1997; Puthenveettil et al., 1996; Stoppler et al., 1998). In this study, E7-mediated apoptosis was determined in negative HF cells and in HPV16 positive SiHa cells (cervical carcinoma cells). The mechanism for E7 modulated apoptosis in these cells was investigated.
pLXSN-E7/neo and pCDNA-neo was stably transfected into HPV-16 negative human fibroblast (HF). HF-E7#8 clone which had higher level of E7 and pRb protein and lower level of p53 protein was selected as the model cell line. On the other hand, SiHa-E7 cell line, constructed by Ms. Chen Ting Ting, and SiHa-neo cell line was also used in this study.
The biological effects of E7 protein in both HF and SiHa cells were assayed by Sulforhodamine B (SRB) survival assay and Sub-G1 apoptotic analysis (Flow cytometry). E7 transfection in HF or SiHa cells led to increased Sub-G1 apoptosis. Furthermore, in response to DNA damage agents (0-8 Gy X-irradiation or 0-1μM cisplatin), both HF -E7 and SiHa -E7 cells were more sensitive than their parental cells because that these two cells had more sub-G1 apoptosis and less survival fraction. That is, E7 had synergistic apoptotic effects in SiHa or HF cells after X-ray or cisplatin treatment. However, no synergistic apoptotic effect of E7 was observed in E7 transfected SiHa cells after SA treatment. In conclusion, E7 protein sensitized HF and SiHa cells to X-irradiation and cisplatin but not SA.
In both HF-E7 and SiHa-E7 cells, E7 protein had been proved its functionality by increasing phosphorylated Rb. E7 protein up-regulated the apoptosis-related protein, p53, ser15-phospho p53, and p21, in HF cells. In contrast, it down-regulated these three proteins expression in SiHa cells. Moreover, all of these proteins increased in X-irradiated HF/HF-E7 cells but p21 decreased in SiHa/SiHa-E7 cells after X-ray or cisplatin. Despite the differences, active form of caspase 3 increased in both HF-E7 and SiHa-E7 cell indicating that E7 mediated caspase 3-dependent apoptosis. All these results indicated that E7 transfection both in HPV16 positive SiHa and HPV16 negative HF cells would increase X-irradiated sub-G1 apoptosis and cell killing, but the molecular mechanisms for pro-apoptosis were not exactly the same in these two cell lines. Therefore, the pro-apoptotic function of E7 in human cells (HF and SiHa) had been investigated in this study.

Reference

Abraham, R.T. 2001. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15:2177-96.
Band, V., J.A. De Caprio, L. Delmolino, V. Kulesa, and R. Sager. 1991. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J Virol. 65:6671-6.
Banin, S., L. Moyal, S. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, and Y. Ziv. 1998. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science. 281:1674-7.
Banks, L., C. Edmonds, and K.H. Vousden. 1990. Ability of the HPV16 E7 protein to bind RB and induce DNA synthesis is not sufficient for efficient transforming activity in NIH3T3 cells. Oncogene. 5:1383-9.
Basile, J.R., V. Zacny, and K. Munger. 2001. The cytokines tumor necrosis factor-alpha (TNF-alpha ) and TNF-related apoptosis-inducing ligand differentially modulate proliferation and apoptotic pathways in human keratinocytes expressing the human papillomavirus-16 E7 oncoprotein. J Biol Chem. 276:22522-8.
Canman, C.E., D.S. Lim, K.A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Appella, M.B. Kastan, and J.D. Siliciano. 1998. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science. 281:1677-9.
Chang, H.Y., and X. Yang. 2000. Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev. 64:821-46.
Chen, Q., F.C. Hung, L. Fromm, and P.A. Overbeek. 2000. Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2. Invest Ophthalmol Vis Sci. 41:4223-31.
Du, J., G.G. Chen, A.C. Vlantis, H. Xu, R.K. Tsang, and A.C. van Hasselt. 2003. The nuclear localization of NFkappaB and p53 is positively correlated with HPV16 E7 level in laryngeal squamous cell carcinoma. J Histochem Cytochem. 51:533-9.
Duensing, S., and K. Munger. 2004. Mechanisms of genomic instability in human cancer: insights from studies with human papillomavirus oncoproteins. Int J Cancer. 109:157-62.
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.
Gillison, M.L., W.M. Koch, R.B. Capone, M. Spafford, W.H. Westra, L. Wu, M.L. Zahurak, R.W. Daniel, M. Viglione, D.E. Symer, K.V. Shah, and D. Sidransky. 2000. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst. 92:709-20.
Goodwin, E.C., and D. DiMaio. 2000. Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc Natl Acad Sci U S A. 97:12513-8.
