GADD45A在肿瘤中的研究进展

GADD45A是细胞生长阻滞和DNA损伤修复基因,在基因组稳定性维持、细胞周期阻滞和细胞凋亡中起着重要的作用,与肿瘤的发生有密切的关系,其表达高低与肿瘤预后有关.本文对GADD45A的结构和功能以及其在肿瘤中的作用进行综述.     

GADD45g 通过抑制E2F1在急性髓系白血病中发挥抑癌作用

[摘要] 目的：检测转录因子E2F1 与生长阻滞和DNA损伤诱导蛋白45g（GADD45g）基因在急性髓系白血病（AML）患者中表达的相关性,探讨GADD45g 是否通过抑制E2F1 诱导AML细胞DNA损伤、凋亡、衰老、周期阻滞和提高药物敏感性。方法：选取2013 年1 月至2016 年12 月中国医学科学院血液病医院初诊为AML患者32 例骨髓标本及AML细胞系U937、HL60、THP-1和Molm-13,用qPCR检测组织和细胞中GADD45g 和E2F1 mRNA的表达水平,并分析其相关性。构建E2F1 过表达载体,并制备重组慢病毒在过表达GADD45g 的Molm-13 和THP-1 细胞中过表达E2F1,通过彗星实验、Annexin V/7AAD流式细胞术、β-半乳糖苷酶染色和PI 染色流式细胞术等确定GADD45g 是否通过抑制E2F1 对AML细胞发挥抑癌作用。结果：AML患者骨髓和细胞系中GADD45g 和E2F1 mRNA的表达呈显著负相关（r=–0.663,P<0.01）。GADD45g 在AML细胞系中过表达显著抑制了E2F1的表达（均P<0.01）。成功构建同时过表达GADD45g 和E2F1 的Molm-13 和THP-1 细胞,与对照组比较,过表达组细胞GADD45g和E2F1 蛋白表达水平均显著升高（均P<0.01）。与过表达GADD45g 的细胞相比,同时过表达GADD45g 和E2F1 细胞的凋亡、衰老率和DNA损伤水平均显著降低（均P<0.01）;在过表达GADD45g 的细胞中过表达E2F1 逆转了GADD45g 诱导的周期阻滞（均P<0.01）,进而降低了过表达GADD45g 对化疗药物的敏感性（均P<0.01）。结论：GADD45g 通过抑制E2F1 在AML中发挥抗肿瘤作用。     

转录因子Sp1在DNA损伤修复中的作用及其与肿瘤治疗的研究进展

细胞受到外界刺激导致DNA损伤时，DNA修复蛋白被激活并发挥功能，受损DNA得到修复。近年来研究表明，转录因子特异性蛋白1(specificity protein 1，Sp1)在DNA损伤修复中起重要作用，是参与调控DNA损伤应答与修复的重要蛋白之一。Sp1在多种肿瘤中高表达，通过调节细胞增殖、迁移和侵袭促进肿瘤的发生和发展，不利于肿瘤治疗。本文首先介绍Sp1特有的结构及其生理功能，由此引入Sp1通过转录依赖方式调控DNA损伤应答，其次介绍Sp1通过非转录依赖方式参与DSB修复，以及Sp1在肿瘤治疗中的研究进展，最后展望研究Sp1的意义及可能具有的应用前景。     

DNA修复蛋白RAD52在同源重组中的作用及其在肿瘤中的研究进展

DNA损伤修复在维持细胞基因组稳定性和机体存活中发挥重要作用。DNA双链断裂（Double strandbreaks,DSBs）是DNA损伤最严重的形式。同源重组修复（homologous recombination repair，HRR）是体内参与DSBs损伤修复的重要机制之一，其中DNA修复蛋白RAD52是体内参与同源重组DNA修复的关键因子。RAD52的表达水平异常与非小细胞肺癌、胃癌、鼻咽癌、乳腺癌等肿瘤的发生、发展相关。本文对DNA修复蛋白RAD52在肿瘤研究中的应用这一热点问题进行综述。     

