载体介导的反义靶向技术在宫颈癌基因治疗中的应用
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摘要
第一部分反义HPV16 E6E7-EGFP重组质粒的构建及其对人宫颈癌细胞凋亡及衰老方面的影响
目的:人乳头瘤病毒16型(human papillomavirus 16, HPV16)的早期基因E6、E7可分别诱导p53的泛素化降解和pRb的高磷酸化失活,与宫颈癌的发生发展关系密切。本研究通过真核表达载体介导HPV16型E6、E7基因反义RNA在SiHa细胞中的表达,以探讨反义RNA能否能降低E6、E7基因的表达,诱导细胞凋亡或衰老发生。
方法:利用pEGFP构建HPV16型E6、E7基因反义RNA的真核表达载体并转染SiHa细胞,利用RT-PCR和Western blot技术检测转染后E6、E7基因的mRNA和蛋白的变化,利用MTT法检测SiHa细胞转染后细胞增殖活性,利用流式细胞仪和激光共聚焦显微镜检测转染后细胞的凋亡以及通过细胞形态学观察及β-gal染色来确定细胞衰老情况。
结果:转染携带HPV16型E6、E7反义RNA的质粒后,SiHa细胞的E6、E7基因的mRNA和蛋白均明显下调;MTT结果显示转染后细胞的增殖活性(0.502±0.050)与转染空载体细胞(1.010±0.055)和未转染细胞(1.283±0.058)比较明显降低(P<0.05);流式细胞仪检测结果示转染后细胞凋亡率(59.3±11.3)%与转染空载体细胞(9.4±1.8)%和未转染细胞(2.1±0.4)%比较有显著性差异(P<0.05)。激光共聚焦显微镜结果示转染后细胞凋亡明显增多;并且HPV16型E6、E7反义RNA使细胞形态学发生改变,细胞变大,变扁平,衰老特异性的β-gal染色阳性明显增多。
结论:HPV16型E6、E7基因反义RNA可降低宫颈癌细胞中E6、E7癌基因的表达,抑制细胞增殖,诱导宫颈癌细胞发生凋亡及衰老。
第二部分利用反义技术抑制HPV18 E6E7的表达及其对宫颈癌HeLa细胞增殖凋亡的影响
目的:构建HPV18型E6E7反义荧光真核表达载体,研究其对宫颈癌HeLa细胞增殖凋亡的影响。
方法:以PCR法扩增HPV18型E6E7区716bp片段并反向克隆到荧光真核表达载体pEGFP-C1,构建重组体pEGFP-HPV18E6E7as(EGFP-18AS)并转染人宫颈癌HeLa细胞。利用RT-PCR和Western blot技术检测转染后E6、E7基因在mRNA和蛋白水平的变化;用MTT法检测转染后细胞的的增殖活性;用流式细胞仪检测转染后细胞的凋亡率;荧光显微镜下观察凋亡细胞的形态学改变。
结果:转染EGFP-18AS重组体后,HeLa细胞中E6、E7基因的mRNA表达和蛋白合成均明显下调;细胞增殖受到抑制;流式显示细胞凋亡率增加;同时在荧光显微镜下经Hoechst染色凋亡细胞出现染色质凝集、边集、碎裂等形态学改变。
结论:重组质粒pEGFP-HPV18E6E7a(sEGFP-18AS)能有效地抑制人宫颈癌HeLa细胞的生长和增殖,诱导其凋亡,证明了反义RNA技术的有效性,为宫颈癌的基因治疗提供了一种新的可能的途径。
第三部分靶向HPV16-E7的短发夹RNA对人宫颈癌SiHa细胞的促凋亡作用
目的:HPV是宫颈癌发生的首要致病因素,其编码的E6和E7癌基因对宫颈病变的恶性转化及恶性表型的维持是至关重要的。针对E6和E7癌基因的分子治疗被证实是可行的,且同时下调E6和E7的表达将具有最有效的抗肿瘤的作用。本研究拟利用RNAi技术抑制E6和E7的表达并观察其对宫颈癌细胞的促凋亡作用。
方法:利用pSIREN-DNR构建靶向HPV16-E7的真核表达载体(pSIREN-16E7)并转染人宫颈癌SiHa细胞,利用RT-PCR和Western blot技术检测转染后E6、E7基因的mRNA和蛋白的变化,利用MTT法检测SiHa细胞转染后细胞增殖活性,利用流式细胞仪检测转染后SiHa细胞的凋亡率。
结果:转染pSIREN-16E7质粒后,SiHa细胞中E6、E7基因在mRNA和蛋白水平的表达均明显下调;MTT结果显示转染-16E7质粒后第三天细胞的存活率明显下降(40.5±3.2%),与转染pSIREN-luc对照质粒(98.7±5.09%)相比统计学上差异有显著性意义(P<0.05);流式细胞仪检测结果示转染pSIREN-16E7后细胞凋亡率(51.3±10.6%)与转染pSIREN-luc(9.5±2.8)%比较明显增加,其差异有统计学意义(P<0.05)。
结论:靶向HPV16-E7的短发夹RNA作为一种新型的基因治疗技术,可有效降低宫颈癌细胞中E6、E7癌基因的表达,抑制细胞增殖,诱导宫颈癌细胞发生凋亡。
PART I Antisense targeting human papillomavirus type 16 E6 andE7 genes contributes to apoptosis and senescence in SiHa cervical carcinoma cells
Objective: Human papillomavirus type 16 (HPV-16) is a high-risk DNA tumour virus involved in the development of cervical carcinomas. Substantial studies have demonstrated that E6 and E7 oncoproteins of HPV-16 could induce cell proliferation and immortalization. Repression of E6 and/or E7 oncogenes may induce cervical cancer cells to undergo apoptosis or senescence. The purpose of this study was to determine whether activation of the p53 and retinoblastoma (Rb) pathway by HPV-16 E6 and E7 repression was responsible for apoptosis and senescence of cervical cancer cells and to explore the potential of an antisense RNA (AS) transcript for gene therapy of cervical cancer.
