E1A基因对人肺腺癌细胞增殖和转移的影响
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摘要
目的：肺癌是我国常见的恶性肿瘤之一，近年来，其发病率不断增高。在我国沿海城市肺癌发病率在男性肿瘤中已占首位。E1A基因约占腺病毒5型基因组最左端1.77kb，可转录产生12S和13S两种mRNA，经不同拼接分别翻译243和289个氨基酸的两种蛋白质。E1A蛋白是多功能转录因子，它能从正、负两种途径调控多种基因的转录。目前，E1A基因抗肿瘤作用的研究，对非小细胞肺癌作为对象研究的较少。本研究旨在探讨E1A基因对人肺腺癌细胞增殖和转移的影响，为人肺腺癌E1A基因治疗的可行性提供理论和实验依据，为基因治疗的临床应用奠定基础。
方法：应用分子克隆技术，构建真核细胞高表达E1A基因的重组质粒pcDNA3-E1A，并对所构建的重组质粒，进行内切酶图谱鉴定和序列分析。脂质体介导将E1A基因转入人肺腺癌细胞系Anip973，经G418筛选获得稳定表达E1A基因的转染细胞Anip973—E1A。通过体内、外实验，观察了其生长速度、倍增时间、软琼脂集落形成能力、对裸鼠的致癌性及肿瘤转移能力等的改变。
结果：构建真核细胞高表达E1A基因的重组质粒pcDNA3—E1A，酶切结果与预计相等，DNA序列分析结果与Gene Bank比较，核酸序列无重排及其它变化。PCR.RT—PCR和免疫细胞化学染色方法证明E1A基因已整合到转染细胞Anip973—E1A中，并稳定表达。体外实验显示，Anip973—E1A细胞生长速度减慢，倍增时间延长，为Anip973—vector细胞的1.36倍，软琼脂集落形成能力显著降低。体内实验显示，Anip973—E1A细胞组裸鼠出瘤时间晚，肿瘤体积小，其中有2只裸鼠至实验结束未触及肿瘤(2／6)，抑瘤率为60.1％。第21天处死裸鼠计数肺转移灶，Anip973，Anip973—vector和Anip973—E1A细胞组肺转移灶数
目分别为16.1士3.2，15.4士3.8和7.3士2.31，转移抑制率为
52.6%，免疫细胞化学染色显示:EIA基因的稳定表达可抑制HER
一2/neu基因的表达。
结论:以上结果初步证明，EIA基因的稳定表达明显抑制人肺腺
癌细胞的恶性表型，降低其对裸鼠的致瘤性，减少肺转移灶形成，
说明EIA基因在肺癌基因治疗中有重要意义，为其临床应用提供
了理论和实验依据。
Lung carcinoma is a common malignant tumor in our country whose morbidity is increasing rapidly in recent years. The initiation and progression of lung cancer are involved in many oncogenes and tumor suppressor genes, such as the deletions of genetic material of 2q and 3q, the over expressions of HER-2/neu gene, the amplification of c-myc gene, the inactivation of function of p53 gene etc, which makes gene prevention and gene therapy possible . The Ad5E1A gene transcribes two mRNAs of 12S and 13S, and codes for two major related proteins of 243 and 289 amino acids by differently splicing. The E1A proteins are nuclear phosphoproteins that are multifunctional transcription factors. The E1A products are positive and negative regulators for the transcription of many cellular genes. At present, non-small cell lung cancer is less as the subject to the study of E1A gene than other. The aim of study is to research the effects of Ad5E1A gene on proliferation and metastasis of the human lung adenocarcinoma (Anip973). The results as follows:
1. Construction of the mammalian expression recombinant pcDNA3-E1A.
A E1A gene recombinant expression vector plasmid was constructed by subcloning a 1. 77kb E1A gene into a
mammalian expression vector pcDNA3 at the sites of Hind III and RamHI, and named after pcDNA3-ElA. The recombinant was cleaved with appropriate endoneuclear and sequenced. The results showed that the orientation of the insert was found to be correct, while no rearrangement was found.
2. Integratuion and expression of EIA gene in Human lung adenocarcinoma cell line (Anip973)
The recombinant pcDNA3-EIA was introduced Anip973 by LipofectinTM Reagent. After the cells resistant to G418, we obtained positive cell clones (Anip973-EIA). PCR, RT-PCR and immunocytochemistry assay showed that EIA gene has been integrated into Anip973-EIA cells and stably expressed.
