E1A基因对鼻咽癌放射增敏作用及机制研究
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摘要
目的探讨腺病毒E1A基因对人鼻咽癌CNE-2Z细胞放射治疗的增敏作用及相关机制。
方法选用人鼻咽癌CNE-2Z细胞为靶细胞,分别用PBS,Ad-β-gal, Ad-E1A作用48小时后,用6MVX线分别给与0、2、4、6和8Gy剂量照射后,用MTT法和流式细胞仪检测三组细胞成活率及细胞周期变化,并进行克隆分析绘制细胞存活曲线,计算放射增敏比来观察E1A基因对人鼻咽癌CNE-2Z细胞放射敏感性的影响,同时用RT-PCR法检测三组细胞的VEGF水平的表达及野生型p53的表达。细胞免疫化学方法检测三组细胞的VEGF蛋白的表达。
结果RT-PCR结果显示Ad-E1A组可以检测到E1A基因的表达。经照射后Ad-E1A组的细胞存活率明显低于PBS对照组和Ad-β-gal对照组。Ad-E1A组的细胞的生长速度明显慢于PBS对照组和Ad-β-gal对照组。细胞存活曲线结果显示Ad-E1A组的CNE-2Z细胞放射增敏比分别为1.37(D0值比)、1.95(Dq值比)、1.46(SF2比)。PBS对照组和Ad-β-gal对照组的细胞照射后细胞存活曲线分析结果显示无差异,D0、Dq、SF2值分别为1.57Gy及1.53Gy、1.82Gy及1.78Gy、0.89及0.82。RT-PCR结果和细胞免疫化学结果显示Ad-E1A组比PBS对照组和Ad-β-gal对照组的人鼻咽癌CNE-2Z细胞的VEGF的表达明显下降,RT-PCR结果显示Ad-E1A组比PBS对照组和Ad-β-gal对照组的野生型p53水平明显上升。流式细胞学实验显示Ad-E1A组+放射治疗组细胞的凋亡明显高于其他组。
结论E1A基因通过下调人鼻咽癌CNE-2Z细胞VEGF的表达和上调野生型p53的表达来增加人鼻咽癌细胞对放射治疗的敏感性。
目的：探讨腺病毒E1A基因对人鼻咽癌动物模型放射增敏的实验研究。
方法：取对数生长期的CNE-2Z细胞5×105/0.2mL,接种于4周龄左右裸鼠右前肢腋部皮下致瘤,第7天裸鼠皮下肿瘤长至直径0.6-0.8cm时,随机分为6组(每组10个)：PBS组,Ad-β-gal组,放射组,Ad-β-gal+放射组,Ad-E1A组,Ad-E1A+放射组。Ad-β-gal组/Ad-E1A组在第2周开始给予荷瘤裸鼠皮下肿瘤内滴度为5×109PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,连续2周,放射组在第3周给予6MV-X照射,每天2Gy,连续5天。Ad-β-gal+放射组/Ad-E1A+放射组在第2周开始,给予荷瘤裸鼠皮下肿瘤内滴度为5×109 PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,连续2周,在第3周给予6MV-X照射,每天2Gy,连续5天。第1次治疗后,每隔4天用卡尺测量1次肿瘤的体积,记录其直径,裸鼠的生存时间。当肿瘤的直径超过2cm时,处死裸鼠,分离肿瘤组织,采用免疫组织化学方法分析VEGF和CD34表达,RT-PCR分析wtp53的表达,Western blot分析VEGF的表达,TUNEL分析细胞凋亡。
结果：Ad-E1A+放射组较其他组能明显延缓移植瘤生长时间,Ad-E1A+放射组裸鼠的肿瘤平均体积比单独放射组小4.7倍,比单独Ad-E1A组的小5.3倍。Ad-E1A+放射组的裸鼠生存率显著高于其他治疗组。Ad-E1A+放射组的VEGF蛋白表达和微血管密度较其他各组明显下降。(P<0.01)。Ad-E1A组和Ad-E1A+放射组的wtp53基因表达明显上调。TUNEL分析结果显示Ad-E1A组和Ad-E1A+放射组及放射组的肿瘤组织内均可明显观察到凋亡细胞。并且,Ad-E1A+放射组肿瘤细胞凋亡数目明显高于Ad-E1A组或单独放射组。
结论：E1A基因通过抑制肿瘤血管的形成和上调肿瘤组织中wtp53的表达及诱导肿瘤细胞的凋亡来提高人鼻咽癌细胞对放射的敏感性。
Objective:The purpose of this study determined the effect and mechanism of Ad-E1A gene therapy in vitro radiosensitivity to nasopharyngeal carcinoma cell.