Method: The antisense RNA directed against HPV-16 E6 and E7 (16AS) was constructed, and its effects on cell apoptosis and senescence of SiHa cervical carcinoma cells harboring HPV-16 were analyzed. The efficiency of 16AS was evaluated with RT-PCR, Western Blotting, flow cytometry analysis, Hoechst 33258 staining, senescent cell morphology observation and senescence associatedβ-galactosidase staining.
Results: The sufficient repression of HPV-16 E6 and E7 oncogenes were achieved in 16AS-transfected SiHa cells, which led to obvious apoptosis and replicative senescence of tumor cells. Furthermore, the downregulation of HPV16 E6 and E7 by 16AS transfection resulted in remarkable increase of both p53 expression and hypophosphorylated p105Rb level in SiHa cells.
Conclusion: These results demonstrate that reduction of E6 and E7 expression is sufficient to induce SiHa cells to undergo apoptosis and senescence, and suggest that transfection of cervical cancer cells with HPV-16 E6 and E7 antisense RNA is a potential approach to treat HPV-16 positive cervical cancers.
PART II Antisense targeting to human papillomavirus (HPV) 18 E6E7 affects the proliferation and apoptosis of human cervical carcinoma HeLa cells
Objective: The eukaryotic fluorescent expression vector carrying antisense human papillomavirus (HPV) 18 E6E7 was constructed and transfected into human cervical carcinoma HeLa cells to investigate its effect on the growth and prolifection of HeLa cells.
Methods: The HPV18 E6E7 716bp was amplified by PCR and then the PCR product was inversely inserted into pEGFP and contructed the recombinant eukaryotic expression plasmid pEGFP-HPV18E6E7as (EGFP-18AS). The recombinant was further transfected into HeLa cells. RT-PCR and western blot were respectively used to detect the mRNA or protein expression of HPV18 E6/E7 in the HeLa cells. The MTT method was performed to dynamically monitor the survived cells and the cell apoptosis was observed by flow cytometer and fluorescence microscope.
Results: The protein and mRNA expression levels of HPV18 E6/E7 in HeLa cells transfected with EGFP-18AS (HeLa/18AS) were both lower than those of the control groups including the HeLa cells transfected with EGFP (HeLa/EGFP) and untreated HeLa cells. The numbers of survived cells in HeLa/18AS cells became significantly lower than those of the control (all P<0.05). The rate of sub-G1 phase was also revealed, the apoptotic rate in HeLa/18AS cells was 47.21%, significantly higher than those of the HeLa/EGFP and untread HeLa cell (14.18% and 3.36% respectively, both P<0.05). Furthermore, an increased number of cells with chromosome condensation and fragmentation were found in HeLa/18AS cells as compared with HeLa/EGFP cells.
Conclusion: The recombinant pEGFP-HPV18E6E7as can effectively inhibit the growth and proliferation of human cervical carcinoma HeLa cells, and further induce the cell apoptosis. The antisense RNA technology was proved to be available, and it might provide a new way to gene therapy of the cervical carcinoma.
PART III RNA interference against HPV16 E7 oncogene leads to apoptosis in SiHa cervical carcinoma cells
Objective: Human papillomavirus type 16 (HPV16), a causative agent of cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a Small interfering RNA (siRNA) targeting HPV16 E7 region to degrade both E6, E6* and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. The shRNA that targets HPV16 E7 was tested in HPV6-positive cell lines to investigate its effect and investigate its mechanism of action.
Method: Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. To examine the effects of pSIREN-16E7 on the expression of E6 and E7 oncogenes and on the cell growth of HPV16-related cervical cancer cells, we applied RT-PCR, Western Blotting, MTT assay, the Annexin V apoptosis assay and flow cytometry in our study.
Results: Using human papillomavirus (HPV) 16-transformed cells as a model system, Our results indicated selective degradation of E6 and E7 mRNAs. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. HPV-negative cells appeared unaffected by the anti-viral siRNAs.
Conclusion: we demonstrated for the first time that HPV16 E7-specific targeting can induce simultaneous E6 and E7 suppression. Therefore, RNAi using E7 shRNA may have potential as a gene-specific therapy for HPV16-related cancers.
