GADD45α参与维持基因组稳定性并发挥肿瘤抑制效应的多样性分子机制

哺乳动物细胞在遭受应激损伤因素刺激时会启动一系列信号传导通路,从而引发细胞周期阻滞、DNA修复或细胞凋亡等效应,这些机制的异常与肿瘤的发生发展密切相关。GADD45α作为生长阻滞及DNA损伤诱导基因编码家族的一员,参与维持基因组稳定性、调控细胞周期行进、DNA损伤修复、细胞衰老及细胞凋亡等多种生物学过程,在肿瘤发生发展和肿瘤抑制反应中具有重要作用。我们简要综述了GADD45α参与维持基因组稳定性并发挥肿瘤抑制效应的分子机制。     

DNA损伤检验点与损伤修复及基因组稳定性

生物有机体基因组DNA经常会受到内源或外源因素的影响而导致结构发生变化,产生损伤;在长期进化过程中,有机体也相应形成了一系列应对与修复损伤DNA,并维持染色体基因组正常结构功能的机制。其中DNA损伤检验点（DNA damage checkpoint）就是在感应DNA损伤的基础上,对损伤感应信号进行转导,或引起细胞周期的暂停,从而使细胞有足够的时间对损伤DNA进行修复,或最终导致细胞发生凋亡。DNA损伤检验点信号转导途径是一个高度保守的信号感应过程,整个途径大致可以分为损伤感应、信号传递及信号效应3个组成部分。其中3-磷脂酰肌醇激酶家族类成员ATM（ataxia-telangiectasia mutated）和ATR（ataxia-telangiectasia and Rad3-related）活性的增加构成整个途径活化的第一步。它们通过激活下游的效应激酶,Chk2／Chk1,通过协同作用许多其他调控细胞周期、DNA复制、DNA损伤修复及细胞凋亡等过程的蛋白质因子来实现细胞对DNA损伤的高度协调反应。近十几年,随着此领域研究的不断深入,人们逐步揭示了DNA损伤检验点途径发生过程中,各种核心组分通过与不同调节因子、效应因子及DNA损伤修复蛋白间的复杂相互作用,以实现监测感应异常DNA结构并实施相应反应的机制;其中,检验点衔接因子（mediators）及染色质结构,尤其是核小体组蛋白的共价修饰在调控ATM／ATR活性,促进ATM／ATR与底物间的相互作用以及介导DNA损伤位点周围染色质区域上多蛋白复合物在时间与空间上的动态形成发挥着重要的作用。同时,人们也开始发现DNA损伤检验点途径与DNA损伤修复、基因组稳定性以及肿瘤发生等过程之间某些内在的联系。该反应途径在通过协调细胞针对DNA损伤做出各种反应的基础上,直接或间接地参与或调控DNA损伤修复过程,并与DNA损伤修复途径协同作用最终保证染色体基凶组结构的完整性,而检验点途径的改变,则会引起基因组不稳定的发生,包括从突变频率的提高到大范围的染色体重排,以及染色体数量的畸变。如：突变发生在肿瘤形成早期,会大大增加肿瘤发生的几率。文章将对DNA损伤检验点途径机制及其对DNA损伤修复、基因组稳定性影响的最新进展进行综述。     

53BP1与DNA双链断裂损伤修复

DNA的精确复制和遗传对维持基因组稳定性有重要作用。DNA双链断裂损伤可能诱导细胞凋亡和染色质重排,在肿瘤的发生发展过程中发挥作用。53BP1是DNA双链断裂修复中的重要调节蛋白质之一,对调控损伤修复平衡和维持基因组稳定性起着重要作用。本文主要对53BP1的结构、生物学功能、信号通路、分子机制和翻译后修饰做一浅显的总结和展望,希望能为53BP1的深入研究提供一些理论基础。     

