放射控导的野生型p53蛋白正反馈基因环路提高肺腺癌细胞对放射敏感性的实验研究
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摘要
背景与目的
放射-基因治疗即同时将具有肿瘤杀伤和辐射诱导特性的基因转入人体细胞内,对肿瘤局部实施照射时诱导肿瘤杀伤基因的表达,造成射线和基因对肿瘤的双重杀伤作用。大量文献报道,Egr-1在照射后可使其下游基因表达迅速增加,其启动子是一种辐射敏感性调控序列cArGs。p53反应元件(pREs)是p53蛋白上调下游基因表达的增强子序列。然而,在常规分割剂量下,辐射敏感性序列诱导的治疗性基因表达强度低且持续时间短,治疗效果不理想。正反馈是基因环路中的一种重要模式,系指由特定基因与目的基因相互作用,构成对目的基因表达放大调控的基因亚网络。本研究将正反馈基因环路引入放射-基因治疗,即将放射敏感性反应元件(cArGs)与p53反应元件(pREs)和p53基因串联构成的正反馈基因环路相耦联,构建放射控导的野生型p53蛋白正反馈基因环路,分析该环路在体外环境的动力学特征及放大效应。进一步利用该基因环路在时间、空间上调控wt-p53基因的表达,研究其在裸鼠体内的抗肿瘤效应,旨在探索既可相对降低等照射剂量,缓解正常组织损伤,又可实现肿瘤放射局部野生型p53基因持续性定位表达,诱导肺癌细胞凋亡的肺癌治疗新途径。
研究方法
1.构建基因环路表达载体及对照组载体
将全基因合成的放射敏感性增强子(E6)、p53反应元件(R4)、以及二者的耦联物(E6R4),分别取代PCI-neo真核表达载体的CMV增强子,但保留核心启动子TATA盒;采用PCR技术,以pcDNA3.1(+)-p53-EGFP质粒为模板,扩增wt-p53 cDNA全长序列;双酶切从双顺反子表达质粒pIRES2-EGFP中获得IRES2-EGFP片段。将上述片段依次克隆于PCI-neo载体相应的多克隆位点,分别构建pE6-p53-EGFP、pR4-p53-EGFP、pE6R4-p53-EGFP表达载体。酶切、测序鉴定目的片段正确性。
2.体外分析放射控导的wt-p53蛋白正反馈基因环路动力学特征
将上述3个重组子和pcDNA3.1(+)-p53-EGFP质粒稳定转染肺腺癌细胞株H1299(p53-/-),以H1299(p53-/-)为对照,用RT-PCR方法分别检测各组细胞wt-p53基因与细胞基因组整合情况。
1) wt-p53蛋白正反馈基因环路的鉴定采用免疫组化法获得Ad5CMV-p53感染H1299(p53-/-)细胞最佳感染复数;以最佳感染复数(MOI=40)感染pR4-p53-EGFP/H1299细胞(V+P组),设质粒组(P组)和单纯病毒感染组(V组)为对照组,Western-blot方法鉴定不同时相点各组细胞wt-p53蛋白表达情况。
2) cArGs放射敏感性的鉴定
Western-blot方法检测pE6-p53-EGFP/H1299细胞在不同放射剂量及同一放射剂量不同时相点wt-p53蛋白表达量和持续时间。
3)放射控导的wt-p53蛋白正反馈基因环路动力学特征检测倒置荧光显微镜及流式细胞仪分析不同放射剂量的EGFP分布情况;Western-blot检测pE6R4-p53-EGFP/H1299细胞在不同放射剂量和同一放射剂量不同时相点的wt-p53蛋白表达量和持续时间。
3.体外分析放射控导的wt-p53蛋白正反馈基因环路的抗癌生物学效应给予治疗组pE6R4-p53-EGFP/H1299细胞和对照组pcDNA3.1(+)-p53-EGFP/H1299细胞、pE6-p53-EGFP/H1299细胞、pR4-p53-EGFP/H1299和H1299(p53-/-)细胞4 Gy 8MV X线照射,分析该环路在放射线诱导下对肺腺癌细胞周期和细胞凋亡率的影响;绘制细胞放射剂量-存活曲线,计算D0值及放射增敏比。
4.分析放射控导的wt-p53蛋白正反馈基因环路对肺腺癌细胞裸鼠移植瘤模型生物学行为的影响
将治疗组pE6R4-p53-EGFP/H1299细胞和对照组pE6-p53-EGFP/H1299、pR4-p53-EGFP/H1299、H1299(p53-/-)细胞分别注射于裸鼠背部皮下,建立裸鼠移植瘤模型;分别给予射线照射,记录治疗开始后肿瘤体积的变化,绘制肿瘤生长曲线;称取瘤重,计算抑瘤率;HE染色观察瘤组织形态结构;免疫组化法检测瘤组织wt-p53表达;将治疗组细胞和对照组细胞成瘤裸鼠,分别给予不同剂量的X线照射,观察照射后肿瘤体积变化,分析计算TCD50及SER。
研究结果
1.成功构建重组质粒
构建了治疗载体pE6R4-p53-EGFP和对照载体pE6-p53-EGFP、pR4-53-EGFP表达载体,酶切和测序鉴定所克隆片段正确。
2.阐明了放射控导的wt-p53蛋白正反馈基因环路动力学特征
RT-PCR方法从4组稳定转染重组质粒的细胞内均扩增出wt-p53基因,说明该基因在转染的细胞中稳定表达。免疫组化法确定Ad5CMV-p53 MOI=40为最佳感染复数。Western-blot分析质粒组(p组)wt-p53蛋白表达随着时相延长无明显差异(p>0.05),病毒组(V组)和病毒感染的质粒组(V+P组) wt-p53蛋白均在感染后72h达高峰,随着时间延长,二者均下降,但后者下降缓慢;3周时,(V+P组)组分别为P组和V组的2.15倍和6.25倍,证明正反馈环路成立。分别用不同剂量照射治疗组pE6R4-p53-EGFP/H1299细胞和对照组pE6-p53-EGFP/H1299细胞,0～4Gy,wt-p53蛋白表达呈增高趋势,4Gy照射时,wt-p53蛋白表达量最高,治疗组是对照组的1.42倍。6Gy照射时,蛋白量均显著下降,治疗组是对照组1.58倍,与该剂量射线对细胞损伤较重相关。4Gy照射后3h,两组细胞wt-p53蛋白均开始升高,12h达高峰,但治疗组是对照组的1.651倍,对照组在36h蛋白基本恢复假照射水平,而治疗组在144h时其蛋白表达量是假照射水平的1.86倍。FCM分析在照射后12h和144h,治疗组pE6R4-p53-EGFP/H1299细胞EGFP荧光强度分别是对照组细胞pCMV-p53-EGFP荧光强度的3.95倍和2.5倍,说明wt-p53蛋白正反馈基因环路在放射控导下可使wt-p53蛋白表达量显著增高。
3.体外证实放射控导的wt-p53蛋白正反馈基因环路策略有显著的抗癌生物学行为有显著影响
4Gy X线照射治疗组和对照组细胞后12h,FCM分析治疗组在照射后发生明显G0/G1期阻滞(75.13±1.42)%。照射后各组细胞凋亡率均高于照射前组,照射后治疗组细胞凋亡率(23.73±0.21%)分别是对照组4组细胞的5.69倍(4.17±0.12)%、1.91倍(12.40±0.20) %、1.51倍(15.67±0.32) %和2.57倍(9.23±0.15) %。分析计算pE6R4-p53-EGFP/ H1299、pE6-p53-EGFP/H1299和H1299(p53-/-)三组细胞D0值分别0.91Gy、1.073 Gy和1.413 Gy,根据D0值计算SER分别为2.63和1.34,说明放射可诱导wt-p53正反馈基因基因环路,上调wt-p53蛋白表达,调控细胞周期和诱导细胞凋亡,该基因环路可增加肺腺癌细胞放射敏感性。
4.体内证实放射控导的wt-p53蛋白正反馈基因环路策略对肺腺癌细胞裸鼠移植瘤模型生物学行为的影响
免疫组化结果显示移植瘤组织中wt-p53蛋白表达定位于细胞核内,pE6R4-p53- EGFP/H1299治疗组wt-p53蛋白表达量(64.8%)显著高于对照组pR4-p53-EGFP/H1299组(4.2%)、pE6-p53-EGFP/H1299组(22.1%)和H1299(p53-/-)组,其中H1299(p53-/-)组无表达。移植瘤生长曲线表明,pE6R4-p53-EGFP/H1299+IR组移植瘤的生长明显受到抑制,抑瘤率(86.41%)明显高于对照组pE6-p53-EGFP/H1299+IR组(70.76%)、pR4- p53-EGFP/H1299+IR组(35.53%)和H1299+IR组(12.58%)。分析计算pE6R4-p53- EGFP/H1299治疗组、pE6-p53-EGFP/H1299和H1299(p53-/-)对照组的荷瘤鼠TCD50分别为12.1 Gy、15.2 Gy和19.4 Gy。根据TCD50计算pE6R4-p53-EGFP/H1299治疗组和pE6-p53-EGFP/H1299组SER分别为1.6和1.28,说明治疗载体组的肿瘤组织对放射最敏感,放射控导的wt-p53蛋白正反馈基因环路明显增加了瘤组织对放射的敏感性。
研究结论
成功构建了放射控导的wt-p53蛋白正反馈基因环路,该基因环路不仅使目的基因的表达水平提高,而且使目的基因在转染细胞内的表达逐步放大和持续表达,较好地解决了目前放射-基因治疗中所遇到的基因表达水平低且短暂的问题。由于基因环路可明显增强细胞中wt-p53蛋白的表达水平,因此能够有效使肿瘤细胞停滞于G1期,诱导细胞凋亡,提高肺癌细胞对放射的敏感性。裸鼠体内移植瘤模型实验进一步证实了这种正反馈基因环路在放射线作用下对肿瘤的杀伤效果。因此,我们的实验结果不仅完善了放射-基因治疗这一新的肿瘤治疗模式,而且对其它基因治疗模式也有一定的借鉴作用。
BACKGROUND AND AIM
Combination of irradiation therapy and gene therapy offers a promising strategy for cancer treatment, which can introduce dual cyto-killing activities derived from the radiation and the transformed gene(s), respectively. There have been several lines of evidence for the existance of irradiation-susceptible sequence(s) in cells such as E6, which acts as an enhancer and up-regulates its target gene expression. However, under the routine fractioned irrdiation dose, the expression of therapeutic gene induced by irradiation-responsive sequence is low and transient, leading to compromised therapeutic effects.
The positive feedback circuit is one of important models of gene feedback circuits in which the target genes are up-regulated by themselves. Thus in this study, we will introduce the positive feedback circuit to the irradiation-gene therapy strategy and verify the effect of this modified strategy on tumor suppression. The irradiation-sensitive response element cArG[CC(A+T)6GC], i.e., E6, will be coupled with p53 response element pREs and wild type p53 gene, establishing an irradiation-controlled wt-p53 positive feedback circuit. The dynamics and the potency of up-regulating target gene wt-p53 of this feedback circuit will be investigated in vitro. Finally, the anti-tumor effects of this feedback circuit in the transplanted tumor nude mouse model will be observed to validate the effects of this circuit to up-regulate the expression of wt-p53 gene spatio-temporally. This research will explore a novel strategy of anti-tumor therapy that can not only decrease the needed irradiation dose thus to relieve the damage of irradiation to normal tissues, but also induce the persisitant expression of wt-p53 at the local tumor site, causing the apoptosis of tumor cells.
