APE1作用相关蛋白及其在肿瘤放射治疗中的作用研究
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摘要
放射治疗(简称放疗)是肿瘤治疗最常用的方法之一。根据世界卫生组织(WHO)的统计显示约有45%的恶性肿瘤可以治愈,其中18%为放疗治愈,化疗仅占5%。与其他治疗方法一样,放疗也存在治疗局限性,特别是对一些中晚期、复发性肿瘤的疗效极差,而最主要原因是肿瘤细胞对放射线产生的抵抗作用。DNA是细胞内对放射线最敏感的靶分子。放射线可以通过直接电离和活性氧分子(ROS)的间接作用致伤DNA,导致DNA碱基损伤、单链或双链断裂从而引起细胞不可逆的生长阻滞(复制性死亡)以及细胞凋亡。几乎所有生物体内都存在着DNA损伤修复系统,这是细胞维持基因组稳定的最重要的防御机制。而对于肿瘤细胞,DNA损伤与修复则是其抵抗放射治疗,或产生对放疗不敏感的重要机制之一。
脱嘌呤/脱嘧啶核酸内切酶(apurinic/ apyrimidinic endonuclease,APE1)是生物大分子功能复合体的一个典范,是DNA碱基切除修复(BER)途径的关键限速酶,具有核酸内切酶及氧化还原双功能,是细胞放射性损伤和化疗药物致伤的重要修复因子。同时,APE1还可以通过氧化还原机制调节多种转录因子的DNA结合活性,进而调节下游靶基因表达,参与肿瘤放化疗抵抗。BTG2(B-cell translocation gene 2)是p53诱导激活的靶基因,是具有抗增殖作用的抑癌性质基因,其对放射引起的细胞损伤十分敏感,是一种早期反应型的“Ionizing Radiation responsive gene”。Nm23-H1 (Non-metastatic protein 23 homolog 1)首先作为肿瘤转移抑制基因被发现,其不仅参与了对肿瘤转移能力的抑制,同时还能利用多种激酶活性参与DNA损伤修复,在调控肿瘤放疗敏感性中发挥作用。前期研究我们通过文献循证和预实验发现BTG2和Nm23-H1两种多功能蛋白可能是APE1相互作用的“新”蛋白,并且在肿瘤放射损伤中发挥协同作用。因此本研究以APE1为核心,通过实验鉴定BTG2和Nm23-H1是APE1相互作用的“新”蛋白,研究它们在不同肿瘤中的表达相关性及其在放射引起的肿瘤细胞损伤后的相互作用特点,进一步丰富APE1、BTG2和Nm23-H1的多功能,为解决临床肿瘤患者的放疗增敏作用提供理论和实验依据。
研究目的
1.明确APE1与BTG2在原发性肝细胞肝癌中的表达及其与细胞周期蛋白之间的关系;
2.探讨APE1与BTG2在肝癌细胞及其细胞电离辐射后的表达相关性,分析APE1与BTG2在肝癌放射损伤中的协同作用;
3.明确APE1与Nm23-H1在非小细胞肺癌中的表达及其与临床病理因素的相关性;
4.探讨APE1在肺癌细胞放射损伤后的表达、定位、DNA损伤修复活性变化及其与Nm23-H1的关系,阐明Nm23-H1通过APE1参与DNA损伤修复的作用机制。
研究内容和方法
1. APE1与BTG2在原发性肝细胞肝癌中的表达及其临床意义:免疫组化方法检测原发性肝癌组织中APE1蛋白表达,分析APE1表达与肝癌临床病理因素的关系;分别通过原位杂交和免疫组化方法检测肝癌组织芯片中BTG2的表达,分析BTG2表达与肝癌临床病理因素的关系。
2. APE1和BTG2的相关性分析及其在肝癌细胞放射损伤中的作用研究:western blot检测不同肝癌细胞株中BTG2及其APE1蛋白的表达;重组人p53腺病毒感染不同肝癌细胞,western blot检测APE1和BTG2蛋白的表达。利用pCMV-BTG2点突变的真核表达载体转染肝癌细胞,western blot检测APE1蛋白表达;分别用rAd-p53腺病毒和Ad5/F35-APE1 siRNA重组腺病毒感染肝癌细胞,western blot检测BTG2与APE1蛋白的表达;免疫共沉淀检测BTG2与APE1在不同肝癌细胞中的相互作用;不同剂量X线照射LO2、HepG2、Hep3B和PLC细胞,western blot检测APE1和BTG2蛋白表达变化;16GyX线照射HepG2、Hep3B细胞和MHCC97-L细胞,免疫荧光染色检测BTG2和APE1亚细胞表达变化情况。
3. APE1与Nm23-H1在肺癌组织中的表达及相关性分析:免疫组化法分别检测非小细胞肺癌组织中Nm23-H1与APE1蛋白的表达,分析Nm23-H1与APE1表达与肺癌临床病理因素的关系。
4. APE1参与肺癌A549细胞放射损伤后的表达、定位、DNA损伤修复活性变化及其与Nm23-H1的相互作用的实验研究:Nm23-H1原核蛋白的鉴定和纯化;利用构建pDC316-EGFP-U6-Nm23-H1siRNA和pEGFP-Nm23-H1真核表达质粒分别转染A549细胞,western blot检测APE1和Nm23-H1蛋白表达;分别用Ad5/F35-APE1 siRNA重组腺病毒感染和pcDNA3.0-APE1真核表达载体转染A549细胞,western blot检测APE1和Nm23-H1蛋白表达。不同剂量X线照射A549细胞后,MTT法检测细胞存活率的改变,western blot和免疫荧光染色检测X线照射后细胞中APE1和Nm23-H1蛋白表达;利用免疫共沉淀和DNA亲和沉淀的方法检测APE1和Nm23-H1蛋白结合情况;采用[γ-32P]ATP标记寡核苷酸法检测APE1和Nm23-H1纯化蛋白AP内切酶活性;EMSA测定经转染pEGFP-Nm23-H1质粒的A549细胞中APE1的DNA结合活性变化。
研究结果
1. 103例HCC组织中,APE1在细胞核和/或细胞浆均可见表达,其中单纯性细胞核表达阳性率为46.6%,细胞核/细胞浆联合表达阳性率为49.51%,单纯性细胞浆表达阳性率为3.88%。在组织学分级中,Ⅲ级APE1细胞浆阳性表达较Ⅰ-Ⅱ级显著增高。BTG2 mRNA在肝细胞组织芯片中阳性表达率为71.19%;肝癌BTG2呈胞浆阳性表达,其阳性率为67.8%,其表达与HCC的病理分级有统计学差异(P<0.05),p53蛋白表达阳性率为44.1%,cyclinD1表达阳性率为59.32%;cyclinE表达阳性率为38.98%。BTG2在肝癌旁组织表达显著高于肝癌组织。
