Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells

We examined whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the promoter of the vascular endothelial growth factor (VEGF) gene that is activated by hypoxia is effective in killing highly metastatic Lewis lung carcinoma A11 cells under hypoxic conditions. We isolated the promoter region encompassing the hypoxia response element (HRE) of the mouse VEGF gene. To assess the hypoxia responsiveness of the VEGF promoter, A11 cells were transiently transfected with luciferase reporter plasmids. Exposure of the transfectants to hypoxia resulted in a 2-3-fold induction of luciferase activity. Deletion of the HRE site abolished VEGF promoter activity under both normoxic and hypoxic conditions. We constructed a retroviral vector harboring the HSV-TK or green fluorescence protein (GFP) gene under the control of the VEGF promoter. A11 cells transfected with vector harboring the VEGF promoter fused to the HSV-TK gene [A11(HRE/TK) cells] were more sensitive to ganciclovir than cells transfected with the control vector harboring the VEGF promoter alone, and the sensitivity of the A11(HRE/TK) cells was increased by exposure to hypoxia followed by reoxygenation. Culturing A11 cells transfected with vector harboring the VEGF promoter fused to the GFP gene under hypoxic conditions resulted in an increase in the expression of GFP. Monitoring GFP expression and vascularity in the A11 transfectant tumors revealed up-regulation of GFP expression in poorly vascularized regions. Administration of ganciclovir to mice bearing s.c. tumors formed by A11(HRE/TK) cells resulted in regression of the tumors. These results suggest a possible application of the suicide gene driven by the VEGF promoter to cancer gene therapy that efficiently targets hypoxic tumor cells.     

Killing of brain tumor cells by hypoxia-responsive element mediated expression of BAX

The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE), which can be activated through hypoxia-inducible factor-1 (HIF-1). We transfected plasmids containing multiple copies of HRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HRE copy number, and the degree of hypoxia.     

受缺氧反应元件调控的荧光素酶报告基因在缺氧肿瘤细胞内的表达活性研究

 目的 研究缺氧反应元件调控的荧光素酶报告基因在肿瘤细胞内的缺氧反应性。方法 将pGL3-6HRE-TATA-luciferase-SV40pa报告载体以Lipofectamine 2000介导瞬时转染Hela S3和HepG2细胞，分别于缺氧（1 % O2）和常氧（21 % O2）培养后检测相对荧光素酶活性。结果 pGL3-6HRE- TATA- luciferase-SV40pa报告载体转染细胞后，缺氧培养条件下相对荧光素酶的活性较常氧培养条件下显著上调，差异具有统计学意义（P＜0.01），并且该缺氧诱导性具有一定的时间依赖性，即Hela S3细胞中，缺氧培养12～24 h，相对荧光素酶的活性上升到了最高值；而HepG2细胞中，缺氧培养36 h，相对荧光素酶的活性上升到了最高值。结论 6HRE人工启动子在肿瘤细胞内具有显著的缺氧诱导性；6HRE人工启动子缺氧诱导性具有一定的时间效应，并且在不同肿瘤细胞中其诱导效应和时间效应可存在着差异。研究结果为实体肿瘤基因治疗奠定了理论和实验基础。     

Hypoxia regulates choline kinase expression through hypoxia-inducible factor-1 alpha signaling in a human prostate cancer model

The intensity of the total choline (tCho) signal in spectroscopic images of tumors is spatially heterogeneous. The likewise heterogeneous physiologic tumor microenvironment may contribute to this heterogeneity. We therefore investigated the relationship between hypoxia, choline metabolites, and choline kinase (Chk) in a human prostate cancer model. Human PC-3 prostate cancer cells were engineered to express enhanced green fluorescent protein (EGFP) under hypoxic conditions. These PC-3-5HRE-EGFP cells were characterized in culture and as tumors transplanted in mice using (1)H magnetic resonance spectroscopy (MRS) and MRS imaging (MRSI) combined with EGFP fluorescence microscopy and imaging. Hypoxic EGFP-fluorescing tumor regions colocalized with regions of high tCho in combined MRSI and optical imaging studies. Cellular phosphocholine (PC) and tCho concentrations as well as Chk expression levels significantly increased following exposure of PC-3 cells to hypoxia. A putative promoter region located 5' of the translation start site of the human chk-alpha gene was cloned and luciferase (Luc)-based reporter vector constructs were generated. Luc reporter assays provided evidence that some of the putative hypoxia response elements (HRE) within this putative chk-alpha promoter region functioned in vitro. Chromatin immunoprecipitation assays using an antibody against hypoxia-inducible factor (HIF)-1 alpha showed that HIF-1 can directly bind this region of the endogenous chk-alpha promoter in hypoxic PC-3-5HRE-EGFP cells. These data suggest that HIF-1 activation of HREs within the putative chk-alpha promoter region can increase Chk-alpha expression within hypoxic environments, consequently increasing cellular PC and tCho levels within these environments.     