METHODS
1. Construction of plasmid of wt-p53 gene positive feedback circuit
The CMV promoter of PCI-neo vector was replaced by six copies of synthetic cArGs (E6) element, p53 response element, or the fusioned E6 element and R4, respectivley. The wt-p53 cDNA was amplifed from the recombinant plasmid pcDNA3.1(+)-p53-EGFP by PCR. The fragment of IRES2-EGFP was prepared from plsmid IRES2-EGFP by double enzyme digestion. The two fragments were inserted into the MCS downstream of enhancer E6, R4, or E6R4 to construct pE6-p53-EGFP, pR4-p53-EGFP, or pE6R4-p53-EGFP plasmids, respectively. These plasmids were verified by both double enzyme digestion and sequencing.
2. Analysis of the dynamic characteristics of positive feedback circuit regulated by irradiation:
The recombinant plasmids and pcDNA3.1(+)-p53-EGFP were transfected into H1299(p53-/-) cells with liposome reagent and the cells stably expressing wt-p53 gene were selected with G418 antibiotics. The expression of wt-p53 gene in these cells was decteted with RT-PCR.
1) Identification of positive feedback wt-p53 gene circuit: H1299(p53-/-) cells were infected by different dose of Ad5CMV-p53 virus, and the optimal MOI was determined by immunohistochemistry. The expression of wt-p53 protein was detcted by Western-blot assay in the pR4-53-EGFP/H1299 group (P group), Ad5CMV-p53/H1299 cell group (V group) and experimental group (V + P group).
2) Identification of radiosensitive cArGs element: The level and the duration of wt-p53 protein expression were detected at different irradiation dose, or different phase with 4Gy.
3) The observation of dynamic characteristics of gene circuit induced by irradiation: The expression level of EGFP was observed with microscope and FCM; The level and the duration of wt-p53 protein expression in pE6R4-p53-EGFP/H1299 cells and control groups were detected by Western-blot after irradiation.
3. Analysis of anti-tumor activity of irradiation-induced wt-p53 positive circuit The cell life cycle and apoptosis caused by the irradiation-induced positive feedback gene circuit were analyzed, after the experimental group pE6R4-p53-EGFP/H1299 and the control group H1299(p53-/-), pE6-p53-EGFP/H1299, pR4-p53-EGFP/H1299, pcDNA3.1 (+)-p53-EGFP/H1299 were exposed to 4Gy radiation dose. D0 and sensitive enhancement ratio (SER) index were caculated from the irradiation dose-survival curve.
4. Anti-tumor effects of irradiation-induced wt-p53 positive feedback circuit on the transplanted lung adenocarcinoma in nude mouse
Nude mouse lung adenocarcinoma transplant model was eastablished by subcutaneous injection of pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299, pR4-p53-EGFP/H1299 and H1299(p53-/-) cells, respectively. Tumor size in these nude mice was measured at the indicated time points post exposure to irradiation, tumor growth curve was drawn. TCD50 and SER value were calculated. At the final experimental stage, the animals were sacrificed and the tumor-inhibition rate was calculated. The morphology of the tumors was observed by HE staning and the expression of wt-p53 protein was detected by immunohistochemistry assay.
RESULTS
1. Construction of recombinant plasmids and transfection to H1299 cells Recombinant plasmid pE6R4-p53/EGFP, and control plasmids pE6-p53/EGFP and pR4-53/EEGFP were constructed and verified by enzymatic digestion and sequencing. After transfection of these plasmids to H1299 cells and selection with G418, wt-p53 mRNA could be amplified from the above 4 groups of transfected cells by RT-PCR method, indicating that stable p53-expressing cells were established.
2. Dynamic characteristics of irradiation-induced wt-p53 positive feedback circuit The optimal multiplicity of infectin (MOI) for Ad5CMV-p53 was identified as 40 by immnohistochemistry assay. Western-blot assays showed that the p53 expression in plasmid-transfected cells did not changed during the experimental period (p>0.05) in contrast, the p53 expression in V-group and V+P group reached the peak at 72h post infection (p.i.) and thereafter decreased gradually. At 3 weeks, the p53 protein expression level in P+V group was 2.15 and 6.25 folds of that in P and V group, respectively, indicating the exsitence of the positive feedback circuit.
When exposed to 0-4Gy of irradiation, pE6R4-p53-EGFP/H1299 cells of therapeutic group and pE6-p53-EGFP/H1299 cells of control group expressed increased wt-p53 protein along with the increment of irradiation doses. The summit level of wt-p53 was observed at 4Gy irradiation in therapeutic group which was 1.42 fold of that in control group, but the expression decreased significantly at 6Gy irradiation, reflecting the severe injury of cells at this dose of irradiation. Thus, 4Gy irradiation was used in following experiments in this study.
At 3h post to 4Gy irradiation, wt-p53 expression could be detected in both groups. The expression level peaked at 12h post irradiation (p.i.) and at this time point, the expression in therapeutic group was 1.651 fold of that in control group. While the wt-p53 expression in control group decreased to baseline at 36h p.i., however, the cells from therapeutic group could express high level of wt-p53 even at 144h p.i., 1.86 fold of that without irradiation. FCM analyses showed that EGFP fluoresence density in therapeutic group was 3.95 and 2.5 folds of the control group at 12h and 144h p.i., respectively, indicating wt-p53 positive feedback circuit could up-regulating wt-p53 expression markedly.
3. Anti-tumor effect of irradiation-induced wt-p53 positive feedback circuit in vitro
At 12h post 4Gy X-ray irradiation, FCM analysis showed that most cells in therapeutic group arrested at G0/G1 stage (75.13±1.42)%, compared with control groups (38.47±0.87)%, (62.57±0.76)%,(51.23±2.41)%, respectively. After irradiation, the cell apoptosis rate in each group was higher than that in cells without irradiation. The apoptosis rate in therapeutic group was (23.73±0.21)%, which was 5.69 folds (4.17±0.12)%, 1.91 folds (12.40±0.20)%, 1.51 folds(15.67±0.32)% and 2.57 folds(9.23±0.15)% of that in four control groups, respectively. The D0 values were 0.91Gy, 1.073 Gy and 1.413 Gy in pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299 and H1299, respectivley. The SER, derived from D0 values, was 2.63 and 1.34, respectively, indicating irradiation could up-regulate wt-p53 expression, regulate cell cycle and induce cell apoptosis, thus this positive feedback circuit could increase the sensitivity of lung adenocarcinoma to irradiation.
4. Effects of the wt-p53 positive feedback circuit on the transplantated tumor in nude mice
Various levels of immunoreactivity for wt-p53 were found in the tissues of therapeutic group and control groups. The expression level of wt-p53 was evaluated as before described in section of materials and methods. Wt-p53 immunostaining was observed in cellular nucleus. Positive wt-p53 staining was increased greatly in H1299/pE6R4-p53-EGFP group (64.8 % ), moderately in H1299/pE6-p53-EGFP group (22.3 % ) and poorly in H1299/pR4-p53 -EGFP group(5.4%). Negative wt-p53 staining was observed in H1299 cell xenograft tumor group. The growth curves of xenograft tumors show that the tumor inhibition ratio in pE6R4-p53-EG FP/H1299/IR group is higher than that in other 3 control groups. The inhibitation radio is 86.41 % , 70.76 % , 35.53 % and 12.58 % in pE6R4-p53-EG FP/H1299+IR group, H1299/pE6-p53-EGFP+IR group, pR4-p53-EG FP/H1299+IR group and H1299+IR group, respectively. The TCD50 values were 12.1 Gy, 15.2 Gy and 19.4 Gy in pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299 and H1299, respectivley. The SER, derived from TCD50 values, was 1.6 and 1.28, respectively, indicating wt-p53 status were significant related to the activation of the positive feedback circuit induced by IR and the radiosensitivity of xenograft tumor in therapeautic group was enhanced significantly.
CONCLUSIONS
In this study, we have constructed a recombinant pladmid containing an irradiation-induced wt-p53 positive feedback circuit, which can lead to much higher expression level of wt-p53 in transfected cells, thus it addressed the problem of current irradiation-gene therapy strategy, i.e., the weak and transient expression of target gene. Because this gene feedback circuit can obviously increase wt-p53 expression in tumor cells, it thus can cause the tumor cells to arrest at G1 stage and induce cell apoptosis, leading to the increased susceptibility of lung tumor to irradiation. This effect of the wt-p53 feedback circuit has been validated further in a transplanted tumor model in nude mice. Therefore, our results have not only improved the irradiation-gene therapy strategy but also could be a reference for other gene therapy strategy.