目的:人乳头瘤病毒16型(human papillomavirus 16, HPV16)的早期基因E6、E7可分别诱导p53的泛素化降解和pRb的高磷酸化失活,与宫颈癌的发生发展关系密切。本研究通过真核表达载体介导HPV16型E6、E7基因反义RNA在SiHa细胞中的表达,以探讨反义RNA能否能降低E6、E7基因的表达,诱导细胞凋亡或衰老发生。
方法:利用pEGFP构建HPV16型E6、E7基因反义RNA的真核表达载体并转染SiHa细胞,利用RT-PCR和Western blot技术检测转染后E6、E7基因的mRNA和蛋白的变化,利用MTT法检测SiHa细胞转染后细胞增殖活性,利用流式细胞仪和激光共聚焦显微镜检测转染后细胞的凋亡以及通过细胞形态学观察及β-gal染色来确定细胞衰老情况。
结果:转染携带HPV16型E6、E7反义RNA的质粒后,SiHa细胞的E6、E7基因的mRNA和蛋白均明显下调;MTT结果显示转染后细胞的增殖活性(0.502±0.050)与转染空载体细胞(1.010±0.055)和未转染细胞(1.283±0.058)比较明显降低(P<0.05);流式细胞仪检测结果示转染后细胞凋亡率(59.3±11.3)%与转染空载体细胞(9.4±1.8)%和未转染细胞(2.1±0.4)%比较有显著性差异(P<0.05)。激光共聚焦显微镜结果示转染后细胞凋亡明显增多;并且HPV16型E6、E7反义RNA使细胞形态学发生改变,细胞变大,变扁平,衰老特异性的β-gal染色阳性明显增多。
结论:HPV16型E6、E7基因反义RNA可降低宫颈癌细胞中E6、E7癌基因的表达,抑制细胞增殖,诱导宫颈癌细胞发生凋亡及衰老。
第二部分利用反义技术抑制HPV18 E6E7的表达及其对宫颈癌HeLa细胞增殖凋亡的影响
目的:构建HPV18型E6E7反义荧光真核表达载体,研究其对宫颈癌HeLa细胞增殖凋亡的影响。
方法:以PCR法扩增HPV18型E6E7区716bp片段并反向克隆到荧光真核表达载体pEGFP-C1,构建重组体pEGFP-HPV18E6E7as(EGFP-18AS)并转染人宫颈癌HeLa细胞。利用RT-PCR和Western blot技术检测转染后E6、E7基因在mRNA和蛋白水平的变化;用MTT法检测转染后细胞的的增殖活性;用流式细胞仪检测转染后细胞的凋亡率;荧光显微镜下观察凋亡细胞的形态学改变。
结果:转染EGFP-18AS重组体后,HeLa细胞中E6、E7基因的mRNA表达和蛋白合成均明显下调;细胞增殖受到抑制;流式显示细胞凋亡率增加;同时在荧光显微镜下经Hoechst染色凋亡细胞出现染色质凝集、边集、碎裂等形态学改变。
结论:重组质粒pEGFP-HPV18E6E7a(sEGFP-18AS)能有效地抑制人宫颈癌HeLa细胞的生长和增殖,诱导其凋亡,证明了反义RNA技术的有效性,为宫颈癌的基因治疗提供了一种新的可能的途径。
第三部分靶向HPV16-E7的短发夹RNA对人宫颈癌SiHa细胞的促凋亡作用
目的:HPV是宫颈癌发生的首要致病因素,其编码的E6和E7癌基因对宫颈病变的恶性转化及恶性表型的维持是至关重要的。针对E6和E7癌基因的分子治疗被证实是可行的,且同时下调E6和E7的表达将具有最有效的抗肿瘤的作用。本研究拟利用RNAi技术抑制E6和E7的表达并观察其对宫颈癌细胞的促凋亡作用。
方法:利用pSIREN-DNR构建靶向HPV16-E7的真核表达载体(pSIREN-16E7)并转染人宫颈癌SiHa细胞,利用RT-PCR和Western blot技术检测转染后E6、E7基因的mRNA和蛋白的变化,利用MTT法检测SiHa细胞转染后细胞增殖活性,利用流式细胞仪检测转染后SiHa细胞的凋亡率。
结果:转染pSIREN-16E7质粒后,SiHa细胞中E6、E7基因在mRNA和蛋白水平的表达均明显下调;MTT结果显示转染-16E7质粒后第三天细胞的存活率明显下降(40.5±3.2%),与转染pSIREN-luc对照质粒(98.7±5.09%)相比统计学上差异有显著性意义(P<0.05);流式细胞仪检测结果示转染pSIREN-16E7后细胞凋亡率(51.3±10.6%)与转染pSIREN-luc(9.5±2.8)%比较明显增加,其差异有统计学意义(P<0.05)。
结论:靶向HPV16-E7的短发夹RNA作为一种新型的基因治疗技术,可有效降低宫颈癌细胞中E6、E7癌基因的表达,抑制细胞增殖,诱导宫颈癌细胞发生凋亡。
PART I Antisense targeting human papillomavirus type 16 E6 andE7 genes contributes to apoptosis and senescence in SiHa cervical carcinoma cells
Objective: Human papillomavirus type 16 (HPV-16) is a high-risk DNA tumour virus involved in the development of cervical carcinomas. Substantial studies have demonstrated that E6 and E7 oncoproteins of HPV-16 could induce cell proliferation and immortalization. Repression of E6 and/or E7 oncogenes may induce cervical cancer cells to undergo apoptosis or senescence. The purpose of this study was to determine whether activation of the p53 and retinoblastoma (Rb) pathway by HPV-16 E6 and E7 repression was responsible for apoptosis and senescence of cervical cancer cells and to explore the potential of an antisense RNA (AS) transcript for gene therapy of cervical cancer.