乳腺癌易感蛋白1在DNA损伤修复中的作用

人类乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)首先是在乳腺癌家族中发现的,是具有遗传倾向的乳腺癌和卵巢癌易感基因,其基因的突变与家族性乳腺癌及卵巢癌的发生有密切联系。BRCA1是一种抑癌基因,其基因产物可以参与维持基因组稳定性的多条细胞信号通路,例如DNA损伤诱导的细胞周期调控、DNA损伤修复、基因转录调节、细胞凋亡、泛素化等重要的细胞活动。本文就近几年来BRCA1在DNA损伤修复中的作用的研究进展作一综述,包括DNA损伤诱导的细胞周期检查点的激活和DNA损伤修复两方面。     

检验点激酶1的研究进展

检验点激酶1(checkpointkinase1,Chk1)为一种进化保守的蛋白激酶,是细胞检验点的转导因子。当电离辐射、紫外线等引起细胞DNA损伤或者DNA复制叉停滞时Chk1活化,诱导细胞产生细胞周期阻滞、DNA修复或细胞凋亡等特征。现对Chk1的结构、功能以及病毒通过Chk1调控宿主细胞周期等方面进行简述。     

表观遗传调控：基于染色质环境的DNA双链断裂修复

DNA双链断裂（DNA double-strand breaks, DSBs）是威胁基因组完整性和细胞存活的最有害的DNA损伤类型。同源重组（homologous recombination,HR）和非同源末端连接（non-homologous end joining,NHEJ）是修复DNA双链断裂的两种主要途径。DSB修复涉及到损伤部位修复蛋白的募集和染色质结构的改变。在DNA双链断裂诱导下,染色质结构的动态变化在时间和空间上受到严格调控,进而对DNA双链断裂修复过程进行精细调节。特定的染色质修饰形成利于修复的染色质状态,有助于DNA双链断裂修复机器的招募、修复途径的选择和DNA损伤检查点的活化;其中修复途径的选择对于基因组稳定性至关重要。修复不当或失败可导致基因组不稳定性,甚至促进肿瘤的发生。本文综述了染色质结构和染色质修饰的动态变化在DSB修复中的重要作用。此外,文章还总结了在癌症治疗中靶向关键染色质调控因子在基因组稳定性维持、肿瘤发生发展以及潜在临床应用价值等方面的进展。     

ATM介导的蛋白磷酸化与DNA损伤的信号转导机制

细胞周期检定点激酶ATM蛋白属于磷酸肌醇3激酶（PI－3K）家族成员,也是哺乳动物细胞BASC高分子蛋白复合物的组成之一。ATM调整由于DNA损伤引发的DNA修复和凋亡通路,该通路主要表现为DNA损伤激活ATM激酶,ATM激酶磷酸化其下游的相应蛋白,使细胞在细胞周期关卡处停滞分裂,主要是G1－S期和G2－M期的阻滞,使损伤的DNA得以修复,当修复失败时,细胞进入凋亡进程。ATM磷酸化的蛋白质很多,如p53,cdc25A,cdc25C等,这些蛋白质对细胞周期关卡调控都非常重要,因此也就证明了ATM在细胞周期调控中的重要作用。     

HCV NS3/4A基因外来表达对Huh7细胞凋亡及DNA损伤应答的影响

NS3/4A是丙型肝炎病毒（hepatitis C virus,HCV）编码的丝氨酸蛋白酶复合体,是病毒完成自身复制周期的必要成分。该研究为调查NS3/4A对细胞凋亡及DNA损伤应答（DNA-damage response,DDR）的影响,在Huh7细胞中表达了外来NS3/4A基因。通过DAPI染色和MTT分析显示,外来表达NS3/4A显著诱导细胞的凋亡和增殖活力的下降。免疫荧光检测结果表明,NS3/4A可明显增加细胞内源性DNA双链断裂（double strand breaks,DSBs）损伤（γH2AX灶点升高）;而进一步用X-ray诱导细胞外源性DSBs损伤后,外来表达NS3/4A的细胞显示出明显的DSBs损伤修复缺陷（减缓的γH2AX灶点消退）。免疫印迹法检测结果显示,NS3/4A可抑制喜树碱（Camptothecin,CPT）诱导的ATM第1 981位丝氨酸的磷酸化（pATM1 981）。以上结果提示,NS3/4A基因外来表达可引起细胞DNA损伤,抑制ATM介导的DSBs损伤修复信号,诱导细胞凋亡通路的活化。     