放射-基因治疗即同时将具有肿瘤杀伤和辐射诱导特性的基因转入人体细胞内,对肿瘤局部实施照射时诱导肿瘤杀伤基因的表达,造成射线和基因对肿瘤的双重杀伤作用。大量文献报道,Egr-1在照射后可使其下游基因表达迅速增加,其启动子是一种辐射敏感性调控序列cArGs。p53反应元件(pREs)是p53蛋白上调下游基因表达的增强子序列。然而,在常规分割剂量下,辐射敏感性序列诱导的治疗性基因表达强度低且持续时间短,治疗效果不理想。正反馈是基因环路中的一种重要模式,系指由特定基因与目的基因相互作用,构成对目的基因表达放大调控的基因亚网络。本研究将正反馈基因环路引入放射-基因治疗,即将放射敏感性反应元件(cArGs)与p53反应元件(pREs)和p53基因串联构成的正反馈基因环路相耦联,构建放射控导的野生型p53蛋白正反馈基因环路,分析该环路在体外环境的动力学特征及放大效应。进一步利用该基因环路在时间、空间上调控wt-p53基因的表达,研究其在裸鼠体内的抗肿瘤效应,旨在探索既可相对降低等照射剂量,缓解正常组织损伤,又可实现肿瘤放射局部野生型p53基因持续性定位表达,诱导肺癌细胞凋亡的肺癌治疗新途径。
研究方法
1.构建基因环路表达载体及对照组载体
将全基因合成的放射敏感性增强子(E6)、p53反应元件(R4)、以及二者的耦联物(E6R4),分别取代PCI-neo真核表达载体的CMV增强子,但保留核心启动子TATA盒;采用PCR技术,以pcDNA3.1(+)-p53-EGFP质粒为模板,扩增wt-p53 cDNA全长序列;双酶切从双顺反子表达质粒pIRES2-EGFP中获得IRES2-EGFP片段。将上述片段依次克隆于PCI-neo载体相应的多克隆位点,分别构建pE6-p53-EGFP、pR4-p53-EGFP、pE6R4-p53-EGFP表达载体。酶切、测序鉴定目的片段正确性。
2.体外分析放射控导的wt-p53蛋白正反馈基因环路动力学特征
将上述3个重组子和pcDNA3.1(+)-p53-EGFP质粒稳定转染肺腺癌细胞株H1299(p53-/-),以H1299(p53-/-)为对照,用RT-PCR方法分别检测各组细胞wt-p53基因与细胞基因组整合情况。
1) wt-p53蛋白正反馈基因环路的鉴定采用免疫组化法获得Ad5CMV-p53感染H1299(p53-/-)细胞最佳感染复数;以最佳感染复数(MOI=40)感染pR4-p53-EGFP/H1299细胞(V+P组),设质粒组(P组)和单纯病毒感染组(V组)为对照组,Western-blot方法鉴定不同时相点各组细胞wt-p53蛋白表达情况。
2) cArGs放射敏感性的鉴定
Western-blot方法检测pE6-p53-EGFP/H1299细胞在不同放射剂量及同一放射剂量不同时相点wt-p53蛋白表达量和持续时间。
3)放射控导的wt-p53蛋白正反馈基因环路动力学特征检测倒置荧光显微镜及流式细胞仪分析不同放射剂量的EGFP分布情况;Western-blot检测pE6R4-p53-EGFP/H1299细胞在不同放射剂量和同一放射剂量不同时相点的wt-p53蛋白表达量和持续时间。
3.体外分析放射控导的wt-p53蛋白正反馈基因环路的抗癌生物学效应给予治疗组pE6R4-p53-EGFP/H1299细胞和对照组pcDNA3.1(+)-p53-EGFP/H1299细胞、pE6-p53-EGFP/H1299细胞、pR4-p53-EGFP/H1299和H1299(p53-/-)细胞4 Gy 8MV X线照射,分析该环路在放射线诱导下对肺腺癌细胞周期和细胞凋亡率的影响;绘制细胞放射剂量-存活曲线,计算D0值及放射增敏比。
4.分析放射控导的wt-p53蛋白正反馈基因环路对肺腺癌细胞裸鼠移植瘤模型生物学行为的影响
将治疗组pE6R4-p53-EGFP/H1299细胞和对照组pE6-p53-EGFP/H1299、pR4-p53-EGFP/H1299、H1299(p53-/-)细胞分别注射于裸鼠背部皮下,建立裸鼠移植瘤模型;分别给予射线照射,记录治疗开始后肿瘤体积的变化,绘制肿瘤生长曲线;称取瘤重,计算抑瘤率;HE染色观察瘤组织形态结构;免疫组化法检测瘤组织wt-p53表达;将治疗组细胞和对照组细胞成瘤裸鼠,分别给予不同剂量的X线照射,观察照射后肿瘤体积变化,分析计算TCD50及SER。
研究结果
1.成功构建重组质粒
构建了治疗载体pE6R4-p53-EGFP和对照载体pE6-p53-EGFP、pR4-53-EGFP表达载体,酶切和测序鉴定所克隆片段正确。
2.阐明了放射控导的wt-p53蛋白正反馈基因环路动力学特征
RT-PCR方法从4组稳定转染重组质粒的细胞内均扩增出wt-p53基因,说明该基因在转染的细胞中稳定表达。免疫组化法确定Ad5CMV-p53 MOI=40为最佳感染复数。Western-blot分析质粒组(p组)wt-p53蛋白表达随着时相延长无明显差异(p>0.05),病毒组(V组)和病毒感染的质粒组(V+P组) wt-p53蛋白均在感染后72h达高峰,随着时间延长,二者均下降,但后者下降缓慢;3周时,(V+P组)组分别为P组和V组的2.15倍和6.25倍,证明正反馈环路成立。分别用不同剂量照射治疗组pE6R4-p53-EGFP/H1299细胞和对照组pE6-p53-EGFP/H1299细胞,0～4Gy,wt-p53蛋白表达呈增高趋势,4Gy照射时,wt-p53蛋白表达量最高,治疗组是对照组的1.42倍。6Gy照射时,蛋白量均显著下降,治疗组是对照组1.58倍,与该剂量射线对细胞损伤较重相关。4Gy照射后3h,两组细胞wt-p53蛋白均开始升高,12h达高峰,但治疗组是对照组的1.651倍,对照组在36h蛋白基本恢复假照射水平,而治疗组在144h时其蛋白表达量是假照射水平的1.86倍。FCM分析在照射后12h和144h,治疗组pE6R4-p53-EGFP/H1299细胞EGFP荧光强度分别是对照组细胞pCMV-p53-EGFP荧光强度的3.95倍和2.5倍,说明wt-p53蛋白正反馈基因环路在放射控导下可使wt-p53蛋白表达量显著增高。
3.体外证实放射控导的wt-p53蛋白正反馈基因环路策略有显著的抗癌生物学行为有显著影响
4Gy X线照射治疗组和对照组细胞后12h,FCM分析治疗组在照射后发生明显G0/G1期阻滞(75.13±1.42)%。照射后各组细胞凋亡率均高于照射前组,照射后治疗组细胞凋亡率(23.73±0.21%)分别是对照组4组细胞的5.69倍(4.17±0.12)%、1.91倍(12.40±0.20) %、1.51倍(15.67±0.32) %和2.57倍(9.23±0.15) %。分析计算pE6R4-p53-EGFP/ H1299、pE6-p53-EGFP/H1299和H1299(p53-/-)三组细胞D0值分别0.91Gy、1.073 Gy和1.413 Gy,根据D0值计算SER分别为2.63和1.34,说明放射可诱导wt-p53正反馈基因基因环路,上调wt-p53蛋白表达,调控细胞周期和诱导细胞凋亡,该基因环路可增加肺腺癌细胞放射敏感性。
4.体内证实放射控导的wt-p53蛋白正反馈基因环路策略对肺腺癌细胞裸鼠移植瘤模型生物学行为的影响
免疫组化结果显示移植瘤组织中wt-p53蛋白表达定位于细胞核内,pE6R4-p53- EGFP/H1299治疗组wt-p53蛋白表达量(64.8%)显著高于对照组pR4-p53-EGFP/H1299组(4.2%)、pE6-p53-EGFP/H1299组(22.1%)和H1299(p53-/-)组,其中H1299(p53-/-)组无表达。移植瘤生长曲线表明,pE6R4-p53-EGFP/H1299+IR组移植瘤的生长明显受到抑制,抑瘤率(86.41%)明显高于对照组pE6-p53-EGFP/H1299+IR组(70.76%)、pR4- p53-EGFP/H1299+IR组(35.53%)和H1299+IR组(12.58%)。分析计算pE6R4-p53- EGFP/H1299治疗组、pE6-p53-EGFP/H1299和H1299(p53-/-)对照组的荷瘤鼠TCD50分别为12.1 Gy、15.2 Gy和19.4 Gy。根据TCD50计算pE6R4-p53-EGFP/H1299治疗组和pE6-p53-EGFP/H1299组SER分别为1.6和1.28,说明治疗载体组的肿瘤组织对放射最敏感,放射控导的wt-p53蛋白正反馈基因环路明显增加了瘤组织对放射的敏感性。
研究结论
成功构建了放射控导的wt-p53蛋白正反馈基因环路,该基因环路不仅使目的基因的表达水平提高,而且使目的基因在转染细胞内的表达逐步放大和持续表达,较好地解决了目前放射-基因治疗中所遇到的基因表达水平低且短暂的问题。由于基因环路可明显增强细胞中wt-p53蛋白的表达水平,因此能够有效使肿瘤细胞停滞于G1期,诱导细胞凋亡,提高肺癌细胞对放射的敏感性。裸鼠体内移植瘤模型实验进一步证实了这种正反馈基因环路在放射线作用下对肿瘤的杀伤效果。因此,我们的实验结果不仅完善了放射-基因治疗这一新的肿瘤治疗模式,而且对其它基因治疗模式也有一定的借鉴作用。
BACKGROUND AND AIM
Combination of irradiation therapy and gene therapy offers a promising strategy for cancer treatment, which can introduce dual cyto-killing activities derived from the radiation and the transformed gene(s), respectively. There have been several lines of evidence for the existance of irradiation-susceptible sequence(s) in cells such as E6, which acts as an enhancer and up-regulates its target gene expression. However, under the routine fractioned irrdiation dose, the expression of therapeutic gene induced by irradiation-responsive sequence is low and transient, leading to compromised therapeutic effects.
The positive feedback circuit is one of important models of gene feedback circuits in which the target genes are up-regulated by themselves. Thus in this study, we will introduce the positive feedback circuit to the irradiation-gene therapy strategy and verify the effect of this modified strategy on tumor suppression. The irradiation-sensitive response element cArG[CC(A+T)6GC], i.e., E6, will be coupled with p53 response element pREs and wild type p53 gene, establishing an irradiation-controlled wt-p53 positive feedback circuit. The dynamics and the potency of up-regulating target gene wt-p53 of this feedback circuit will be investigated in vitro. Finally, the anti-tumor effects of this feedback circuit in the transplanted tumor nude mouse model will be observed to validate the effects of this circuit to up-regulate the expression of wt-p53 gene spatio-temporally. This research will explore a novel strategy of anti-tumor therapy that can not only decrease the needed irradiation dose thus to relieve the damage of irradiation to normal tissues, but also induce the persisitant expression of wt-p53 at the local tumor site, causing the apoptosis of tumor cells.
METHODS
1. Construction of plasmid of wt-p53 gene positive feedback circuit
The CMV promoter of PCI-neo vector was replaced by six copies of synthetic cArGs (E6) element, p53 response element, or the fusioned E6 element and R4, respectivley. The wt-p53 cDNA was amplifed from the recombinant plasmid pcDNA3.1(+)-p53-EGFP by PCR. The fragment of IRES2-EGFP was prepared from plsmid IRES2-EGFP by double enzyme digestion. The two fragments were inserted into the MCS downstream of enhancer E6, R4, or E6R4 to construct pE6-p53-EGFP, pR4-p53-EGFP, or pE6R4-p53-EGFP plasmids, respectively. These plasmids were verified by both double enzyme digestion and sequencing.
2. Analysis of the dynamic characteristics of positive feedback circuit regulated by irradiation:
The recombinant plasmids and pcDNA3.1(+)-p53-EGFP were transfected into H1299(p53-/-) cells with liposome reagent and the cells stably expressing wt-p53 gene were selected with G418 antibiotics. The expression of wt-p53 gene in these cells was decteted with RT-PCR.
1) Identification of positive feedback wt-p53 gene circuit: H1299(p53-/-) cells were infected by different dose of Ad5CMV-p53 virus, and the optimal MOI was determined by immunohistochemistry. The expression of wt-p53 protein was detcted by Western-blot assay in the pR4-53-EGFP/H1299 group (P group), Ad5CMV-p53/H1299 cell group (V group) and experimental group (V + P group).
2) Identification of radiosensitive cArGs element: The level and the duration of wt-p53 protein expression were detected at different irradiation dose, or different phase with 4Gy.
3) The observation of dynamic characteristics of gene circuit induced by irradiation: The expression level of EGFP was observed with microscope and FCM; The level and the duration of wt-p53 protein expression in pE6R4-p53-EGFP/H1299 cells and control groups were detected by Western-blot after irradiation.
3. Analysis of anti-tumor activity of irradiation-induced wt-p53 positive circuit The cell life cycle and apoptosis caused by the irradiation-induced positive feedback gene circuit were analyzed, after the experimental group pE6R4-p53-EGFP/H1299 and the control group H1299(p53-/-), pE6-p53-EGFP/H1299, pR4-p53-EGFP/H1299, pcDNA3.1 (+)-p53-EGFP/H1299 were exposed to 4Gy radiation dose. D0 and sensitive enhancement ratio (SER) index were caculated from the irradiation dose-survival curve.
