缺氧/辐射双敏感性启动子的构建与肺癌放射—基因治疗实验研究
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摘要
利用放射线诱导治疗基因表达是近年来提出的一种肿瘤治疗新策略，即放射-基因治疗（Radio-genetic therapy），将辐射敏感性调控序列与对肿瘤具有治疗作用的基因相耦联，赋予其放射诱导特性，转入肿瘤细胞，在对肿瘤局部实施放疗时诱导肿瘤治疗基因表达，通过射线和基因双重作用杀伤肿瘤。这一疗法在已有的研究中显示出良好的应用前景。但仍存在明显的不足，辐射诱导的治疗基因表达水平低且持续短暂，尤其在临床常规放疗剂量下，不能满足治疗需要；在活体内，辐射敏感性调控序列的放射诱导活性往往低于体外培养细胞，可能与实体肿瘤细胞缺氧有关。对这些问题目前尚缺乏有效的解决办法，影响了肿瘤放射-基因治疗的疗效和应用。
缺氧引起细胞发生一系列分子改变，缺氧反应元件（Hypoxia response elements，HREs）是介导细胞缺氧反应的重要调控序列，是一缺氧敏感性增强子，通过与缺氧诱导因子-1（Hypoxia inducible factor-1，HIF-1）特异性结合诱导下游基因表达。实体瘤是一缺氧微环境，HIF-1α在肿瘤组织高表达，因此，实体瘤具有激活HRE所需的条件。为此，本研究设想用HRE反应元件增强辐射敏感性调控序列在缺氧条件下的转录活性，尤其是增强在实体瘤内的辐射敏感性，以克服上述放射-基因治疗存在的不足。
早期生长反应基因-1（Early growth response gene-1，Egr-1）启动子是一种辐射敏感性调控序列，广泛用于放射-基因治疗。为探讨以上思路的可行性，本研究进行了以下实验：①构建Egr-1启动子调控的荧光素酶（Luc）报告基因载体，转染肺癌A549细胞，给以2、4、6、8、10Gy γ-线照射，观察照射后0、3、8、12、24、36h各时相点Luc表达水平，鉴定Egr-1启动子的放射诱导活性；检测以上剂量组在0.1%、0.5%、1%、2.5%、5%、10%等氧浓度下照射后8h的Luc表达水平，观察缺氧对Egr-1启动子辐射诱导活性的影响。进一步检测不同氧浓度下照射后过氧化氢（H2O2）产量的变化，观察缺氧对照射后氧自由基产量的影响，探讨缺氧对Egr-1启动子辐射诱导活性的作用机理。②利用基因重组技术将HRE序列与Egr-1启动子相串联，构建HRE-Egr启动子及其调控的Luc报告基因载体。转染A549细胞，给以与上相同剂量的γ-线照射，并检测照射后与上相同时相点Luc表达水平，观察HRE-Egr启动子的放射敏感性；检测以上剂量组在与上相同氧浓度下照射后8h的Luc表达水平，观察HRE-Egr启动子的缺氧敏感性，以及缺氧对其辐射诱导活性的影响。进一步检测在1%氧浓度下各
剂量组照射后报告基因Luc表达的动态变化，观察缺氧对HRE-Egr启动子转录活性持续时间的影响。③构建分别由Egr-1和HRE-Egr启动子调控的抑瘤素M(OSM)（一种可抑制肺癌生长的细胞因子）表达载体，通过脂质体介导转染，G418加压筛选，获得阳性克隆。分为对照组、照射组（6 Gy）、缺氧组（1% O2）、照射+缺氧组（6 Gy + 1% O2）。分别用ELISA法和免疫荧光法检测OSM表达情况；流式细胞仪分析细胞周期改变；通过细胞辐射剂量-存活曲线分析D0值、氧增比（OER）、增敏比（SER）；利用TUNEL法和透射电镜观察细胞凋亡情况。④利用BALB/C裸小鼠建立移植瘤模型，根据接种细胞分为A549细胞组（n=5）、A549/pEO组(含Egr-1启动子) （n=5）、A549/pHEO组（含HRE-Egr启动子）（n=5）。待肿瘤长至一定体积，分别给以总量6 Gy（2 Gy/次，隔日一次）局部照射，其中A549细胞组设一组未经照射作为对照。观察指标包括肿瘤生长曲线、组织学改变、OSM表达水平、TCD50(50%肿瘤控制所需剂量)等。主要结果和结论如下：
1．将Egr-1启动子置于Luc基因上游，构建了Egr-1启动子报告载体，转染A549细胞。结果表明，在γ-线照射下，Luc表达活性迅速显著增强，并呈现明显的照射剂量依赖性，证明本实验中Egr-1启动子具有辐射诱导活性；进一步在不同氧浓度下实施照射，结果发现，当氧浓度<2.5%时，Egr-1启动子的辐射诱导活性随氧浓度的降低而减弱；并且照射后的H2O2产量也呈现类似的变化特征。这一结果表明了缺氧可导致Egr-1启动子辐射诱导活性下降，并提示可能与H2O2等活性氧的产量减少有关。
2．成功构建了HRE-Egr启动子及其调控的Luc报告基因载体pHEL。转染A549细胞，结果发现，在常氧下，HRE-Egr启动子在照射后的表达特征与Egr-1启动子相似；在氧浓度≤2.5%时，HRE-Egr启动子的表达活性明显升高，并于1%氧浓度时达最高值，在2 Gy照射时A549/pHEL细胞Luc表达量为2282±89 ng/L， 是同样条件下Egr-1启动子活性（172±20 ng/L）的13倍多。以上结果说明，嵌合型HRE-Egr启动子具有辐射和缺氧双重敏感性，并且缺氧对辐射诱导活性具有明显的增强作用。结果还发现，在缺氧条件下，HRE-Egr启动子调控的报告基因Luc表达活性持续时间明显延长，在2 Gy照射后，Lu表达活性维持36h，明显长于常氧下的12h。
3．分别用Egr-1和HRE-Egr调控OSM表达，成功构建了辐射敏感性OSM表达载体pEO和缺氧/辐射双敏感性表达载体pHEO。经DOTAP脂质体介导，G418筛选，获得阳性克隆A549/pEO、A549/pHEO。结果发现，在照射合并缺氧时，A549/pHEO细胞OSM表达水平（694.97±73.72 pg/ml）明显高于A549/pEO细胞（98.97±7.95 pg/ml）?