BRCA1在DNA损伤应激反应中的研究进展

乳腺癌易感基因BRCA1与乳腺癌、卵巢癌等肿瘤的发生紧密相关。此外,BRCA1还参与了许多重要的细胞信号通路,包括DNA损伤所诱发的细胞应激反应,如DNA损伤导致的细胞周期检验点的激活、DNA损伤修复、染色体重塑及细胞凋亡等。本文对BRCA1在DNA损伤应激反应中的研究进展,尤其是它在DNA双链断裂修复的两种模式（非同源末端连接和同源重组）中的作用作一综述。     

不同增殖状态下CNE-2细胞株DNA损伤修复相关基因表达差异的观察

目的:了解不同增殖状态鼻咽癌细胞(CNE-2)基因表达差异,探讨鼻咽癌放射治疗中加速再增殖的机制.方法:对不同增殖状态的CNE-2细胞,采用基因芯片技术、实时荧光定量PCR检测其DNA损伤修复相关基因的表达差异.结果:CNE-2细胞增殖活性最高时相(2 Gy连续照射第3天)与最低时相(2 Gy连续照射第5天)表达有差异的DNA损伤修复相关基因6条,占检测基因总数的5.9%(6/102),其中细胞增殖活性增高时上调基因为CDC25A,下调基因为DDIT3、GADD45、CDKN1A、BNIP3和FOXO3A.应用相对定量荧光实时PCR技术,对部分差异表达>2倍的基因(CCNE1、CDC25A、CDKN1A、DDIT3、GADD45和BNIP3)进行了mRNA水平的验证,结果显示实时PCR实验结果与芯片结果具有良好的一致性.结论:DNA损伤修复的某些基因如CDC25A、CDKN1A、DDIT3、GADD45、BNIP3和FOXO3A等可能参与了CNE-2细胞放射治疗过程中发生的加速再增殖过程,加速再增殖可能是一个复杂的、多基因协同作用的结果.     

GADD45g基因在急性髓系白血病细胞中的低表达及其抑癌特性

目的： 检测生长阻滞和DNA损伤诱导蛋白45g（growth arrest and DNA damage inducible protein 45g,GADD45g）基因在 急性髓系白血病（acute myeloid leukemia, AML）患者骨髓标本和细胞系中的表达情况及其表达水平与AML患者预后的关系,探 究过表达GADD45g对AML细胞增殖、凋亡、衰老、周期、分化和药物敏感性的影响。 方法： 选取中国医学科学院血液病医院 2013年1月至2016年12月初次诊断为AML患者的骨髓标本共27例,用Real-time PCR和Western blotting检测AML患者、正常对 照骨髓单个核细胞以及 AML 细胞系中 GADD45g mRNA 和蛋白水平的表达情况。在两个相互独立的数据集 GSE10358 和 GSE425-GPL317中分析GADD45g表达与AML患者的总生存率（overall survival,OS）和无事件生存率（event-free survival,EFS） 的相关性。构建GADD45g过表达慢病毒并感染AML细胞系U937、THP-1和Molm-13,通过生长曲线、集落形成、β-半乳糖苷酶 染色、流式细胞术分析Annexin V/7AAD染色和PI染色等方法分析GADD45g过表达对AML细胞增殖、克隆形成、衰老、凋亡、周 期、分化和化疗药物敏感性的影响。 结果： GADD45g在AML患者和AML细胞系中的表达水平显著低于正常对照（均P< 0.01）。低表达 GADD45g 的 AML 患者的 OS（P<0.05）和 EFS 较高表达患者显著缩短（均 P<0.05）。在 AML 细胞系中过表达 GADD45g能显著抑制细胞增殖、集落形成,显著促进细胞的凋亡、衰老、周期阻滞和分化,增强了细胞对化疗药物敏感性（P<0.05 或P<0.01）。 结论： GADD45g基因在AML患者骨髓组织和AML细胞系中低表达,对AML细胞系有显著的全方位的抑制作用, 其表达水平对AML具有预后判断价值。     

Rad51C 在DNA 损伤修复中的研究进展*

细胞毒性物质及电离辐射等易致细胞DNA 损伤,真核生物中DNA 双链断裂（double strand breaks,DSBs）修复的主要通路是同源重组（homologous recombination,HR）。 Rad51C 蛋白作为HR通路的关键因子,其表达异常可致DNA 损伤修复的失调,引起基因组的不稳定,最终导致肿瘤的发生发展。近年来随着对Rad51C 基因的研究,发现Rad51C 可能会成为恶性肿瘤治疗的潜在靶点。本文就Rad51C 在DNA 损伤修复及放疗中作用的研究进展进行综述。      