Methods:The human nasopharyngeal carcinoma CNE-2Z cell lines were investigated. The recombinant adenovirus vector containing E1A gene was used for this study. After CNE-2Z cells was treated with PBS, Ad-β-gal and Ad-E1A for 48h, the three groups were irradiated in different doses at 0、2、4、6 and 8Gy, The cytotoxicity was determined by MTT assay and cell cycle was analysis by flow cytometry. Clone forming assays were carried out, cell survival curves were drawn and the sensitivity enhancing ratio (SER) was calculated. The VEGF expression was evaluated by RT-PCR assay and immunocytochemical analysis. The wtp53 express was evaluated by RT-PCR.
Results:RT-PCR confirmed that E1A gene had been integrated into positively transfected cells and stably expressed. Significant cell deaths by IR were observed in a dose dependent manner in the three group CNE-2Z cells. After transduction of the E1A gene into CNE-2Z cells, the sensitivity of these cells to radiation was enhanced than the PBS treated group and Ad-β-gal treated group. Cell growth inhibition in Ad-E1A treated group by IR was strongly enhanced than Ad-β-gal treated group and PBS treated group. Cell survival curves showed that the SER of Do, Dq and SF2 value was 1.37,1.95 and 1.46 with Ad-E1A treated group The Do, Dq and SF2 value was 1.57Gy,1.82Gy,0.89 in PBS treated group and 1.53Gy,1.78Gy,0.82 in Ad-β-gal treated group,respectively. RT-PCR assay and immunocytochemical analysis showed VEGF expression was downregulation in Ad-E1A treated group. Moreover, the expression of wtp53 gene was markedly enhanced in Ad-E1A treated group. The Flow cytometry showed more apotosis can be detected in the cells treated with Ad-E1A plus radiotherapy than those of the other treated groups.
Conclusions:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma cell sensitivity to the radiotherapy by down regulated VEGF expression and enhanced the expression of wtp53.
Objective:To determine the effect of Ad-E1A gene therapy in vivo radiosensitivity to nasopharyngeal carcinoma.
Methods:CNE-2Z cells (2 x 105) were injected subcutaneously into nude mice resulted in tumor development (1-3 mm) 6 days later. The tumor-bearing mice were then randomly divided into six groups (10 mice per group) for PBS treatment or treatment with radiotherapy, Ad-E1 A, or Ad-β-gal alone or radiotherapy in combination with Ad-E1A or Ad-β-gal. Animals were treated with Ad-E1A or Ad-β-gal (5 x 109 plaque forming units) by intratumoral injection twice weekly for 2 weeks at beginning of week 2. Animals treated with radiotherapy in combination with Ad-E1A or Ad-β-gal were received 2 Gy radiotherapy daily for 5 days following the first week of treatment with Ad-E1A or Ad-β-gal. Control animals received PBS therapy or radiotherapy only after tumor cells were injected. When the sizes of tumors exceeded 2 cm, the mice were killed and the tumors underwent immunohistochemical analysis for VEGF and CD34 expression and RT-PCR analysis for wtp53 expression and TUNEL assay for apoptosis and Western blot analysis for VEGF expression
Results:The growth delay time (TGD) was longest in the Ad-E1A plus radiotherapy group. Tumors treated with Ad-E1A plus radiotherapy were 4.7-fold smaller than those treated with radiotherapy alone and 5.3-fold smaller than those treated with Ad-E1A alone. The survival rate of tumor-bearing mice treated with Ad-E1A plus radiotherapy was significantly higher than that of other treated groups. The vessel density and the VEGF expression were significantly lower in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-E1A alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy (P<0.01). wtp53 expression were significantly increased in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy. TUNEL staining revealing apoptosis can be detected in Ad-E1A group, radiotherapy group, Ad-E1A plus radiotherapy group, and more apoptosis can be detected in the tumors treated with Ad-E1A plus radiotherapy than those of the other treated groups.
