喉癌基因治疗的临床前研究
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摘要
喉癌是一种较常见的恶性肿瘤，严重危害人类的健康。在我国，喉癌约占全身肿瘤的2％，占头颈部肿瘤的11—12％。目前临床上常用的治疗方法(手术、放疗和化疗)都存在一定的局限性，因此需要探索一种有效治疗喉癌的的新方法。军事医学科学院二所三室构建的携带人野生型p53基因和肿瘤免疫相关基因B7—1、GM—CSF的重组腺病毒(命名为BB—102)感染喉癌原代细胞后，能明显抑制癌细胞的增殖并诱导其凋亡，而且体外能诱导自体外周血淋巴细胞生成肿瘤杀伤性CTL细胞。本研究在此基础上，研究BB—102对种植喉癌细胞系的裸鼠的治疗效果，为进一步走向临床提供新的治疗方案。同时，我们还探讨了p14~(ARF)对喉癌细胞的抑制作用及对内源性p53的影响，以便加强自体p53水平，从而为进一步防治喉癌提供实验依据。据此，本研究完成以下工作：
1．腺病毒的扩增与质量检定
在293细胞中分别扩增BB—102和携带绿色荧光蛋白基因的对照重组病毒Ad-GFP；以氯化铯密度梯度超速离心法纯化病毒；以蚀斑分析法测定病毒滴度；按照《中国生物制品规则》之通则，检测重组腺病毒BB—102的质量。结果表明：Ad-GFP和BB—102的滴度分别为1.1×10~(11)、2.4×10~(10)；重组腺病毒BB—102的质量符合《中国生物制品规则》的要求；重组腺病毒介导的目的基因可以在喉癌细胞中有效表达。
2．重组腺病毒介导人野生型p53、B7—1和GM—CSF基因对喉癌的临床前研究
建立喉癌裸鼠模型，瘤内注射BB—102、Ad-GFP和PBS，在裸鼠体内进行人喉癌的基因治疗的临床前研究，对肿瘤组织进行测量、病理学检查、
摘 要
免疫组化等研究。结果表明：在实验组与对照组之间经多因素析因方差分析，
肿瘤重量和瘤体积具有显著性差异。经治疗后，实验组肿瘤中未发现大变个
P53的表达，Ki67阳性表达率极低，悦明BB—102在喉癌细胞中有效表达并
能显著抑制喉癌细胞在裸鼠体内增殖。同时光镜和透射电镜下可见肿瘤细胞
的坏死、i）M亡及炎性细胞浸润。
3．pl4叩’基因抑制喉癌细胞生长及其对内源性p53表达的影响
采用基因转染技术，将全长野生型 PI4“”CDNA转染到人喉鳞状细胞癌
HepZ细胞中，利用流式细胞仪、RT—PCR和 WesternEloting等万法研究其
对细胞周期、内源性野生型p53表达的影响。结果显示：转染N叩’基因的
Hep习细胞的生长受到明显抑制，其克隆形成能力为转染空载体的 5 7 ％。细胞
分析表明，转染刊“”CDNA48 ’J＼时后，处在＊G；和 G。／M期的细胞皆增加了
一倍。同时，Western Bloting分析结果表明，内源性野生型p53表达明显卜
升。
根据以上比较实验，我们得到如下结论：
BB－102在喉癌基回治疗中具有较好的应用前景，可望发展成临床卜的一
种抗癌剂。同时，pl4“”基因的失活可能是喉癌发生过程中的一个重要因素，
野生型p14的恢复，上调喉癌HepZ细胞中内源性野生型p53的表达并抑制
喉癌细胞生长，进一步发挥p53肿瘤抑制作用，有可能成为临床基因治疗的
又一新靶点。
Laryngeal carcinoma is a very common malignant tumor, which is seriously detrimental to human health. It has been estimated that the incidence of laryngocarcinoma is 2% of all human tumors and 11-12% of the head and neck tumors. Conventional therapeutic methods such as surgical treatment ,radiotherapy and chemotherapy are being used currently, but all have certain limitations. It therefore ,is needed to develop a new kind of therapy for effective treatment of laryngeal carcinoma.
In Department of Experimental Hematology of Beijing Institute of Radiation Medicine, they constructed a recombinant adenovirus carrying human wild -type p53 gene and tumor immunity-related genes -B7-1 and GM-CSF, named.as BB-102, and observed that proliferation of the human laryngocarcinoma cells was inhibited markedly and apoptosis was induced after infection with BB-102.Besides,such infected cells could also induce in vitro autologous peripheral blood lymphocytes to transform into tumor-killing cytotoxic T lymphocyte (CTL).Hence, in the present study the effect on cotransduction of human wide-type p53,B7-l and GM-CSF genes mediated by recombinant adenovirus on nude mice model bearing laryngocarcinoma were studied to explore the biological value in the therapy of human laryngeal carcinoma. Along with the study, we also explore the inhibitory effect of p!4ARF on the cell growth of laryngeal carcinoma and the expression on endogenous p53 to intensify the autologous level of p53 , thus providing experimental evidence for new therapeutic measures of laryngeal carcinoma.
Construction and production of recombinant adenoviruses
Recombinant plasmids pBB-102 (containing human wild-type p53, GM-CSF and B7-1 genes) were constructed by molecular cloning. Recombinant adenoviruses BB-102 were constructed by homologous recombination in 293 cells. BB-102 and Ad-GFP (control virus) were amplified in 293 cells on a large scale and purified by ultra-centrifugation in CsCl step gradient solutions. The viral particles were determined by OD260nm and the purity was evaluated by the ratio of OD260 nm/OD280 nm. The viral liters were determined by plaque assays.