Gadd45蛋白家族在肿瘤发生发展中的作用

 Gadd45蛋白是一种响应环境的应激蛋白，在DNA修复、细胞周期调控及衰老、基因毒性应激反应等多种细胞功能中起着重要的调节作用，该类蛋白包括α，β和γ三种亚型。大量研究结果显示，它们在肿瘤细胞中参与了多种信号通路的调控，与肿瘤的发生及发展关系密切，其表达改变与肿瘤预后息息相关。本文就近年来Gadd45蛋白家族与肿瘤发生发展的研究进展进行简要介绍与阐述。     

GADD45β和survivin基因在人原发性肝癌组织中的蛋白表达及意义

目的 探讨GADD45β和survivin基因在人原发性肝癌(HCC)组织中的蛋白表达及意义.方法 收集不同分化程度的HCC组织标本68例,正常肝组织标本20例,采用免疫组化S-P法检测GADD45β和survivin基因的蛋白表达.结果 GADD45β基因与survivin基因在HCC组织中的蛋白表达率分别为38.24%(26/68)和75.00%(51/68),两者的表达均与HCC的病理分化程度、临床分期有关(P＜0.05),survivin基因的表达还与门脉癌栓有关(P＜0.05).随着survivin基因在HCC组织中蛋白表达率的升高,GADD45β基因蛋白表达强度明显下降,两者的表达呈负相关(r=-0.454,P＜0.05).结论 GADD45β基因在HCC发生发展中起抑制作用,GADD45β基因表达缺失是 HCC 发生发展的重要因素之一;survivin基因的异常表达在肝癌的发生发展中起促进作用.联合检测 GADD45β基因和survivin基因的蛋白表达有助于判断HCC的恶性程度和HCC患者的预后.     

GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity

In response to DNA damage, the cell cycle checkpoint is an important biological event in maintaining genomic fidelity. Gadd45, a p53-regulated and DNA damage inducible protein, has recently been demonstrated to play a role in the G2-M checkpoint in response to DNA damage. In the current study, we further investigated the biochemical mechanism(s) involved in the GADD45-activated cell cycle G2-M arrest. Using the tetracycline-controlled system (tet-off), we established GADD45-inducible lines in HCT116 (wild-type p53) and Hela (inactivated p53 status) cells. Following inducible expression of the Gadd45 protein, cell growth was strongly suppressed in both HCT116 and Hela cells. Interestingly, HCT116 cells revealed a significant G2-M arrest but Hela cells failed to arrest at the G2-M phases, indicating that the GADD45-activated G2-M arrest requires normal p53 function. The GADD45-induced G2-M arrest was observed independent of p38 kinase activity. Importantly, induction of Gadd45 protein resulted in a reduction of nuclear cyclin B1 protein, whose nuclear localization is critical for the completion of G2-M transition. The reduced nuclear cyclin B1 levels correlated with inhibition of Cdc2/cyclin B1 kinase activity. Additionally, overexpression of cyclin B1 substantially abrogated the GADD45-induced cell growth suppression. Therefore, GADD45 inhibition of Cdc2 kinase activity through alteration of cyclin B1 subcellular localization may be an essential step in the GADD45-induced cell cycle G2-M arrest and growth suppression.     

Decitabine-Induced Demethylation of 5′ CpG Island in GADD45A Leads to Apoptosis in Osteosarcoma Cells

GADD45 genes are epigenetically inactivated in various types of cancer and tumor cell lines. To date, defects of the GADD45 gene family have not been implicated in osteosarcoma (OS) oncogenesis, and the role of this pathway in regulating apoptosis in this tumor is unknown. The therapeutic potential of Gadd45 in OS emerged when our previous studies showed that GADD45A was reexpressed by treatment with the demethylation drug decitabine. In this study, we analyze the OS cell lines MG63 and U2OS and show that on treatment with decitabine, a significant loss of DNA methylation of GADD45A was associated with elevated expression and induction of apoptosis. In vivo affects of decitabine treatment in mice showed that untreated control xenografts exhibited low nuclear staining for Gadd45a protein, whereas the nuclei from xenografts in decitabine-treated mice exhibited increased amounts of protein and elevated apoptosis. To show the specificity of this gene for decitabine-induced apoptosis in OS, GADD45A mRNAs were disrupted using short interference RNA, and the ability of the drug to induce apoptosis was reduced. Understanding the role of demethylation of GADD45A in reexpression of this pathway and restoration of apoptotic control is important for understanding OS oncogenesis and for more targeted therapeutic approaches.     