Conclusion:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma sensitivity to the radiotherapy by down-regulating VEGF expression increasing wtp53 expression and inducing apoptosis. These findings may pave the way for efficient radiation-gene therapy to NPC in future.
方法选用人鼻咽癌CNE-2Z细胞为靶细胞,分别用PBS,Ad-β-gal, Ad-E1A作用48小时后,用6MVX线分别给与0、2、4、6和8Gy剂量照射后,用MTT法和流式细胞仪检测三组细胞成活率及细胞周期变化,并进行克隆分析绘制细胞存活曲线,计算放射增敏比来观察E1A基因对人鼻咽癌CNE-2Z细胞放射敏感性的影响,同时用RT-PCR法检测三组细胞的VEGF水平的表达及野生型p53的表达。细胞免疫化学方法检测三组细胞的VEGF蛋白的表达。
结果RT-PCR结果显示Ad-E1A组可以检测到E1A基因的表达。经照射后Ad-E1A组的细胞存活率明显低于PBS对照组和Ad-β-gal对照组。Ad-E1A组的细胞的生长速度明显慢于PBS对照组和Ad-β-gal对照组。细胞存活曲线结果显示Ad-E1A组的CNE-2Z细胞放射增敏比分别为1.37(D0值比)、1.95(Dq值比)、1.46(SF2比)。PBS对照组和Ad-β-gal对照组的细胞照射后细胞存活曲线分析结果显示无差异,D0、Dq、SF2值分别为1.57Gy及1.53Gy、1.82Gy及1.78Gy、0.89及0.82。RT-PCR结果和细胞免疫化学结果显示Ad-E1A组比PBS对照组和Ad-β-gal对照组的人鼻咽癌CNE-2Z细胞的VEGF的表达明显下降,RT-PCR结果显示Ad-E1A组比PBS对照组和Ad-β-gal对照组的野生型p53水平明显上升。流式细胞学实验显示Ad-E1A组+放射治疗组细胞的凋亡明显高于其他组。
结论E1A基因通过下调人鼻咽癌CNE-2Z细胞VEGF的表达和上调野生型p53的表达来增加人鼻咽癌细胞对放射治疗的敏感性。
目的：探讨腺病毒E1A基因对人鼻咽癌动物模型放射增敏的实验研究。
方法：取对数生长期的CNE-2Z细胞5×105/0.2mL,接种于4周龄左右裸鼠右前肢腋部皮下致瘤,第7天裸鼠皮下肿瘤长至直径0.6-0.8cm时,随机分为6组(每组10个)：PBS组,Ad-β-gal组,放射组,Ad-β-gal+放射组,Ad-E1A组,Ad-E1A+放射组。Ad-β-gal组/Ad-E1A组在第2周开始给予荷瘤裸鼠皮下肿瘤内滴度为5×109PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,连续2周,放射组在第3周给予6MV-X照射,每天2Gy,连续5天。Ad-β-gal+放射组/Ad-E1A+放射组在第2周开始,给予荷瘤裸鼠皮下肿瘤内滴度为5×109 PFU/50μL的Ad-E1A/Ad-β-gal注射,每周2次,连续2周,在第3周给予6MV-X照射,每天2Gy,连续5天。第1次治疗后,每隔4天用卡尺测量1次肿瘤的体积,记录其直径,裸鼠的生存时间。当肿瘤的直径超过2cm时,处死裸鼠,分离肿瘤组织,采用免疫组织化学方法分析VEGF和CD34表达,RT-PCR分析wtp53的表达,Western blot分析VEGF的表达,TUNEL分析细胞凋亡。
结果：Ad-E1A+放射组较其他组能明显延缓移植瘤生长时间,Ad-E1A+放射组裸鼠的肿瘤平均体积比单独放射组小4.7倍,比单独Ad-E1A组的小5.3倍。Ad-E1A+放射组的裸鼠生存率显著高于其他治疗组。Ad-E1A+放射组的VEGF蛋白表达和微血管密度较其他各组明显下降。(P<0.01)。Ad-E1A组和Ad-E1A+放射组的wtp53基因表达明显上调。TUNEL分析结果显示Ad-E1A组和Ad-E1A+放射组及放射组的肿瘤组织内均可明显观察到凋亡细胞。并且,Ad-E1A+放射组肿瘤细胞凋亡数目明显高于Ad-E1A组或单独放射组。
结论：E1A基因通过抑制肿瘤血管的形成和上调肿瘤组织中wtp53的表达及诱导肿瘤细胞的凋亡来提高人鼻咽癌细胞对放射的敏感性。
Objective:The purpose of this study determined the effect and mechanism of Ad-E1A gene therapy in vitro radiosensitivity to nasopharyngeal carcinoma cell.