4. Anti-tumor effects of irradiation-induced wt-p53 positive feedback circuit on the transplanted lung adenocarcinoma in nude mouse
Nude mouse lung adenocarcinoma transplant model was eastablished by subcutaneous injection of pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299, pR4-p53-EGFP/H1299 and H1299(p53-/-) cells, respectively. Tumor size in these nude mice was measured at the indicated time points post exposure to irradiation, tumor growth curve was drawn. TCD50 and SER value were calculated. At the final experimental stage, the animals were sacrificed and the tumor-inhibition rate was calculated. The morphology of the tumors was observed by HE staning and the expression of wt-p53 protein was detected by immunohistochemistry assay.
RESULTS
1. Construction of recombinant plasmids and transfection to H1299 cells Recombinant plasmid pE6R4-p53/EGFP, and control plasmids pE6-p53/EGFP and pR4-53/EEGFP were constructed and verified by enzymatic digestion and sequencing. After transfection of these plasmids to H1299 cells and selection with G418, wt-p53 mRNA could be amplified from the above 4 groups of transfected cells by RT-PCR method, indicating that stable p53-expressing cells were established.
2. Dynamic characteristics of irradiation-induced wt-p53 positive feedback circuit The optimal multiplicity of infectin (MOI) for Ad5CMV-p53 was identified as 40 by immnohistochemistry assay. Western-blot assays showed that the p53 expression in plasmid-transfected cells did not changed during the experimental period (p>0.05) in contrast, the p53 expression in V-group and V+P group reached the peak at 72h post infection (p.i.) and thereafter decreased gradually. At 3 weeks, the p53 protein expression level in P+V group was 2.15 and 6.25 folds of that in P and V group, respectively, indicating the exsitence of the positive feedback circuit.
When exposed to 0-4Gy of irradiation, pE6R4-p53-EGFP/H1299 cells of therapeutic group and pE6-p53-EGFP/H1299 cells of control group expressed increased wt-p53 protein along with the increment of irradiation doses. The summit level of wt-p53 was observed at 4Gy irradiation in therapeutic group which was 1.42 fold of that in control group, but the expression decreased significantly at 6Gy irradiation, reflecting the severe injury of cells at this dose of irradiation. Thus, 4Gy irradiation was used in following experiments in this study.
At 3h post to 4Gy irradiation, wt-p53 expression could be detected in both groups. The expression level peaked at 12h post irradiation (p.i.) and at this time point, the expression in therapeutic group was 1.651 fold of that in control group. While the wt-p53 expression in control group decreased to baseline at 36h p.i., however, the cells from therapeutic group could express high level of wt-p53 even at 144h p.i., 1.86 fold of that without irradiation. FCM analyses showed that EGFP fluoresence density in therapeutic group was 3.95 and 2.5 folds of the control group at 12h and 144h p.i., respectively, indicating wt-p53 positive feedback circuit could up-regulating wt-p53 expression markedly.
3. Anti-tumor effect of irradiation-induced wt-p53 positive feedback circuit in vitro
At 12h post 4Gy X-ray irradiation, FCM analysis showed that most cells in therapeutic group arrested at G0/G1 stage (75.13±1.42)%, compared with control groups (38.47±0.87)%, (62.57±0.76)%,(51.23±2.41)%, respectively. After irradiation, the cell apoptosis rate in each group was higher than that in cells without irradiation. The apoptosis rate in therapeutic group was (23.73±0.21)%, which was 5.69 folds (4.17±0.12)%, 1.91 folds (12.40±0.20)%, 1.51 folds(15.67±0.32)% and 2.57 folds(9.23±0.15)% of that in four control groups, respectively. The D0 values were 0.91Gy, 1.073 Gy and 1.413 Gy in pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299 and H1299, respectivley. The SER, derived from D0 values, was 2.63 and 1.34, respectively, indicating irradiation could up-regulate wt-p53 expression, regulate cell cycle and induce cell apoptosis, thus this positive feedback circuit could increase the sensitivity of lung adenocarcinoma to irradiation.
4. Effects of the wt-p53 positive feedback circuit on the transplantated tumor in nude mice
Various levels of immunoreactivity for wt-p53 were found in the tissues of therapeutic group and control groups. The expression level of wt-p53 was evaluated as before described in section of materials and methods. Wt-p53 immunostaining was observed in cellular nucleus. Positive wt-p53 staining was increased greatly in H1299/pE6R4-p53-EGFP group (64.8 % ), moderately in H1299/pE6-p53-EGFP group (22.3 % ) and poorly in H1299/pR4-p53 -EGFP group(5.4%). Negative wt-p53 staining was observed in H1299 cell xenograft tumor group. The growth curves of xenograft tumors show that the tumor inhibition ratio in pE6R4-p53-EG FP/H1299/IR group is higher than that in other 3 control groups. The inhibitation radio is 86.41 % , 70.76 % , 35.53 % and 12.58 % in pE6R4-p53-EG FP/H1299+IR group, H1299/pE6-p53-EGFP+IR group, pR4-p53-EG FP/H1299+IR group and H1299+IR group, respectively. The TCD50 values were 12.1 Gy, 15.2 Gy and 19.4 Gy in pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299 and H1299, respectivley. The SER, derived from TCD50 values, was 1.6 and 1.28, respectively, indicating wt-p53 status were significant related to the activation of the positive feedback circuit induced by IR and the radiosensitivity of xenograft tumor in therapeautic group was enhanced significantly.
CONCLUSIONS
In this study, we have constructed a recombinant pladmid containing an irradiation-induced wt-p53 positive feedback circuit, which can lead to much higher expression level of wt-p53 in transfected cells, thus it addressed the problem of current irradiation-gene therapy strategy, i.e., the weak and transient expression of target gene. Because this gene feedback circuit can obviously increase wt-p53 expression in tumor cells, it thus can cause the tumor cells to arrest at G1 stage and induce cell apoptosis, leading to the increased susceptibility of lung tumor to irradiation. This effect of the wt-p53 feedback circuit has been validated further in a transplanted tumor model in nude mice. Therefore, our results have not only improved the irradiation-gene therapy strategy but also could be a reference for other gene therapy strategy.
引文
1. Daniel JC, Smythe WR. Gene therapy of lung cancer[J]. Semin Surg Oncol. 2003;21(3):196-204。
2. Argiris A, Liptay M, LaCombe M. et al. A phase I/II trial of induction chemotherapy with carboplatin and gemcitabine followed by concurrent vinorelbine and paclitaxel with chest radiation in patients with stage III non-small cell lung cancer[J]. Lung Cancer. 2004 Aug;45(2):243-53.
3. Weichselbaum RR, Hallahan DE, Beckett MA, et al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells [J]. Cancer Res, 1994, 54(16):4266-4269.
4. Olga G, Simon S. Tumor Hypoxia and Targeted Gene Therapy[J]. Int Rev Cytol, 2007,257:181-212.
5. Weichselbaum R R, Kufe D W, Advani S J, et al. Molecular targeting of gene therapy and radio therapy[J]. Acta Oncol, 2001;40(6):735-738.
6. Ahmed MM. Regulation of radiation-induced apoptosis by early growth response-1 gene in solid tumors[J]. Curr Cancer Drug Targets. 2004 Feb;4(1):43-52.
7. Ma HB, Wang XJ, Di ZL, et al. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro[J]. World J Gastroenterol. 2007 Aug 21;13(31):4214-8.
8. Hollstein M, Rice K, Greenblatt MS, et al. Database of p53 gene somatic mutations in human tumors and cell lines[J]. Nucleic Acids Res. 1994 Sep;22(17):3551-5.
9. Donehower LA, Harvey M, Slagle BL, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors[J]. Nature. 1992 Mar 19;356(6366):215-21. 10. Jacks T, Remington L, Williams BO, et al. Tumor spectrum analysis in p53-mutant mice[J]. Curr Biol. 1994 Jan 1;4(1):1-7.
11. Malkin D, Li FP, Strong LC, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms[J]. Science. 1990 Nov 30;250(4985): 1233-8.
12. Srivastava S, Zou ZQ, Pirollo K, et al .Germ-line transmission of a mutated p53 gene ina cancer-prone family with Li-Fraumeni syndrome[J]. Nature. 1990 Dec 20-27;348 (6303):747-9.
13. Lebedeva IV, Su ZZ, Sarkar D, et al. Restoring apoptosis as a strategy for cancer gene therapy: focus on p53 and mda-7[J]. Semin Cancer Biol. 2003;13(2):169-78.
14. Kufe D, Weichselbaum R. Radiation therapy: activation for gene transcription and the development of genetic radiotherapy-therapeutic strategies in oncology[J]. Cancer Biol Ther. 2003;2(4):326-9.
15. Scott SD, Marples B, Hendry JH, et al. A radiation-controlled molecular switch for use in gene therapy of cancer[J]. Gene Ther. 2000 Jul;7(13):1121-5.
16. Wall ME, Hlavacek WS, Savageau MA. Design of gene circuits: lessons from bacteria[J]. Nat Rev Genet. 2004;5(1):34-42.
17. Feng XJ, Hooshangi S, Chen D, et al. Optimizing genetic circuits by global sensitivity analysis[J]. Biophys J. 2004 ;87(4):2195-202.
18. Peng Hua-zheng, Pan Jian-wei, Zhu Mu-yuan. The progress of gene networks [J]. Prog Biochem Biophys, 2001, 28 ( 6):815-818.
19. Shmulevich I, Dougherty ER, Kim S, et al. Probabilistic Boolean networks: a rule-based uncertainty model for gene regulatory networks[J]. Bioinformatics, 2002, 18 (2):261-274.
20. Nir F, Michal L, Iftach N, et al. Using bayesian networks to analyze expression data[J]. Comp Biol, 2000, 7(3):601-620.
21. Heinemann M, Panke S. Synthetic biology--putting engineering into biology[J]. Bioinformatics, 2006, 22(22):2790-2799.
22. Maeda YT, Sano M. Regulatory dynamics of synthetic gene networks with positive feedback[J]. J Mol Biol. 2006 Jun 16;359(4):1107-24
23. Kobayashi T, Chen L, Aihara K. Modeling genetic switches with positive feedback circuits[J]. J Theor Biol. 2003;221(3):379-99.
24. Karig D, Weiss R. Signal-amplifying genetic circuit enables in vivo observation of weak promoter activation in the Rhl quorum sensing system[J]. Biotechnol Bioeng. 2005,89(1):46-50.
25. Kaznessis YN. Models for synthetic biology[J]. BMC Syst Biol. 2007 Nov 6;1:47.
26. Fu P. A perspective of synthetic biology: assembling building blocks for novel functions[J]. Biotechnol J. 2006 Jun;1(6):690-9.
27. Nakamura Y. Isolation of p53-target genes and their functional analysis[J]. Cancer Sci. 2004;95(1):7-11.
28. Datta R, Rubin E, Sukhatme V, et al. Ionizing radiation activates transcription of the EGR-1 gene via CArG elements[J]. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21): 10149-53.