2. APE1在不同肝癌细胞中表达均较高未有显著性差异;而BTG2在肝癌细胞PLC及其MHCC97-H中的表达较低,相对于LO2中的表达具有显著性差异。APE1表达在感染重组人p53腺病毒后有降低的趋势,特别是在HepG2和MHCC97-L中,降低趋势显著,而BTG2的表达增加。在HepG2细胞中,当敲低APE1表达后,BTG2的表达降低。免疫共沉淀显示BTG2与APE1蛋白在LO2细胞和HepG2细胞中相互结合。经过不同剂量X线照射后48hr,不同肝癌细胞中BTG2蛋白表达较高,有随剂量增加而表达增加的趋势;在2-8Gy照射后48小时,不同肝癌细胞中APE1表达处于高水平,但16Gy照射后其表达降低,特别在PLC细胞中降低显著。16Gy的X线照射不同肝癌细胞后,8hr内BTG2的核/浆表达具有变化,其浆表达在1-4hr之间增加,8hr后恢复到原来状态甚至浆表达降低,特别是在具有侵袭力的MHCC97-L细胞中,其1hr浆表达增加显著,8hr恢复到原来水平;而细胞中APE1在上述时间点核浆表达未见明显改变。
3.免疫组化显示APE1蛋白在非小细胞肺癌中呈胞核表达、胞浆表达或核浆共同表达。30例非小细胞肺癌患者中APE1核表达(nAPE1)和浆表达(cAPE1)与肺癌病理类型有显著性差异,但与性别、年龄、吸烟和组织学分级无明显相关性,胞浆表达APE1与放疗疗效之间具有明确的反向关系,表达越高疗效越差,胞核APE1的表达与放疗疗效没有相关性;Nm23-H1蛋白主要定位于细胞胞浆,胞核有少量表达。Nm23-H1在30例肺癌患者中的表达与性别、吸烟和肺癌病理类型有显著性差异,与年龄和组织学分级无明显相关性。Nm23-H1表达高的放疗疗效差,表达低的疗效好,具有一定的相关趋势。
4.在A549细胞中分别敲低和增加APE1后,Nm23-H1表达显著降低和增加,表达趋势一致;而分别敲低和增加Nm23-H1后,APE1表达降低和增加,但无显著性差异。放射诱导A549细胞中Nm23-H1和APE1表达增高,随放射时间和剂量的增加而增加,并具有相似的表达趋势;放射诱导A549细胞后Nm23-H1由正常状态下胞浆表达向胞核表达转运趋势,而APE1则由胞核表达为主逐渐变为胞核、胞浆都有分布,两者亚细胞定位有相反的变化特点;His-pull down和免疫共沉淀实验显示Nm23-H1与APE1是相互结合的蛋白,而DNA亲和沉淀实验提示放射诱导可以促进Nm23-H1与APE1共同结合于AP位点形成复合体;Nm23-H1蛋白本身不具有AP内切酶活性,但是可以增强APE1的AP内切酶的活性。
结论
1.在肝细胞中APE1蛋白细胞浆表达/BTG2蛋白高表达与HCC进展相关;BTG2在肝癌中表达异常与cyclinD1和cyclinE的高表达密切相关。
2. APE1与BTG2在肝癌细胞及其细胞电离辐射后表达具有相关性,它们相互结合可能共同参与放射引起的肝癌细胞DNA损伤修复作用。
3. APE1核/浆表达及Nm23-H1的表达与非小细胞肺癌病理类型相关;而APE1浆表达和Nm23-H1高表达是本组30例非小细胞肺癌患者放疗疗效和预后判定的生物学标记。
4. APE1与Nm23-H1蛋白共同结合于AP位点,并通过刺激APE1的AP核酸内切酶活性修复放射诱导引起的肺癌细胞DNA损伤。
Ionizing radiation (IR) used as a therapy (radiotherapy) belongs, besides surgery and conventional cytotoxic chemotherapy, to the three major cancer treatment modalities. More than 45% of the cancer patients are cured during their treatment and about 18% of cancers are cured with radiotherapy, but only 5% of the patients for chemotherapy. Compared with other treatment, the major limitations of Radiotherapy are not adequate for the majority of advanced or recurrent cancer patients because of the remarkable resistance of tumor cells to radiation. The consequences of IR in the human body are mainly induction of DNA damage either by direct ionization of DNA or indirectly by the generation of free radicals. The free radicals due to their unpaired electron are highly unstable and reactive. Since most of the cell is constituted from water, free radicals induced by IR are produced predominantly by the degradation of water molecules. As a consequence, reactive oxygen species (ROS) are generated. These free oxygenated radicals are strong oxidants that damage macromolecules such as DNA, but also cellular proteins and lipids. And one of the important mechanisms of the resistance of tumor cells to radiation are DNA damage and repair.