Method: The antisense RNA directed against HPV-16 E6 and E7 (16AS) was constructed, and its effects on cell apoptosis and senescence of SiHa cervical carcinoma cells harboring HPV-16 were analyzed. The efficiency of 16AS was evaluated with RT-PCR, Western Blotting, flow cytometry analysis, Hoechst 33258 staining, senescent cell morphology observation and senescence associatedβ-galactosidase staining.
Results: The sufficient repression of HPV-16 E6 and E7 oncogenes were achieved in 16AS-transfected SiHa cells, which led to obvious apoptosis and replicative senescence of tumor cells. Furthermore, the downregulation of HPV16 E6 and E7 by 16AS transfection resulted in remarkable increase of both p53 expression and hypophosphorylated p105Rb level in SiHa cells.
Conclusion: These results demonstrate that reduction of E6 and E7 expression is sufficient to induce SiHa cells to undergo apoptosis and senescence, and suggest that transfection of cervical cancer cells with HPV-16 E6 and E7 antisense RNA is a potential approach to treat HPV-16 positive cervical cancers.
PART II Antisense targeting to human papillomavirus (HPV) 18 E6E7 affects the proliferation and apoptosis of human cervical carcinoma HeLa cells
Objective: The eukaryotic fluorescent expression vector carrying antisense human papillomavirus (HPV) 18 E6E7 was constructed and transfected into human cervical carcinoma HeLa cells to investigate its effect on the growth and prolifection of HeLa cells.
Methods: The HPV18 E6E7 716bp was amplified by PCR and then the PCR product was inversely inserted into pEGFP and contructed the recombinant eukaryotic expression plasmid pEGFP-HPV18E6E7as (EGFP-18AS). The recombinant was further transfected into HeLa cells. RT-PCR and western blot were respectively used to detect the mRNA or protein expression of HPV18 E6/E7 in the HeLa cells. The MTT method was performed to dynamically monitor the survived cells and the cell apoptosis was observed by flow cytometer and fluorescence microscope.
Results: The protein and mRNA expression levels of HPV18 E6/E7 in HeLa cells transfected with EGFP-18AS (HeLa/18AS) were both lower than those of the control groups including the HeLa cells transfected with EGFP (HeLa/EGFP) and untreated HeLa cells. The numbers of survived cells in HeLa/18AS cells became significantly lower than those of the control (all P<0.05). The rate of sub-G1 phase was also revealed, the apoptotic rate in HeLa/18AS cells was 47.21%, significantly higher than those of the HeLa/EGFP and untread HeLa cell (14.18% and 3.36% respectively, both P<0.05). Furthermore, an increased number of cells with chromosome condensation and fragmentation were found in HeLa/18AS cells as compared with HeLa/EGFP cells.
Conclusion: The recombinant pEGFP-HPV18E6E7as can effectively inhibit the growth and proliferation of human cervical carcinoma HeLa cells, and further induce the cell apoptosis. The antisense RNA technology was proved to be available, and it might provide a new way to gene therapy of the cervical carcinoma.
PART III RNA interference against HPV16 E7 oncogene leads to apoptosis in SiHa cervical carcinoma cells
Objective: Human papillomavirus type 16 (HPV16), a causative agent of cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a Small interfering RNA (siRNA) targeting HPV16 E7 region to degrade both E6, E6* and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. The shRNA that targets HPV16 E7 was tested in HPV6-positive cell lines to investigate its effect and investigate its mechanism of action.
Method: Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. To examine the effects of pSIREN-16E7 on the expression of E6 and E7 oncogenes and on the cell growth of HPV16-related cervical cancer cells, we applied RT-PCR, Western Blotting, MTT assay, the Annexin V apoptosis assay and flow cytometry in our study.
Results: Using human papillomavirus (HPV) 16-transformed cells as a model system, Our results indicated selective degradation of E6 and E7 mRNAs. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. HPV-negative cells appeared unaffected by the anti-viral siRNAs.
Conclusion: we demonstrated for the first time that HPV16 E7-specific targeting can induce simultaneous E6 and E7 suppression. Therefore, RNAi using E7 shRNA may have potential as a gene-specific therapy for HPV16-related cancers.
引文
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10. Cho C W, Poo H, Cho Y S, et al. HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing [J]. Exp Mol Med, 2002,34(2):159-166.
11.牛晓宇,彭芝兰,王和. RNA干扰抑制宫颈癌Caski细胞株HPV16 E6基因的研究[J].癌症, 2004,23(11): 1257-1262.
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4.陈庆云,卞美璐,陈志华,等.宫颈癌癌前病变中人乳头状瘤病毒16整合状态的检测.中华医学杂志, 2005,85:400-404.
5. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer, 2002,2:342-350.
6.曹雪涛,顾健人,刘德培,等.我国基因治疗的研究前景与战略重点.中华医学杂志, 2001,81:705-708.
7. Shillitoe EJ. Papillomaviruses as targets for cancer gene therapy. Cancer Gene Ther, 2006,13:445-450.
8. DiPaolo JA, Alvarez-Salas LM. Advances in the development of therapeutic nucleic acids against cervical cancer. Expert Opin Biol Ther, 2004,4:1251-1264.
9. Choo CK, Ling MT, Suen CK et al. Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells. Gynecol Oncol, 2000,78:293-301.
10. Wu S, Meng L, Wang S, et al. Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA. Clin Cancer Res, 2006,12:2032-2037.