29. Thiel G, Cibelli G. Regulation of life and death by the zinc finger transcription factor Egr-1[J]. J Cell Physiol. 2002,193: 287-292.
30. Tsurushima H, Yuan X, Dillehay LE, et al. Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors[J]. Cancer Gene Ther. 2007 Dec;14(12):1002.
31. Dulic V, Kaufmann WK, Wilson SJ, et al. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest[J]. Cell. 1994 Mar 25;76(6):1013-23.
32. Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases[J]. Cell. 1993 Nov 19;75(4):805-16.
33. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817-25.
34. van der Velden AW, Thomas AA. The role of the 5' untranslated region of an mRNA in translation regulation during development[J]. Int J Biochem Cell Biol. 1999 Jan;31(1):87-106.
35. Hennecke M, Kwissa M, Metzger K, et al. Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs[J]. Nucleic Acids Res. 2001 Aug 15;29(16):3327-34.
36. Mizuguchi H, Xu Z, Ishii-Watabe A, et al. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector[J]. Mol Ther. 2000 Apr;1(4):376-82.
37. Cornelis S, Vandenbranden M, Ruysschaert JM. Role of intracellular cationic liposome-DNA complex dissociation in transfection mediated by cationic lipids[J].DNA Cell Biol. 2002, 21(2): 91-97.
38. Dalby B, Cates S, Harris A, et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications[J]. Methods. 2004,33(2):95-103.
39. Ladurner AG. Rheostat control of gene expression by metabolites[J]. Mol Cell. 2006 Oct 6;24(1):1-11.
40. Tsai-Morris CH, Cao XM, Sukhatme VP. 5' flanking sequence and genomic structure of Egr-1, a murine mitogen inducible zinc finger encoding gene[J]. Nucleic Acids Res. 1988 Sep 26;16(18):8835-46.
41. Ahmed MM, Venkatasubbarao K, Fruitwala SM, et al. EGR-1 induction is required for maximal radiosensitivity in A375-C6 melanoma cells[J]. J Biol Chem. 1996 Nov 15;271(46):29231-7.
42. Takahashi T, Namiki Y, Ohno T. Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with radioisotopes[J]. Hum Gene Ther. 1997 May 1;8(7):827-33.
43. Hasty J, McMillen D, Collins JJ, et al. Engineered gene circuits[J].Nature. 2002 Nov 14;420(6912):224-30.
44. Weiss R, Basu S, Hooshangi S, et a1.Genetic circuit building blocks for cellular computation, communications and signal processing[J].Nat Comp. 2003,6(2):1-40.
45. Kramer BP, Fischer CF, ussenegger M. BioLogic gates enable logical transcription control in mammalian cells[J]. Biotech Bioeng. 2004, 87(4):478-484.
46. Wong WW, Liao JC. The design of intracellular oscillators that interact with metabolism[J]. Cell Mol Life Sci, 2006, 63(11):1215-1220.
47. Peng Z. current status of gendicine in China:recombinant human Ad-p53 agent for treatment of cancers[J].Hum Gene Ther. 2005, 16(9):1016-1027.
48.沈瑜,糜福顺.肿瘤放射生物学[J].中国医药科技出版社. 2002, 178-183.
49. Graziano SL, Tatum A, Herndon JE, et al. Use of neuroendocrine markers, p53, and HER2 to predict response to chemotherapy in patients with stage III non-small cell lung cancer: a Cancer and Leukemia Group B study[J]. Lung Cancer. 2001 Aug-Sep;33(2-3):115-23.
50. Maniwa Y, Yoshimura M, Obayashi C, et al. Association of p53 gene mutation andtelomerase activity in resectable non-small cell lung cancer[J]. Chest. 2001,Aug;120(2): 589-94.
51. Malkin D. The role of p53 in human cancer[J]. J Neurooncol. 2001 Feb;51(3):231-43.
52. Yarnold J. Molecular aspects of cellular responses to radiotherapy[J]. Radiother Oncol. 1997 Jul;44(1):1-7.
53. Kagawa S, Fujiwara T, Hizuta A, et al. p53 expression overcomes p21WAF1/CIP1- mediated G1 arrest and induces apoptosis in human cancer cells[J]. Oncogene. 1997 Oct 16;15(16):1903-9.
54. McIlwrath AJ, Vasey PA, Ross GM, et al. Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity[J]. Cancer Res. 1994 Jul 15;54(14):3718-22.
55. Khanna KK, Keating KE, Kozlov S, et al. ATM associates with and phosphorylates p53: mapping the region of interaction[J]. Nat Genet. 1998 Dec;20(4):398-400.
56. Gomez-Lazaro M, Fernandez-Gomez FJ, Jordán J. p53: twenty five years understanding the mechanism of genome protection[J]. J Physiol Biochem. 2004 Dec;60(4):287-307.
57. Lu X. p53: a heavily dictated dictator of life and death[J]. Curr Opin Genet Dev. 2005 Feb;15(1):27-33.
58. Castillo JP, Kowalik TF. Human cytomegalovirus immediate early proteins and cell growth control[J]. Gene. 2002 May 15;290(1-2):19-34.
59. Kerr JF, Wyllie AH, Currie AR. Apoptosis : a basix biological phenomenon with wide-ranging implifications in tissue kinetics[J]. Br J Cancer,1972,26(4):239-57.
60. Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer[J]. Nature. 2001 May 17;411(6835):342-8.
61. Duan X, Zhang H, Liu B, et al. Apoptosis of murine melanoma cells induced by heavy-ion radiation combined with Tp53 gene transfer[J]. Int J Radiat Biol. 2008 Mar;84(3):211-7.
62. Wakasa T, Inoue T, Kawai N, et al. The combination of ionizing radiation and expression of a wild type p53 gene via recombinant adenovirus induced a prominent tumour suppressing effect in human oral squamous cell carcinoma[J]. Br J Radiol. 2002Aug;75(896):657-62.
63. Huh JJ, Wolf JK, Fightmaster DL, et al. Transduction of adenovirus-mediated wild-type p53 after radiotherapy in human cervical cancer cells[J]. Gynecol Oncol. 2003 May;89(2):243-50.
64. Alvarez S, DranéP, Meiller A, et al. A comprehensive study of p53 transcriptional activity in thymus and spleen of gamma irradiated mouse: high sensitivity of genes involved in the two main apoptotic pathways[J]. Int J Radiat Biol. 2006 Nov;82(11):761-70.
65. Miller LD, Smeds J, George J,et al. An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival[J]. Proc Natl Acad Sci U S A. 2005 Sep 20;102(38):13550-5. Epub 2005 Sep 2.
66. Lacroix M, Toillon RA, Leclercq G. p53 and breast cancer, an update[J]. Endocr Relat Cancer. 2006 Jun;13(2):293-325. Review.
67. Menendez D, Inga A, Resnick MA. The biological impact of the human master regulator p53 can be altered by mutations that change the spectrum and expression of its target genes[J]. Mol Cell Biol. 2006 Mar;26(6):2297-308.
68. Chen MF, Chen WC, Wu CT,et al. p53 status is a major determinant of effects of decreasing peroxiredoxin I expression on tumor growth and response of lung cancer cells to treatment[J]. Int J Radiat Oncol Biol Phys. 2006 Dec 1;66(5):1461-72.
69. Seong J, Oh HJ, Kim J, Identification of proteins that regulate radiation-induced apoptosis in murine tumors with wild type p53[J]. J Radiat Res (Tokyo). 2007 Sep;48(5):435-41.
70. Sah NK, Munshi A, Nishikawa T. Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity[J]. Mol Cancer Ther. 2003 Nov;2(11):1223-31.
71. Yamazaki Y, Chiba I, Hirai A, et al. Radioresistance in oral squamous cell carcinoma with p53 DNA contact mutation[J]. Am J Clin Oncol. 2003 Oct;26(5):124-9.
72. Seung LP, Mauceri HJ, Beckett MA, et al. Genetic radiotherapy overcomes tumor resistance to cytotoxic agents[J]. Cancer Res. 1995 Dec 1;55(23):5561-5.
73. Wang WD, Chen ZT, Li DZ, et al. HSV-TK gene therapy of lung adenocarcinomaxenografts using a hypoxia/radiation dual-sensitive promoter[J]. Ai Zheng. 2004 Jul;23(7):788-93. Chinese.
74. Salloum RM, Saunders MP, Mauceri HJ, et al. Dual induction of the Epo-Egr- TNF-alpha- plasmid in hypoxic human colon adenocarcinoma produces tumor growth delay[J]. Am Surg. 2003 Jan;69(1):24-7.
75. Gupta VK, Park JO, Jaskowiak NT, et al. Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma[J]. Ann Surg Oncol. 2002 Jun;9(5):500-4.
76. Greco O, Powell TM, Marples B, et al. Gene therapy vectors containing CArG elements from the Egr1 gene are activated by neutron irradiation, cisplatin and doxorubicin[J]. Cancer Gene Ther. 2005 Jul;12(7):655-62.
77. Mezhir JJ, Smith KD, Posner MC, et al. Ionizing radiation: a genetic switch for cancer therapy[J]. Cancer Gene Ther. 2006 Jan 1;13(1):1-6.,
78. Dennis E, Hallahan, Helena J. et al. Spatial and temporal control of gene therapy using ionizing radiation[J]. Nature Medicine. 1995, 1:786 - 791
79. Jiang W, Liao Y, Zhao S, et al. Role of enhanced radiosensitivity and the tumor- specific suicide gene vector in gene therapy of nasopharyngeal carcinoma[J]. J Radiat Res (Tokyo). 2007 May;48(3):211-8.
80. Greco O, Joiner MC, Doleh A, et al. Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer[J]. Gene Ther. 2006 Feb;13(3):206-15.
81. Gu Z, Flemington C, Chittenden T, et al. ei24, a p53 response gene involved in growth suppression and apoptosis[J]. Mol Cell Biol. 2000 Jan;20(1):233-41.
82. Harms KL, Chen X. The C terminus of p53 family proteins is a cell fate determinant[J]. Mol Cell Biol. 2005 Mar;25(5):2014-30.
1. Weichselbaum RR, Hallahan DE, Sukhatme VP, et.al. Gene therapy targeted by ionizing radiation[J]. Int J Radiat Oncol Biol Phys. 1992;24(3):565-7. Review.
2. Lei N, Heckert LL. Sp1 and Egr1 regulate transcription of the Dmrt1 gene in Sertoli cells[J].Biol Reprod. 2002 Mar;66(3):675-84
3. Tourtellotte WG, Nagarajan R, Bartke A, et al. Functional compensation by Egr4 in Egr1-dependent luteinizing hormone regulation and Leydig cell steroidogenesis[J].Mol Cell Biol. 2000 Jul;20(14):5261-8.
4. Papanikolaou NA, Sabban EL. Ability of Egr1 to activate tyrosine hydroxylase transcription in PC12 cells. Cross-talk with AP-1 factors[J].J Biol Chem. 2000 Sep 1;275(35):26683-9
5. Zaiou M, Azrolan N, Hayek T, et al. The full induction of human apoprotein A-I gene expression by the experimental nephrotic syndrome in transgenic mice depends oncis-acting elements in the proximal 256 base-pair promoter region and the trans-acting factor early growth response factor[J].J Clin Invest. 1998 Apr 15;101(8):1699-707.