Radio-genetic therapy is a novel strategy for cancer treatment, in which an ionizing radiation-inducible regulatory sequence is linked with an adjuvant tumor-therapeutic cytokine sequence, and transfected into tumor cells. The expression of therapeutic gene, therefore, will be induced by radiation in cancer radiotherapy. The transfected cancer cells will be destroyed by both radiation and the radiation-inducible gene. Although some beneficial effects for tumor therapy have been reported in a recent clinical trial, a few shortcomings still exist in radio-genetic therapy, which include the expression level of therapeutic genes induced by radiation is too low to completely eradicate the tumor, especially under the routine clinical dose of radiation; and the sensitivity to radiation of radio-inducible sequences in in vivo tumor tissues is less than that of in vitro cultured cells. These shortcomings will decrease the therapeutic effects of radio-genetic therapy on tumors.
The cellular adaptive response to hypoxia involves a series of modulations of biochemical and pathophysiological processes such as glucose homeostasis, angiogenesis, vascular permeability and inflammation. HIF-1(hypoxia inducible factor-1) is the core mediator of hypoxic responses. To modulate related gene expression, HIF-1 specifically binds to hypoxia-responsive elements(HREs), enhancers containing the same core sequence in several hypoxia-regulated genes. The HRE/HIF regulatory system was shown to be common to all mammalian cells including human cells tested to date, and HIF-1α subunit was found to be overexpressed in 68% of the human tumors analysed. The high frequency of HIF-1 expression in numerous human tumors with diverse tissue origins provides a possible therapeutic target for HRE-directed gene therapy of tumor cells in hypoxic environments. It has been demonstrated that expression of promoters which were linked with HRE could be enhanced under hypoxic conditions. Solid tumors are characterized by hypoxia which can activate the HRE effectively. Therefore, we want to utilize the HRE to enhance the transcriptional activity of radiation-inducible promoters in
the hypoxic environment of tumors to try to solve the problems with radio-genetic therapy mentioned above.