11. Sherman L, Alloul N. Human papillomavirus type 16 expresses a variety of alternatively spliced mRNAs putatively encoding the E2 protein. Virology 1992;191: 953-9.
1. Parkin, D. M., Bray, F., Ferlay, J., and Pisani, P. (2005). Global cancer statistics, 2002. CA Cancer J. Clin. 55: 74-108.
2. Walboomers J. M., Jacobs M. V., Manos M. M., Bosch F. X., Kummer J. A., Shah K. V. et al. (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189: 12-19.
3. Bosch FX, Lorincz A, Munoz N, et al. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol, 2002, 55(4): 244-265.
4. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990; 248: 76-9.
5. Munger K, Basile JR, Duensing S, Eichten A, Gonzalez SL, Grace M, Zacny VL. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene 2001; 20: 7888-98.
6. He Y, Huang L. Growth inhibition of human papillomavirus 16 DNA-positive mouse tumor by antisense RNA transcribed from U6 promoter. Cancer Res 1997;57: 3993-9.
7. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411: 494-8.
8. Sherman L, Alloul N. Human papillomavirus type 16 expresses a variety of alternatively spliced mRNAs putatively encoding the E2 protein. Virology 1992; 191: 953-9.
9. Yoshinouchi M, Yamada T, Kizaki M, Fen J, Koseki T, Ikeda Y, Nishihara T, Yamato K. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol Ther 2003;8: 762-8.
10. Gu W, Putral L, Hengst K, Minto K, Saunders NA, Leggatt G, McMillan NA. Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes.Cancer Gene Ther 2006;13: 1023-32.
11. Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 2002;21: 6041-8.
[1] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55: 74-108.
[2] Waggoner SE. Cervical cancer. Lancet 2003;361: 2217-25.
[3] Munger K, Baldwin A, Edwards KM, Hayakawa H, Nguyen CL, Owens M, Grace M, Huh K. Mechanisms of human papillomavirus-induced oncogenesis. J Virol 2004;78: 11451-60.
[4] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189: 12-9.
[5] Lai HC, Sun CA, Yu MH, Chen HJ, Liu HS, Chu TY. Favorable clinical outcome of cervical cancers infected with human papilloma virus type 58 and related types. Int J Cancer 1999;84: 553-7.
[6] Stoler MH. Human papillomaviruses and cervical neoplasia: a model for carcinogenesis. Int J Gynecol Pathol 2000;19: 16-28.
[7] Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348: 518-27.
[8] Cornelison TL. Human papillomavirus genotype 16 vaccines for cervical cancer prophylaxis and treatment. Curr Opin Oncol 2000;12: 466-73.
[9] zur Hausen H. Viruses in human cancers. Science 1991;254: 1167-73.
[10] Crum CP. Contemporary theories of cervical carcinogenesis: the virus, the host, and the stem cell. Mod Pathol 2000;13: 243-51.
[11] Marchetti B, Ashrafi GH, Tsirimonaki E, O'Brien PM, Campo MS. The bovine papillomavirus oncoprotein E5 retains MHC class I molecules in the Golgi apparatus and prevents their transport to the cell surface. Oncogene 2002;21: 7808-16.
[12] Finley D, Ciechanover A, Varshavsky A. Ubiquitin as a central cellular regulator. Cell1984;37: 43-55.
[13] Tommasino M, Accardi R, Caldeira S, Dong W, Malanchi I, Smet A, Zehbe I. The role of TP53 in Cervical carcinogenesis. Hum Mutat 2003;21: 307-12.
[14] Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, Breuer J, Leigh IM, Matlashewski G, Banks L. Role of a p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 1998;393: 229-34.
[15] Singh L, Gao Q, Kumar A, Gotoh T, Wazer DE, Band H, Feig LA, Band V. The high-risk human papillomavirus type 16 E6 counters the GAP function of E6TP1 toward small Rap G proteins. J Virol 2003;77: 1614-20.
[16] Gao Q, Srinivasan S, Boyer SN, Wazer DE, Band V. The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation. Mol Cell Biol 1999;19: 733-44.
[17] Ronco LV, Karpova AY, Vidal M, Howley PM. Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes & Development 1998;12: 2061-2072.
[18] Burkhart BA, Alcorta DA, Chiao C, Isaacs JS, Barrett JC. Two Posttranscriptional Pathways That Regulate p21Cip1Waf1Sdi1 Are Identified by HPV16-E6 Interaction and Correlate with Life Span and Cellular Senescence. Experimental Cell Research 1999;247: 168-175.
[19] Mergny JL, Riou JF, Mailliet P, Teulade-Fichou MP, Gilson E. Natural and pharmacological regulation of telomerase. Nucleic Acids Research 2002;30: 839-865.
[20] Ohta Y, Tsutsumi K, Kikuchi K, Yasumoto S. Two distinct human uterine cervical epithelial cell lines established after transfection with human papillomavirus 16 DNA. Jpn J Cancer Res 1997;88: 644-51.
[21] Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266: 2011.
[22] Harley CB. Telomere loss: mitotic clock or genetic time bomb? Mutat Res 1991;256: 271-82.
[23] Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345: 458-460.
[24] Das K, Bohl J, Vande Pol SB. Identification of a second transforming function in bovine papillomavirus type 1 E6 and the role of E6 interactions with paxillin, E6BP, and E6AP. J Virol 2000;74: 812-6.