The apurinic endonuclease 1/redox factor-1 (APE1) is a master regulator of cellular response to oxidative stress and plays a central role in the maintenance of genome stability acting as the major apurinic/apyrimidinic(AP)-endonuclease in the DNA base excision repair (BER) pathway that copes with DNA damage caused by both endogenous and exogenous agents. As importantly, APE1 also functions as a redox agent maintaining transcription factors involved in cancer promotion and progression in an active reduced state, and recent studies identify it as an excellent target for sensitizing tumor cells to radiotherapy and chemotherapy. B-cell translocation gene 2(BTG2) is an early growth response gene whose promoter contains p53-binding sites and is regulated by p53. It is also an antiproliferative gene and plays an important role in the suppression of carcinogenesis. Several lines of evidence suggest that the expression of BTG2 relates with the sensitivity to ionizing radiation, and BTG2 is also called Ionizing Radiation responsive gene. Nm23-H1 was initially identified as a putative metastasis suppressor on the basis of its reduced expression in certain highly metastatic cell lines and tumors. Non-metastatic protein 23 homolog 1(Nm23-H1) is also a multifunctional enzyme, although its enzymatic activity provided no evidence for a role as a metastasis suppressor in tumor progression. Extensive studies using Nm23-H1 proteins have shown that they participate in the regulation of a broad spectrum of cellular responses including development, differentiation, proliferation, apoptosis and DNA synthesis. All these studies suggest that Nm23-H1 may participate in the DNA repair of human cells. Much evidence-based literature has showed that BTG2 and Nm23-H1 might be involved in DNA repair to resistance of tumor cells to radiation and may interact with APE1. The aim of this study was to evaluate the major functions of APE1 and a detailed comprehension of the molecular targets such as BTG2 and Nm23-H1 of APE1 functions, allowed establishment of ionizing radiation of cell models to inspect and characterize in better detail the major functions of these genes.
Objective
1. To evaluate the expression of APE1 and BTG2 compared with p53/ cyclinD1/ cyclinE and clinicopathologic parameters in HCC.
2. To further elucidate the correlation of APE1 and BTG2 expression in different human liver cancer cell lines irradiated with X-rays.
3. To investigate the expression of APE1 and Nm23-H1 in non-small-cell lung carcinoma (NSCLC) compared with clinicopathologic parameters and the curative effects.
4. To further elucidate the role of Nm23-H1 and APE1 in the human lung cancer A549 cell line irradiated with X-rays.
Materials and Methods
1. Expression feature of APE1 and BTG2 and its significance in HCC: The expression of BTG2 mRNA on tissue microarray (TMA) of HCC via in situ hybridization. Expression of APE1 and BTG2 protein was detected by S-P immunohistochemical method in normal liver tissue and HCC.
2. Relation between APE1 and BTG2 in different liver cancer cells after IR: The expression of human APE1 and BTG2 protein was detected after Ad5/F35-APE1 siRNA and Ad-p53 were transfered into different liver cancer cells by western blot analysis. The interaction between APE1 and BTG2 was demonstrated by co-immunoprecipitation assays. Western blot was used to analysis the interaction between these two proteins at different time points after irradiating different human liver cancer cells with different doses of X-rays. Subcellular distribution of APE1 and BTG2 was also examined by laser confocal microscopy at 0-8h in IR treated MHCC97-L, HepG2 and Hep3B cell.
3. Expression of APE1 and Nm23-H1 compared with clinicopathologic parameters and the curative effects in NSCLC : Expression of APE1 and Nm23-H1 protein was detected by immunohistochemical method in 30 patients with NSCLC. Statistical analyses were performed using a statistical software package (SPSS for Windows). The relevant variables were studied by univariate and multivariate statistical techniques. Survival difference was analyzed by the log-rank test. Multivariate analyses were performed using Cox proportional hazards model. The survival curves were drawn according to Kaplan-Meier method. To calculate statistical significance between categorical variables, we used Chi-square.
4. Study of the role of Nm23-H1 and APE1 in the human lung cancer A549 cell line irradiated with X-rays: Western blot was used to detected the expression of these two proteins at different time points after irradiating human lung cancer A549 cells with different doses of X-rays. The subcellular distribution pattern of Nm23-H1 and APE1 in A549 cells after irradiation was also examined by laser confocal microscopy at different time points in IR treated A549 cell. To determine whether Nm23-H1 expression correlated with APE1 expression, we detected the expression of Nm23-H1 in A549 cells treated with Ad5/F35-APE1 siRNA by western blotting.The interaction between Nm23-H1 and APE1 was demonstrated by His-pull down and co-immunoprecipitation assays. The presence of Nm23-H1/APE1 complex in X-rays-irradiated A549 cells was also detected by DNA affinity precipitation analysis of a DNA fragment containing an AP site. The AP endonuclease activities of APE1 and Nm23-H1 were evaluated by a well-characterized oligonucleotide cleavage assay. Electrophoretic mobility shift assays (EMSAs) were performed using APE1, nuclear proteins of A549 cells transfected with different doses of pEGFP-Nm23-H1 plasmid, and oligonucleotides in DNA affinity precipitation analysis .