Cellular responses to ionizing radiation include the transcriptional induction of immediate early genes such as early growth response-1(Egr-1) which may enhance cell death after ionizing radiation. The radiosensitivity of the Egr-1 promoter has been demonstrated, and widely used in radio-genetic therapy. To address the feasibility of hypoxia-specific enhancement of expression of the Egr-1 promoter induced by ionizing radiation, experiments have been carried out as follows:①First, a reporter vector containing the luciferase gene under the control of the Egr-1 promoter was constructed, and transfected into the human adenocarcinoma cell line A549. After exposure to doses of 2、4、6、8 or 10 Gy γ-radiation using a 60Co source, the expression level of luciferase in cell extracts at 0h、3h、8h、12h、24h and 36h post-irradiation was analysed. The expression level of luciferase and H2O2 production in the cells was also detected at 8h post-radiation in trasfected cells with exposure to above doses of radiation under 0.1%、0.5%、1%、2.5%、5% or 10% oxygen concentration. ②An HRE-Egr chimeric promoter was constructed, in which a 0.3 kb fragment of the human vascular endothelial growth factor 5`-flanking sequence containing HRE was fused to the Egr-1 promoter. The chimeric promoter was then inserted into the upstream of the luciferase gene in a reporter vector. Luciferase activity was determined in cell extracts after exposure to the same doses of radiation and oxygen concentrations as shown above. Furthermore, the dynamic alterations in HRE-Egr promoter activity were observed after the cells were incubated in 1% O2 and irradiated. ③The OSM(Oncostatin M) expression vectors pEO and pHEO, in which the Egr-1 promoter or HRE-Egr-1 promoter was inser
缺氧引起细胞发生一系列分子改变，缺氧反应元件（Hypoxia response elements，HREs）是介导细胞缺氧反应的重要调控序列，是一缺氧敏感性增强子，通过与缺氧诱导因子-1（Hypoxia inducible factor-1，HIF-1）特异性结合诱导下游基因表达。实体瘤是一缺氧微环境，HIF-1α在肿瘤组织高表达，因此，实体瘤具有激活HRE所需的条件。为此，本研究设想用HRE反应元件增强辐射敏感性调控序列在缺氧条件下的转录活性，尤其是增强在实体瘤内的辐射敏感性，以克服上述放射-基因治疗存在的不足。
早期生长反应基因-1（Early growth response gene-1，Egr-1）启动子是一种辐射敏感性调控序列，广泛用于放射-基因治疗。为探讨以上思路的可行性，本研究进行了以下实验：①构建Egr-1启动子调控的荧光素酶（Luc）报告基因载体，转染肺癌A549细胞，给以2、4、6、8、10Gy γ-线照射，观察照射后0、3、8、12、24、36h各时相点Luc表达水平，鉴定Egr-1启动子的放射诱导活性；检测以上剂量组在0.1%、0.5%、1%、2.5%、5%、10%等氧浓度下照射后8h的Luc表达水平，观察缺氧对Egr-1启动子辐射诱导活性的影响。进一步检测不同氧浓度下照射后过氧化氢（H2O2）产量的变化，观察缺氧对照射后氧自由基产量的影响，探讨缺氧对Egr-1启动子辐射诱导活性的作用机理。②利用基因重组技术将HRE序列与Egr-1启动子相串联，构建HRE-Egr启动子及其调控的Luc报告基因载体。转染A549细胞，给以与上相同剂量的γ-线照射，并检测照射后与上相同时相点Luc表达水平，观察HRE-Egr启动子的放射敏感性；检测以上剂量组在与上相同氧浓度下照射后8h的Luc表达水平，观察HRE-Egr启动子的缺氧敏感性，以及缺氧对其辐射诱导活性的影响。进一步检测在1%氧浓度下各
剂量组照射后报告基因Luc表达的动态变化，观察缺氧对HRE-Egr启动子转录活性持续时间的影响。③构建分别由Egr-1和HRE-Egr启动子调控的抑瘤素M(OSM)（一种可抑制肺癌生长的细胞因子）表达载体，通过脂质体介导转染，G418加压筛选，获得阳性克隆。分为对照组、照射组（6 Gy）、缺氧组（1% O2）、照射+缺氧组（6 Gy + 1% O2）。分别用ELISA法和免疫荧光法检测OSM表达情况；流式细胞仪分析细胞周期改变；通过细胞辐射剂量-存活曲线分析D0值、氧增比（OER）、增敏比（SER）；利用TUNEL法和透射电镜观察细胞凋亡情况。④利用BALB/C裸小鼠建立移植瘤模型，根据接种细胞分为A549细胞组（n=5）、A549/pEO组(含Egr-1启动子) （n=5）、A549/pHEO组（含HRE-Egr启动子）（n=5）。待肿瘤长至一定体积，分别给以总量6 Gy（2 Gy/次，隔日一次）局部照射，其中A549细胞组设一组未经照射作为对照。观察指标包括肿瘤生长曲线、组织学改变、OSM表达水平、TCD50(50%肿瘤控制所需剂量)等。主要结果和结论如下：
1．将Egr-1启动子置于Luc基因上游，构建了Egr-1启动子报告载体，转染A549细胞。结果表明，在γ-线照射下，Luc表达活性迅速显著增强，并呈现明显的照射剂量依赖性，证明本实验中Egr-1启动子具有辐射诱导活性；进一步在不同氧浓度下实施照射，结果发现，当氧浓度<2.5%时，Egr-1启动子的辐射诱导活性随氧浓度的降低而减弱；并且照射后的H2O2产量也呈现类似的变化特征。这一结果表明了缺氧可导致Egr-1启动子辐射诱导活性下降，并提示可能与H2O2等活性氧的产量减少有关。
2．成功构建了HRE-Egr启动子及其调控的Luc报告基因载体pHEL。转染A549细胞，结果发现，在常氧下，HRE-Egr启动子在照射后的表达特征与Egr-1启动子相似；在氧浓度≤2.5%时，HRE-Egr启动子的表达活性明显升高，并于1%氧浓度时达最高值，在2 Gy照射时A549/pHEL细胞Luc表达量为2282±89 ng/L， 是同样条件下Egr-1启动子活性（172±20 ng/L）的13倍多。以上结果说明，嵌合型HRE-Egr启动子具有辐射和缺氧双重敏感性，并且缺氧对辐射诱导活性具有明显的增强作用。结果还发现，在缺氧条件下，HRE-Egr启动子调控的报告基因Luc表达活性持续时间明显延长，在2 Gy照射后，Lu表达活性维持36h，明显长于常氧下的12h。
3．分别用Egr-1和HRE-Egr调控OSM表达，成功构建了辐射敏感性OSM表达载体pEO和缺氧/辐射双敏感性表达载体pHEO。经DOTAP脂质体介导，G418筛选，获得阳性克隆A549/pEO、A549/pHEO。结果发现，在照射合并缺氧时，A549/pHEO细胞OSM表达水平（694.97±73.72 pg/ml）明显高于A549/pEO细胞（98.97±7.95 pg/ml）?