[25] Borysiewicz LK, Fiander A, Nimako M, Man S, Wilkinson GW, Westmoreland D, Evans AS, Adams M, Stacey SN, Boursnell ME. A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 1996;347: 1523-7.
[26] Rosales C, Graham VV, Rosas GA, Merchant H, Rosales R. A recombinant vaccinia virus containing the papilloma E2 protein promotes tumor regression by stimulating macrophage antibody-dependent cytotoxicity. Cancer Immunol Immunother 2000;49: 347-60.
[27] Cope JU, Hildesheim A, Schiffman MH, Manos MM, Lorincz AT, Burk RD, Glass AG, Greer C, Buckland J, Helgesen K. Comparison of the hybrid capture tube test and PCR for detection of human papillomavirus DNA in cervical specimens. J Clin Microbiol 1997;35: 2262-5.
[28] Harro CD, Pang YY, Roden RB, Hildesheim A, Wang Z, Reynolds MJ, Mast TC, Robinson R, Murphy BR, Karron RA. Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine. J Natl Cancer Inst 2001;93: 284-92.
[29] Jochmus I, Schafer K, Faath S, Muller M, Gissmann L. Chimeric virus-like particles of the human papillomavirus type 16 (HPV 16) as a prophylactic and therapeutic vaccine. Arch Med Res 1999;30: 269-74.
[30] De Marco F, Hallez S, Brulet JM, Gesche F, Marzano P, Flamini S, Marcante ML,Venuti A. DNA vaccines against HPV-16 E7-expressing tumour cells. Anticancer Res 2003;23: 1449-54.
[31] He Z, Wlazlo AP, Kowalczyk DW, Cheng J, Xiang ZQ, Giles-Davis W, Ertl HC. Viral recombinant vaccines to the E6 and E7 antigens of HPV-16. Virology 2000;270: 146-161.
[32] Wakabayashi MT, Da Silva DM, Potkul RK, Kast WM. Comparison of Human Papillomavirus Type 16 L1 Chimeric Virus-Like Particles versus L1/L2 Chimeric Virus-Like Particles in Tumor Prevention. Intervirology 2002;45: 300-307.
[33] Ling M, Kanayama M, Roden R, Wu TC. Preventive and therapeutic vaccines for human papillomavirus-associated cervical cancers. Journal of Biomedical Science 2000;7: 341-356.
[34] Santin AD, Hermonat PL, Ravaggi A, Bellone S, Roman JJ, Jayaprabhu S, Pecorelli S, Parham GP, Cannon MJ. Expression of CD56 by human papillomavirus E7-specific CD8+ cytotoxic T lymphocytes correlates with increased intracellular perforin expression and enhanced cytotoxicity against HLA-A2-matched cervical tumor cells. Clin Cancer Res 2001;7: 804s-810s.
[35] Santin AD, Hermonat PL, Ravaggi A, Chiriva-Internati M, Zhan D, Pecorelli S, Parham GP, Cannon MJ. Induction of Human Papillomavirus-Specific CD4+ and CD8+ Lymphocytes by E7-Pulsed Autologous Dendritic Cells in Patients with Human Papillomavirus Type 16-and 18-Positive Cervical Cancer. Journal of Virology 1999;73: 5402-5410.
[36] Thornburg C, Boczkowski D, Gilboa E, Nair SK. Induction of cytotoxic T lymphocytes with dendritic cells transfected with human papillomavirus E6 and E7 RNA: implications for cervical cancer immunotherapy. J Immunother 2000;23: 412-418.
[37] Rossi JL, Gissman L, Jansen K, Muller M. Assembly of infectious human papillomavirus type 16 virus-like particle in Saccharomyces cerevisiae. Hum Gene Ther 2000;11: 1165-1176.
2. Ledwaba T, Dlamini Z, Naicker S, et al. Molecular genetics of human cervical cancer: role of papillomavirus and the apoptotic cascade [J]. Biol Chem, 2004,385(8):671-682.
3. Cho CW, Poo H, Cho YS, Cho MC, Lee KA, Lee SJ, Park SN, Kim IK, Jung YK, Choe YK, Yeom YI, Choe IS, Yoon DY. HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing. Exp Mol Med 2002; 34: 159-66.
4. DeFilippis RA, Goodwin EC, Wu L, DiMaio D. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. J Virol 2003; 77: 1551-63.
5. Rorke E A. Antisense human papillomavirus (HPV) E6/E7 expression, reduced stability of epidermal growth factor, and diminished growth of HPV-positive tumor cells [J]. J Natl Cancer Inst, 1997, 89(17):1243-1246.
6. Burd E M. Human papillomavirus and cervical cancer [J]. Clin Microbiol Rev, 2003,16(1):1-17.
7. Nomine Y, Masson M, Charbonnier S, et al. Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirus-mediated pathogenesis [J]. Mol Cell, 2006, 21(5):665-678.
8. DiPaolo J A, Alvarez-Salas L M. Advances in the development of therapeutic nucleic acids against cervical cancer [J]. Expert Opin Biol Ther, 2004,4(8):1251-1264.
9. Choo C K, Ling M T, Suen C K, et al. Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells [J]. Gynecol Oncol, 2000,78(3 Pt 1):293-301.
10. Cho C W, Poo H, Cho Y S, et al. HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing [J]. Exp Mol Med, 2002,34(2):159-166.
11.牛晓宇,彭芝兰,王和. RNA干扰抑制宫颈癌Caski细胞株HPV16 E6基因的研究[J].癌症, 2004,23(11): 1257-1262.