6. Bahouth SW, Beauchamp MJ, Vu KN. Reciprocal regulation of beta(1)-adrenergic receptor gene transcription by Sp1 and early growth response gene 1: induction of EGR-1 inhibits the expression of the beta(1)-adrenergic receptor gene[J].Mol Pharmacol. 2002 Feb;61(2):379-90.
7. Madden SL, Cook DM, Morris JF, et al. Transcriptional repression mediated by the WT1 Wilms tumor gene product[J].Science. 1991 Sep 27;253(5027):1550-3.
8. Drummond IA, Madden SL, Rohwer-Nutter P, et al. Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1[J].Science. 1992 Jul 31;257(5070):674-8.
9. Wang ZY, Madden SL, Deuel TF, et al. The Wilms' tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene[J].J Biol Chem. 1992 Nov 5;267(31):21999-2002.
10. Ahmed MM, Sells SF, Venkatasubbarao K, et al. Ionizing radiation-inducible apoptosis in the absence of p53 linked to transcription factor EGR-1[J].J Biol Chem. 1997 Dec 26;272(52):33056-61
11. Hengartner MO.Apoptosis:death cycle and swiss army knives[J].Nature, 1998,391: 441-442.
12. Kuwana T, Smith JJ, Muzio M, et al. Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome[J].J Biol Chem. 1998 Jun 26;273(26):16589-94
13. Nagata S. Apoptosis by death factor[J]. Cell. 1997 Feb 7;88(3):355-65.
14. Das A, Chendil D, Dey S, et al. Ionizing radiation down-regulates p53 protein in primary Egr-1-/- mouse embryonic fibroblast cells causing enhanced resistance to apoptosis [J].J Biol Chem. 2001 Feb 2;276(5):3279-86. Epub 2000 Oct 16
15. Lane DP et al. T antigen is bound to a host protein in SV40 transformed cell [J].Nature. 1979 278 (5701), 261-3
16. Buckbinder L, Talbott R, Velasco-Miguel S, et al. Induction of the growth inhibitorIGF-binding protein 3 by p53[J]. Nature. 1995 Oct 19;377(6550):646-9.
17. Miyashita T, Harigai M, Hanada M, et al. Identification of a p53-dependent negative response element in the bcl-2 gene[J]. Cancer Res. 1994 Jun 15;54(12):3131-5.
18. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene[J].Cell. 1995,Jan 27;80(2):293-9.
19. Owen-Schaub LB, Zhang W, Cusack JC, et al. Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression[J]. Mol Cell Biol. 1995 Jun;15(6):3032-40.
20. Lee,J.M;Bernstein,A.p53 mutations increase resistance to ionzing radiation[J]. Proc.Natl.Acad.Sci.USA ,1993,90,5742-5746.
21. Ko LJ, Prives C. p53: puzzle and paradigm[J].Genes Dev. 1996 May 1;10(9):1054-72.
22. Nair P, Muthukkumar S, Sells SF, et.al. Early growth response-1-dependent apoptosis is mediated by p53[J].J Biol Chem. 1997 Aug 8;272(32):20131-8.
23. Weichselbaum RR, Hallahan DE, Beckett MA, et.al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells[J]. Cancer Res. 1994 Aug 15;54(16):4266-9.
24. Gupta VK, Park JO, Jaskowiak NT, et.al Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma[J].Ann Surg Oncol. 2002 Jun;9(5):500-4.
25. Staba MJ, Mauceri HJ, Kufe DW, et.al Adenoviral TNF-alpha gene therapy and radiation damage tumor vasculature in a human malignant glioma xenograft[J].Gene Ther. 1998 Mar;5(3):293-300.
26. 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[J].Hum Gene Ther. 1998 Jul 1;9(10):1393-4.
27. 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[J]. Hum Gene Ther. 1999 Jun 10;10(9):1509-19.
28. Joki T, Nakamura M, Ohno T, et.al. Activation of the radiosensitive EGR-1 promoterinduces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir[J].Hum Gene Ther. 1995 Dec;6(12):1507-13.
29. Marples B, Greco O. et.al. Molecular approaches to chemo-radiotherapy[J].Eur J Cancer. 2002 Jan;38(2):231-9.
30.王卫东、陈正堂、李德志等.缺氧/辐射双敏感性启动子调控HSV-TK表达对肺癌移植瘤的作用[J].癌症.2004.23(7):788-793.
31. Zhu H, Jackson T, Bunn HF. Detecting and responding to hypoxia[J].Nephrol Dial Transplant. 2002;17 Suppl 1:3-7.
32. Greco O, Marples B, Dachs GU, et al. Novel chimeric gene promoters responsive to hypoxia and ionizing radiation[J].Gene Ther. 2002 Oct;9(20):1403-11.
33. Wu CM, Li XY, Huang TH. Anti-tumor effect of pEgr-IFNgamma gene-radiotherapy in B16 melanoma-bearing mice [J]. World J Gastroenterol. 2004 Oct 15;10(20):3011-5.
34. Yang Y, Liu SZ, Fu SB.et al. Anti-tumor effects of pNEgr-mIL-12 recombinant plasmid induced by X-irradiation and its mechanisms [J].Biomed Environ Sci. 2004 Jun; 17(2):135-43.
35. Ma HB, Hu HT, Di ZL, et al. Association of cyclin D1, p16 and retinoblastoma protein expressions with prognosis and metastasis of gallbladder carcinoma[J].World J Gastroenterol. 2005 Feb 7;11(5):744-7
[1]. Olga G, Simon S. Tumor Hypoxia and Targeted Gene Therapy[J]. Int Rev Cytol, 2007,257:181-212.
[2]. Weichselbaum R R, Kufe D W, Advani S J, et al. Molecular targeting of gene therapy and radio therapy[J]. Acta Oncol, 2001;40(6):735-738.
[3]. Ahmed MM. Regulation of radiation-induced apoptosis by early growth response-1 gene in solid tumors[J]. Curr Cancer Drug Targets. 2004 Feb;4(1):43-52.
[4]. Ma HB, Wang XJ, Di ZL, et al. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro[J]. World J Gastroenterol. 2007 Aug 21;13(31):4214-8.
[5]. Kufe D, Weichselbaum R. Radiation therapy: activation for gene transcription and the development of genetic radiotherapy-therapeutic strategies in oncology[J]. Cancer Biol Ther. 2003;2(4):326-9.
[6]. Gu Z, Flemington C, Chittenden T, et al. ei24, a p53 response gene involved in growth suppression and apoptosis[J]. Mol Cell Biol. 2000 Jan;20(1):233-41.
[7]. Tsurushima H, Yuan X, Dillehay LE, et al. Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors[J]. Cancer Gene Ther. 2007 Dec;14(12):1002.
[8]. Nakamura Y. Isolation of p53-target genes and their functional analysis[J]. Cancer Sci. 2004;95(1):7-11.
[9]. Dulic V, Kaufmann WK, Wilson SJ, et al. p53-dependent inhibition of cyclin- dependent kinase activities in human fibroblasts during radiation-induced G1 arrest[J]. Cell. 1994 Mar 25;76(6):1013-23.
[10]. Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases[J]. Cell. 1993 Nov 19;75(4):805-16.
[11]. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817-25.
[12]. Malkin D. The role of p53 in human cancer[J]. J Neurooncol. 2001 Feb;51(3):231-43.
[13]. Lebedeva IV, Su ZZ, Sarkar D, et al. Restoring apoptosis as a strategy for cancer gene therapy: focus on p53 and mda-7[J]. Semin Cancer Biol. 2003;13(2):169-78.
[14]. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin. 2002 Jan-Feb;52(1):23-47..
[15]. Cortes-funes H.(2002) New treatment approaches for lung cancer and impact on survival. Semin oncol 29 (suppl 8):26-29.
[16]. Ferreira CG, Span SW, Peters GJ, Kruyt FA, Giaccone G. Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460. Cancer Res. 2000 Dec 15;60(24):7133-41.
[17]. Weichselbaum RR, Hallahan DE, Beckett MA, et al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells [J]. Cancer Res, 1994, 54(16):4266-4269.
[18]. Hennecke M, Kwissa M, Metzger K, et al. Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs[J]. Nucleic Acids Res. 2001 Aug 15;29(16):3327-34.
[19]. Scott SD, Marples B, Hendry JH, et al. A radiation-controlled molecular switch for use in gene therapy of cancer[J]. Gene Ther. 2000 Jul;7(13):1121-5.
[20]. Greco O, Joiner MC, Doleh A, et al. Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer[J]. Gene Ther. 2006 Feb;13(3):206-15.
[21]. Harms KL, Chen X. The C terminus of p53 family proteins is a cell fate determinant[J]. Mol Cell Biol. 2005 Mar;25(5):2014-30.
2. Argiris A, Liptay M, LaCombe M. et al. A phase I/II trial of induction chemotherapy with carboplatin and gemcitabine followed by concurrent vinorelbine and paclitaxel with chest radiation in patients with stage III non-small cell lung cancer[J]. Lung Cancer. 2004 Aug;45(2):243-53.
3. Weichselbaum RR, Hallahan DE, Beckett MA, et al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells [J]. Cancer Res, 1994, 54(16):4266-4269.
4. Olga G, Simon S. Tumor Hypoxia and Targeted Gene Therapy[J]. Int Rev Cytol, 2007,257:181-212.
5. Weichselbaum R R, Kufe D W, Advani S J, et al. Molecular targeting of gene therapy and radio therapy[J]. Acta Oncol, 2001;40(6):735-738.
6. Ahmed MM. Regulation of radiation-induced apoptosis by early growth response-1 gene in solid tumors[J]. Curr Cancer Drug Targets. 2004 Feb;4(1):43-52.
7. Ma HB, Wang XJ, Di ZL, et al. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro[J]. World J Gastroenterol. 2007 Aug 21;13(31):4214-8.
8. Hollstein M, Rice K, Greenblatt MS, et al. Database of p53 gene somatic mutations in human tumors and cell lines[J]. Nucleic Acids Res. 1994 Sep;22(17):3551-5.
9. Donehower LA, Harvey M, Slagle BL, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors[J]. Nature. 1992 Mar 19;356(6366):215-21. 10. Jacks T, Remington L, Williams BO, et al. Tumor spectrum analysis in p53-mutant mice[J]. Curr Biol. 1994 Jan 1;4(1):1-7.
11. Malkin D, Li FP, Strong LC, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms[J]. Science. 1990 Nov 30;250(4985): 1233-8.
12. Srivastava S, Zou ZQ, Pirollo K, et al .Germ-line transmission of a mutated p53 gene ina cancer-prone family with Li-Fraumeni syndrome[J]. Nature. 1990 Dec 20-27;348 (6303):747-9.
13. Lebedeva IV, Su ZZ, Sarkar D, et al. Restoring apoptosis as a strategy for cancer gene therapy: focus on p53 and mda-7[J]. Semin Cancer Biol. 2003;13(2):169-78.
14. Kufe D, Weichselbaum R. Radiation therapy: activation for gene transcription and the development of genetic radiotherapy-therapeutic strategies in oncology[J]. Cancer Biol Ther. 2003;2(4):326-9.