Preclinical study of recombinant adenovirus in the treatment of human laryngeal squamous carcinoma
Nude mice model bearing laryngocarcinoma were established using human laryngeal squamous carcinoma cell line (Hep-2). Large amounts of recombinant adenovirus were injected into the tumor. Changes in carcinoma administrated with recombinant adenovirus were observed under light and electron microscopes. The difference between experimental and control groups was statistically significant. The necrosis of the tumor cells and inflammatory cellular infiltration were found under light and electron microscopes. The morphology of cells infected with BB-102 was analyzed for evidence of apoptosis by transmission electron microscopy. It has been shown that wild type P53 protein can inhibit cell growth and induce apoptosis, while B7-1 and GM-CSF have no such effects. So the high level expression of p53 gene mediated by BB-102 is reasonably the major cause of cell growth inhibition and apoptosis.
Effect of p!4ARF gene on cell growth of human laryngeal tumor cells and expression of endogenous p53 protein
p!4ARFcDNA was transferred to the cell line Hep-2 of squamous cell carcinoma of the larynx by gene transfer to study the cell cycles and the expression of endogenous wild type p53 by using flow cytometry, RT-PCR and Western-blotting. Expression of p!4ARF significantly affected the Hep-2 cell growth. The clone-forming efficiency of the Hep-2 cells transferred p!4ARF was 57%,compared with empty vector pcDNA3. The numbers of cells transferred p!4ARF at the Go/G,,G2/M phase as twice as the control after 48 hours transferred with p!4ARF cDNA . The expression of endogenous wild type p53 significantly enhanced.
As mentioned above, the results showed that BB-102 has significant efficacy on suppressing tumor cell growth and inducing their apoptosis, which suggested that BB-102 might be further devel
1．腺病毒的扩增与质量检定
在293细胞中分别扩增BB—102和携带绿色荧光蛋白基因的对照重组病毒Ad-GFP；以氯化铯密度梯度超速离心法纯化病毒；以蚀斑分析法测定病毒滴度；按照《中国生物制品规则》之通则，检测重组腺病毒BB—102的质量。结果表明：Ad-GFP和BB—102的滴度分别为1.1×10~(11)、2.4×10~(10)；重组腺病毒BB—102的质量符合《中国生物制品规则》的要求；重组腺病毒介导的目的基因可以在喉癌细胞中有效表达。
2．重组腺病毒介导人野生型p53、B7—1和GM—CSF基因对喉癌的临床前研究
建立喉癌裸鼠模型，瘤内注射BB—102、Ad-GFP和PBS，在裸鼠体内进行人喉癌的基因治疗的临床前研究，对肿瘤组织进行测量、病理学检查、
摘 要
免疫组化等研究。结果表明：在实验组与对照组之间经多因素析因方差分析，
肿瘤重量和瘤体积具有显著性差异。经治疗后，实验组肿瘤中未发现大变个
P53的表达，Ki67阳性表达率极低，悦明BB—102在喉癌细胞中有效表达并
能显著抑制喉癌细胞在裸鼠体内增殖。同时光镜和透射电镜下可见肿瘤细胞
的坏死、i）M亡及炎性细胞浸润。
3．pl4叩’基因抑制喉癌细胞生长及其对内源性p53表达的影响
采用基因转染技术，将全长野生型 PI4“”CDNA转染到人喉鳞状细胞癌
HepZ细胞中，利用流式细胞仪、RT—PCR和 WesternEloting等万法研究其
对细胞周期、内源性野生型p53表达的影响。结果显示：转染N叩’基因的
Hep习细胞的生长受到明显抑制，其克隆形成能力为转染空载体的 5 7 ％。细胞
分析表明，转染刊“”CDNA48 ’J＼时后，处在＊G；和 G。／M期的细胞皆增加了
一倍。同时，Western Bloting分析结果表明，内源性野生型p53表达明显卜
升。
根据以上比较实验，我们得到如下结论：
BB－102在喉癌基回治疗中具有较好的应用前景，可望发展成临床卜的一
种抗癌剂。同时，pl4“”基因的失活可能是喉癌发生过程中的一个重要因素，
野生型p14的恢复，上调喉癌HepZ细胞中内源性野生型p53的表达并抑制
喉癌细胞生长，进一步发挥p53肿瘤抑制作用，有可能成为临床基因治疗的
又一新靶点。
Laryngeal carcinoma is a very common malignant tumor, which is seriously detrimental to human health. It has been estimated that the incidence of laryngocarcinoma is 2% of all human tumors and 11-12% of the head and neck tumors. Conventional therapeutic methods such as surgical treatment ,radiotherapy and chemotherapy are being used currently, but all have certain limitations. It therefore ,is needed to develop a new kind of therapy for effective treatment of laryngeal carcinoma.
In Department of Experimental Hematology of Beijing Institute of Radiation Medicine, they constructed a recombinant adenovirus carrying human wild -type p53 gene and tumor immunity-related genes -B7-1 and GM-CSF, named.as BB-102, and observed that proliferation of the human laryngocarcinoma cells was inhibited markedly and apoptosis was induced after infection with BB-102.Besides,such infected cells could also induce in vitro autologous peripheral blood lymphocytes to transform into tumor-killing cytotoxic T lymphocyte (CTL).Hence, in the present study the effect on cotransduction of human wide-type p53,B7-l and GM-CSF genes mediated by recombinant adenovirus on nude mice model bearing laryngocarcinoma were studied to explore the biological value in the therapy of human laryngeal carcinoma. Along with the study, we also explore the inhibitory effect of p!4ARF on the cell growth of laryngeal carcinoma and the expression on endogenous p53 to intensify the autologous level of p53 , thus providing experimental evidence for new therapeutic measures of laryngeal carcinoma.