3. Effects of adenovirus type 5 EIA gene on proliferation and metastasis of human lung adenocarcinoma cells.
The characteristics of the EIA expressing Anip973-EIA cells were studied in virto and viro. The expressing change of HER-2/neu gene was investigated, too. The results showed that (1) the growth rate of Anip973-EIA cells was diminished and their doubling-time increased; (2)the colony-forming efficiency of Anip973-EIA cells was inhibited significantly;(3)immunocytochemistry analysis also showed that the products of HER-2neu gene have decreased in EIA-expressing cells;(4)tumorigencity and metastasis in nude mice of Anip973-EIA were markedly suppressed. With these, we conclude that EIA gene is able to significantly suppress the malignant phenotye of human lung adenocarcinoma and the expression of HER-2/neu gene.
The above results showed that EIA gene possesses important effects on gene therapy of human lung carcinoma.
Our study provided data for gene therapy of tumor and possible of using EIA gene to improve tumor treatment.
方法：应用分子克隆技术，构建真核细胞高表达E1A基因的重组质粒pcDNA3-E1A，并对所构建的重组质粒，进行内切酶图谱鉴定和序列分析。脂质体介导将E1A基因转入人肺腺癌细胞系Anip973，经G418筛选获得稳定表达E1A基因的转染细胞Anip973—E1A。通过体内、外实验，观察了其生长速度、倍增时间、软琼脂集落形成能力、对裸鼠的致癌性及肿瘤转移能力等的改变。
结果：构建真核细胞高表达E1A基因的重组质粒pcDNA3—E1A，酶切结果与预计相等，DNA序列分析结果与Gene Bank比较，核酸序列无重排及其它变化。PCR.RT—PCR和免疫细胞化学染色方法证明E1A基因已整合到转染细胞Anip973—E1A中，并稳定表达。体外实验显示，Anip973—E1A细胞生长速度减慢，倍增时间延长，为Anip973—vector细胞的1.36倍，软琼脂集落形成能力显著降低。体内实验显示，Anip973—E1A细胞组裸鼠出瘤时间晚，肿瘤体积小，其中有2只裸鼠至实验结束未触及肿瘤(2／6)，抑瘤率为60.1％。第21天处死裸鼠计数肺转移灶，Anip973，Anip973—vector和Anip973—E1A细胞组肺转移灶数
目分别为16.1士3.2，15.4士3.8和7.3士2.31，转移抑制率为
52.6%，免疫细胞化学染色显示:EIA基因的稳定表达可抑制HER
一2/neu基因的表达。
结论:以上结果初步证明，EIA基因的稳定表达明显抑制人肺腺
癌细胞的恶性表型，降低其对裸鼠的致瘤性，减少肺转移灶形成，
说明EIA基因在肺癌基因治疗中有重要意义，为其临床应用提供
了理论和实验依据。
Lung carcinoma is a common malignant tumor in our country whose morbidity is increasing rapidly in recent years. The initiation and progression of lung cancer are involved in many oncogenes and tumor suppressor genes, such as the deletions of genetic material of 2q and 3q, the over expressions of HER-2/neu gene, the amplification of c-myc gene, the inactivation of function of p53 gene etc, which makes gene prevention and gene therapy possible . The Ad5E1A gene transcribes two mRNAs of 12S and 13S, and codes for two major related proteins of 243 and 289 amino acids by differently splicing. The E1A proteins are nuclear phosphoproteins that are multifunctional transcription factors. The E1A products are positive and negative regulators for the transcription of many cellular genes. At present, non-small cell lung cancer is less as the subject to the study of E1A gene than other. The aim of study is to research the effects of Ad5E1A gene on proliferation and metastasis of the human lung adenocarcinoma (Anip973). The results as follows:
1. Construction of the mammalian expression recombinant pcDNA3-E1A.
A E1A gene recombinant expression vector plasmid was constructed by subcloning a 1. 77kb E1A gene into a
mammalian expression vector pcDNA3 at the sites of Hind III and RamHI, and named after pcDNA3-ElA. The recombinant was cleaved with appropriate endoneuclear and sequenced. The results showed that the orientation of the insert was found to be correct, while no rearrangement was found.
2. Integratuion and expression of EIA gene in Human lung adenocarcinoma cell line (Anip973)
The recombinant pcDNA3-EIA was introduced Anip973 by LipofectinTM Reagent. After the cells resistant to G418, we obtained positive cell clones (Anip973-EIA). PCR, RT-PCR and immunocytochemistry assay showed that EIA gene has been integrated into Anip973-EIA cells and stably expressed.