Results
1. There were the nuclear APE1, the cytoplasmic APE1 and the nuclear-cytoplasmic APE1 in HCC-tissues. Expression of nuclear APE1/ cytoplasmic APE1/ nuclear-cytoplasmic APE1, was positive in 46.6/3.88/49.51 percent in HCC-tissues, respectively. The cytoplasmic APE1 expression implied a higher tumor grade significantly.BTG2 mRNA was expressed in 71.19% HCC and BTG2 protein expression was 67.8% in HCC-tissues respectively. BTG2 expression was predominantly detected in the cytoplasm of tumour cells and the mRNA/protein expression correlated significantly with the increasing tumor grade. Expression of p53/cyclin-D1/cyclin-E was positive in 44.1/59.32/38.98 percent in HCC-tissues, respectively. There was a significant difference of BTG2 expression between normal liver tissue and HCC tissues.
2. The expression of APE1 was higher in all of the liver cancer cells but the BTG2 expression was significantly higher in PLC and MHCC97-L cell than in LO2 cell. rAd-p53 enhanced the expression of BTG2 protein in all of liver cancer cells, but APE1 expression was decreased in all of liver cancer cells, especially in HepG2 and MHCC97-L cell. Ad5/F35-APE1 siRNA suppressed significantly the expression of APE1 and BTG2 in HepG2. Co-immunoprecipitation assays indicated BTG2 binds to APE1 in LO2 and HepG2 cell. The overexpression of BTG2 protein in different liver cells was induced by irradiation in a dose- dependent manner. Although the expression of APE1 protein was higher after 4 and 16 Gy X-rays irradiation at 48 hrs, the APE1 expression was significantly decreased after 16 Gy X-rays irradiation, especially in PLC cell. After irradiation with 16Gy X-rays in MHCC97-L, HepG2 and Hep3B cell, BTG2 which was initially localized in the cytoplasm of these cells was gradually increased at 1-4 hrs whereas was gradually decreased to control level at 8 hrs, especially in MHCC97-L cell. But APE1 which was mainly localized in the nuclei of cells was not changed at the same time point.
3. APE1 was detected in nucleus, cytoplasm and nucleus to cytoplasm of NSCLC tissues by immunohistochemical staining technique. The positive rate of APE1 nuclear expression and cytoplasmic expression were correlated significantly with pathological type in 30 patients of NSCLC. There were not relation between the expression of APE1 and other clinicopathologic parameters. The expression of APE1 correlated to radiotherapy which affected overall survival(OS) and the cytoplasmic overexpression of APE1 was associated with poor prognosis of NSCLC. The nuclear expression of APE1 was not relationship with curative effects. Nm23-H1 was examined mainly in cytoplasm of NSCLC tissues by immunohistochemical staining technique. The expression of Nm23-H1 in 30 patients of NSCLC was correlated significantly to clinicopathologic parameters including Gender, Smokeing and pathological type. the expression of Nm23-H1 no correlated with other clinicopathologic parameters including differention grading and Age. The expression of Nm23-H1 correlated to the curative effects of radiotherapy and the overexpression of Nm23-H1 was associated with poor prognosis of NSCLC.
4. The overexpression of APE1 and Nm23-H1 proteins in A549 cells was induced by irradiation in a dose- and time-dependent manner. The subcellular distribution pattern of APE1 and Nm23-H1 in A549 cells after irradiation was reversed. After irradiation with different doses of X-rays, APE1 which was initially localized in the nuclei of A549 cells was gradually increased in the cytoplasm whereas Nm23-H1 which was mainly localized in the cytoplasm was gradually increased in the nuclei of these cells. Altered Nm23-H1 expression correlates with changes in APE1 expression, detected by knockdown and overexpression.The interaction between Nm23-H1 and APE1 was demonstrated by His-pull down and co-immunoprecipitation assays. Nm23-H1 binds with APE1 to mediate AP sites and may therefore participate in the DNA BER repair pathway. Although the AP endonuclease activity of Nm23-H1 protein was too weak to be detected, the AP endonuclease activity of APE1 protein was increased with the increased expression of Nm23-H1 protein. a mobility shift clearly occurs in the presence of APE1 and nuclear proteins of A549 cells. Besides, as the doses of pEGFP-Nm23-H1 plasmid transfected into the cells were increased, formation of the APE1-DNA complex was also increased.
Conclusion
1. The overexpression of cytoplasmic APE1 and BTG2 protein in liver cells may play a pivotal role in the carcinogenesis and progression of HCC. Aberrant expression of BTG2 in hepatocellular carcinoma is associated with overexpression of cyclinD1/cyclinE.
2. The expression of APE1 correlated to BTG2 expression in different human liver cancer cells and ionizing radiation of these cells. BTG2 protein binds to APE1 and an interaction between BTG2 and APE1 proteins may be a factor involved in a DNA repair pathway in human liver cancer cells following damage by ionizing radiation.