t（6；11）染色体易位肾细胞癌中Alpha基因片段的克隆及启动子活性分析

  目的   构建包含不同Alpha基因片段的重组报告质粒和Alpha1-TFEB融合基因表达质粒，并评价Alpha基因启动子活性。   方法   利用软件对Alpha基因进行启动子预测；采用PCR技术扩增出5种不同长度的Alpha基因片段和正常TFEB基因启动子序列pTFEB，构建含Alpha基因片段和pTFEB的重组报告质粒；采用脂质体转染法将其分别转染至人胚肾293T细胞；通过荧光素酶活性检测确定Alpha基因的启动子活性。同时构建含Alpha1-TFEB融合基因表达质粒，转染293T细胞后，采用Western blot法检测转染前后TFEB蛋白的表达水平。   结果   成功构建含不同Alpha基因片段和pTFEB的重组报告质粒。与pGL3-Basic质粒转染组进行比较，Alpha1、Alpha2、Alpha3、Alpha4、Alpha5质粒转染组的荧光素酶活性显著增高（P＜0.01）；与正常TFEB基因启动子进行比较，Alpha1、Alpha2、Alpha5的荧光素酶活性明显增高（P＜0.01）；与pGL3-Enhancer质粒转染组进行比较，pGL3-Enhancer-Alpha1-TFEB表达质粒组TFEB蛋白的表达明显升高。   结论   t（6；11）染色体易位肾细胞癌中Alpha基因具有启动子活性，可促进TFEB表达，Alpha5片段最强活性区域位于643~693位碱基序列。      

人脂肪酸合成酶FAS启动子的克隆及其在几种肿瘤细胞中的转录活性分析

目的:克隆人脂肪酸合成酶基因FAS启动子的序列,构建重组荧光素酶表达载体pGL3-FAS,并分析其在几种肿瘤细胞中的转录活性,为其作为肿瘤靶向基因治疗的工具提供依据。方法:以人基因组DNA为模板,PCR扩增FAS启动子区680bp活性序列,经测序鉴定正确后,克隆至荧光素酶表达载体pGL3-enhancer中,构建成重组荧光素酶表达载体pGL3-FAS。将pGL3-FAS通过脂质体转染胃癌细胞系SGC-7901、人宫颈癌细胞系Hela、人乳腺癌细胞系SKBR3、人肝癌细胞系HepG2以及NIH3T3成纤维细胞系中,应用荧光素酶检测系统测定荧光素酶活性,通过内参校正得到相对转录活性。结果:成功扩增出大小约为680bp的FAS启动子序列,经测序鉴定与Genebank报道的一致。经酶切鉴定,成功构建重组荧光素酶表达载体pGL3-FAS。瞬时转染pGL3-FAS,发现其在SGC-7901、Hela、SKBR3、HepG2细胞中均具有较强的荧光素酶活性,且荧光素酶活性高于强启动子SV40驱动的pGL3-control载体;而在正常成纤维细胞中,转录活性较低。结论:FAS启动子在肿瘤细胞中具有强转录活性,而在正常细胞中转录活性很低,具有良好的肿瘤靶向性,为肿瘤靶向基因治疗提供理论依据。     