Radio-genetic therapy is a novel strategy for cancer treatment, in which an ionizing radiation-inducible regulatory sequence is linked with an adjuvant tumor-therapeutic cytokine sequence, and transfected into tumor cells. The expression of therapeutic gene, therefore, will be induced by radiation in cancer radiotherapy. The transfected cancer cells will be destroyed by both radiation and the radiation-inducible gene. Although some beneficial effects for tumor therapy have been reported in a recent clinical trial, a few shortcomings still exist in radio-genetic therapy, which include the expression level of therapeutic genes induced by radiation is too low to completely eradicate the tumor, especially under the routine clinical dose of radiation; and the sensitivity to radiation of radio-inducible sequences in in vivo tumor tissues is less than that of in vitro cultured cells. These shortcomings will decrease the therapeutic effects of radio-genetic therapy on tumors.
The cellular adaptive response to hypoxia involves a series of modulations of biochemical and pathophysiological processes such as glucose homeostasis, angiogenesis, vascular permeability and inflammation. HIF-1(hypoxia inducible factor-1) is the core mediator of hypoxic responses. To modulate related gene expression, HIF-1 specifically binds to hypoxia-responsive elements(HREs), enhancers containing the same core sequence in several hypoxia-regulated genes. The HRE/HIF regulatory system was shown to be common to all mammalian cells including human cells tested to date, and HIF-1α subunit was found to be overexpressed in 68% of the human tumors analysed. The high frequency of HIF-1 expression in numerous human tumors with diverse tissue origins provides a possible therapeutic target for HRE-directed gene therapy of tumor cells in hypoxic environments. It has been demonstrated that expression of promoters which were linked with HRE could be enhanced under hypoxic conditions. Solid tumors are characterized by hypoxia which can activate the HRE effectively. Therefore, we want to utilize the HRE to enhance the transcriptional activity of radiation-inducible promoters in
the hypoxic environment of tumors to try to solve the problems with radio-genetic therapy mentioned above.
Cellular responses to ionizing radiation include the transcriptional induction of immediate early genes such as early growth response-1(Egr-1) which may enhance cell death after ionizing radiation. The radiosensitivity of the Egr-1 promoter has been demonstrated, and widely used in radio-genetic therapy. To address the feasibility of hypoxia-specific enhancement of expression of the Egr-1 promoter induced by ionizing radiation, experiments have been carried out as follows:①First, a reporter vector containing the luciferase gene under the control of the Egr-1 promoter was constructed, and transfected into the human adenocarcinoma cell line A549. After exposure to doses of 2、4、6、8 or 10 Gy γ-radiation using a 60Co source, the expression level of luciferase in cell extracts at 0h、3h、8h、12h、24h and 36h post-irradiation was analysed. The expression level of luciferase and H2O2 production in the cells was also detected at 8h post-radiation in trasfected cells with exposure to above doses of radiation under 0.1%、0.5%、1%、2.5%、5% or 10% oxygen concentration. ②An HRE-Egr chimeric promoter was constructed, in which a 0.3 kb fragment of the human vascular endothelial growth factor 5`-flanking sequence containing HRE was fused to the Egr-1 promoter. The chimeric promoter was then inserted into the upstream of the luciferase gene in a reporter vector. Luciferase activity was determined in cell extracts after exposure to the same doses of radiation and oxygen concentrations as shown above. Furthermore, the dynamic alterations in HRE-Egr promoter activity were observed after the cells were incubated in 1% O2 and irradiated. ③The OSM(Oncostatin M) expression vectors pEO and pHEO, in which the Egr-1 promoter or HRE-Egr-1 promoter was inser
引文
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20. Greco O, Patterson AV, Dachs G. Can gene therapy overcome the problem of hypoxia in radiotherapy ? J Radiat Res, 2000;41:201-212.