3. The expression of nuclear/cytoplasmic APE1 and Nm23-H1 was correlated with pathological type of NSCLC. The overexpression of cytoplasmic APE1 and Nm23-H1 can be a potential marker for poor prognosis prediction of NSCLC patients after radiotherapy screening.
4. Nm23-H1 protein binds to APE1 at AP sites and stimulates AP endonuclease activity following ionizing radiation of the human lung cancer A549 cells.
脱嘌呤/脱嘧啶核酸内切酶(apurinic/ apyrimidinic endonuclease,APE1)是生物大分子功能复合体的一个典范,是DNA碱基切除修复(BER)途径的关键限速酶,具有核酸内切酶及氧化还原双功能,是细胞放射性损伤和化疗药物致伤的重要修复因子。同时,APE1还可以通过氧化还原机制调节多种转录因子的DNA结合活性,进而调节下游靶基因表达,参与肿瘤放化疗抵抗。BTG2(B-cell translocation gene 2)是p53诱导激活的靶基因,是具有抗增殖作用的抑癌性质基因,其对放射引起的细胞损伤十分敏感,是一种早期反应型的“Ionizing Radiation responsive gene”。Nm23-H1 (Non-metastatic protein 23 homolog 1)首先作为肿瘤转移抑制基因被发现,其不仅参与了对肿瘤转移能力的抑制,同时还能利用多种激酶活性参与DNA损伤修复,在调控肿瘤放疗敏感性中发挥作用。前期研究我们通过文献循证和预实验发现BTG2和Nm23-H1两种多功能蛋白可能是APE1相互作用的“新”蛋白,并且在肿瘤放射损伤中发挥协同作用。因此本研究以APE1为核心,通过实验鉴定BTG2和Nm23-H1是APE1相互作用的“新”蛋白,研究它们在不同肿瘤中的表达相关性及其在放射引起的肿瘤细胞损伤后的相互作用特点,进一步丰富APE1、BTG2和Nm23-H1的多功能,为解决临床肿瘤患者的放疗增敏作用提供理论和实验依据。
研究目的
1.明确APE1与BTG2在原发性肝细胞肝癌中的表达及其与细胞周期蛋白之间的关系;
2.探讨APE1与BTG2在肝癌细胞及其细胞电离辐射后的表达相关性,分析APE1与BTG2在肝癌放射损伤中的协同作用;
3.明确APE1与Nm23-H1在非小细胞肺癌中的表达及其与临床病理因素的相关性;
4.探讨APE1在肺癌细胞放射损伤后的表达、定位、DNA损伤修复活性变化及其与Nm23-H1的关系,阐明Nm23-H1通过APE1参与DNA损伤修复的作用机制。
研究内容和方法
1. APE1与BTG2在原发性肝细胞肝癌中的表达及其临床意义:免疫组化方法检测原发性肝癌组织中APE1蛋白表达,分析APE1表达与肝癌临床病理因素的关系;分别通过原位杂交和免疫组化方法检测肝癌组织芯片中BTG2的表达,分析BTG2表达与肝癌临床病理因素的关系。
2. APE1和BTG2的相关性分析及其在肝癌细胞放射损伤中的作用研究:western blot检测不同肝癌细胞株中BTG2及其APE1蛋白的表达;重组人p53腺病毒感染不同肝癌细胞,western blot检测APE1和BTG2蛋白的表达。利用pCMV-BTG2点突变的真核表达载体转染肝癌细胞,western blot检测APE1蛋白表达;分别用rAd-p53腺病毒和Ad5/F35-APE1 siRNA重组腺病毒感染肝癌细胞,western blot检测BTG2与APE1蛋白的表达;免疫共沉淀检测BTG2与APE1在不同肝癌细胞中的相互作用;不同剂量X线照射LO2、HepG2、Hep3B和PLC细胞,western blot检测APE1和BTG2蛋白表达变化;16GyX线照射HepG2、Hep3B细胞和MHCC97-L细胞,免疫荧光染色检测BTG2和APE1亚细胞表达变化情况。
3. APE1与Nm23-H1在肺癌组织中的表达及相关性分析:免疫组化法分别检测非小细胞肺癌组织中Nm23-H1与APE1蛋白的表达,分析Nm23-H1与APE1表达与肺癌临床病理因素的关系。
4. APE1参与肺癌A549细胞放射损伤后的表达、定位、DNA损伤修复活性变化及其与Nm23-H1的相互作用的实验研究:Nm23-H1原核蛋白的鉴定和纯化;利用构建pDC316-EGFP-U6-Nm23-H1siRNA和pEGFP-Nm23-H1真核表达质粒分别转染A549细胞,western blot检测APE1和Nm23-H1蛋白表达;分别用Ad5/F35-APE1 siRNA重组腺病毒感染和pcDNA3.0-APE1真核表达载体转染A549细胞,western blot检测APE1和Nm23-H1蛋白表达。不同剂量X线照射A549细胞后,MTT法检测细胞存活率的改变,western blot和免疫荧光染色检测X线照射后细胞中APE1和Nm23-H1蛋白表达;利用免疫共沉淀和DNA亲和沉淀的方法检测APE1和Nm23-H1蛋白结合情况;采用[γ-32P]ATP标记寡核苷酸法检测APE1和Nm23-H1纯化蛋白AP内切酶活性;EMSA测定经转染pEGFP-Nm23-H1质粒的A549细胞中APE1的DNA结合活性变化。
研究结果
1. 103例HCC组织中,APE1在细胞核和/或细胞浆均可见表达,其中单纯性细胞核表达阳性率为46.6%,细胞核/细胞浆联合表达阳性率为49.51%,单纯性细胞浆表达阳性率为3.88%。在组织学分级中,Ⅲ级APE1细胞浆阳性表达较Ⅰ-Ⅱ级显著增高。BTG2 mRNA在肝细胞组织芯片中阳性表达率为71.19%;肝癌BTG2呈胞浆阳性表达,其阳性率为67.8%,其表达与HCC的病理分级有统计学差异(P<0.05),p53蛋白表达阳性率为44.1%,cyclinD1表达阳性率为59.32%;cyclinE表达阳性率为38.98%。BTG2在肝癌旁组织表达显著高于肝癌组织。