PC-1基因在肿瘤组织中的表达及其启动子区的克隆和活性分析

背景与目的：PC－1是在骨转移和雄激素非依赖的前列腺癌细胞株C4－2中高表达的新基因。本文拟研究PC－1基因在多种人肿瘤组织和正常组织中的表达水平,并对该基因的启动子区进行克隆及活性分析。方法：以PC－1基因特异性DNA序列为探针与10对肿瘤和正常组织的总RNA进行杂交;以C4－2细胞基因组DNA为模板,PCR扩增PC－1基因翻译起始位点上游DNA序列。PCR产物定向克隆到含荧光素酶报告基因的载体pGL3-Basic上,将重组质粒瞬时转染C4－2细胞,荧光素酶定量分析检测启动子活性。结果：多肿瘤组织中PC－1基因的表达水平明显高于相应的正常组织中的表达;翻译起始位点上游340bp的片段没有启动子活性,而ATG前1099bp、1337bp,1579bp,1831bp和4939bp长度的片段都表现出启动子的功能活性。结论：PC－1基因在人前列腺癌以及多种肿瘤组织中被特异激活,表明该基因的功能可能和肿瘤的发生有关;研究的初步结果表明4939bp长度的启动子活性最强,以1831bp和4939bp之间可能含有转录增强子元件。     

Enhanced efficacy of radiation-induced gene therapy in mice bearing lung adenocarcinoma xenografts using hypoxia responsive elements

The aim of the present study was to investigate whether the hypoxia responsive element (HRE) could be used to enhance suicide gene (HSV-tk) expression and tumoricidal activity in radiation-controlled gene therapy of human lung adenocarcinoma xenografts. A chimeric promoter, HRE-Egr, was generated by directly linking a 0.3-kb fragment of HRE to a 0.6-kb human Egr-1 promoter. Retroviral vectors containing luciferase or the HSV-tk gene driven by Egr-1 or HRE-Egr were constructed. A human adenocarcinoma cell line (A549) was stably transfected with the above vectors using the lipofectamine method. The sensitivity of transfected cells to prodrug ganciclovir (GCV) and cell survival rates were analyzed after exposure to a dose of 2 Gy radiation and hypoxia (1%). In vivo, tumor xenografts in BALB/c mice were transfected with the constructed retroviruses and irradiated to a total dose of 6 Gy, followed by GCV treatment (20 mg/kg for 14 days). When the HSV-tk gene controlled by the HRE-Egr promoter was introduced into A549 cells by a retroviral vector, the exposure to 1% O(2) and 2 Gy radiation induced significant enhancement of GCV cytotoxicity to the cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors infected with the hybrid promoter-containing virus gradually disappeared after GCV administration and radiation. These results indicate that HRE can enhance transgene expression and tumoricidal activity in HSV-tk gene therapy controlled by ionizing radiation in hypoxic human lung adenocarcinoma.     

HIF1A induces expression of the WASF3 metastasis-associated gene under hypoxic conditions

The WASF3 (WAVE3) gene is an important mediator of cell motility, invasion and metastasis and is expressed at high levels in some advanced stage tumors. In our survey of breast cancer cells, we now demonstrate that exposure to hypoxic conditions increases WASF3 expression levels in MDA231, SKBR3 and MCF7 cells. The WASF3 promoter region contains HIF1A response elements (HRE). ChIP assays demonstrate that HIF1A binds to these HRE elements in the promoter region, and luciferase reporter assays using the WASF3 gene minimal promoter shows that hypoxia results in its upregulation. Phosphorylation of WASF3 is required for its ability to affect invasion and increased phosphoactivation of WASF3 is also seen in cells challenged with hypoxia. These cells also show increased motility in the scratch wound assay. Cells in which WASF3 has been knocked down show no response to hypoxia as expected, implicating the specificity of the hypoxic response to WASF3. Overall, these experiments demonstrate WASF3 is a HIF1A-regulated gene and suggests a mechanism to explain the observation of elevated expression of WASF3 in advanced stage tumors.     