21. Brown JM. The hypoxia cell: A target for selective cancer therapy. Cancer Res, 1999;59:5863-5870.
22. Wenger R, Gassman M. Oxygen and the hypoxia inducible factor 1. Biol Chem, 1997;378:609-616.
23. O`Rourke JF, Dachs GU, Gleadle JM, et al. Hypoxia response elements. Oncol Res, 1997;9:327-332.
24. Blancher C, Harris A. The molecular basis of the hypoxia responsive pathway:tumor hypoxia as a therapy target. Cancer Met Rev, 1998;17:187-194.
25. Airley RE, Mrpharms MR, Monaghan JE, et al. Hypoxia and diseases: opportunities for novel diagnostic and therapeutic prodrug strategies. Pharmaceutical Journal, 2000;264:666-673.
26. Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human α-fetoprotein promoter. Cancer Res, 2001;61:3016-3021.
27. Modlich U, Pugh CW, Bicknell R. Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers. Cancer Ther, 2000;7:896-902.
Shibata T, Giaccia AJ, Brown JM. Development of a hypoxia-responsive vector for
28. tumor specific gene therapy. Gene Ther, 2000;7:493-498.
29. Binley K, Iqball S, Kingsman S, et al. An adenoviral vector regulated by hypoxia for the treatment of ischemic disease and cancer. Gene Ther, 1999;6:1721-1727.
30. Boast K, Binley K, Iqball S, et al. Characterization of physiologically regulated vectors for the treatment of ischemic disease. Hum Gene Ther, 1999;10:2197-2208.
31. 蒙国照, 吴名耀. 即刻早期基因Egr-1与肿瘤研究. 国外医学分子生物学分册, 2001; 23（3）：178-180.
32. Datta R, Taneja N, Sukhatme VP, et al. Reactive oxygen intermediates target CC(A/T)6GG sequence to mediate activation the early growth response 1 transcription factor gene by ionizing radiation. Proc Natl Acad Sci USA, 1993;90:2419-2424.
33. 吕星, 邢瑞云, 孙志贤, 等. 小鼠Egr-1基因调控序列的克隆及其辐射诱导特性的鉴定. 中国肿瘤生物治疗杂志, 1998; 5（1）：16-19.
34. Manome Y, Kunieda T, Wen PY, et al. Transgene expression in malignant glioma using a replication-defective adenoviral vector containing the Egr-1 promoter: activation by ionizing radiation or uptake of radioactive iododeoxyuridine. Hum Gene Ther. 1998 ;9(10):1409-17.
35. Advani S, Chmura SJ, Weichselbaum RR. Radiogenetic therapy: on the interaction of viral therapy and ionizing radiation for improving local control of tumors. Seminars in Oncology, 1997;24(6):633-638.
36. 历兴君, 夏爱娣, 陈诗书, 等. Egr-1启动子在肿瘤基因治疗中的应用. 生命科学, 2002; 4（1）：53-55.
37. Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst. 2001;93(4):266-76.
38. Nordsmark M, Alsner J, Keller J, et al. Hypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutations. Br J Cancer. 2001 ;84(8):1070-5.
39. Takahashi T, Namiki Y, Ohno T. Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with radioisotopes. Hum Gene Ther. 1997;8(7):827-33.
40. Mauceri HJ, Hanna NN, Wayne JD, et al. Tumor necrosis factor alpha (TNF-alpha) gene therapy targeted by ionizing radiation selectively damages tumor vasculature. Cancer Res. 1996;56(19):4311-4.
41. 梁国栋，主编. 最新分子生物学实验指南. 北京：科学出版社，2002，200-201.
王卫东，陈正堂，段玉忠，等. 一种定量研究细胞缺氧的简易模型. 中国病理生理
42. 杂志，2003，待刊.
43. 庞战军，周玫，陈瑗，著. 自由基医学研究方法. 北京：人民卫生出版社，2000，18-19.
44. Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982;2(9):1044-51.
45. Wood KV. Marker proteins for gene expression. Curr Opin Biotechnol. 1995;6(1):50-8.
46. de Wet JR, Wood KV, DeLuca M, et al. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987;7(2):725-37.
47. de Wet JR, Wood KV, Helinski DR, DeLuca M. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci U S A. 1985;82(23):7870-3.
48. Wood KV, de Wet JR, Dewji N, DeLuca M. Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns. Biochem Biophys Res Commun. 1984;124(2):592-6.
49. Pazzagli M, Devine JH, Peterson DO, Baldwin TO. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.
Anal Biochem. 1992;204(2):315-23.
50. Alam J, Cook JL. Reporter genes: application to the study of mammalian gene transcription. Anal Biochem. 1990;188(2):245-54.
51. Schneider M, Ow DW, Howell SH. The in vivo pattern of firefly luciferase expression in transgenic plants. Plant Mol Biol. 1990;14(6):935-47.
52. Horsman MR. Measurement of tumor oxygenation. Int J Radiat Oncol Biol Phys. 1998;42(4):701-4.
53. Lyng H, Sundfor K, Rofstad EK. Oxygen tension in human squamous cell carcinoma and adenocarcinoma of the uterine cervix. Adv Exp Med Biol. 1998;454:635-41.
