Checkpoint bypass and cell viability

DNA damage impairs cell growth by delaying or preventing critical processes such as DNA replication and chromosome segregation. In normal proliferating cells, initiation of these processes is controlled by genetically-defined pathways known as checkpoints. Tumors often acquire mutations that disable checkpoints and cancer cells can therefore progress unimpeded into S-phase, through G2 and into mitosis with chromosomal DNA damage. Checkpoint bypass in cancer cells is associated with cell death and loss of proliferative capacity and therefore is believed to contribute to the efficacy of DNA-damaging therapies. Are cancer cell clones that bypass checkpoints invariably more sensitive to DNA damage than checkpoint-proficient cells in normal tissues? We present evidence that the inherent survival of damaged human cells can be surprisingly independent of checkpoint control.     

HDAC5 is required for maintenance of pericentric heterochromatin, and controls cell-cycle progression and survival of human cancer cells

Histone deacetylases (HDACs) form a family of enzymes, which have fundamental roles in the epigenetic regulation of gene expression and contribute to the growth, differentiation, and apoptosis of cancer cells. In this study, we further investigated the biological function of HDAC5 in cancer cells. We found HDAC5 is associated with actively replicating pericentric heterochromatin during late S phase. We demonstrated that specific depletion of HDAC5 by RNA interference resulted in profound changes in the heterochromatin structure and slowed down ongoing replication forks. This defect in heterochromatin maintenance and assembly are sensed by DNA damage checkpoint pathways, which triggered cancer cells to autophagy and apoptosis, and arrested their growth both in vitro and in vivo. Finally, we also demonstrated that HDAC5 depletion led to enhanced sensitivity of DNA to DNA-damaging agents, suggesting that heterochromatin de-condensation induced by histone HDAC5 silencing may enhance the efficacy of cytotoxic agents that act by targeting DNA in vitro. Together, these results highlighted for the first time an unrecognized link between HDAC5 and the maintenance/assembly of heterochromatin structure, and demonstrated that its specific inhibition might contribute to increase the efficacy of DNA alteration-based cancer therapies in clinic.     

DNA修复——基因组稳定性护卫机制的原创与发现之旅

基因组DNA是遗传的物质基础,编码的信息指导生物种系的复制延续、生命体的生长发育和代谢活动。无论是在外环境因素的应激压力下还是处于正常状态,DNA损伤时刻在发生,由此,DNA损伤修复作为重要的细胞内在机制,在维护基因组稳定性、降低癌症等人类系列重大疾病风险中发挥了不可替代作用。三位科学家汤姆·林达尔(Tomas Lindahl)、阿齐兹·桑贾尔（Aziz Sancar）、保罗·莫德里奇（Paul Modrich）因发现和揭示DNA修复及其机制的杰出贡献,获得2015年诺贝尔化学奖。本文综述了三位获奖者分别在DNA损伤的碱基切除修复、核苷酸切除修复和错配修复研究中的原创发现,以及相应的修复通路机制的描绘。此3种修复通路,主要是针对紫外线和化学物所致DNA的碱基损伤、嘧啶二聚体及加合物或者DNA复制过程中发生的碱基错误配对的修复。恰巧,2015年拉斯克基础医学研究奖授予的两位科学家,也因他们揭示了DNA损伤应答现象和机制研究的重大贡献而获奖,本文也呈现了获奖者的关键性科学发现。最后,简要展望了中国DNA损伤修复领域的发展。     

DNA repair: exploiting the Fanconi anemia pathway as a potential therapeutic target

DNA repair is an active cellular process to respond to constant DNA damage caused by metabolic processes and environmental factors. Since the outcome of DNA damage is generally adverse and long term effects may contribute to oncogenesis, cells have developed a variety of DNA repair mechanisms, which operate depending on the type of DNA damage inflicted. At least 15 Fanconi anemia (FA) proteins interact in a common pathway involved in homologous recombination. Inherited homozygous mutations in any of these FA genes cause a rare disease, Fanconi anemia, characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility. Heterozygous germline FA mutations predispose to various types of cancer. In addition, somatic FA mutations have been identified in diverse cancer types. Evidence exists that cells deficient in the FA pathway become dependent on alternative pathways for survival. Additional inhibition of such alternative pathways is thus expected to result in cell death, creating a relationship of synthetic lethality. Identifying these relationships can reveal yet unknown mechanisms of DNA repair and new targets for therapy.     

Replication fork dynamics and the DNA damage response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.     