Methods:The human nasopharyngeal carcinoma CNE-2Z cell lines were investigated. The recombinant adenovirus vector containing E1A gene was used for this study. After CNE-2Z cells was treated with PBS, Ad-β-gal and Ad-E1A for 48h, the three groups were irradiated in different doses at 0、2、4、6 and 8Gy, The cytotoxicity was determined by MTT assay and cell cycle was analysis by flow cytometry. Clone forming assays were carried out, cell survival curves were drawn and the sensitivity enhancing ratio (SER) was calculated. The VEGF expression was evaluated by RT-PCR assay and immunocytochemical analysis. The wtp53 express was evaluated by RT-PCR.
Results:RT-PCR confirmed that E1A gene had been integrated into positively transfected cells and stably expressed. Significant cell deaths by IR were observed in a dose dependent manner in the three group CNE-2Z cells. After transduction of the E1A gene into CNE-2Z cells, the sensitivity of these cells to radiation was enhanced than the PBS treated group and Ad-β-gal treated group. Cell growth inhibition in Ad-E1A treated group by IR was strongly enhanced than Ad-β-gal treated group and PBS treated group. Cell survival curves showed that the SER of Do, Dq and SF2 value was 1.37,1.95 and 1.46 with Ad-E1A treated group The Do, Dq and SF2 value was 1.57Gy,1.82Gy,0.89 in PBS treated group and 1.53Gy,1.78Gy,0.82 in Ad-β-gal treated group,respectively. RT-PCR assay and immunocytochemical analysis showed VEGF expression was downregulation in Ad-E1A treated group. Moreover, the expression of wtp53 gene was markedly enhanced in Ad-E1A treated group. The Flow cytometry showed more apotosis can be detected in the cells treated with Ad-E1A plus radiotherapy than those of the other treated groups.
Conclusions:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma cell sensitivity to the radiotherapy by down regulated VEGF expression and enhanced the expression of wtp53.
Objective:To determine the effect of Ad-E1A gene therapy in vivo radiosensitivity to nasopharyngeal carcinoma.