Construction and production of recombinant adenoviruses
Recombinant plasmids pBB-102 (containing human wild-type p53, GM-CSF and B7-1 genes) were constructed by molecular cloning. Recombinant adenoviruses BB-102 were constructed by homologous recombination in 293 cells. BB-102 and Ad-GFP (control virus) were amplified in 293 cells on a large scale and purified by ultra-centrifugation in CsCl step gradient solutions. The viral particles were determined by OD260nm and the purity was evaluated by the ratio of OD260 nm/OD280 nm. The viral liters were determined by plaque assays.
Preclinical study of recombinant adenovirus in the treatment of human laryngeal squamous carcinoma
Nude mice model bearing laryngocarcinoma were established using human laryngeal squamous carcinoma cell line (Hep-2). Large amounts of recombinant adenovirus were injected into the tumor. Changes in carcinoma administrated with recombinant adenovirus were observed under light and electron microscopes. The difference between experimental and control groups was statistically significant. The necrosis of the tumor cells and inflammatory cellular infiltration were found under light and electron microscopes. The morphology of cells infected with BB-102 was analyzed for evidence of apoptosis by transmission electron microscopy. It has been shown that wild type P53 protein can inhibit cell growth and induce apoptosis, while B7-1 and GM-CSF have no such effects. So the high level expression of p53 gene mediated by BB-102 is reasonably the major cause of cell growth inhibition and apoptosis.
Effect of p!4ARF gene on cell growth of human laryngeal tumor cells and expression of endogenous p53 protein
p!4ARFcDNA was transferred to the cell line Hep-2 of squamous cell carcinoma of the larynx by gene transfer to study the cell cycles and the expression of endogenous wild type p53 by using flow cytometry, RT-PCR and Western-blotting. Expression of p!4ARF significantly affected the Hep-2 cell growth. The clone-forming efficiency of the Hep-2 cells transferred p!4ARF was 57%,compared with empty vector pcDNA3. The numbers of cells transferred p!4ARF at the Go/G,,G2/M phase as twice as the control after 48 hours transferred with p!4ARF cDNA . The expression of endogenous wild type p53 significantly enhanced.
As mentioned above, the results showed that BB-102 has significant efficacy on suppressing tumor cell growth and inducing their apoptosis, which suggested that BB-102 might be further devel
引文
1． Agarwal, M.L., Taylor, W.R., Chernov, M.V., Chernova, O.B., Stark, G.R. (1998) . The p53 network. J Bio Chem, 273: 1-4．
2． Akagi, J., Hodge, J.W., McLaughlin, J.P., Gritz, L., Mazzara, G., Kufe, D., Schlom, J., Kantor, J.A. (1997) . Therapeutic antitumor response after immunization with an admixture of recombinant vaccinia viruses expressing a modified MUC1 gene and the murine T-cell costimulatory molecule B7． J Immunother, 20: 38-47．
3． Alexander, R.M., William, H.M., Kelly, H., et al. (1994) . Cytokine-mediated gene therapy for cancer. Ann Surg Oncol, 1: 436-450．
4． Bargonetti, J., Friedmann, P.N., Kem, S.E., et al. (1991) . Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell, 65: 1083-1091．
5． Baskar, S. (1996) . Gene-modified tumor cells as a cellular vaccine. Cancer Immunol Immunother, 43: 165-173．
6． Boyle JO, John H, Koch W. The incidence of P53 mutations increases with progression of head and neck cancer. Cancer Res,1993,53:4477-4480
7． Caplen, N.J., Higginbotham, J.N., Scheel, J.R., Vahanian, N., Yoshida, Y., Hamada, H., Blaese, R.M., Ramsey, W.J. (1999) . Adeno-retroviral chimeric viruses as in vivo transducing agents. Gene Ther, 6: 454-459．
8． Crystal, R.G. (1995) . Transfer of genes to humans: early lessons and obstacles to success. Science, 270: 404-410．
9． Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V, Hamada, H., Pardoll, D., Mulligan, R.C. (1993) . Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent specific and long-lasting antitumor immunity. Proc Natl Acad Sci USA, 90: 3539-3543．
10． Drew, M.P. (1998) Cancer vaccines. Nature Medicine Vaccine, Supplement 4: 525-531．
11． Duro,D., Bernard, O., Valle, V., et al, A new type of p16INK4A/MTS1 gene transcript expressed in B-cell malignancies. Oncogene, 1995,11,21-29．
12． Egilmez NK, Cuenca R, Yokota SJ, et al. (1996) . In vivo cytokine gene therapy of human tumor xenografts in SCID mice by liposome-mediated DNA delivery. Gene Ther, 3:607
13． Fan, S., Smith, M.L., Rivet, D.J., et al. (1995) . Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res. 55, 1649-1654．
14． Fields, S., Jang, S.J. (1990) . Presence of a potent transcription activating sequence in the p53 protein. Science, 249: 1046-1049．
15． Fujiwara, T., Grimm, E.A., Mukhopadhyay, T., et al. (1994) . Induction of chemosensitivity in human lung cancer cells in view by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res, 54: 2287-2291．
16． Gilboa, E., Smita, K.N., Lyerly, H.K. (1998) . Immunotherapy of cancer with dendritic-cell-based vaccines. Cancer Immunol Immunother, 46: 82-87．
17． Graham, F.L., Smiley, J., Russell, W.C., Nairn, R. (1977) . Characteristics of a human cell line transformed by DNA from human adenovirus type 5． J Gen Virol, 36: 59-74．
18． Hawkins, D.S., Demers, G.W., Galloway, D.A. (1996) . Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents. Cancer Res, 56: 892-898．
19． Hermonat PL, Muzyczka N. (1984) . Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA, 81: 6466
20． Herrmann, F. (1995) . Cancer gene therapy: principles, problems and perspectives. J Mol Med, 73: 157-163．
21． Hogge, G.S., et al. (1998) . Development of human granulocyte-macrophage colony-stimulating factor-transfected tumor cell vaccines for the treatment of spontaneous canine cancer. Hum Gene Ther, 9: 1851-1861．
22． Hollstein, M, Sidransky, D., Vogelstein, B., Harris, C.C. (1991) . p53 mutations in human cancers. Science, 253: 49-53．
23． Houbiers JG, Nijman HW, Burg SH, et al. In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53． Eur J Immunol. 1993,23:2072-2077
24． Howard, B., Burrascano, M., McCallister, T., Chong, K., Gangavalli, R., Severinsson L, Jolly, D.J., Darrow, T., Vervaert, C., Abdel-Wahab, Z., Siegler, H.F., Barber, J.R. (1994) .