A novel unidirectional cross-talk from the insulin-like growth factor-I receptor to leptin receptor in human breast cancer cells

Obesity is a major risk factor for the development and progression of breast cancer. Increased circulating levels of the obesity-associated hormones leptin and insulin-like growth factor-I (IGF-I) and overexpression of the leptin receptor (Ob-R) and IGF-I receptor (IGF-IR) have been detected in a majority of breast cancer cases and during obesity. Due to correlations between increased leptin, Ob-R, IGF-I, and IGF-IR in breast cancer, we hypothesized that molecular interactions may exist between these two signaling pathways. Coimmunoprecipitation and immunoblotting showed that IGF-IR and Ob-R interact in the breast cancer cell lines MDA-MB-231, MCF7, BT474, and SKBR3. Stimulation of cells with IGF-I promoted Ob-R phosphorylation, which was blocked by IGF-IR kinase inhibition. In addition, IGF-I activated downstream signaling molecules in the leptin receptor and IGF-IR pathways. In contrast to IGF-I, leptin did not induce phosphorylation of IGF-IR, indicating that receptor cross-signaling is unidirectional, occurring from IGF-IR to Ob-R. Our results show, for the first time, a novel interaction and cross-talk between the IGF-I and leptin receptors in human breast cancer cells.     

A review and appraisal of the DNA damage theory of ageing

Given the central role of DNA in life, and how ageing can be seen as the gradual and irreversible breakdown of living systems, the idea that damage to the DNA is the crucial cause of ageing remains a powerful one. DNA damage and mutations of different types clearly accumulate with age in mammalian tissues. Human progeroid syndromes resulting in what appears to be accelerated ageing have been linked to defects in DNA repair or processing, suggesting that elevated levels of DNA damage can accelerate physiological decline and the development of age-related diseases not limited to cancer. Higher DNA damage may trigger cellular signalling pathways, such as apoptosis, that result in a faster depletion of stem cells, which in turn contributes to accelerated ageing. Genetic manipulations of DNA repair pathways in mice further strengthen this view and also indicate that disruption of specific pathways, such as nucleotide excision repair and non-homologous end joining, is more strongly associated with premature ageing phenotypes. Delaying ageing in mice by decreasing levels of DNA damage, however, has not been achieved yet, perhaps due to the complexity inherent to DNA repair and DNA damage response pathways. Another open question is whether DNA repair optimization is involved in the evolution of species longevity, and we suggest that the way cells from different organisms respond to DNA damage may be crucial in species differences in ageing. Taken together, the data suggest a major role of DNA damage in the modulation of longevity, possibly through effects on cell dysfunction and loss, although understanding how to modify DNA damage repair and response systems to delay ageing remains a crucial challenge.     

Apoptosis-like yeast cell death in response to DNA damage and replication defects

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.     

Anchorage-independent activation of mitogen-activated protein kinase through phosphatidylinositol-3 kinase by insulin-like growth factor I

Insulin-like growth factor I (IGF-I) is a well-established mitogen in human breast cancer cells. We show here that human breast cancer MCF-7 cells, which were prevented from attaching to the substratum and were floating in medium, responded to IGF-I and initiated DNA synthesis. The addition of IGF-I to floating cells induced activation of protein kinase B (PKB)/Akt, as to cells attached to the substratum. In addition, mitogen-activated protein kinase (MAPK)/extracellular response kinase (ERK) and its upstream kinases, ERK kinase (MEK) and Raf-1, were activated by IGF-I in floating cells. While the IGF-I-induced activation of PKB/Akt was inhibited by PI3-K inhibitor LY294002 but not by MEK inhibitor PD98059, the activation of both MEK and ERK by IGF-I was inhibited by both. These findings suggest that the IGF-I signal that leads to stimulation of DNA synthesis of MCF-7 cells is transduced to ERK through PI3-K, only when they are anchorage-deficient.     

Saturated fatty acids modulate cell response to DNA damage: implication for their role in tumorigenesis

DNA damage triggers a network of signaling events that leads to cell cycle arrest or apoptosis. This DNA damage response acts as a mechanism to prevent cancer development. It has been reported that fatty acids (FAs) synthesis is increased in many human tumors while inhibition of fatty acid synthase (FASN) could suppress tumor growth. Here we report that saturated fatty acids (SFAs) play a negative role in DNA damage response. Palmitic acid, as well as stearic acid and myristic acid, compromised the induction of p21 and Bax expression in response to double stranded breaks and ssDNA, while inhibition or knockdown of FASN enhanced these cellular events. SFAs appeared to regulate p21 and Bax expression via Atr-p53 dependent and independent pathways. These effects were only observed in primary mouse embryonic fibroblasts and osteoblasts, but not in immortalized murine NIH3T3, or transformed HCT116 and MCF-7 cell lines. Accordingly, SFAs showed some positive effects on proliferation of MEFs in response to DNA damage. These results suggest that SFAs, by negatively regulating the DNA damage response pathway, might promote cell transformation, and that increased synthesis of SFAs in precancer/cancer cells might contribute to tumor progression and drug resistance.     