ATM调节体细胞重编程

共济失调–毛细血管扩张突变(ataxia telangiectasia mutated,ATM)蛋白属于磷脂酰肌醇-3-激酶相关激酶家族(phosphatidylinositol-3-kinase related kinase family,PIKK)成员,是DNA损伤的感应器并将DNA损伤信号传递到下游修复蛋白,从而启动DNA修复、细胞周期阻滞和细胞凋亡等一系列事件,进而维持细胞基因组完整性。近期的研究揭示,ATM参与了体细胞重编程过程,当ATM完全缺失后显著影响诱导多能干细胞(induced pluripotent stem cells,i PS cells)获得以及染色质的稳定;ATM还通过参与重编程过程中的染色质重塑进而调控体细胞重编程。ATM下游的效应因子p53和H2AX(histone 2A member X)等在重编程引起的细胞周期阻滞和凋亡中发挥重要作用。该文重点探讨了ATM及其下游细胞因子在参与调控体细胞重编程过程中的作用机制。     

衰老或肿瘤：癌基因诱导的双向性

在大部分的肿瘤中都发现有癌基因的活化,癌基因的活化被认为是导致肿瘤发生的重要原因．然而,在野生型细胞内,癌基因的活化可以诱导细胞衰老,称为癌基因诱导的细胞衰老(oncogene-induced senescence, OIS),从而抑制进一步的肿瘤发生．因而,癌基因的活化具有诱导衰老或肿瘤的双向性．DNA损伤调控反应(DNA damage checkpoint response, DDR)是细胞应对DNA损伤时感应损伤,从而延迟或阻滞细胞周期进程的一种分子信号传递通路,是诱导细胞衰老的重要机制．癌基因的活化可以引发DNA损伤信号的产生,从而激活DDR,诱导细胞衰老．在DDR异常时,癌基因的激活可引发DNA的过度复制与细胞的过度增殖,并导致基因组不稳定性的积累,最终导致肿瘤发生．DDR的完整性决定了癌基因诱导的双向性．DDR在癌基因诱导中的重要作用,提示了保持和恢复DDR的完整性可以作为肿瘤预防和治疗的新方向．     

Behind the wheel and under the hood: Functions of cyclin-dependent kinases in response to DNA damage

Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair – homologous recombination (HR) and non-homologous end joining (NHEJ) – are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs – as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function – could be balanced to produce a coordinated and effective response to DNA damage.     

miRNA response to DNA damage

Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that miRNAs play a crucial role in regulation of DNA damage response. In this review, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage.     

DNA damage-dependent cell cycle checkpoints and genomic stability

In response to genotoxic stress, which can be caused by environmental or endogenous genotoxic insults such as ionizing or ultraviolet radiation, various chemicals and reactive cellular metabolites, cell cycle checkpoints which slow down or arrest cell cycle progression can be activated, allowing the cell to repair or prevent the transmission of damaged or incompletely replicated chromosomes. Checkpoint machineries can also initiate pathways leading to apoptosis and the removal of a damaged cell from a tissue. The balance between cell cycle arrest and damage repair on one hand and the initiation of cell death, on the other hand, could determine if cellular or DNA damage is compatible with cell survival or requires cell elimination by apoptosis. Defects in these processes may lead to hypersensitivity to cellular stress, and susceptibility to DNA damage, genomic defects, and resistance to apoptosis, which characterize cancer cells. In this article, we have noted recent studies of DNA damage-dependent cell cycle checkpoints, which may be significant in preventing genomic instability.     