15. Scott SD, Marples B, Hendry JH, et al. A radiation-controlled molecular switch for use in gene therapy of cancer[J]. Gene Ther. 2000 Jul;7(13):1121-5.
16. Wall ME, Hlavacek WS, Savageau MA. Design of gene circuits: lessons from bacteria[J]. Nat Rev Genet. 2004;5(1):34-42.
17. Feng XJ, Hooshangi S, Chen D, et al. Optimizing genetic circuits by global sensitivity analysis[J]. Biophys J. 2004 ;87(4):2195-202.
18. Peng Hua-zheng, Pan Jian-wei, Zhu Mu-yuan. The progress of gene networks [J]. Prog Biochem Biophys, 2001, 28 ( 6):815-818.
19. Shmulevich I, Dougherty ER, Kim S, et al. Probabilistic Boolean networks: a rule-based uncertainty model for gene regulatory networks[J]. Bioinformatics, 2002, 18 (2):261-274.
20. Nir F, Michal L, Iftach N, et al. Using bayesian networks to analyze expression data[J]. Comp Biol, 2000, 7(3):601-620.
21. Heinemann M, Panke S. Synthetic biology--putting engineering into biology[J]. Bioinformatics, 2006, 22(22):2790-2799.
22. Maeda YT, Sano M. Regulatory dynamics of synthetic gene networks with positive feedback[J]. J Mol Biol. 2006 Jun 16;359(4):1107-24
23. Kobayashi T, Chen L, Aihara K. Modeling genetic switches with positive feedback circuits[J]. J Theor Biol. 2003;221(3):379-99.
24. Karig D, Weiss R. Signal-amplifying genetic circuit enables in vivo observation of weak promoter activation in the Rhl quorum sensing system[J]. Biotechnol Bioeng. 2005,89(1):46-50.
25. Kaznessis YN. Models for synthetic biology[J]. BMC Syst Biol. 2007 Nov 6;1:47.
26. Fu P. A perspective of synthetic biology: assembling building blocks for novel functions[J]. Biotechnol J. 2006 Jun;1(6):690-9.
27. Nakamura Y. Isolation of p53-target genes and their functional analysis[J]. Cancer Sci. 2004;95(1):7-11.
28. Datta R, Rubin E, Sukhatme V, et al. Ionizing radiation activates transcription of the EGR-1 gene via CArG elements[J]. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21): 10149-53.
29. Thiel G, Cibelli G. Regulation of life and death by the zinc finger transcription factor Egr-1[J]. J Cell Physiol. 2002,193: 287-292.
30. Tsurushima H, Yuan X, Dillehay LE, et al. Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors[J]. Cancer Gene Ther. 2007 Dec;14(12):1002.
31. Dulic V, Kaufmann WK, Wilson SJ, et al. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest[J]. Cell. 1994 Mar 25;76(6):1013-23.
32. Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases[J]. Cell. 1993 Nov 19;75(4):805-16.
33. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817-25.
34. van der Velden AW, Thomas AA. The role of the 5' untranslated region of an mRNA in translation regulation during development[J]. Int J Biochem Cell Biol. 1999 Jan;31(1):87-106.
35. Hennecke M, Kwissa M, Metzger K, et al. Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs[J]. Nucleic Acids Res. 2001 Aug 15;29(16):3327-34.
36. Mizuguchi H, Xu Z, Ishii-Watabe A, et al. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector[J]. Mol Ther. 2000 Apr;1(4):376-82.
37. Cornelis S, Vandenbranden M, Ruysschaert JM. Role of intracellular cationic liposome-DNA complex dissociation in transfection mediated by cationic lipids[J].DNA Cell Biol. 2002, 21(2): 91-97.
38. Dalby B, Cates S, Harris A, et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications[J]. Methods. 2004,33(2):95-103.
39. Ladurner AG. Rheostat control of gene expression by metabolites[J]. Mol Cell. 2006 Oct 6;24(1):1-11.
40. Tsai-Morris CH, Cao XM, Sukhatme VP. 5' flanking sequence and genomic structure of Egr-1, a murine mitogen inducible zinc finger encoding gene[J]. Nucleic Acids Res. 1988 Sep 26;16(18):8835-46.
41. Ahmed MM, Venkatasubbarao K, Fruitwala SM, et al. EGR-1 induction is required for maximal radiosensitivity in A375-C6 melanoma cells[J]. J Biol Chem. 1996 Nov 15;271(46):29231-7.
42. Takahashi T, Namiki Y, Ohno T. Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with radioisotopes[J]. Hum Gene Ther. 1997 May 1;8(7):827-33.
43. Hasty J, McMillen D, Collins JJ, et al. Engineered gene circuits[J].Nature. 2002 Nov 14;420(6912):224-30.
44. Weiss R, Basu S, Hooshangi S, et a1.Genetic circuit building blocks for cellular computation, communications and signal processing[J].Nat Comp. 2003,6(2):1-40.
45. Kramer BP, Fischer CF, ussenegger M. BioLogic gates enable logical transcription control in mammalian cells[J]. Biotech Bioeng. 2004, 87(4):478-484.
46. Wong WW, Liao JC. The design of intracellular oscillators that interact with metabolism[J]. Cell Mol Life Sci, 2006, 63(11):1215-1220.
47. Peng Z. current status of gendicine in China:recombinant human Ad-p53 agent for treatment of cancers[J].Hum Gene Ther. 2005, 16(9):1016-1027.
48.沈瑜,糜福顺.肿瘤放射生物学[J].中国医药科技出版社. 2002, 178-183.
49. Graziano SL, Tatum A, Herndon JE, et al. Use of neuroendocrine markers, p53, and HER2 to predict response to chemotherapy in patients with stage III non-small cell lung cancer: a Cancer and Leukemia Group B study[J]. Lung Cancer. 2001 Aug-Sep;33(2-3):115-23.
50. Maniwa Y, Yoshimura M, Obayashi C, et al. Association of p53 gene mutation andtelomerase activity in resectable non-small cell lung cancer[J]. Chest. 2001,Aug;120(2): 589-94.
51. Malkin D. The role of p53 in human cancer[J]. J Neurooncol. 2001 Feb;51(3):231-43.
52. Yarnold J. Molecular aspects of cellular responses to radiotherapy[J]. Radiother Oncol. 1997 Jul;44(1):1-7.
53. Kagawa S, Fujiwara T, Hizuta A, et al. p53 expression overcomes p21WAF1/CIP1- mediated G1 arrest and induces apoptosis in human cancer cells[J]. Oncogene. 1997 Oct 16;15(16):1903-9.
54. McIlwrath AJ, Vasey PA, Ross GM, et al. Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity[J]. Cancer Res. 1994 Jul 15;54(14):3718-22.
55. Khanna KK, Keating KE, Kozlov S, et al. ATM associates with and phosphorylates p53: mapping the region of interaction[J]. Nat Genet. 1998 Dec;20(4):398-400.
56. Gomez-Lazaro M, Fernandez-Gomez FJ, Jordán J. p53: twenty five years understanding the mechanism of genome protection[J]. J Physiol Biochem. 2004 Dec;60(4):287-307.
57. Lu X. p53: a heavily dictated dictator of life and death[J]. Curr Opin Genet Dev. 2005 Feb;15(1):27-33.
58. Castillo JP, Kowalik TF. Human cytomegalovirus immediate early proteins and cell growth control[J]. Gene. 2002 May 15;290(1-2):19-34.
59. Kerr JF, Wyllie AH, Currie AR. Apoptosis : a basix biological phenomenon with wide-ranging implifications in tissue kinetics[J]. Br J Cancer,1972,26(4):239-57.
60. Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer[J]. Nature. 2001 May 17;411(6835):342-8.
61. Duan X, Zhang H, Liu B, et al. Apoptosis of murine melanoma cells induced by heavy-ion radiation combined with Tp53 gene transfer[J]. Int J Radiat Biol. 2008 Mar;84(3):211-7.
62. Wakasa T, Inoue T, Kawai N, et al. The combination of ionizing radiation and expression of a wild type p53 gene via recombinant adenovirus induced a prominent tumour suppressing effect in human oral squamous cell carcinoma[J]. Br J Radiol. 2002Aug;75(896):657-62.
63. Huh JJ, Wolf JK, Fightmaster DL, et al. Transduction of adenovirus-mediated wild-type p53 after radiotherapy in human cervical cancer cells[J]. Gynecol Oncol. 2003 May;89(2):243-50.
64. Alvarez S, DranéP, Meiller A, et al. A comprehensive study of p53 transcriptional activity in thymus and spleen of gamma irradiated mouse: high sensitivity of genes involved in the two main apoptotic pathways[J]. Int J Radiat Biol. 2006 Nov;82(11):761-70.
65. Miller LD, Smeds J, George J,et al. An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival[J]. Proc Natl Acad Sci U S A. 2005 Sep 20;102(38):13550-5. Epub 2005 Sep 2.
66. Lacroix M, Toillon RA, Leclercq G. p53 and breast cancer, an update[J]. Endocr Relat Cancer. 2006 Jun;13(2):293-325. Review.
67. Menendez D, Inga A, Resnick MA. The biological impact of the human master regulator p53 can be altered by mutations that change the spectrum and expression of its target genes[J]. Mol Cell Biol. 2006 Mar;26(6):2297-308.
68. Chen MF, Chen WC, Wu CT,et al. p53 status is a major determinant of effects of decreasing peroxiredoxin I expression on tumor growth and response of lung cancer cells to treatment[J]. Int J Radiat Oncol Biol Phys. 2006 Dec 1;66(5):1461-72.
69. Seong J, Oh HJ, Kim J, Identification of proteins that regulate radiation-induced apoptosis in murine tumors with wild type p53[J]. J Radiat Res (Tokyo). 2007 Sep;48(5):435-41.
70. Sah NK, Munshi A, Nishikawa T. Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity[J]. Mol Cancer Ther. 2003 Nov;2(11):1223-31.
71. Yamazaki Y, Chiba I, Hirai A, et al. Radioresistance in oral squamous cell carcinoma with p53 DNA contact mutation[J]. Am J Clin Oncol. 2003 Oct;26(5):124-9.
72. Seung LP, Mauceri HJ, Beckett MA, et al. Genetic radiotherapy overcomes tumor resistance to cytotoxic agents[J]. Cancer Res. 1995 Dec 1;55(23):5561-5.
73. Wang WD, Chen ZT, Li DZ, et al. HSV-TK gene therapy of lung adenocarcinomaxenografts using a hypoxia/radiation dual-sensitive promoter[J]. Ai Zheng. 2004 Jul;23(7):788-93. Chinese.
74. Salloum RM, Saunders MP, Mauceri HJ, et al. Dual induction of the Epo-Egr- TNF-alpha- plasmid in hypoxic human colon adenocarcinoma produces tumor growth delay[J]. Am Surg. 2003 Jan;69(1):24-7.
75. Gupta VK, Park JO, Jaskowiak NT, et al. Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma[J]. Ann Surg Oncol. 2002 Jun;9(5):500-4.
76. Greco O, Powell TM, Marples B, et al. Gene therapy vectors containing CArG elements from the Egr1 gene are activated by neutron irradiation, cisplatin and doxorubicin[J]. Cancer Gene Ther. 2005 Jul;12(7):655-62.