Methods:CNE-2Z cells (2 x 105) were injected subcutaneously into nude mice resulted in tumor development (1-3 mm) 6 days later. The tumor-bearing mice were then randomly divided into six groups (10 mice per group) for PBS treatment or treatment with radiotherapy, Ad-E1 A, or Ad-β-gal alone or radiotherapy in combination with Ad-E1A or Ad-β-gal. Animals were treated with Ad-E1A or Ad-β-gal (5 x 109 plaque forming units) by intratumoral injection twice weekly for 2 weeks at beginning of week 2. Animals treated with radiotherapy in combination with Ad-E1A or Ad-β-gal were received 2 Gy radiotherapy daily for 5 days following the first week of treatment with Ad-E1A or Ad-β-gal. Control animals received PBS therapy or radiotherapy only after tumor cells were injected. When the sizes of tumors exceeded 2 cm, the mice were killed and the tumors underwent immunohistochemical analysis for VEGF and CD34 expression and RT-PCR analysis for wtp53 expression and TUNEL assay for apoptosis and Western blot analysis for VEGF expression
Results:The growth delay time (TGD) was longest in the Ad-E1A plus radiotherapy group. Tumors treated with Ad-E1A plus radiotherapy were 4.7-fold smaller than those treated with radiotherapy alone and 5.3-fold smaller than those treated with Ad-E1A alone. The survival rate of tumor-bearing mice treated with Ad-E1A plus radiotherapy was significantly higher than that of other treated groups. The vessel density and the VEGF expression were significantly lower in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-E1A alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy (P<0.01). wtp53 expression were significantly increased in the tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-β-gal alone or Ad-β-gal plus radiotherapy. TUNEL staining revealing apoptosis can be detected in Ad-E1A group, radiotherapy group, Ad-E1A plus radiotherapy group, and more apoptosis can be detected in the tumors treated with Ad-E1A plus radiotherapy than those of the other treated groups.
Conclusion:E1A gene therapy can effectively enhance the nasopharyngeal carcinoma sensitivity to the radiotherapy by down-regulating VEGF expression increasing wtp53 expression and inducing apoptosis. These findings may pave the way for efficient radiation-gene therapy to NPC in future.
引文
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[2]屠规益,唐平章.我国头颈肿瘤外科的发展轨迹.中华耳鼻咽喉科杂志,1998,33：259-260
[3]Nakajima T, Morita K, Ohi N, et al. Degradation of topoisomerase Ⅱα during adenovirus E1A-induced apoptosis is mediated by the activation of the ubiquitin proteolysis system. J Biol Chem,1996,271:24842-24849
[4]吴旻.基因治疗的历史现状与未来.中国肿瘤生物治疗杂志,1995,2(1)：1-5
[5]Roth JA. Gene therapy for cancer:what have we done and where are we going. NCIJ,1997,89(1):21-39
[6]Chinnadurai G. Adenovirus E1A as a tumor suppressor gene. Oncogene,1992,7: 1255-1258
[7]Frisch SM. Reversal of malignancy by the adenovirus E1A gene. Mutation Res, 1996,350:261-266
[8]Chang JY, Xia W, Shao R, et al. Inhibition of intratracheal lung cancer development by systemic delivery of E1A. Oncogene,1996,13:1405-1412
[9]Yu DH, Hung MC. The erbB-2 gene as a cancer therapeutic target and the tumor metastasis suppressing function of E1A. Cancer Metast Rev,1998,17:195-202
[10]Yu D, Wang SS, Dulski KM, et al. c-erbB-2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties. Cancer Res.1994 Jun 15;54(12):3260-3266