54. Adam MF, Dorie MJ, Brown JM. Oxygen tension measurements of tumors growing in mice. Int J Radiat Oncol Biol Phys. 1999;45(1):171-80.
55. Hallahan DE, Mauceri HJ, Seung LP, et al. Spatial and temporal control of gene therapy using ionizing radiation. Nat Med. 1995;1(8):786-91.
Gashler A, Sukhatme VP. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol.
56. 1995;50:191-224.
57. Rauscher FJ 3rd. Tumor suppressor genes which encode transcriptional repressors: studies on the EGR and Wilms' tumor (WT1) gene products. Adv Exp Med Biol. 1993;348:23-9.
58. Thiel G, Cibelli G. Regulation of life and death by the zinc finger transcription factor Egr-1.J Cell Physiol. 2002 ;193(3):287-92.
59. Liu C, Calogero A, Ragona G, Adamson E, Mercola D. EGR-1, the reluctant suppression factor: EGR-1 is known to function in the regulation of growth, differentiation, and also has significant tumor suppressor activity and a mechanism involving the induction of TGF-beta1 is postulated to account for this suppressor activity. Crit Rev Oncog. 1996;7(1-2):101-25.
60. Staba MJ, Mauceri HJ, Kufe DW, et al. Adenoviral TNF-alpha gene therapy and radiation damage tumor vasculature in a human malignant glioma xenograft. Gene Ther. 1998;5(3):293-300.
61. Khachigian LM, Collins T. Early growth response factor 1: a pleiotropic mediator of inducible gene expression. J Mol Med. 1998;76(9):613-6.
62. Datta R, Rubin E, Sukhatme VP, et al. Ionizing radiation activates trnscription of the Egr-1 gene via CArG elements. Proc Natl Acad Sci USA, 1992;89:10149-10154.
63. 夏寿萱，主编. 放射生物学. 北京：军事医学科学出版社，1998，12-15.
64. 沈瑜，糜福顺. 肿瘤放射生物学. 北京：中国医药科技出版社，2002，14-20.
65. 刘克良，姜德志，主编. 放射损伤与防护学. 北京：原子能出版社，1994，30-38.
66. Meyer RG, Kupper JH, Kandolf R, et al. Early growth response-1 gene (Egr-1) promoter induction by ionizing radiation in U87 malignant glioma cells in vitro. Eur J Biochem. 2002;269(1):337-46.
67. Yan SF, Lu J, Zou YS, et al. Hypoxia-associated induction of early growth response-1 gene expression. J Biol Chem. 1999;274(21):15030-40.
68. Lo LW, Cheng JJ, Chiu JJ, et al. Endothelial exposure to hypoxia induces Egr-1 expression involving PKCalpha-mediated Ras/Raf-1/ERK1/2 pathway. J Cell Physiol. 2001;188(3):304-12.
2. 孙燕，主编. 内科肿瘤学. 北京：人民卫生出版社，2001，640-673.
3. Weichselbaum RR, Hallahan DE, Sukhatme VP，et al. Gene targeted by ionizing radiation. Int J Radiat Oncol Biol Phys, 1992;24:565-567.
4. Hallahan DE，Sukhatme VP，Sherman ML, et al. Protein kinase C mediated x-ray inducibility of nuclear signal transducers Egr-1 and Jun. Proc Natl Acad Sci USA, 1991;88:2156-2160.
5. Sherman ML, Datta R, Hallahan DE, et al. Ionizing radiation regulates expression of the c-jun protooncogene. Proc Natl Acad Sci USA, 1990;87:5663-5666.
6. Brach MA, Hass R, Sherman ML, et al. Ionizing radiation induces expression and binding activity of the nuclear factor kappa B. J Clin Invest, 1991;88:691-695.
7. Sukhatme VP, Kartha S, Toback FG, et al. A novel early growth response gene rapidly induced by fibroblast, epithelial cell and lymphocyte mitogenes. Oncogene Res, 1987;1:343-348.
8. Tsai-Morris CH, Cao X, Sukhatme VP. 5`flanking sequence and genomic structure of Egr-1: a murine mitogen inducible zinc finger encoding gene. Nucleic Acid Res, 1988;16:8835-8840.
9. Weichselbaum RR, Hallahan D, Fuks Z, Kufe D. Radiation induction of immediate early genes: effectors of the radiation-stress response. Int J Radiat Oncol Biol Phys. 1994;30(1):229-34.
10. Gubits RM, Geard CR, Schiff PB. Expression of immediate early genes after treatment of human astrocytoma cells with radiation and taxol. Int J Radiat Oncol Biol Phys. 1993;27(3):637-42.
11. Weichselbaum RR, Hallahan DE, Beckett MA, et al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells. Cancer Res, 1994;54:4266-4269.