3. Effects of adenovirus type 5 EIA gene on proliferation and metastasis of human lung adenocarcinoma cells.
The characteristics of the EIA expressing Anip973-EIA cells were studied in virto and viro. The expressing change of HER-2/neu gene was investigated, too. The results showed that (1) the growth rate of Anip973-EIA cells was diminished and their doubling-time increased; (2)the colony-forming efficiency of Anip973-EIA cells was inhibited significantly;(3)immunocytochemistry analysis also showed that the products of HER-2neu gene have decreased in EIA-expressing cells;(4)tumorigencity and metastasis in nude mice of Anip973-EIA were markedly suppressed. With these, we conclude that EIA gene is able to significantly suppress the malignant phenotye of human lung adenocarcinoma and the expression of HER-2/neu gene.
The above results showed that EIA gene possesses important effects on gene therapy of human lung carcinoma.
Our study provided data for gene therapy of tumor and possible of using EIA gene to improve tumor treatment.
引文
1．吴旻．基疗研究的历史、现状与未来。中国肿瘤生物治疗杂志，1995，2：1-6．
2. Rogenbery SA, Aebersold P, Cometta K et al. Gene transfer into humans-inmunotherapy of patients with advanced melanoma, using tumor infiltrating lymobocytes modified by retroviral gene transduction. New England Jourel of Medicine, 1990; 323:570-576
3. Anderon WF. Human gene therapy. Science, 1992; 256:809-814
4. Xindrt KT. Human gene marker/therapy clinical protocols. Human Gene Therapy, 1998; 9:2805-2811.
5. Nation Research council. Mapping and sequencing the Human Genome. National Academic press. 1998.
6．李连弟，鲁凤珠，张思维等，中国恶性肿瘤死亡率20年变化趋势和近期预测分析．中华肿瘤杂志，1997；19：3-5．
7．孙燕，肺癌病因、诊断、治疗中的困惑和展望．癌症杂志，1-5．
8. Sander A, Nemunaitis J, Dehuam C, et al. Phase Ⅲ study of cisplatin (c) with or without gemcitabine(G) in patients with advanced non-small cell lung cancer (NSCLC). Proc Am soc clin Oncol, 1998: 17:454-460.
9．孙燕，刘丽影，范魁生等．紫衫醇治疗中晚期恶性肿瘤(121例)．中国新药杂志，1996；5：252-255．
10．吕永杰，程书均(导师)．人支气管上皮细胞癌变早期的分子遗传学变化研究．1995(博士论文)，中国医学科学院肿瘤研究所图书馆．
11. Steven M, Frisch SH. Antioncogenic effect of adenovirus
E1A in human tumor cells. 191, 88:9077-9081.
12. Chinnadurai G. Adenovirus E1A as a tumor suppressor gene. Oncogene, 1992: 7:1255-1261.
13. Ruley H. Adenovirus early region enables viral and cellular transforming genes to transform primary cells in culture. Nature, 1983: 304:602-606.
14. Houweling A, Van den Elsen P, Van den Eb p. Partial transformation of primary rat cells by leftmost 4.5% fragment of adenovirus 5 DNA. Virology, 1980; 105:537-550.
15. Debroach U, Yakiharus Jana R, et al. C-terminal domain of the adenovirus E1A oncogene product is required for induction of cytotoxic T Lymphocytes and tumor-specific trams plantation immunity Virology, 1989: 173:606-641.
16. Kimelman D, Miller ST, Porter D, et al. EIA region of the human adenovirus and of the highly oncogenic simian adenovirus T over closely related. J. Viro, 1985; 2:399-344.
17. Nevins J. adenovirus EIA-dependent transactivation of transcription. Cancer Biob, 1990; 1:57-67.
18．张大昕、千新来、赵清正等．EIA基因对肿瘤细胞放身敏感性的影响．癌症，2000：19：197-199．
19．卢圣栋．现代分子生物学实验技术．北京：高等教育出版社．1993．
20. Weinberg R A. How cancer Arises. Scientific American, 275:32-40.
21. Veno NT, Yu DH, Hung MC Chemosensitization of HER-2/neu over expressing human breast cancer cells to paclitacel by Adenovirus type 5 E1A. Oncogene, 1997,
15:8953-8960.
22. Kotin R M, Siniscalco M, Samulski P J, et al. site specific in tegration by adeno-associ ated virus. Proc Natl Acal Sol USA 1990: 87:2211-2215.