77. Mezhir JJ, Smith KD, Posner MC, et al. Ionizing radiation: a genetic switch for cancer therapy[J]. Cancer Gene Ther. 2006 Jan 1;13(1):1-6.,
78. Dennis E, Hallahan, Helena J. et al. Spatial and temporal control of gene therapy using ionizing radiation[J]. Nature Medicine. 1995, 1:786 - 791
79. Jiang W, Liao Y, Zhao S, et al. Role of enhanced radiosensitivity and the tumor- specific suicide gene vector in gene therapy of nasopharyngeal carcinoma[J]. J Radiat Res (Tokyo). 2007 May;48(3):211-8.
80. Greco O, Joiner MC, Doleh A, et al. Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer[J]. Gene Ther. 2006 Feb;13(3):206-15.
81. Gu Z, Flemington C, Chittenden T, et al. ei24, a p53 response gene involved in growth suppression and apoptosis[J]. Mol Cell Biol. 2000 Jan;20(1):233-41.
82. Harms KL, Chen X. The C terminus of p53 family proteins is a cell fate determinant[J]. Mol Cell Biol. 2005 Mar;25(5):2014-30.
1. Weichselbaum RR, Hallahan DE, Sukhatme VP, et.al. Gene therapy targeted by ionizing radiation[J]. Int J Radiat Oncol Biol Phys. 1992;24(3):565-7. Review.
2. Lei N, Heckert LL. Sp1 and Egr1 regulate transcription of the Dmrt1 gene in Sertoli cells[J].Biol Reprod. 2002 Mar;66(3):675-84
3. Tourtellotte WG, Nagarajan R, Bartke A, et al. Functional compensation by Egr4 in Egr1-dependent luteinizing hormone regulation and Leydig cell steroidogenesis[J].Mol Cell Biol. 2000 Jul;20(14):5261-8.
4. Papanikolaou NA, Sabban EL. Ability of Egr1 to activate tyrosine hydroxylase transcription in PC12 cells. Cross-talk with AP-1 factors[J].J Biol Chem. 2000 Sep 1;275(35):26683-9
5. Zaiou M, Azrolan N, Hayek T, et al. The full induction of human apoprotein A-I gene expression by the experimental nephrotic syndrome in transgenic mice depends oncis-acting elements in the proximal 256 base-pair promoter region and the trans-acting factor early growth response factor[J].J Clin Invest. 1998 Apr 15;101(8):1699-707.
6. Bahouth SW, Beauchamp MJ, Vu KN. Reciprocal regulation of beta(1)-adrenergic receptor gene transcription by Sp1 and early growth response gene 1: induction of EGR-1 inhibits the expression of the beta(1)-adrenergic receptor gene[J].Mol Pharmacol. 2002 Feb;61(2):379-90.
7. Madden SL, Cook DM, Morris JF, et al. Transcriptional repression mediated by the WT1 Wilms tumor gene product[J].Science. 1991 Sep 27;253(5027):1550-3.
8. Drummond IA, Madden SL, Rohwer-Nutter P, et al. Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1[J].Science. 1992 Jul 31;257(5070):674-8.
9. Wang ZY, Madden SL, Deuel TF, et al. The Wilms' tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene[J].J Biol Chem. 1992 Nov 5;267(31):21999-2002.
10. Ahmed MM, Sells SF, Venkatasubbarao K, et al. Ionizing radiation-inducible apoptosis in the absence of p53 linked to transcription factor EGR-1[J].J Biol Chem. 1997 Dec 26;272(52):33056-61
11. Hengartner MO.Apoptosis:death cycle and swiss army knives[J].Nature, 1998,391: 441-442.
12. Kuwana T, Smith JJ, Muzio M, et al. Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome[J].J Biol Chem. 1998 Jun 26;273(26):16589-94
13. Nagata S. Apoptosis by death factor[J]. Cell. 1997 Feb 7;88(3):355-65.
14. Das A, Chendil D, Dey S, et al. Ionizing radiation down-regulates p53 protein in primary Egr-1-/- mouse embryonic fibroblast cells causing enhanced resistance to apoptosis [J].J Biol Chem. 2001 Feb 2;276(5):3279-86. Epub 2000 Oct 16
15. Lane DP et al. T antigen is bound to a host protein in SV40 transformed cell [J].Nature. 1979 278 (5701), 261-3
16. Buckbinder L, Talbott R, Velasco-Miguel S, et al. Induction of the growth inhibitorIGF-binding protein 3 by p53[J]. Nature. 1995 Oct 19;377(6550):646-9.
17. Miyashita T, Harigai M, Hanada M, et al. Identification of a p53-dependent negative response element in the bcl-2 gene[J]. Cancer Res. 1994 Jun 15;54(12):3131-5.
18. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene[J].Cell. 1995,Jan 27;80(2):293-9.
19. Owen-Schaub LB, Zhang W, Cusack JC, et al. Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression[J]. Mol Cell Biol. 1995 Jun;15(6):3032-40.
20. Lee,J.M;Bernstein,A.p53 mutations increase resistance to ionzing radiation[J]. Proc.Natl.Acad.Sci.USA ,1993,90,5742-5746.
21. Ko LJ, Prives C. p53: puzzle and paradigm[J].Genes Dev. 1996 May 1;10(9):1054-72.
22. Nair P, Muthukkumar S, Sells SF, et.al. Early growth response-1-dependent apoptosis is mediated by p53[J].J Biol Chem. 1997 Aug 8;272(32):20131-8.
23. Weichselbaum RR, Hallahan DE, Beckett MA, et.al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells[J]. Cancer Res. 1994 Aug 15;54(16):4266-9.
24. Gupta VK, Park JO, Jaskowiak NT, et.al Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma[J].Ann Surg Oncol. 2002 Jun;9(5):500-4.
25. Staba MJ, Mauceri HJ, Kufe DW, et.al Adenoviral TNF-alpha gene therapy and radiation damage tumor vasculature in a human malignant glioma xenograft[J].Gene Ther. 1998 Mar;5(3):293-300.
26. 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[J].Hum Gene Ther. 1998 Jul 1;9(10):1393-4.
27. 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[J]. Hum Gene Ther. 1999 Jun 10;10(9):1509-19.
28. Joki T, Nakamura M, Ohno T, et.al. Activation of the radiosensitive EGR-1 promoterinduces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir[J].Hum Gene Ther. 1995 Dec;6(12):1507-13.
29. Marples B, Greco O. et.al. Molecular approaches to chemo-radiotherapy[J].Eur J Cancer. 2002 Jan;38(2):231-9.
30.王卫东、陈正堂、李德志等.缺氧/辐射双敏感性启动子调控HSV-TK表达对肺癌移植瘤的作用[J].癌症.2004.23(7):788-793.
31. Zhu H, Jackson T, Bunn HF. Detecting and responding to hypoxia[J].Nephrol Dial Transplant. 2002;17 Suppl 1:3-7.
32. Greco O, Marples B, Dachs GU, et al. Novel chimeric gene promoters responsive to hypoxia and ionizing radiation[J].Gene Ther. 2002 Oct;9(20):1403-11.
33. Wu CM, Li XY, Huang TH. Anti-tumor effect of pEgr-IFNgamma gene-radiotherapy in B16 melanoma-bearing mice [J]. World J Gastroenterol. 2004 Oct 15;10(20):3011-5.
34. Yang Y, Liu SZ, Fu SB.et al. Anti-tumor effects of pNEgr-mIL-12 recombinant plasmid induced by X-irradiation and its mechanisms [J].Biomed Environ Sci. 2004 Jun; 17(2):135-43.
35. Ma HB, Hu HT, Di ZL, et al. Association of cyclin D1, p16 and retinoblastoma protein expressions with prognosis and metastasis of gallbladder carcinoma[J].World J Gastroenterol. 2005 Feb 7;11(5):744-7
[1]. Olga G, Simon S. Tumor Hypoxia and Targeted Gene Therapy[J]. Int Rev Cytol, 2007,257:181-212.
[2]. Weichselbaum R R, Kufe D W, Advani S J, et al. Molecular targeting of gene therapy and radio therapy[J]. Acta Oncol, 2001;40(6):735-738.
[3]. Ahmed MM. Regulation of radiation-induced apoptosis by early growth response-1 gene in solid tumors[J]. Curr Cancer Drug Targets. 2004 Feb;4(1):43-52.
[4]. Ma HB, Wang XJ, Di ZL, et al. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro[J]. World J Gastroenterol. 2007 Aug 21;13(31):4214-8.
[5]. Kufe D, Weichselbaum R. Radiation therapy: activation for gene transcription and the development of genetic radiotherapy-therapeutic strategies in oncology[J]. Cancer Biol Ther. 2003;2(4):326-9.
[6]. Gu Z, Flemington C, Chittenden T, et al. ei24, a p53 response gene involved in growth suppression and apoptosis[J]. Mol Cell Biol. 2000 Jan;20(1):233-41.
[7]. Tsurushima H, Yuan X, Dillehay LE, et al. Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors[J]. Cancer Gene Ther. 2007 Dec;14(12):1002.
[8]. Nakamura Y. Isolation of p53-target genes and their functional analysis[J]. Cancer Sci. 2004;95(1):7-11.
[9]. Dulic V, Kaufmann WK, Wilson SJ, et al. p53-dependent inhibition of cyclin- dependent kinase activities in human fibroblasts during radiation-induced G1 arrest[J]. Cell. 1994 Mar 25;76(6):1013-23.
[10]. Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases[J]. Cell. 1993 Nov 19;75(4):805-16.
[11]. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817-25.
[12]. Malkin D. The role of p53 in human cancer[J]. J Neurooncol. 2001 Feb;51(3):231-43.
[13]. Lebedeva IV, Su ZZ, Sarkar D, et al. Restoring apoptosis as a strategy for cancer gene therapy: focus on p53 and mda-7[J]. Semin Cancer Biol. 2003;13(2):169-78.
[14]. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin. 2002 Jan-Feb;52(1):23-47..
[15]. Cortes-funes H.(2002) New treatment approaches for lung cancer and impact on survival. Semin oncol 29 (suppl 8):26-29.
[16]. Ferreira CG, Span SW, Peters GJ, Kruyt FA, Giaccone G. Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460. Cancer Res. 2000 Dec 15;60(24):7133-41.
[17]. Weichselbaum RR, Hallahan DE, Beckett MA, et al. Gene therapy targeted by radiation preferentially radiosensitizes tumor cells [J]. Cancer Res, 1994, 54(16):4266-4269.
[18]. Hennecke M, Kwissa M, Metzger K, et al. Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs[J]. Nucleic Acids Res. 2001 Aug 15;29(16):3327-34.
[19]. Scott SD, Marples B, Hendry JH, et al. A radiation-controlled molecular switch for use in gene therapy of cancer[J]. Gene Ther. 2000 Jul;7(13):1121-5.
[20]. Greco O, Joiner MC, Doleh A, et al. Hypoxia- and radiation-activated Cre/loxP 'molecular switch' vectors for gene therapy of cancer[J]. Gene Ther. 2006 Feb;13(3):206-15.
[21]. Harms KL, Chen X. The C terminus of p53 family proteins is a cell fate determinant[J]. Mol Cell Biol. 2005 Mar;25(5):2014-30.