[11]Tsai CM, Yu D, Chang KT, et al. Enhanced chemoresistance by elevation of p185neu Levels in HER-2/neu-transfected human lung cancer cell. J Natl Cancer Inst,1995,87:682-684
[12]Chen IH, Chang JT, Liao CT, et al. Prognostic significance of EGFR and Her-2 in oral cavity cancer in betel quid prevalent area cancer prognosis. Br J Cancer. 2003,18; 89(4):681-686
[13]Chang JY, Xia W, Shao R, et al. The tumor suppression activity of E1A in HER-2/neu-overexpressing breast cancer. Oncogene,1997 14:561-568
[14]Frisch SM, Dolter KE. Adenovirus E1A-mediated tumor suppression by a c-erbB-2/neu-independent mechanism. Cancer Res,1995,55:5551-5555
[15]Ricardo SP, Quintanilla M, Cano A, et al. Cancinoma cell lines become sensitive to DNA-damaging agents by the expression of the adenovirus E1A gene. Oncogene,1996,13:1083-1092
[16]Chen MJ, Holskin B, Strickler J, et al. Induction by E1A oncogene expression of cellular susceptibility to lysis by TNF. Nature,1987,330:581-584
[17]Steeg PS, Bevilacqa G, Pozzatti R, et al. Altered expression of nm23, a gene associated with low tumor metastatic potential, during adenovirus 2 E1A inhibition of experimental metastasis. Cancer Res,1988,48:6550-6554
[18]Lane DP. P53, guardian of the genome. Nature,1992,358:15-16
[19]Low SW, Ruley HE, Jacks T, et al. P53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell,1993,74:957-967
[20]Querido E, Teokoro JG, Branton PE. Accumulation of p53 induced by adenovirus El A proteins requires regions involved in the stimulation of DNA synthesis. J Virol,1997,71:3526-3533
[21]Teodoro JG, Shore GC, Branton PE. Adenovirus E1A proteins induced apoptosis by both p53-independent and p53-dependent mechanisms. Oncogene,1995,11: 467-474
[22]Sanchez PR, Quintanilla M, Cano A, et al. Carcinoma cell lines become sensitive to DNA-damaging agents by the expression of the adenovirus E1A gene. Oncogene,1996,13:1083-1092
[23]Bracey TS, Miller JC, Paraskeva C.γ-Radiation induced apoptosis in human colorectal adenoma and carcinoma cell lines can occur in the absence of wild type p53. Oncogene,1995,10:2391-2396
[24]Ko LJ, Prives C. P53 puzzle and paradigm. Genes Dev,1996,10:1054-1072
[25]Shao RP, Karunagaran D, Zhou BH, et al. Inhibition of nuclear factor-KB activity is involved in E1A mediated sensitization of radiation-induced apoptosis. J Biol
Chem,1997,272 (52):32739-32742
[26]Wang CY, Mayo MW, Baldwin AB, et al. TNF and cancer therapy-induced apoptosis:potentiation by inhibition of NF-κB. Science,1996,274:784-787
[27]Gilmore TD, Koedood M, Piffat KA, et al. Rel/NF-κB/IκB proteins and cancer. Oncogene,1996,13:1367-1378
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[31]Hunt KK, Deng J, Liu TJ, et al. Adenovirus mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian carcinoma cell line and does not require p53. Cancer Res,1997,57:4722-4726
[32]Ikeda MA, Jakoi L, Nevins JR. A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.Proc Natl Acad Sci USA,1996,93:3215-3220
[33]Xia W, Lan YK, Zhang HZ, et al.Strong correlation between c-erbB-2 overexpression and overall survival of patients with oral squamous cell carcinoma.Clin Cancer Res,1997,3:3-9
[34]Hou L, Shi D, Tu SM, et al.Oral cancer progression and c-erbB-2/neu proto-oncogene expression.Cancer Letters,1992,65:215-220