Retrovirus-mediated gene transfer of the human γ-IFN gene: a therapy for cancer. Ann New York Acad Sci, 716: 167-187．
25． Huschtscha Li and Reddel RR. p16(INK4a) and the control of cellular proliferative life span. Carcinogenesis, 1999, 20, 921-926．
26． Kamijo T; Weber JD; Zambetti G; et al . Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2． Proc Natl Acad Sci USA, 1998, 95, 8292-8297
27． Kamijo T; Zindy F; Roussel MF; et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell, 1997, 91, 649-659
28． Knudson, A.G., Upton, A.C. (1990) . Tumor suppressor gene workshop. Cancer Res, 50: 6765．
29． Komata, T., Tanaka, R., Yamamoto, K., Tazunu, O., Ono, K., Yoshida, S., Takahashi, M. (1997) . B7-1 (CD80) -transfected human glioma cells and interleukin-12 directly stimulate allogeneic CD8+ T cells. J Immunother, 20: 256-264．
30． Kourea HP, Orlow I, Scheithauer BW, et al, Deletions of the INK4Agene occur in malignant peripheral nerve sheath tumors but not in neurofibromas. Am J. Pathol., 1999,155(6) , :1855-1860．
31． Lane DP. P53,guardian of the genome.Nature,1992,358:15-16
32． Leimig, T., Brenner, M., Ramsey, J., Vanin, E., Blaese, M., Dilloo, D. (1996) . High-efficiency transduction of freshly isolated human tumor cells using adenoviral interleukin-2 vectors. Hum Gene Ther, 7: 1233-1239．
33． Lowe, S.W., Ruley, H.Z., Jacks, T., et al. (1993) . p53-dependent apoptosis modulates the cytocoxicity of anticancer agents. Cell, 74: 957-967．
34． Mao, L., Merlo,A., Bedi,G., et al. A novel p16INK4A transcript. Cancer Res. 1995,55,2995-2997．
35． Marconi P, Krisky D, Oligino T, et al. (1996) . Replication-defective herpes simplex virus vectors for gene transfer in vivo. Proc Natl Acad Sci USA, 93: 11319
36． Mario, P.C., Guido, F. (1996) . Immunotherapy I : Cytokine gene transfer strategies. Cancer Metast Rev, 15: 317-328．
37． Mewrcer, W.E. (1992) . Cell cycle regulation and the p53 tumor suppressor protein. Crit Rev Eukaruoe Gene Expr, 2: 251-263．
38． Miller, A.D. (1992) . Human gene therapy comes of age. Nature, 357: 455-460．
39． Miyoshi H, Takahashi M, Gage FH, et al. (1997) . Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA, 94: 10319．
40． Montenarh, M. (1992) . Biochemical, immunological, and functional aspects of the growth-suppressor/oncoprotein p53． Crit Rev Oncol, 3: 233-256．
41． Montenarh, M. (1992) . Biochemical, immunological, and functional aspects of the growth-suppressor/oncoprotein p53． Crit Rev Oncol, 3: 233-256．
42． Moss B. (1996) . Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci USA, 93: 11341．
43． Mulligan, R.C. (1993) . The basic science of gene therapy. Science, 260: 926-932．
44． Naldini L, Blomer U, Gage FH, et al. (1996) . Efficient transfer, integration and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA, 93: 11382．
45． Nigro JM,Baker SJ, Preisinger AC. Mutations in the P53 gene occur in diverse human tumor types.Nature,1989,342:705-708
46． Omori, F., Messner, H.A., Ye, C., Gronda, M.V., O'Neill, J.P., Atkins, H., Heng, H.H.Q. (1999) . Nontargeted stable integration of recombinant adeno-associated virus into human leukemia and lymphoma cell lines as evaluated by fluorescence in situ hybridization. Hum Gene Ther, 10: 537-543．
47． Orlow I, LaRue H, Osman I, et al, Deletions of the INK4Agene in superficial bladder tumors, Association with recurrence. Am J. Pathol., 1999,155(1) , :105-113．
48． Paillard, F. (1998) . Cancer gene therapy annual conference 1997: trend and news. Hum Gene Ther, 9: 283-286．
49． Parney, I.F., Petruk, K.C., Zhang, C., Farr-Jones, M., Sykes, D.B., Chang, L.J. (1997) . Granulocyte-Macrophage colony-stimulating factor and B7-2 combination immunogene therapy in an allogeneic Hu-PBL-SCID/Beige mouse-human glioblastoma multiform model. Hum Gene Ther, 8: 1073-1085．
50． Peter, F.S., Lawrence, S.Y. (1996) . Immunotherapy II : Antigen, receptors and costimulation. Cancer Metast Rev, 15: 317-328．
51． Poeschla E, Corbeau P, Wong-Steel F. (1996) . Development of HIV vectors for anti-HIV gene therapy. Proc Natl Acad Sci USA, 93: 11395．
52． Qiu ZH,Lao MF, Wu Chu-tse.(2001) Co-transfer of human wild-type p53 and