hTERT启动子的克隆及hTERT启动子/SV40增强子在食管癌细胞中的转录活性

目的 克隆hTERT启动子核心序列,研究hTERT启动子/SV40增强子在食管癌细胞中的联合转录活性。 方法 以人基因组DNA为模板,PCR扩增hTERT启动子核心片段;将其分别插入荧光素酶基因报告质粒pGL3-Basic和pGL3-Enhancer中,构建hTERT启动子调控的表达载体pGL3-hTERTp和由hTERT启动子/SV40增强子联合调控的表达载体pGL3-hTERTp-SV40en,将上述重组质粒分别瞬时转染食管癌细胞Eca-109、EC1和人胚肺成纤维细胞MRC-5,用荧光素酶检测试剂盒检测转染细胞中荧光素酶基因的表达水平并以此计算hTERT启动子和hTERT启动子/SV40增强子在各种细胞中的转录活性。结果 克隆出长213 bp的hTETR启动子核心片段,DNA测序结果与GenBank中hTERT启动子的碱基序列完全一致;成功构建真核表达载体pGL3-hTERTp和pGL3-hTERTp-SV40en;hTETR启动子在食管癌细胞Eca-109和EC1中均有转录活性,在MRC-5细胞中无明显转录活性;hTERT启动子/SV40增强子在食管癌细胞Eca-109和EC1中的转录活性显著高于hTETR启动子的单独转录活性。结论 hTERT启动子在食管癌细胞中具有靶向性转录活性,SV40增强子能显著增强hTERT启动子在食管癌细胞中的转录活性,有可能作为肿瘤靶向性基因治疗的转录调控元件。     

survivin启动子的克隆及在HeLa细胞中的特异生物活性

目的: 构建带有survivin启动子的pGL3Basic真核表达载体，探讨survivin启动子在HeLa细胞中的特异表达活性。 方法: 采用PCR技术扩增survivin启动子，插入pGL3Basic载体，构建携带survivin启动子的pGL3Basic真核表达载体（pGL3Basic/ Surp）。纯化pGL3Basic/ Surp质粒，用脂质体法转染HeLa细胞和正常血管内皮细胞ECV304，48 h后收集转染细胞与荧光素酶底物反应，检测荧光素酶活性。 结果: 成功克隆1 kb survivin基因启动子，并构建了携带有survivin基因启动子的pGL3Basic真核表达载体，转染后的Hela细胞荧光素酶活性为2074.2±78.5，而ECV304荧光素酶活性为9.7±1.1。 结论： 成功克隆的survivin启动子在HeLa细胞中表现出较高的肿瘤特异性活性，为进一步开发肿瘤的靶向基因治疗奠定了基础     

LCRG1基因5′端调控区域的克隆及活性测定

背景与目的:LCRG1基因的调控机制至今不清楚,本研究拟克隆LCRG1基因的转录起始区上游序列,寻找与其表达有关的调控序列。方法:利用生物信息学技术预测LCRG1基因5′端调控区域的启动子活性区域,采用PCR方法从人基因组DNA中扩增LCRG1基因5′端调控区域1.776kb(-1191bp～ 585bp)片段,并将其构建到荧光素酶报告基因pGL3basic载体中。与内参照质粒PhRL-SV40共转染COS7细胞,通过双荧光素酶活性检验测定其启动子活性。结果:成功扩增LCRG1基因5′端调控区域1.776kb(-1191bp～ 585bp)片段,测序正确;pGL3-1776启动子活性大约为阳性对照组pGL3-control的0.16倍,为阴性组pGL3basic的35倍。结论:成功克隆LCRG1基因5′端调控区域1.776kb(-1191bp～ 585bp),该片段具有启动子活性。     

人类生存蛋白启动子的克隆及其在淋巴瘤细胞中的转录活性研究

  目的　克隆人类生存蛋白（Survivin）核心启动子,研究Survivin启动子在人类淋巴瘤细胞Ramos和健康人肝脏细胞Chang Liver中的转录活性。方法　以人类肠基因组DNA为模板,PCR扩增Survivin启动子987 bp片段,将其通过酶切位点连入pGL3-Basic载体构建pGL3-Survivin荧光素酶报告基因载体,通过脂质体转染法转染Ramos和Chang Liver 细胞,通过检测荧光素酶表达水平,比较Survivin启动子在这两种细胞中的转录活性。结果　成功克隆出987 bp的Survivin启动子;双酶切、PCR检测和DNA测序证实pGL3-Survivin载体构建成功;荧光素酶活性检查显示：Survivin启动子在Ramos细胞中的转录活性为阳性对照CMV启动子活性的4.5 %,明显高于在Chang Liver细胞中的0.19 %,在淋巴瘤细胞中具有较高特异性,且在淋巴瘤细胞中的活性明显高于阴性对照pGL3-Basic的0.086 %。结论　Survivin启动子在淋巴瘤细胞中具有较高的特异性,可以作为淋巴瘤细胞基因转染的肿瘤特异性启动子使用。