[35]Beckhardt RN,Kiyokawa N,Xi L, et al.HER-2/neu oncogene characterization in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg, 1995,121:1265-1270
[36]Karja V,Syrjanen S,Kataja V, et al.c-erbB-2 oncogene expression in salivary land tumors.ORL J Otorhinolaryngol Relat Spec,1994,56:206-212
[37]王琪,韩德民,刘等.头颈部肿瘤的P53基因研究进展.国外医学耳鼻咽喉科 学分册,1996,20：4-7
[38]Liu M,Lawson QDelos M, et al.Expression of E-cadherin adhesion molecule in vocal cord carcinomas.Eur Arch Otorhinolaryngol,1997,254:417-421
[39]王强,郭敏,孙连玉.nm23-H1基因在喉癌组织中表达的研究.中华耳鼻咽喉科杂志,1996,31：198-200
[40]Yoo GH,Ensley J,Jacobs J, et al.Intratumora ElAgene therapy for patientswith unresectable and recurrent head and neck cancer.Proc AACR,1998,39:322-324
[41]Kirn D,Nemunaitis J,Ganly I, et al. A phase Ⅱ trial of intratumoral injection with an E1B-deleted adenovirus, ONYX-015, in patient with recurrent, refractory head and neck Cancer.Proc ASCO,1998,17:391-394
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[1]屠规益,韩德民.当前头颈肿瘤科研应着重临床实用.中华耳鼻咽喉科杂志,1997,32：131-132
[2]屠规益,唐平章.我国头颈肿瘤外科的发展轨迹.中华耳鼻咽喉科杂志,1998,33：259-260
[3]Nakajima T, Morita K, Ohi N, et al. Degradation of topoisomerase Ⅱα during adenovirus E1A-induced apoptosis is mediated by the activation of the ubiquitin proteolysis system. J Biol Chem,1996,271:24842-24849
[4]吴旻.基因治疗的历史现状与未来.中国肿瘤生物治疗杂志,1995,2(1)：1-5
[5]Roth JA. Gene therapy for cancer:what have we done and where are we going. NCIJ,1997,89(1):21-39
[6]Chinnadurai G. Adenovirus E1A as a tumor suppressor gene. Oncogene,1992,7: 1255-1258
[7]Frisch SM. Reversal of malignancy by the adenovirus E1A gene. Mutation Res, 1996,350:261-266
[8]Chang JY, Xia W, Shao R, et al. Inhibition of intratracheal lung cancer development by systemic delivery of E1A. Oncogene,1996,13:1405-1412
[9]Yu DH, Hung MC. The erbB-2 gene as a cancer therapeutic target and the tumor metastasis suppressing function of E1A. Cancer Metast Rev,1998,17:195-202
[10]Yu D, Wang SS, Dulski KM, et al. c-erbB-2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties. Cancer Res.1994 Jun 15;54(12):3260-3266
[11]Tsai CM, Yu D, Chang KT, et al. Enhanced chemoresistance by elevation of p185neu Levels in HER-2/neu-transfected human lung cancer cell. J Natl Cancer Inst,1995,87:682-684
[12]Chen IH, Chang JT, Liao CT, et al. Prognostic significance of EGFR and Her-2 in oral cavity cancer in betel quid prevalent area cancer prognosis. Br J Cancer. 2003,18; 89(4):681-686
[13]Chang JY, Xia W, Shao R, et al. The tumor suppression activity of E1A in HER-2/neu-overexpressing breast cancer. Oncogene,1997 14:561-568
[14]Frisch SM, Dolter KE. Adenovirus E1A-mediated tumor suppression by a c-erbB-2/neu-independent mechanism. Cancer Res,1995,55:5551-5555
[15]Ricardo SP, Quintanilla M, Cano A, et al. Cancinoma cell lines become sensitive to DNA-damaging agents by the expression of the adenovirus E1A gene. Oncogene,1996,13:1083-1092
[16]Chen MJ, Holskin B, Strickler J, et al. Induction by E1A oncogene expression of cellular susceptibility to lysis by TNF. Nature,1987,330:581-584
[17]Steeg PS, Bevilacqa G, Pozzatti R, et al. Altered expression of nm23, a gene associated with low tumor metastatic potential, during adenovirus 2 E1A inhibition of experimental metastasis. Cancer Res,1988,48:6550-6554
[18]Lane DP. P53, guardian of the genome. Nature,1992,358:15-16