Gulliver, G.A., R.L. Herber, A. Liem, and P.F. Lambert. 1997. Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice. J Virol. 71:5905-14.
Halbert, C.L., G.W. Demers, and D.A. Galloway. 1992. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells. J Virol. 66:2125-34.
Hall-Jackson, C.A., D.A. Cross, N. Morrice, and C. Smythe. 1999. ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK. Oncogene. 18:6707-13.
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.
Hickman, E.S., S. Bates, and K.H. Vousden. 1997. Perturbation of the p53 response by human papillomavirus type 16 E7. J Virol. 71:3710-8.
Hiebert, S.W., G. Packham, D.K. Strom, R. Haffner, M. Oren, G. Zambetti, and J.L. Cleveland. 1995. E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis. Mol Cell Biol. 15:6864-74.
Hurlin, P.J., P. Kaur, P.P. Smith, N. Perez-Reyes, R.A. Blanton, and J.K. McDougall. 1991. Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype. Proc Natl Acad Sci U S A. 88:570-4.
Irwin, M., M.C. Marin, A.C. Phillips, R.S. Seelan, D.I. Smith, W. Liu, E.R. Flores, K.Y. Tsai, T. Jacks, K.H. Vousden, and W.G. Kaelin, Jr. 2000. Role for the p53 homologue p73 in E2F-1-induced apoptosis. Nature. 407:645-8.
Jewers, R.J., P. Hildebrandt, J.W. Ludlow, B. Kell, and D.J. McCance. 1992. Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes. J Virol. 66:1329-35.
Kastan, M.B., and D.S. Lim. 2000. The many substrates and functions of ATM. Nat Rev Mol Cell Biol. 1:179-86.
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.
Kowalik, T.F., J. DeGregori, J.K. Schwarz, and J.R. Nevins. 1995. E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis. J Virol. 69:2491-500.
Lee, J.E., C.Y. Kim, A.J. Giaccia, and R.G. Giffard. 1998. The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury. Brain Res. 795:10-6.
Lissy, N.A., P.K. Davis, M. Irwin, W.G. Kaelin, and S.F. Dowdy. 2000. A common E2F-1 and p73 pathway mediates cell death induced by TCR activation. Nature. 407:642-5.
Macleod, K.F., Y. Hu, and T. Jacks. 1996. Loss of Rb activates both p53-dependent and independent cell death pathways in the developing mouse nervous system. Embo J. 15:6178-88.
Munger, K., J.R. Basile, S. Duensing, A. Eichten, S.L. Gonzalez, M. Grace, and V.L. Zacny. 2001. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene. 20:7888-98.
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.
Puthenveettil, J.A., S.M. Frederickson, and C.A. Reznikoff. 1996. Apoptosis in human papillomavirus16 E7-, but not E6-immortalized human uroepithelial cells. Oncogene. 13:1123-31.
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.
Scheffner, M., B.A. Werness, J.M. Huibregtse, A.J. Levine, and P.M. Howley. 1990. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 63:1129-36.
Shay, J.W., W.E. Wright, D. Brasiskyte, and B.A. Van der Haegen. 1993. E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. Oncogene. 8:1407-13.
Shiloh, Y. 2001. ATM and ATR: networking cellular responses to DNA damage. Curr Opin Genet Dev. 11:71-7.
Stanley, M.A. 2001. Human papillomavirus and cervical carcinogenesis. Best Pract Res Clin Obstet Gynaecol. 15:663-76.
Stiewe, T., and B.M. Putzer. 2000. Role of the p53-homologue p73 in E2F1-induced apoptosis. Nat Genet. 26:464-9.
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.
Thompson, D.A., V. Zacny, G.S. Belinsky, M. Classon, D.L. Jones, R. Schlegel, and K. Munger. 2001. The HPV E7 oncoprotein inhibits tumor necrosis factor alpha-mediated apoptosis in normal human fibroblasts. Oncogene. 20:3629-40.
Vigo, E., H. Muller, E. Prosperini, G. Hateboer, P. Cartwright, M.C. Moroni, and K. Helin. 1999. CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase. Mol Cell Biol. 19:6379-95.
Wazer, D.E., X.L. Liu, Q. Chu, Q. Gao, and V. Band. 1995. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7. Proc Natl Acad Sci U S A. 92:3687-91.
Wells, S.I., D.A. Francis, A.Y. Karpova, J.J. Dowhanick, J.D. Benson, and P.M. Howley. 2000. Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways. Embo J. 19:5762-71.
Werness, B.A., A.J. Levine, and P.M. Howley. 1990. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 248:76-9.
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.
Ziebold, U., T. Reza, A. Caron, and J.A. Lees. 2001. E2F3 contributes both to the inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev. 15:386-91.
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.