granulocyte-macrophage colony-stimulating factor genes via recombinant adenovirus induces apoptosis and enhances immunogenecity in laryngeal cancer cells. Cancer Letters 167,25-32
53． Quelle DE; Zindy F; Ashmun RA; et al. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell, 1995,83,993-1000
54． Richard, G.V., Heung, C. (1996) . Immunotherapy III: Combinatorial molecular immunotherapy-a synthesis and suggestions. Cancer Metast Rev, 15: 317-328．
55． Robbins, P.D., Tahara, H., Ghivizzani, S.C. (1998) . Viral vectors for gene therapy. Tibtech January, 16: 35-40．
56． Roizman B. (1996) . The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors. Proc Natl Acad Sci USA, 93: 11307．
57． Roth, J.A., Cristiano, R.J. (1997) . Gene therapy for cancer: what have we done and where are we going? J Natl Cancer, 89: 21-38．
58． Samulski RJ, Chang LS, Shenk T. (1989) . Heper-free stock of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol, 63: 3822．
59． Sanchez Cespedes M, Reed AL, Buta M, et al, Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancers. Oncogene, 1999,18(43) :5843-5849．
60． Schmitt CA, McCurrach ME, de Stanchina E, et al. INK4A/ARF mutations accelarate lymphomagenesis and promote chemoresistance by disabling p53． Genes Dev., 1999,13(20) :2670-2677．
61． Schultze, J., Nadler, L.M., Gribben, J.G. (1996) . B7-mediated costimulation and the immune response. Blood Rev, 10: 111-127．
62． Sharpless NE and DePinho RA. The INK4A/ARF locus and its two gene products. Curr Opin Genet Dev, 1999, 9, 22-30
63． Scott, R.E. (1997) . Differentiation, differentiation/gene therapy and cancer. Pharmacol Ther, 73:51-65．
64． Shaw, P., Bovey, R., Tardy, S., et al. (1992) . Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci USA, 89: 4495-4499．
65． Smith KT, Shepherd AJ, Boyd JE, et al. (1996) . Gene delivery systems for use in gene therapy: an overview of quality assurance and safety issues. Gene Ther, 3: 190．
66． Somers, K.D., Schechter, G.L. (1992) . Genetic alterations in head and neck cancer. Otolaryngol Clin North Am, 25: 1065-1071．
67． Stott FJ; Bates S; James MC; et al . The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2． EMBO J, 1998, 17,5001-5014
68． Taniguchi T, Chikatsu N, Takahashi S, et al, Expression of p16INK4A and p14ARF in hematological malignancies, Leukemia, 1999,13(11) :1760-1769．
69． Tao W and Levine AJ. P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2． Proc Natl Acad Sci USA, 1999, 96, 6937-6941
70． Theobald M, Biggs J, Dittmer D, et al. Targeting p53 as a general tumor antigen. Proc Natl Acad Sci USA,1995,92:11993-11997．
71． Tominaga, O., Hamelin, R., Remvikos, Y., Salmon, R.J., Thomas, G. (1992) . p53 from basic research to clinical applications. Crit Rev Oncol, 3: 257-282．
72． Trapnell, B.C. (1993) . Adenoviral vectors for gene transfer. Adv Drug Deliv Rev, 12: 185-199．
73． Verma, I.M., Somla, N. (1997) . Gene therapy-promises, problems and prospects. Nature, 389:238-242．
74． Wahl, A.F., Donaldson, K.L., Fairchild, C., et al. (1996) . Loss of normal p53 function confers sensitization to taxol by increasing G2/M arrest and apoptosis. Nat Med, 2: 72-79．
75． Weinberg, R.A. (1991) . Tumor suppressor genes. Science, 254: 1138-1146．
76． Weinberg, R.A. (1992) . The integration of molecular genetics into cancer management. Cancer, 70: 1653-1658．
77． Xu, G.W.K., Sun, Z.T., Forrester, K., et al. (1996) . Tissue-specific growth suppression and chemosensitivity promotion in human hepatocelluar carcinoma cells by retroviral-mediated transfer of the wild type p53 gene. Hepatology, 24: 1264-1268．
78． Yang, S., Vervaert, C.E., Seigler, H.F., Darrow, T.L. (1999) . Tumor cells cotransduced with B7． 1 and r-IFN induce effective rejection of established parental tumor. Gene Ther, 6: 253-262．
79. Yu,J.S.,Burwick,J.A.,Dranoff,G.,Breakefield,X.O.(1997).Gene therapy for metastatic brain tumors by vaccination with granulocyte-macrophage colony-stimulating factor-transduced tumor cells.Hum Gene Ther,8:1065-1072.
80. Zhang Y;Xiong Y and Yarbrough WG.ARF promotes MDM2 degradation and stabilizes p53:ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways.Cell,1998,92,725-734
81. Zufferey,R.,Nagy,D.,Mandel,R.J.,et al.(1997).Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.Nat Biotech,15:871.