[19]Low SW, Ruley HE, Jacks T, et al. P53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell,1993,74:957-967
[20]Querido E, Teokoro JG, Branton PE. Accumulation of p53 induced by adenovirus El A proteins requires regions involved in the stimulation of DNA synthesis. J Virol,1997,71:3526-3533
[21]Teodoro JG, Shore GC, Branton PE. Adenovirus E1A proteins induced apoptosis by both p53-independent and p53-dependent mechanisms. Oncogene,1995,11: 467-474
[22]Sanchez PR, Quintanilla M, Cano A, et al. Carcinoma cell lines become sensitive to DNA-damaging agents by the expression of the adenovirus E1A gene. Oncogene,1996,13:1083-1092
[23]Bracey TS, Miller JC, Paraskeva C.γ-Radiation induced apoptosis in human colorectal adenoma and carcinoma cell lines can occur in the absence of wild type p53. Oncogene,1995,10:2391-2396
[24]Ko LJ, Prives C. P53 puzzle and paradigm. Genes Dev,1996,10:1054-1072
[25]Shao RP, Karunagaran D, Zhou BH, et al. Inhibition of nuclear factor-KB activity is involved in E1A mediated sensitization of radiation-induced apoptosis. J Biol
Chem,1997,272 (52):32739-32742
[26]Wang CY, Mayo MW, Baldwin AB, et al. TNF and cancer therapy-induced apoptosis:potentiation by inhibition of NF-κB. Science,1996,274:784-787
[27]Gilmore TD, Koedood M, Piffat KA, et al. Rel/NF-κB/IκB proteins and cancer. Oncogene,1996,13:1367-1378
[28]Liu ZG, Hau H, Goeddel DV, et al. Disection of TNF receptor 1 effector functions:JNK activation is not linked apoptosis while NF-κB activation prevents cell death. Cell,1996,87:565-576
[29]Haaskogan DA, Kogan SC, Levi D, et al. Inhibition of apoptosis by the retinoblastoma gene product. EMBO J,1995,14:461-472
[30]Field SJ, Tsai FY, Kuo F, et al. E2F-lfunctions in mice to pro-mote apoptosis and suppress proliferation. Cell,1996,85:549-561
[31]Hunt KK, Deng J, Liu TJ, et al. Adenovirus mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian carcinoma cell line and does not require p53. Cancer Res,1997,57:4722-4726
[32]Ikeda MA, Jakoi L, Nevins JR. A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.Proc Natl Acad Sci USA,1996,93:3215-3220
[33]Xia W, Lan YK, Zhang HZ, et al.Strong correlation between c-erbB-2 overexpression and overall survival of patients with oral squamous cell carcinoma.Clin Cancer Res,1997,3:3-9
[34]Hou L, Shi D, Tu SM, et al.Oral cancer progression and c-erbB-2/neu proto-oncogene expression.Cancer Letters,1992,65:215-220
[35]Beckhardt RN,Kiyokawa N,Xi L, et al.HER-2/neu oncogene characterization in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg, 1995,121:1265-1270
[36]Karja V,Syrjanen S,Kataja V, et al.c-erbB-2 oncogene expression in salivary land tumors.ORL J Otorhinolaryngol Relat Spec,1994,56:206-212
[37]王琪,韩德民,刘等.头颈部肿瘤的P53基因研究进展.国外医学耳鼻咽喉科 学分册,1996,20：4-7
[38]Liu M,Lawson QDelos M, et al.Expression of E-cadherin adhesion molecule in vocal cord carcinomas.Eur Arch Otorhinolaryngol,1997,254:417-421
[39]王强,郭敏,孙连玉.nm23-H1基因在喉癌组织中表达的研究.中华耳鼻咽喉科杂志,1996,31：198-200
[40]Yoo GH,Ensley J,Jacobs J, et al.Intratumora ElAgene therapy for patientswith unresectable and recurrent head and neck cancer.Proc AACR,1998,39:322-324
[41]Kirn D,Nemunaitis J,Ganly I, et al. A phase Ⅱ trial of intratumoral injection with an E1B-deleted adenovirus, ONYX-015, in patient with recurrent, refractory head and neck Cancer.Proc ASCO,1998,17:391-394