12. Wu S, Meng L, Wang S, et al. Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA[J]. Clin Cancer Res, 2006, 12 (7 Pt 1): 2032-2037.
1. Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer, 1999,80:827-841.
2. Andersson S, Rylander E, Larsson B et al. The role of human papillomavirus in cervical adenocarcinoma carcinogenesis. Eur J Cancer, 2001,37:246-250.
3. Burd EM. Human papillomavirus and cervical cancer. Clin Microbiol Rev, 2003,16:1-17.
4.陈庆云,卞美璐,陈志华,等.宫颈癌癌前病变中人乳头状瘤病毒16整合状态的检测.中华医学杂志, 2005,85:400-404.
5. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer, 2002,2:342-350.
6.曹雪涛,顾健人,刘德培,等.我国基因治疗的研究前景与战略重点.中华医学杂志, 2001,81:705-708.
7. Shillitoe EJ. Papillomaviruses as targets for cancer gene therapy. Cancer Gene Ther, 2006,13:445-450.
8. DiPaolo JA, Alvarez-Salas LM. Advances in the development of therapeutic nucleic acids against cervical cancer. Expert Opin Biol Ther, 2004,4:1251-1264.
9. Choo CK, Ling MT, Suen CK et al. Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells. Gynecol Oncol, 2000,78:293-301.
10. Wu S, Meng L, Wang S, et al. Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA. Clin Cancer Res, 2006,12:2032-2037.
11. Sherman L, Alloul N. Human papillomavirus type 16 expresses a variety of alternatively spliced mRNAs putatively encoding the E2 protein. Virology 1992;191: 953-9.
1. Parkin, D. M., Bray, F., Ferlay, J., and Pisani, P. (2005). Global cancer statistics, 2002. CA Cancer J. Clin. 55: 74-108.
2. Walboomers J. M., Jacobs M. V., Manos M. M., Bosch F. X., Kummer J. A., Shah K. V. et al. (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189: 12-19.
3. Bosch FX, Lorincz A, Munoz N, et al. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol, 2002, 55(4): 244-265.
4. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990; 248: 76-9.
5. Munger K, Basile JR, Duensing S, Eichten A, Gonzalez SL, Grace M, Zacny VL. Biological activities and molecular targets of the human papillomavirus E7 oncoprotein. Oncogene 2001; 20: 7888-98.
6. He Y, Huang L. Growth inhibition of human papillomavirus 16 DNA-positive mouse tumor by antisense RNA transcribed from U6 promoter. Cancer Res 1997;57: 3993-9.
7. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411: 494-8.
8. Sherman L, Alloul N. Human papillomavirus type 16 expresses a variety of alternatively spliced mRNAs putatively encoding the E2 protein. Virology 1992; 191: 953-9.
9. Yoshinouchi M, Yamada T, Kizaki M, Fen J, Koseki T, Ikeda Y, Nishihara T, Yamato K. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol Ther 2003;8: 762-8.
10. Gu W, Putral L, Hengst K, Minto K, Saunders NA, Leggatt G, McMillan NA. Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes.Cancer Gene Ther 2006;13: 1023-32.
11. Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 2002;21: 6041-8.
[1] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55: 74-108.
[2] Waggoner SE. Cervical cancer. Lancet 2003;361: 2217-25.
[3] Munger K, Baldwin A, Edwards KM, Hayakawa H, Nguyen CL, Owens M, Grace M, Huh K. Mechanisms of human papillomavirus-induced oncogenesis. J Virol 2004;78: 11451-60.
[4] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189: 12-9.
[5] Lai HC, Sun CA, Yu MH, Chen HJ, Liu HS, Chu TY. Favorable clinical outcome of cervical cancers infected with human papilloma virus type 58 and related types. Int J Cancer 1999;84: 553-7.
[6] Stoler MH. Human papillomaviruses and cervical neoplasia: a model for carcinogenesis. Int J Gynecol Pathol 2000;19: 16-28.
[7] Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348: 518-27.
[8] Cornelison TL. Human papillomavirus genotype 16 vaccines for cervical cancer prophylaxis and treatment. Curr Opin Oncol 2000;12: 466-73.
[9] zur Hausen H. Viruses in human cancers. Science 1991;254: 1167-73.
[10] Crum CP. Contemporary theories of cervical carcinogenesis: the virus, the host, and the stem cell. Mod Pathol 2000;13: 243-51.
[11] Marchetti B, Ashrafi GH, Tsirimonaki E, O'Brien PM, Campo MS. The bovine papillomavirus oncoprotein E5 retains MHC class I molecules in the Golgi apparatus and prevents their transport to the cell surface. Oncogene 2002;21: 7808-16.
[12] Finley D, Ciechanover A, Varshavsky A. Ubiquitin as a central cellular regulator. Cell1984;37: 43-55.
[13] Tommasino M, Accardi R, Caldeira S, Dong W, Malanchi I, Smet A, Zehbe I. The role of TP53 in Cervical carcinogenesis. Hum Mutat 2003;21: 307-12.
[14] Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, Breuer J, Leigh IM, Matlashewski G, Banks L. Role of a p53 polymorphism in the development of human papillomavirus-associated cancer. Nature 1998;393: 229-34.