Methylation‐mediated repression of GADD45A and GADD45G expression in gastric cardia adenocarcinoma

The growth arrest DNA damage‐inducible gene (GADD45) family, which is composed of GADD45A, GADD45B, and GADD45G, may play similar but not identical roles in tumorigenesis. Genetic changes associated with or responsible for their dysregulation are in general uncommon. This study was to detect the role of GADD45 gene family in gastric cardia adenocarcinoma (GCA) and the relationship of GADD45A and GADD45G methylation to a series of pathological parameters in a large GCA sample, in order to elucidate more information on the role of GADD45 gene family with regard to the pathogenesis of GCA. Decreased mRNA and protein expression of GADD45A and GADD45G but not GADD45B were found in 138 GCA tumor tissues. The methylation frequency of 5′ 4 CpG region located in distal promoter of GADD45A and proximal promoter of GADD45G in GCA tumor tissues was significantly higher than that in corresponding normal tissues. The expression levels of GADD45A and GADD45G were inversely correlated with methylation levels. GADD45B expression was not correlated with GCA patients survival, while GADD45A and GADD45G methylation status and protein expression were independently associated with GCA patients' survival. These results suggest that GADD45A and GADD45G gene may act as functional tumor suppressor but being frequently inactivated epigenetically in patients with GCA. Silencing of GADD45A and GADD45G, negative regulator of cell growth, is most likely responsible for conferring a selective growth advantage during GCA evolution and outgrowth.     

P53 and its implication in apoptosis (review)

The tumor suppressor protein p53 is one of the most important manipulators of the cell cycle. By interaction with targets such as WAF1/CIP1, mdm2 or GADD45 p53 functions as a negative or positive regulator of the cell cycle. After DNA damage p53 is implicated in growth arrest of cells in the G(1) phase of the cell cycle and in DNA repair and therefore p53 is regarded as a 'guardian of the genome'. Besides this protective role p53 participates in processes where cells are driven into programmed cell death. This controlled process called apoptosis is composed of a cascade of events which are dominantly influenced by p53. p53 plays a role in the initiation of cell suicide, directly or by interaction with cellular targets. A possible clinical implication of apoptosis and the implication of p53 in this process will be discussed.     

GADD45α alleviates the CDDP resistance of SK-OV3/cddp cells via redox-mediated DNA damage

Epithelial ovarian cancer has the highest mortality rate of all malignant ovarian cancer types. Great progress has been made in the treatment of ovarian cancer in recent years. However, drug resistance has led to a low level of 5-year survival rate of epithelial ovarian cancer, and the molecular mechanism of which remains unknown. The aim of the present study was to identify the role of redox status in the cisplatin (CDDP) resistance of ovarian cancer. CDDP-resistant SK-OV3 (SK-OV3/cddp) cells were prepared and their reactive oxygen species and glutathione levels were investigated. The effects of hydrogen peroxide on the CDDP sensitivity of the SK-OV3/cddp cells and their expression levels of the redox-associated protein growth arrest and DNA damage 45a (GADD45α) were also investigated. In addition, the impact of GADD45α overexpression on cell viability was evaluated in vitro and in vivo, and the levels of Ser-139 phosphorylated H2A histone family member X (γ-H2AX), which is associated with DNA damage, were detected. The results suggested that redox status affected the drug resistance of the ovarian cancer cells by increasing the expression of GADD45α. The overexpression of GADD45α reversed the CDDP resistance of the SK-OV3/cddp cells and increased the level of γ-H2AX. In conclusion, GADD45α alleviated the CDDP resistance of SK-OV3/cddp cells via the induction of redox-mediated DNA damage.