12. Seung LP, Mauceri HJ, Michael A, et al. Genetic radiotherapy overcomes tumor resistance to cytotoxic agents. Cancer Res, 1995;55:5561-5565.
13. Joki T, Nakamura M, Ohno T. Activation of the radiosensitive Egr-1 promoter induces expression of the herpes simples virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir. Hum Gene Ther, 1995;6:1507-1513.
Kim JH, Kim SH, Brown SL, et al. Selective enhancement by an antiviral agent of the
14. radiation induced vell killing of human glioma cells transduced with HSV-TK gene. Cancer Res, 1994;54:6053-6056.
15. Kawashita Y, Ohtsuru A, Kaneda Y, et al. Regression of hepatocellular carcinoma in vitro and in vivo by radiosensitizing suicide gene therapy under the inducible and spatial control of radiation. Hum Gene Ther, 1999;10:1509-1519.
16. 吕星，邢瑞云，路萍，等. OSM对黑色素瘤的抑制作用与基因-放射治疗效果.中国肿瘤生物治疗杂志，20001；8（1）：27-30.
17. Scott SD, Marples B, Hendry JH, et al. A radiation-controlled molecular switch for use in gene therapy of cancer. Gene Ther, 2000,7:1121-1125.
18. Weichselbaum RR, Kufe DW, Advani SJ et al. Molecular targeting of gene therapy and radiotherapy. Acta Oncologia, 2001;40(6):735-738.
19. Brown JM, Giaccia AJ. The unique physiology of solid tumor: opportunities(and problems) for cancer therapy. Cancer Res, 1998; 58:1408-1416.
20. Greco O, Patterson AV, Dachs G. Can gene therapy overcome the problem of hypoxia in radiotherapy ? J Radiat Res, 2000;41:201-212.
21. Brown JM. The hypoxia cell: A target for selective cancer therapy. Cancer Res, 1999;59:5863-5870.
22. Wenger R, Gassman M. Oxygen and the hypoxia inducible factor 1. Biol Chem, 1997;378:609-616.
23. O`Rourke JF, Dachs GU, Gleadle JM, et al. Hypoxia response elements. Oncol Res, 1997;9:327-332.
24. Blancher C, Harris A. The molecular basis of the hypoxia responsive pathway:tumor hypoxia as a therapy target. Cancer Met Rev, 1998;17:187-194.
25. Airley RE, Mrpharms MR, Monaghan JE, et al. Hypoxia and diseases: opportunities for novel diagnostic and therapeutic prodrug strategies. Pharmaceutical Journal, 2000;264:666-673.
26. Ido A, Uto H, Moriuchi A, et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human α-fetoprotein promoter. Cancer Res, 2001;61:3016-3021.
27. Modlich U, Pugh CW, Bicknell R. Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers. Cancer Ther, 2000;7:896-902.
Shibata T, Giaccia AJ, Brown JM. Development of a hypoxia-responsive vector for
28. tumor specific gene therapy. Gene Ther, 2000;7:493-498.
29. Binley K, Iqball S, Kingsman S, et al. An adenoviral vector regulated by hypoxia for the treatment of ischemic disease and cancer. Gene Ther, 1999;6:1721-1727.
30. Boast K, Binley K, Iqball S, et al. Characterization of physiologically regulated vectors for the treatment of ischemic disease. Hum Gene Ther, 1999;10:2197-2208.
31. 蒙国照, 吴名耀. 即刻早期基因Egr-1与肿瘤研究. 国外医学分子生物学分册, 2001; 23（3）：178-180.
32. Datta R, Taneja N, Sukhatme VP, et al. Reactive oxygen intermediates target CC(A/T)6GG sequence to mediate activation the early growth response 1 transcription factor gene by ionizing radiation. Proc Natl Acad Sci USA, 1993;90:2419-2424.
33. 吕星, 邢瑞云, 孙志贤, 等. 小鼠Egr-1基因调控序列的克隆及其辐射诱导特性的鉴定. 中国肿瘤生物治疗杂志, 1998; 5（1）：16-19.
34. Manome Y, Kunieda T, Wen PY, et al. Transgene expression in malignant glioma using a replication-defective adenoviral vector containing the Egr-1 promoter: activation by ionizing radiation or uptake of radioactive iododeoxyuridine. Hum Gene Ther. 1998 ;9(10):1409-17.
35. Advani S, Chmura SJ, Weichselbaum RR. Radiogenetic therapy: on the interaction of viral therapy and ionizing radiation for improving local control of tumors. Seminars in Oncology, 1997;24(6):633-638.
36. 历兴君, 夏爱娣, 陈诗书, 等. Egr-1启动子在肿瘤基因治疗中的应用. 生命科学, 2002; 4（1）：53-55.
37. Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst. 2001;93(4):266-76.
38. Nordsmark M, Alsner J, Keller J, et al. Hypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutations. Br J Cancer. 2001 ;84(8):1070-5.