23．王文阁，王波，史历等．插入外源程序性白细胞介素2基因的肿瘤细胞的抗肿瘤抗转移作用．中国肿瘤生物治疗杂志，1995：2：25-28．
24. Frisch SM, Dolter KE. Adenovirus E1A-mediated tumor suppression by a c-erb-2/neu-independent mechanism. Cancer Res, 1995: 55:5551-5555.
25．千新来，赵清正，王争．腺病毒5型EIA基因对永生化人支气管上皮细胞生长的抑制作用及其机理．中国生物化学与分子生物学报，20-2000；16：156-161．
2. Rogenbery SA, Aebersold P, Cometta K et al. Gene transfer into humans-inmunotherapy of patients with advanced melanoma, using tumor infiltrating lymobocytes modified by retroviral gene transduction. New England Jourel of Medicine, 1990; 323:570-576
3. Anderon WF. Human gene therapy. Science, 1992; 256:809-814
4. Xindrt KT. Human gene marker/therapy clinical protocols. Human Gene Therapy, 1998; 9:2805-2811.
5. Nation Research council. Mapping and sequencing the Human Genome. National Academic press. 1998.
6．李连弟，鲁凤珠，张思维等，中国恶性肿瘤死亡率20年变化趋势和近期预测分析．中华肿瘤杂志，1997；19：3-5．
7．孙燕，肺癌病因、诊断、治疗中的困惑和展望．癌症杂志，1-5．
8. Sander A, Nemunaitis J, Dehuam C, et al. Phase Ⅲ study of cisplatin (c) with or without gemcitabine(G) in patients with advanced non-small cell lung cancer (NSCLC). Proc Am soc clin Oncol, 1998: 17:454-460.
9．孙燕，刘丽影，范魁生等．紫衫醇治疗中晚期恶性肿瘤(121例)．中国新药杂志，1996；5：252-255．
10．吕永杰，程书均(导师)．人支气管上皮细胞癌变早期的分子遗传学变化研究．1995(博士论文)，中国医学科学院肿瘤研究所图书馆．
11. Steven M, Frisch SH. Antioncogenic effect of adenovirus
E1A in human tumor cells. 191, 88:9077-9081.
12. Chinnadurai G. Adenovirus E1A as a tumor suppressor gene. Oncogene, 1992: 7:1255-1261.
13. Ruley H. Adenovirus early region enables viral and cellular transforming genes to transform primary cells in culture. Nature, 1983: 304:602-606.
14. Houweling A, Van den Elsen P, Van den Eb p. Partial transformation of primary rat cells by leftmost 4.5% fragment of adenovirus 5 DNA. Virology, 1980; 105:537-550.
15. Debroach U, Yakiharus Jana R, et al. C-terminal domain of the adenovirus E1A oncogene product is required for induction of cytotoxic T Lymphocytes and tumor-specific trams plantation immunity Virology, 1989: 173:606-641.
16. Kimelman D, Miller ST, Porter D, et al. EIA region of the human adenovirus and of the highly oncogenic simian adenovirus T over closely related. J. Viro, 1985; 2:399-344.
17. Nevins J. adenovirus EIA-dependent transactivation of transcription. Cancer Biob, 1990; 1:57-67.
18．张大昕、千新来、赵清正等．EIA基因对肿瘤细胞放身敏感性的影响．癌症，2000：19：197-199．
19．卢圣栋．现代分子生物学实验技术．北京：高等教育出版社．1993．
20. Weinberg R A. How cancer Arises. Scientific American, 275:32-40.
21. Veno NT, Yu DH, Hung MC Chemosensitization of HER-2/neu over expressing human breast cancer cells to paclitacel by Adenovirus type 5 E1A. Oncogene, 1997,
15:8953-8960.
22. Kotin R M, Siniscalco M, Samulski P J, et al. site specific in tegration by adeno-associ ated virus. Proc Natl Acal Sol USA 1990: 87:2211-2215.
23．王文阁，王波，史历等．插入外源程序性白细胞介素2基因的肿瘤细胞的抗肿瘤抗转移作用．中国肿瘤生物治疗杂志，1995：2：25-28．
24. Frisch SM, Dolter KE. Adenovirus E1A-mediated tumor suppression by a c-erb-2/neu-independent mechanism. Cancer Res, 1995: 55:5551-5555.
25．千新来，赵清正，王争．腺病毒5型EIA基因对永生化人支气管上皮细胞生长的抑制作用及其机理．中国生物化学与分子生物学报，20-2000；16：156-161．