82．奥斯伯 F，金斯顿 RE，赛德曼 JG．等，编著．精编分子生物学实验指南[M]．北京：科学出版社，1998：29—108。
83．陈诗书。抗癌基因的产物及其作用。陈诗书、汤雪明主编，医学细胞与分子生物学：512—515。上海医科大学出版社，1995。
84．高志，毛祖彝，高家让。(1997)头颈部鳞状细胞癌的抑癌基因研究。国外医学—口腔学分册，24：20—23。
85．侯亚非，孙宗棠，Appella E等。北方汉族人群HLA—A2亚型分布及p53的合成肽体外诱导CTL反应。中华微生物学和免疫学杂志，1999，19：47—50。
86．卢大儒，邱信芳，薛京伦。(1996)基因转移研究进展。生物工程进展，16：5。
87．卢欣。抑癌基因 p53。肿瘤分子生物学研究进展—’96北京国际肿瘤分子生物学学术年会论文汇编：1—10。军事医学科学出版社，1996。
88．费声重。喉癌。李树玲主编，头颈肿瘤；650—667。天津科学技术出版社，1993。
89．沈关心，毕爱华。肿瘤免疫学。毕爱华主编，医学免疫学；240—252。人民军医出版社，1995。
90．王会贤，王玲。肿瘤免疫。钱玉昆主编，临床免疫学；88—94。北京医科大学中国协和医科大学联合出版社，1994。
91．王征旭，吴祖泽，肿瘤基因工程瘤苗研究进展。国外医学肿瘤学分册，1998，25(1)：1-3。
92．张维维。腺病毒载体及其在基因治疗上的应用。肿瘤分子生物学研究进展—’96北京国际肿瘤分子生物学学术年会论文汇编；1—10。军事医学科学出版社，1996。
93．朱学军，于益芝，曹雪涛。(1996)肿瘤的“自杀基因”疗法研究进展。国外医学—肿瘤学分册，23：36。
2． Akagi, J., Hodge, J.W., McLaughlin, J.P., Gritz, L., Mazzara, G., Kufe, D., Schlom, J., Kantor, J.A. (1997) . Therapeutic antitumor response after immunization with an admixture of recombinant vaccinia viruses expressing a modified MUC1 gene and the murine T-cell costimulatory molecule B7． J Immunother, 20: 38-47．
3． Alexander, R.M., William, H.M., Kelly, H., et al. (1994) . Cytokine-mediated gene therapy for cancer. Ann Surg Oncol, 1: 436-450．
4． Bargonetti, J., Friedmann, P.N., Kem, S.E., et al. (1991) . Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell, 65: 1083-1091．
5． Baskar, S. (1996) . Gene-modified tumor cells as a cellular vaccine. Cancer Immunol Immunother, 43: 165-173．
6． Boyle JO, John H, Koch W. The incidence of P53 mutations increases with progression of head and neck cancer. Cancer Res,1993,53:4477-4480
7． Caplen, N.J., Higginbotham, J.N., Scheel, J.R., Vahanian, N., Yoshida, Y., Hamada, H., Blaese, R.M., Ramsey, W.J. (1999) . Adeno-retroviral chimeric viruses as in vivo transducing agents. Gene Ther, 6: 454-459．
8． Crystal, R.G. (1995) . Transfer of genes to humans: early lessons and obstacles to success. Science, 270: 404-410．
9． Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V, Hamada, H., Pardoll, D., Mulligan, R.C. (1993) . Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent specific and long-lasting antitumor immunity. Proc Natl Acad Sci USA, 90: 3539-3543．
10． Drew, M.P. (1998) Cancer vaccines. Nature Medicine Vaccine, Supplement 4: 525-531．
11． Duro,D., Bernard, O., Valle, V., et al, A new type of p16INK4A/MTS1 gene transcript expressed in B-cell malignancies. Oncogene, 1995,11,21-29．
12． Egilmez NK, Cuenca R, Yokota SJ, et al. (1996) . In vivo cytokine gene therapy of human tumor xenografts in SCID mice by liposome-mediated DNA delivery. Gene Ther, 3:607
13． Fan, S., Smith, M.L., Rivet, D.J., et al. (1995) . Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res. 55, 1649-1654．
14． Fields, S., Jang, S.J. (1990) . Presence of a potent transcription activating sequence in the p53 protein. Science, 249: 1046-1049．
15． Fujiwara, T., Grimm, E.A., Mukhopadhyay, T., et al. (1994) . Induction of chemosensitivity in human lung cancer cells in view by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res, 54: 2287-2291．
16． Gilboa, E., Smita, K.N., Lyerly, H.K. (1998) . Immunotherapy of cancer with dendritic-cell-based vaccines. Cancer Immunol Immunother, 46: 82-87．
17． Graham, F.L., Smiley, J., Russell, W.C., Nairn, R. (1977) . Characteristics of a human cell line transformed by DNA from human adenovirus type 5． J Gen Virol, 36: 59-74．
18． Hawkins, D.S., Demers, G.W., Galloway, D.A. (1996) . Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents. Cancer Res, 56: 892-898．
19． Hermonat PL, Muzyczka N. (1984) . Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA, 81: 6466
20． Herrmann, F. (1995) . Cancer gene therapy: principles, problems and perspectives. J Mol Med, 73: 157-163．
21． Hogge, G.S., et al. (1998) . Development of human granulocyte-macrophage colony-stimulating factor-transfected tumor cell vaccines for the treatment of spontaneous canine cancer. Hum Gene Ther, 9: 1851-1861．
22． Hollstein, M, Sidransky, D., Vogelstein, B., Harris, C.C. (1991) . p53 mutations in human cancers. Science, 253: 49-53．
23． Houbiers JG, Nijman HW, Burg SH, et al. In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53． Eur J Immunol. 1993,23:2072-2077
24． Howard, B., Burrascano, M., McCallister, T., Chong, K., Gangavalli, R., Severinsson L, Jolly, D.J., Darrow, T., Vervaert, C., Abdel-Wahab, Z., Siegler, H.F., Barber, J.R. (1994) .