Crosstalk between Phosphoinositide 3-kinase/Akt signaling pathway with DNA damage response and oxidative stress in cancer

The phosphatidylinositol 3-kinases (PI3K)/Akt signaling pathway is one of the well-characterized and most important signaling pathways activated in response to DNA damage. This review discusses the most recent discoveries on the involvement of PI3K/Akt signaling pathway in cancer development, as well as stimulation of some important signaling networks involved in the maintenance of cellular homeostasis upon DNA damage, with an exploration of how PI3K/Akt signaling pathway contributes to the regulation of modulators and effectors underlying DNA damage response, the intricate, protein-based signal transduction network, which decides between cell cycle arrest, DNA repair, and apoptosis, the elimination of irreparably damaged cells to maintain homeostasis. The review continues by looking at the interplay between cell cycle checkpoints, checking the repair of damage inflicted to the DNA before entering DNA replication to facilitate DNA synthesis, and PI3K/Akt signaling pathway. We then investigate the challenges the cells overcome to ameliorate damages induced by oxidative activities, for example, the recruitment of many pathways and factors to maintain integrity and hemostasis. Finally, the review provides a discussion of how cells use the PI3K/Akt signaling pathway to regulate the balance between these networks.     

"ATR activation in response to ionizing radiation: still ATM territory"

ABSTRACT : Unrepaired DNA double-strand breaks (DSBs) are a major cause for genomic instability. Therefore, upon detection of a DSB a rapid response must be assembled to coordinate the proper repair/signaling of the lesion or the elimination of cells with unsustainable amounts of DNA damage. Three members of the PIKK family of protein kinases -ATM, ATR and DNA-PKcs- take the lead and initiate the signaling cascade emanating from DSB sites. Whereas DNA-PKcs activity seems to be restricted to the phosphorylation of targets involved in DNA repair, ATM and ATR phosphorylate a broad spectrum of cell cycle regulators and DNA repair proteins. In the canonical model, ATM and ATR are activated by two different types of lesions and signal through two independent and alternate pathways. Specifically, ATR is activated by various forms of DNA damage, including DSBs, arising at stalled replication forks ("replication stress"), and ATM is responsible for the signaling of DSBs that are not associated with the replication machinery throughout the cell cycle. Recent evidence suggests that this model might be oversimplified and that coordinated crosstalk between ATM and ATR activation routes goes on at the core of the DNA damage response.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Triplex-Induced DNA Damage Response

Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage.     

Polo-like kinases and Chk2 at the interface of DNA damage checkpoint pathways and mitotic regulation

The cell cycle controls processes of DNA replication and segregation of replicated DNA into two daughter cells. These processes are coordinated by multiple signaling pathways, which employ many protein kinases. The members of the family of Polo-like protein kinases are among these key cell cycle regulators. In response to DNA damage and inhibited DNA replication, DNA structure checkpoints delay cell cycle progression to provide cells with time for repair of damaged DNA and protect it from more severe damage. These effects are achieved by affecting key players of the basic cell cycle regulation of the cells with damaged DNA. This review is focused on the interplay between Chk2, a bona fide checkpoint protein kinase, and Polo-like kinases.     

Shaping chromatin for repair

To counteract the adverse effects of various DNA lesions, cells have evolved an array of diverse repair pathways to restore DNA structure and to coordinate repair with cell cycle regulation. Chromatin changes are an integral part of the DNA damage response, particularly with regard to the types of repair that involve assembly of large multiprotein complexes such as those involved in double strand break (DSB) repair and nucleotide excision repair (NER). A number of phosphorylation, acetylation, methylation, ubiquitylation and chromatin remodeling events modulate chromatin structure at the lesion site. These changes demarcate chromatin neighboring the lesion, afford accessibility and binding surfaces to repair factors and provide on-the-spot means to coordinate repair and damage signaling. Thus, the hierarchical assembly of repair factors at a double strand break is mostly due to their regulated interactions with posttranslational modifications of histones. A large number of chromatin remodelers are required at different stages of DSB repair and NER. Remodelers physically interact with proteins involved in repair processes, suggesting that chromatin remodeling is a requisite for repair factors to access the damaged site. Together, recent findings define the roles of histone post-translational modifications and chromatin remodeling in the DNA damage response and underscore possible differences in the requirements for these events in relation to the chromatin context.     

Deficiency in DNA damage response,a new characteristic of cells infected with latent HIV-1

Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.