[15] Singh L, Gao Q, Kumar A, Gotoh T, Wazer DE, Band H, Feig LA, Band V. The high-risk human papillomavirus type 16 E6 counters the GAP function of E6TP1 toward small Rap G proteins. J Virol 2003;77: 1614-20.
[16] Gao Q, Srinivasan S, Boyer SN, Wazer DE, Band V. The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation. Mol Cell Biol 1999;19: 733-44.
[17] Ronco LV, Karpova AY, Vidal M, Howley PM. Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes & Development 1998;12: 2061-2072.
[18] Burkhart BA, Alcorta DA, Chiao C, Isaacs JS, Barrett JC. Two Posttranscriptional Pathways That Regulate p21Cip1Waf1Sdi1 Are Identified by HPV16-E6 Interaction and Correlate with Life Span and Cellular Senescence. Experimental Cell Research 1999;247: 168-175.
[19] Mergny JL, Riou JF, Mailliet P, Teulade-Fichou MP, Gilson E. Natural and pharmacological regulation of telomerase. Nucleic Acids Research 2002;30: 839-865.
[20] Ohta Y, Tsutsumi K, Kikuchi K, Yasumoto S. Two distinct human uterine cervical epithelial cell lines established after transfection with human papillomavirus 16 DNA. Jpn J Cancer Res 1997;88: 644-51.
[21] Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266: 2011.
[22] Harley CB. Telomere loss: mitotic clock or genetic time bomb? Mutat Res 1991;256: 271-82.
[23] Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345: 458-460.
[24] Das K, Bohl J, Vande Pol SB. Identification of a second transforming function in bovine papillomavirus type 1 E6 and the role of E6 interactions with paxillin, E6BP, and E6AP. J Virol 2000;74: 812-6.
[25] Borysiewicz LK, Fiander A, Nimako M, Man S, Wilkinson GW, Westmoreland D, Evans AS, Adams M, Stacey SN, Boursnell ME. A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 1996;347: 1523-7.
[26] Rosales C, Graham VV, Rosas GA, Merchant H, Rosales R. A recombinant vaccinia virus containing the papilloma E2 protein promotes tumor regression by stimulating macrophage antibody-dependent cytotoxicity. Cancer Immunol Immunother 2000;49: 347-60.
[27] Cope JU, Hildesheim A, Schiffman MH, Manos MM, Lorincz AT, Burk RD, Glass AG, Greer C, Buckland J, Helgesen K. Comparison of the hybrid capture tube test and PCR for detection of human papillomavirus DNA in cervical specimens. J Clin Microbiol 1997;35: 2262-5.
[28] Harro CD, Pang YY, Roden RB, Hildesheim A, Wang Z, Reynolds MJ, Mast TC, Robinson R, Murphy BR, Karron RA. Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine. J Natl Cancer Inst 2001;93: 284-92.
[29] Jochmus I, Schafer K, Faath S, Muller M, Gissmann L. Chimeric virus-like particles of the human papillomavirus type 16 (HPV 16) as a prophylactic and therapeutic vaccine. Arch Med Res 1999;30: 269-74.
[30] De Marco F, Hallez S, Brulet JM, Gesche F, Marzano P, Flamini S, Marcante ML,Venuti A. DNA vaccines against HPV-16 E7-expressing tumour cells. Anticancer Res 2003;23: 1449-54.
[31] He Z, Wlazlo AP, Kowalczyk DW, Cheng J, Xiang ZQ, Giles-Davis W, Ertl HC. Viral recombinant vaccines to the E6 and E7 antigens of HPV-16. Virology 2000;270: 146-161.
[32] Wakabayashi MT, Da Silva DM, Potkul RK, Kast WM. Comparison of Human Papillomavirus Type 16 L1 Chimeric Virus-Like Particles versus L1/L2 Chimeric Virus-Like Particles in Tumor Prevention. Intervirology 2002;45: 300-307.
[33] Ling M, Kanayama M, Roden R, Wu TC. Preventive and therapeutic vaccines for human papillomavirus-associated cervical cancers. Journal of Biomedical Science 2000;7: 341-356.
[34] Santin AD, Hermonat PL, Ravaggi A, Bellone S, Roman JJ, Jayaprabhu S, Pecorelli S, Parham GP, Cannon MJ. Expression of CD56 by human papillomavirus E7-specific CD8+ cytotoxic T lymphocytes correlates with increased intracellular perforin expression and enhanced cytotoxicity against HLA-A2-matched cervical tumor cells. Clin Cancer Res 2001;7: 804s-810s.
[35] Santin AD, Hermonat PL, Ravaggi A, Chiriva-Internati M, Zhan D, Pecorelli S, Parham GP, Cannon MJ. Induction of Human Papillomavirus-Specific CD4+ and CD8+ Lymphocytes by E7-Pulsed Autologous Dendritic Cells in Patients with Human Papillomavirus Type 16-and 18-Positive Cervical Cancer. Journal of Virology 1999;73: 5402-5410.
[36] Thornburg C, Boczkowski D, Gilboa E, Nair SK. Induction of cytotoxic T lymphocytes with dendritic cells transfected with human papillomavirus E6 and E7 RNA: implications for cervical cancer immunotherapy. J Immunother 2000;23: 412-418.
[37] Rossi JL, Gissman L, Jansen K, Muller M. Assembly of infectious human papillomavirus type 16 virus-like particle in Saccharomyces cerevisiae. Hum Gene Ther 2000;11: 1165-1176.