Retrovirus-mediated gene transfer of the human γ-IFN gene: a therapy for cancer. Ann New York Acad Sci, 716: 167-187．
25． Huschtscha Li and Reddel RR. p16(INK4a) and the control of cellular proliferative life span. Carcinogenesis, 1999, 20, 921-926．
26． Kamijo T; Weber JD; Zambetti G; et al . Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2． Proc Natl Acad Sci USA, 1998, 95, 8292-8297
27． Kamijo T; Zindy F; Roussel MF; et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell, 1997, 91, 649-659
28． Knudson, A.G., Upton, A.C. (1990) . Tumor suppressor gene workshop. Cancer Res, 50: 6765．
29． Komata, T., Tanaka, R., Yamamoto, K., Tazunu, O., Ono, K., Yoshida, S., Takahashi, M. (1997) . B7-1 (CD80) -transfected human glioma cells and interleukin-12 directly stimulate allogeneic CD8+ T cells. J Immunother, 20: 256-264．
30． Kourea HP, Orlow I, Scheithauer BW, et al, Deletions of the INK4Agene occur in malignant peripheral nerve sheath tumors but not in neurofibromas. Am J. Pathol., 1999,155(6) , :1855-1860．
31． Lane DP. P53,guardian of the genome.Nature,1992,358:15-16
32． Leimig, T., Brenner, M., Ramsey, J., Vanin, E., Blaese, M., Dilloo, D. (1996) . High-efficiency transduction of freshly isolated human tumor cells using adenoviral interleukin-2 vectors. Hum Gene Ther, 7: 1233-1239．
33． Lowe, S.W., Ruley, H.Z., Jacks, T., et al. (1993) . p53-dependent apoptosis modulates the cytocoxicity of anticancer agents. Cell, 74: 957-967．
34． Mao, L., Merlo,A., Bedi,G., et al. A novel p16INK4A transcript. Cancer Res. 1995,55,2995-2997．
35． Marconi P, Krisky D, Oligino T, et al. (1996) . Replication-defective herpes simplex virus vectors for gene transfer in vivo. Proc Natl Acad Sci USA, 93: 11319
36． Mario, P.C., Guido, F. (1996) . Immunotherapy I : Cytokine gene transfer strategies. Cancer Metast Rev, 15: 317-328．
37． Mewrcer, W.E. (1992) . Cell cycle regulation and the p53 tumor suppressor protein. Crit Rev Eukaruoe Gene Expr, 2: 251-263．
38． Miller, A.D. (1992) . Human gene therapy comes of age. Nature, 357: 455-460．
39． Miyoshi H, Takahashi M, Gage FH, et al. (1997) . Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA, 94: 10319．
40． Montenarh, M. (1992) . Biochemical, immunological, and functional aspects of the growth-suppressor/oncoprotein p53． Crit Rev Oncol, 3: 233-256．
41． Montenarh, M. (1992) . Biochemical, immunological, and functional aspects of the growth-suppressor/oncoprotein p53． Crit Rev Oncol, 3: 233-256．
42． Moss B. (1996) . Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci USA, 93: 11341．
43． Mulligan, R.C. (1993) . The basic science of gene therapy. Science, 260: 926-932．
44． Naldini L, Blomer U, Gage FH, et al. (1996) . Efficient transfer, integration and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA, 93: 11382．
45． Nigro JM,Baker SJ, Preisinger AC. Mutations in the P53 gene occur in diverse human tumor types.Nature,1989,342:705-708
46． Omori, F., Messner, H.A., Ye, C., Gronda, M.V., O'Neill, J.P., Atkins, H., Heng, H.H.Q. (1999) . Nontargeted stable integration of recombinant adeno-associated virus into human leukemia and lymphoma cell lines as evaluated by fluorescence in situ hybridization. Hum Gene Ther, 10: 537-543．
47． Orlow I, LaRue H, Osman I, et al, Deletions of the INK4Agene in superficial bladder tumors, Association with recurrence. Am J. Pathol., 1999,155(1) , :105-113．
48． Paillard, F. (1998) . Cancer gene therapy annual conference 1997: trend and news. Hum Gene Ther, 9: 283-286．
49． Parney, I.F., Petruk, K.C., Zhang, C., Farr-Jones, M., Sykes, D.B., Chang, L.J. (1997) . Granulocyte-Macrophage colony-stimulating factor and B7-2 combination immunogene therapy in an allogeneic Hu-PBL-SCID/Beige mouse-human glioblastoma multiform model. Hum Gene Ther, 8: 1073-1085．
50． Peter, F.S., Lawrence, S.Y. (1996) . Immunotherapy II : Antigen, receptors and costimulation. Cancer Metast Rev, 15: 317-328．
51． Poeschla E, Corbeau P, Wong-Steel F. (1996) . Development of HIV vectors for anti-HIV gene therapy. Proc Natl Acad Sci USA, 93: 11395．
52． Qiu ZH,Lao MF, Wu Chu-tse.(2001) Co-transfer of human wild-type p53 and
granulocyte-macrophage colony-stimulating factor genes via recombinant adenovirus induces apoptosis and enhances immunogenecity in laryngeal cancer cells. Cancer Letters 167,25-32
53． Quelle DE; Zindy F; Ashmun RA; et al. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell, 1995,83,993-1000
54． Richard, G.V., Heung, C. (1996) . Immunotherapy III: Combinatorial molecular immunotherapy-a synthesis and suggestions. Cancer Metast Rev, 15: 317-328．