39. Takahashi T, Namiki Y, Ohno T. Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with radioisotopes. Hum Gene Ther. 1997;8(7):827-33.
40. Mauceri HJ, Hanna NN, Wayne JD, et al. Tumor necrosis factor alpha (TNF-alpha) gene therapy targeted by ionizing radiation selectively damages tumor vasculature. Cancer Res. 1996;56(19):4311-4.
41. 梁国栋，主编. 最新分子生物学实验指南. 北京：科学出版社，2002，200-201.
王卫东，陈正堂，段玉忠，等. 一种定量研究细胞缺氧的简易模型. 中国病理生理
42. 杂志，2003，待刊.
43. 庞战军，周玫，陈瑗，著. 自由基医学研究方法. 北京：人民卫生出版社，2000，18-19.
44. Gorman CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982;2(9):1044-51.
45. Wood KV. Marker proteins for gene expression. Curr Opin Biotechnol. 1995;6(1):50-8.
46. de Wet JR, Wood KV, DeLuca M, et al. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987;7(2):725-37.
47. de Wet JR, Wood KV, Helinski DR, DeLuca M. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci U S A. 1985;82(23):7870-3.
48. Wood KV, de Wet JR, Dewji N, DeLuca M. Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns. Biochem Biophys Res Commun. 1984;124(2):592-6.
49. Pazzagli M, Devine JH, Peterson DO, Baldwin TO. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.
Anal Biochem. 1992;204(2):315-23.
50. Alam J, Cook JL. Reporter genes: application to the study of mammalian gene transcription. Anal Biochem. 1990;188(2):245-54.
51. Schneider M, Ow DW, Howell SH. The in vivo pattern of firefly luciferase expression in transgenic plants. Plant Mol Biol. 1990;14(6):935-47.
52. Horsman MR. Measurement of tumor oxygenation. Int J Radiat Oncol Biol Phys. 1998;42(4):701-4.
53. Lyng H, Sundfor K, Rofstad EK. Oxygen tension in human squamous cell carcinoma and adenocarcinoma of the uterine cervix. Adv Exp Med Biol. 1998;454:635-41.
54. Adam MF, Dorie MJ, Brown JM. Oxygen tension measurements of tumors growing in mice. Int J Radiat Oncol Biol Phys. 1999;45(1):171-80.
55. Hallahan DE, Mauceri HJ, Seung LP, et al. Spatial and temporal control of gene therapy using ionizing radiation. Nat Med. 1995;1(8):786-91.
Gashler A, Sukhatme VP. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol.
56. 1995;50:191-224.
57. Rauscher FJ 3rd. Tumor suppressor genes which encode transcriptional repressors: studies on the EGR and Wilms' tumor (WT1) gene products. Adv Exp Med Biol. 1993;348:23-9.
58. Thiel G, Cibelli G. Regulation of life and death by the zinc finger transcription factor Egr-1.J Cell Physiol. 2002 ;193(3):287-92.
59. Liu C, Calogero A, Ragona G, Adamson E, Mercola D. EGR-1, the reluctant suppression factor: EGR-1 is known to function in the regulation of growth, differentiation, and also has significant tumor suppressor activity and a mechanism involving the induction of TGF-beta1 is postulated to account for this suppressor activity. Crit Rev Oncog. 1996;7(1-2):101-25.
60. Staba MJ, Mauceri HJ, Kufe DW, et al. Adenoviral TNF-alpha gene therapy and radiation damage tumor vasculature in a human malignant glioma xenograft. Gene Ther. 1998;5(3):293-300.
61. Khachigian LM, Collins T. Early growth response factor 1: a pleiotropic mediator of inducible gene expression. J Mol Med. 1998;76(9):613-6.
62. Datta R, Rubin E, Sukhatme VP, et al. Ionizing radiation activates trnscription of the Egr-1 gene via CArG elements. Proc Natl Acad Sci USA, 1992;89:10149-10154.
63. 夏寿萱，主编. 放射生物学. 北京：军事医学科学出版社，1998，12-15.
64. 沈瑜，糜福顺. 肿瘤放射生物学. 北京：中国医药科技出版社，2002，14-20.
65. 刘克良，姜德志，主编. 放射损伤与防护学. 北京：原子能出版社，1994，30-38.
66. Meyer RG, Kupper JH, Kandolf R, et al. Early growth response-1 gene (Egr-1) promoter induction by ionizing radiation in U87 malignant glioma cells in vitro. Eur J Biochem. 2002;269(1):337-46.
67. Yan SF, Lu J, Zou YS, et al. Hypoxia-associated induction of early growth response-1 gene expression. J Biol Chem. 1999;274(21):15030-40.
68. Lo LW, Cheng JJ, Chiu JJ, et al. Endothelial exposure to hypoxia induces Egr-1 expression involving PKCalpha-mediated Ras/Raf-1/ERK1/2 pathway. J Cell Physiol. 2001;188(3):304-12.