2. APE1在不同肝癌细胞中表达均较高未有显著性差异;而BTG2在肝癌细胞PLC及其MHCC97-H中的表达较低,相对于LO2中的表达具有显著性差异。APE1表达在感染重组人p53腺病毒后有降低的趋势,特别是在HepG2和MHCC97-L中,降低趋势显著,而BTG2的表达增加。在HepG2细胞中,当敲低APE1表达后,BTG2的表达降低。免疫共沉淀显示BTG2与APE1蛋白在LO2细胞和HepG2细胞中相互结合。经过不同剂量X线照射后48hr,不同肝癌细胞中BTG2蛋白表达较高,有随剂量增加而表达增加的趋势;在2-8Gy照射后48小时,不同肝癌细胞中APE1表达处于高水平,但16Gy照射后其表达降低,特别在PLC细胞中降低显著。16Gy的X线照射不同肝癌细胞后,8hr内BTG2的核/浆表达具有变化,其浆表达在1-4hr之间增加,8hr后恢复到原来状态甚至浆表达降低,特别是在具有侵袭力的MHCC97-L细胞中,其1hr浆表达增加显著,8hr恢复到原来水平;而细胞中APE1在上述时间点核浆表达未见明显改变。
3.免疫组化显示APE1蛋白在非小细胞肺癌中呈胞核表达、胞浆表达或核浆共同表达。30例非小细胞肺癌患者中APE1核表达(nAPE1)和浆表达(cAPE1)与肺癌病理类型有显著性差异,但与性别、年龄、吸烟和组织学分级无明显相关性,胞浆表达APE1与放疗疗效之间具有明确的反向关系,表达越高疗效越差,胞核APE1的表达与放疗疗效没有相关性;Nm23-H1蛋白主要定位于细胞胞浆,胞核有少量表达。Nm23-H1在30例肺癌患者中的表达与性别、吸烟和肺癌病理类型有显著性差异,与年龄和组织学分级无明显相关性。Nm23-H1表达高的放疗疗效差,表达低的疗效好,具有一定的相关趋势。
4.在A549细胞中分别敲低和增加APE1后,Nm23-H1表达显著降低和增加,表达趋势一致;而分别敲低和增加Nm23-H1后,APE1表达降低和增加,但无显著性差异。放射诱导A549细胞中Nm23-H1和APE1表达增高,随放射时间和剂量的增加而增加,并具有相似的表达趋势;放射诱导A549细胞后Nm23-H1由正常状态下胞浆表达向胞核表达转运趋势,而APE1则由胞核表达为主逐渐变为胞核、胞浆都有分布,两者亚细胞定位有相反的变化特点;His-pull down和免疫共沉淀实验显示Nm23-H1与APE1是相互结合的蛋白,而DNA亲和沉淀实验提示放射诱导可以促进Nm23-H1与APE1共同结合于AP位点形成复合体;Nm23-H1蛋白本身不具有AP内切酶活性,但是可以增强APE1的AP内切酶的活性。
结论
1.在肝细胞中APE1蛋白细胞浆表达/BTG2蛋白高表达与HCC进展相关;BTG2在肝癌中表达异常与cyclinD1和cyclinE的高表达密切相关。
2. APE1与BTG2在肝癌细胞及其细胞电离辐射后表达具有相关性,它们相互结合可能共同参与放射引起的肝癌细胞DNA损伤修复作用。
3. APE1核/浆表达及Nm23-H1的表达与非小细胞肺癌病理类型相关;而APE1浆表达和Nm23-H1高表达是本组30例非小细胞肺癌患者放疗疗效和预后判定的生物学标记。
4. APE1与Nm23-H1蛋白共同结合于AP位点,并通过刺激APE1的AP核酸内切酶活性修复放射诱导引起的肺癌细胞DNA损伤。
Ionizing radiation (IR) used as a therapy (radiotherapy) belongs, besides surgery and conventional cytotoxic chemotherapy, to the three major cancer treatment modalities. More than 45% of the cancer patients are cured during their treatment and about 18% of cancers are cured with radiotherapy, but only 5% of the patients for chemotherapy. Compared with other treatment, the major limitations of Radiotherapy are not adequate for the majority of advanced or recurrent cancer patients because of the remarkable resistance of tumor cells to radiation. The consequences of IR in the human body are mainly induction of DNA damage either by direct ionization of DNA or indirectly by the generation of free radicals. The free radicals due to their unpaired electron are highly unstable and reactive. Since most of the cell is constituted from water, free radicals induced by IR are produced predominantly by the degradation of water molecules. As a consequence, reactive oxygen species (ROS) are generated. These free oxygenated radicals are strong oxidants that damage macromolecules such as DNA, but also cellular proteins and lipids. And one of the important mechanisms of the resistance of tumor cells to radiation are DNA damage and repair.