55． Robbins, P.D., Tahara, H., Ghivizzani, S.C. (1998) . Viral vectors for gene therapy. Tibtech January, 16: 35-40．
56． Roizman B. (1996) . The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors. Proc Natl Acad Sci USA, 93: 11307．
57． Roth, J.A., Cristiano, R.J. (1997) . Gene therapy for cancer: what have we done and where are we going? J Natl Cancer, 89: 21-38．
58． Samulski RJ, Chang LS, Shenk T. (1989) . Heper-free stock of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol, 63: 3822．
59． Sanchez Cespedes M, Reed AL, Buta M, et al, Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancers. Oncogene, 1999,18(43) :5843-5849．
60． Schmitt CA, McCurrach ME, de Stanchina E, et al. INK4A/ARF mutations accelarate lymphomagenesis and promote chemoresistance by disabling p53． Genes Dev., 1999,13(20) :2670-2677．
61． Schultze, J., Nadler, L.M., Gribben, J.G. (1996) . B7-mediated costimulation and the immune response. Blood Rev, 10: 111-127．
62． Sharpless NE and DePinho RA. The INK4A/ARF locus and its two gene products. Curr Opin Genet Dev, 1999, 9, 22-30
63． Scott, R.E. (1997) . Differentiation, differentiation/gene therapy and cancer. Pharmacol Ther, 73:51-65．
64． Shaw, P., Bovey, R., Tardy, S., et al. (1992) . Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci USA, 89: 4495-4499．
65． Smith KT, Shepherd AJ, Boyd JE, et al. (1996) . Gene delivery systems for use in gene therapy: an overview of quality assurance and safety issues. Gene Ther, 3: 190．
66． Somers, K.D., Schechter, G.L. (1992) . Genetic alterations in head and neck cancer. Otolaryngol Clin North Am, 25: 1065-1071．
67． Stott FJ; Bates S; James MC; et al . The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2． EMBO J, 1998, 17,5001-5014
68． Taniguchi T, Chikatsu N, Takahashi S, et al, Expression of p16INK4A and p14ARF in hematological malignancies, Leukemia, 1999,13(11) :1760-1769．
69． Tao W and Levine AJ. P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2． Proc Natl Acad Sci USA, 1999, 96, 6937-6941
70． Theobald M, Biggs J, Dittmer D, et al. Targeting p53 as a general tumor antigen. Proc Natl Acad Sci USA,1995,92:11993-11997．
71． Tominaga, O., Hamelin, R., Remvikos, Y., Salmon, R.J., Thomas, G. (1992) . p53 from basic research to clinical applications. Crit Rev Oncol, 3: 257-282．
72． Trapnell, B.C. (1993) . Adenoviral vectors for gene transfer. Adv Drug Deliv Rev, 12: 185-199．
73． Verma, I.M., Somla, N. (1997) . Gene therapy-promises, problems and prospects. Nature, 389:238-242．
74． Wahl, A.F., Donaldson, K.L., Fairchild, C., et al. (1996) . Loss of normal p53 function confers sensitization to taxol by increasing G2/M arrest and apoptosis. Nat Med, 2: 72-79．
75． Weinberg, R.A. (1991) . Tumor suppressor genes. Science, 254: 1138-1146．
76． Weinberg, R.A. (1992) . The integration of molecular genetics into cancer management. Cancer, 70: 1653-1658．
77． Xu, G.W.K., Sun, Z.T., Forrester, K., et al. (1996) . Tissue-specific growth suppression and chemosensitivity promotion in human hepatocelluar carcinoma cells by retroviral-mediated transfer of the wild type p53 gene. Hepatology, 24: 1264-1268．
78． Yang, S., Vervaert, C.E., Seigler, H.F., Darrow, T.L. (1999) . Tumor cells cotransduced with B7． 1 and r-IFN induce effective rejection of established parental tumor. Gene Ther, 6: 253-262．
79. Yu,J.S.,Burwick,J.A.,Dranoff,G.,Breakefield,X.O.(1997).Gene therapy for metastatic brain tumors by vaccination with granulocyte-macrophage colony-stimulating factor-transduced tumor cells.Hum Gene Ther,8:1065-1072.
80. Zhang Y;Xiong Y and Yarbrough WG.ARF promotes MDM2 degradation and stabilizes p53:ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways.Cell,1998,92,725-734
81. Zufferey,R.,Nagy,D.,Mandel,R.J.,et al.(1997).Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.Nat Biotech,15:871.
82．奥斯伯 F，金斯顿 RE，赛德曼 JG．等，编著．精编分子生物学实验指南[M]．北京：科学出版社，1998：29—108。
83．陈诗书。抗癌基因的产物及其作用。陈诗书、汤雪明主编，医学细胞与分子生物学：512—515。上海医科大学出版社，1995。
84．高志，毛祖彝，高家让。(1997)头颈部鳞状细胞癌的抑癌基因研究。国外医学—口腔学分册，24：20—23。
85．侯亚非，孙宗棠，Appella E等。北方汉族人群HLA—A2亚型分布及p53的合成肽体外诱导CTL反应。中华微生物学和免疫学杂志，1999，19：47—50。
86．卢大儒，邱信芳，薛京伦。(1996)基因转移研究进展。生物工程进展，16：5。
87．卢欣。抑癌基因 p53。肿瘤分子生物学研究进展—’96北京国际肿瘤分子生物学学术年会论文汇编：1—10。军事医学科学出版社，1996。
88．费声重。喉癌。李树玲主编，头颈肿瘤；650—667。天津科学技术出版社，1993。
89．沈关心，毕爱华。肿瘤免疫学。毕爱华主编，医学免疫学；240—252。人民军医出版社，1995。
90．王会贤，王玲。肿瘤免疫。钱玉昆主编，临床免疫学；88—94。北京医科大学中国协和医科大学联合出版社，1994。
91．王征旭，吴祖泽，肿瘤基因工程瘤苗研究进展。国外医学肿瘤学分册，1998，25(1)：1-3。
92．张维维。腺病毒载体及其在基因治疗上的应用。肿瘤分子生物学研究进展—’96北京国际肿瘤分子生物学学术年会论文汇编；1—10。军事医学科学出版社，1996。
93．朱学军，于益芝，曹雪涛。(1996)肿瘤的“自杀基因”疗法研究进展。国外医学—肿瘤学分册，23：36。