The apurinic endonuclease 1/redox factor-1 (APE1) is a master regulator of cellular response to oxidative stress and plays a central role in the maintenance of genome stability acting as the major apurinic/apyrimidinic(AP)-endonuclease in the DNA base excision repair (BER) pathway that copes with DNA damage caused by both endogenous and exogenous agents. As importantly, APE1 also functions as a redox agent maintaining transcription factors involved in cancer promotion and progression in an active reduced state, and recent studies identify it as an excellent target for sensitizing tumor cells to radiotherapy and chemotherapy. B-cell translocation gene 2(BTG2) is an early growth response gene whose promoter contains p53-binding sites and is regulated by p53. It is also an antiproliferative gene and plays an important role in the suppression of carcinogenesis. Several lines of evidence suggest that the expression of BTG2 relates with the sensitivity to ionizing radiation, and BTG2 is also called Ionizing Radiation responsive gene. Nm23-H1 was initially identified as a putative metastasis suppressor on the basis of its reduced expression in certain highly metastatic cell lines and tumors. Non-metastatic protein 23 homolog 1(Nm23-H1) is also a multifunctional enzyme, although its enzymatic activity provided no evidence for a role as a metastasis suppressor in tumor progression. Extensive studies using Nm23-H1 proteins have shown that they participate in the regulation of a broad spectrum of cellular responses including development, differentiation, proliferation, apoptosis and DNA synthesis. All these studies suggest that Nm23-H1 may participate in the DNA repair of human cells. Much evidence-based literature has showed that BTG2 and Nm23-H1 might be involved in DNA repair to resistance of tumor cells to radiation and may interact with APE1. The aim of this study was to evaluate the major functions of APE1 and a detailed comprehension of the molecular targets such as BTG2 and Nm23-H1 of APE1 functions, allowed establishment of ionizing radiation of cell models to inspect and characterize in better detail the major functions of these genes.
Objective
1. To evaluate the expression of APE1 and BTG2 compared with p53/ cyclinD1/ cyclinE and clinicopathologic parameters in HCC.
2. To further elucidate the correlation of APE1 and BTG2 expression in different human liver cancer cell lines irradiated with X-rays.
3. To investigate the expression of APE1 and Nm23-H1 in non-small-cell lung carcinoma (NSCLC) compared with clinicopathologic parameters and the curative effects.
4. To further elucidate the role of Nm23-H1 and APE1 in the human lung cancer A549 cell line irradiated with X-rays.
Materials and Methods
1. Expression feature of APE1 and BTG2 and its significance in HCC: The expression of BTG2 mRNA on tissue microarray (TMA) of HCC via in situ hybridization. Expression of APE1 and BTG2 protein was detected by S-P immunohistochemical method in normal liver tissue and HCC.
2. Relation between APE1 and BTG2 in different liver cancer cells after IR: The expression of human APE1 and BTG2 protein was detected after Ad5/F35-APE1 siRNA and Ad-p53 were transfered into different liver cancer cells by western blot analysis. The interaction between APE1 and BTG2 was demonstrated by co-immunoprecipitation assays. Western blot was used to analysis the interaction between these two proteins at different time points after irradiating different human liver cancer cells with different doses of X-rays. Subcellular distribution of APE1 and BTG2 was also examined by laser confocal microscopy at 0-8h in IR treated MHCC97-L, HepG2 and Hep3B cell.
3. Expression of APE1 and Nm23-H1 compared with clinicopathologic parameters and the curative effects in NSCLC : Expression of APE1 and Nm23-H1 protein was detected by immunohistochemical method in 30 patients with NSCLC. Statistical analyses were performed using a statistical software package (SPSS for Windows). The relevant variables were studied by univariate and multivariate statistical techniques. Survival difference was analyzed by the log-rank test. Multivariate analyses were performed using Cox proportional hazards model. The survival curves were drawn according to Kaplan-Meier method. To calculate statistical significance between categorical variables, we used Chi-square.
4. Study of the role of Nm23-H1 and APE1 in the human lung cancer A549 cell line irradiated with X-rays: Western blot was used to detected the expression of these two proteins at different time points after irradiating human lung cancer A549 cells with different doses of X-rays. The subcellular distribution pattern of Nm23-H1 and APE1 in A549 cells after irradiation was also examined by laser confocal microscopy at different time points in IR treated A549 cell. To determine whether Nm23-H1 expression correlated with APE1 expression, we detected the expression of Nm23-H1 in A549 cells treated with Ad5/F35-APE1 siRNA by western blotting.The interaction between Nm23-H1 and APE1 was demonstrated by His-pull down and co-immunoprecipitation assays. The presence of Nm23-H1/APE1 complex in X-rays-irradiated A549 cells was also detected by DNA affinity precipitation analysis of a DNA fragment containing an AP site. The AP endonuclease activities of APE1 and Nm23-H1 were evaluated by a well-characterized oligonucleotide cleavage assay. Electrophoretic mobility shift assays (EMSAs) were performed using APE1, nuclear proteins of A549 cells transfected with different doses of pEGFP-Nm23-H1 plasmid, and oligonucleotides in DNA affinity precipitation analysis .
Results
1. There were the nuclear APE1, the cytoplasmic APE1 and the nuclear-cytoplasmic APE1 in HCC-tissues. Expression of nuclear APE1/ cytoplasmic APE1/ nuclear-cytoplasmic APE1, was positive in 46.6/3.88/49.51 percent in HCC-tissues, respectively. The cytoplasmic APE1 expression implied a higher tumor grade significantly.BTG2 mRNA was expressed in 71.19% HCC and BTG2 protein expression was 67.8% in HCC-tissues respectively. BTG2 expression was predominantly detected in the cytoplasm of tumour cells and the mRNA/protein expression correlated significantly with the increasing tumor grade. Expression of p53/cyclin-D1/cyclin-E was positive in 44.1/59.32/38.98 percent in HCC-tissues, respectively. There was a significant difference of BTG2 expression between normal liver tissue and HCC tissues.
2. The expression of APE1 was higher in all of the liver cancer cells but the BTG2 expression was significantly higher in PLC and MHCC97-L cell than in LO2 cell. rAd-p53 enhanced the expression of BTG2 protein in all of liver cancer cells, but APE1 expression was decreased in all of liver cancer cells, especially in HepG2 and MHCC97-L cell. Ad5/F35-APE1 siRNA suppressed significantly the expression of APE1 and BTG2 in HepG2. Co-immunoprecipitation assays indicated BTG2 binds to APE1 in LO2 and HepG2 cell. The overexpression of BTG2 protein in different liver cells was induced by irradiation in a dose- dependent manner. Although the expression of APE1 protein was higher after 4 and 16 Gy X-rays irradiation at 48 hrs, the APE1 expression was significantly decreased after 16 Gy X-rays irradiation, especially in PLC cell. After irradiation with 16Gy X-rays in MHCC97-L, HepG2 and Hep3B cell, BTG2 which was initially localized in the cytoplasm of these cells was gradually increased at 1-4 hrs whereas was gradually decreased to control level at 8 hrs, especially in MHCC97-L cell. But APE1 which was mainly localized in the nuclei of cells was not changed at the same time point.
3. APE1 was detected in nucleus, cytoplasm and nucleus to cytoplasm of NSCLC tissues by immunohistochemical staining technique. The positive rate of APE1 nuclear expression and cytoplasmic expression were correlated significantly with pathological type in 30 patients of NSCLC. There were not relation between the expression of APE1 and other clinicopathologic parameters. The expression of APE1 correlated to radiotherapy which affected overall survival(OS) and the cytoplasmic overexpression of APE1 was associated with poor prognosis of NSCLC. The nuclear expression of APE1 was not relationship with curative effects. Nm23-H1 was examined mainly in cytoplasm of NSCLC tissues by immunohistochemical staining technique. The expression of Nm23-H1 in 30 patients of NSCLC was correlated significantly to clinicopathologic parameters including Gender, Smokeing and pathological type. the expression of Nm23-H1 no correlated with other clinicopathologic parameters including differention grading and Age. The expression of Nm23-H1 correlated to the curative effects of radiotherapy and the overexpression of Nm23-H1 was associated with poor prognosis of NSCLC.
4. The overexpression of APE1 and Nm23-H1 proteins in A549 cells was induced by irradiation in a dose- and time-dependent manner. The subcellular distribution pattern of APE1 and Nm23-H1 in A549 cells after irradiation was reversed. After irradiation with different doses of X-rays, APE1 which was initially localized in the nuclei of A549 cells was gradually increased in the cytoplasm whereas Nm23-H1 which was mainly localized in the cytoplasm was gradually increased in the nuclei of these cells. Altered Nm23-H1 expression correlates with changes in APE1 expression, detected by knockdown and overexpression.The interaction between Nm23-H1 and APE1 was demonstrated by His-pull down and co-immunoprecipitation assays. Nm23-H1 binds with APE1 to mediate AP sites and may therefore participate in the DNA BER repair pathway. Although the AP endonuclease activity of Nm23-H1 protein was too weak to be detected, the AP endonuclease activity of APE1 protein was increased with the increased expression of Nm23-H1 protein. a mobility shift clearly occurs in the presence of APE1 and nuclear proteins of A549 cells. Besides, as the doses of pEGFP-Nm23-H1 plasmid transfected into the cells were increased, formation of the APE1-DNA complex was also increased.
Conclusion
1. The overexpression of cytoplasmic APE1 and BTG2 protein in liver cells may play a pivotal role in the carcinogenesis and progression of HCC. Aberrant expression of BTG2 in hepatocellular carcinoma is associated with overexpression of cyclinD1/cyclinE.
2. The expression of APE1 correlated to BTG2 expression in different human liver cancer cells and ionizing radiation of these cells. BTG2 protein binds to APE1 and an interaction between BTG2 and APE1 proteins may be a factor involved in a DNA repair pathway in human liver cancer cells following damage by ionizing radiation.
3. The expression of nuclear/cytoplasmic APE1 and Nm23-H1 was correlated with pathological type of NSCLC. The overexpression of cytoplasmic APE1 and Nm23-H1 can be a potential marker for poor prognosis prediction of NSCLC patients after radiotherapy screening.
4. Nm23-H1 protein binds to APE1 at AP sites and stimulates AP endonuclease activity following ionizing radiation of the human lung cancer A549 cells.
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