The genetics of the hereditary xeroderma pigmentosum syndrome

All living organisms are constantly exposed to endogenous or exogenous agents that can cause damage to the genomic DNA, leading to the loss of stable genetic information. Fortunately, all cells are equipped with numerous classes of DNA repair pathways which are able to correct many kinds of DNA damage such as bulky adducts, oxidative lesions, single- and double-strand breaks and mismah.The importance of these DNA repair processes is attested by the existence of several rare but dramatic hereditary diseases caused by defects in one of their repair pathways. These diseases are usually associated with early onset of malignancies confirming the direct relationship between unrepaired DNA lesions, mutations or chromosomal modifications and cancer incidence. Among these hereditary diseases the UV-hypersensitive ones have been particularly well studied and the xeroderma pigmentosum (XP) is probably the best known syndrome up to now in terms of genetics and biochemistry.     

DNA Repair and Genome Maintenance in Bacillus subtilis

Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis.     

Berberine attenuates XRCC1‐mediated base excision repair and sensitizes breast cancer cells to the chemotherapeutic drugs

Berberine (BBR) is a natural isoquinoline alkaloid, which is used in traditional medicine for its anti‐microbial, anti‐protozoal, anti‐diarrhoeal activities. Berberine interacts with DNA and displays anti‐cancer activities, yet its effects on cellular DNA repair and on synthetic treatments with chemotherapeutic drugs remain unclear. In this study, we investigated the effects of BBR on DNA repair and on sensitization of breast cancer cells to different types of DNA damage anti‐tumoural drugs. We found BBR arrested cells in the cell cycle S phase and induced DNA breaks. Cell growth analysis showed BBR sensitized MDA‐MB‐231 cells to cisplatin, camptothecin and methyl methanesulfonate; however, BBR had no synergistic effects with hydroxurea and olaparib. These results suggest BBR only affects specific DNA repair pathways. Western blot showed BBR down‐regulated XRCC1 expressions, and the rescued XRCC1 recovered the resistance of cancer cells to BBR. Therefore, we conclude that BBR interferes with XRCC1‐mediated base excision repair to sensitize cancer cells to chemotherapeutic drugs. These finding can contribute to understanding the effects of BBR on cellular DNA repair and the clinical employment of BBR in treatment of breast cancer.     

Causes and Consequences of the DNA Damage Response

A prerequisite for maintaining genome stability in all cell types is the accurate repair and efficient signaling of DNA double strand breaks (DSBs). It is believed that DSBs are initially detected by damage sensors that trigger the activation of transducing kinases. These transducers amplify the damage signal, which is then relayed to effector proteins, which regulate the progression of the cell cycle, DNA repair and apoptosis. Errors in the execution of the repair and/or signaling of DSBs can give rise to multi-systemic disorders characterized by tissue degeneration, infertility, immune system dysfunction, age-related pathologies and cancer. This special Spotlight issue of Cell Cycle highlights recent advances in our understanding of the biology and significance of the DNA damage response. A range of issues are addressed including mechanistic ones: what is the aberrant DNA structure that triggers the activation of the checkpoint - how does chromatin structure influence the recruitment of repair and checkpoint proteins- how does chromosomal instability contribute to the evolution of cancer. In addition, questions related to the physiology of the DNA damage response in normal and abnormal cells is explored: what is the in vivo consequence of altering specific amino acids in a DNA damage sensor- does DNA damage accumulation in stem cells cause aging- how is neurodegeneration linked to deficiencies in specific DNA repair pathways, and finally, what is the biological basis for selection of aberrant DNA damage responses in cancer cells?     

IGFs stimulate zebrafish cell proliferation by activating MAP kinase and PI3-kinase-signaling pathways

Insulin-like growth factor (IGF)-I and -II have been cloned from a number of teleost species, but their cellular actions in fish are poorly defined. In this study, we show that both IGF-I and -II stimulated zebrafish embryonic cell proliferation and DNA synthesis in a concentration-dependent manner, whereas insulin had little mitogenic activity. Affinity cross-linking and immunoblotting studies revealed the presence of IGF receptors with the characteristics of the mammalian type I IGF receptor. Competitive binding assay results indicated that the binding affinities of the zebrafish IGF-I receptors to IGF-I, IGF-II, and insulin are 1.9, 2.6, and >190 nM, indicating that IGF-I and -II bind to the IGF-I receptor(s) with approximately equal high affinity. To further investigate the cellular mechanism of IGF actions, we have studied the effects of IGFs on two major signal transduction pathways: mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase). IGFs activated MAPK in zebrafish embryonic cells in a dose-dependent manner. This activation occurred within 5 min of IGF-I stimulation and disappeared after 1 h. IGF-I also caused a concentration-dependent activation of protein kinase B, a downstream target of PI3 kinase, this activation being sustained for several hours. Inhibition of MAPK activation by the MAPK kinase inhibitor PD-98059 inhibited the IGF-I-stimulated DNA synthesis. Similarly, use of the PI3 kinase inhibitor LY-294002 also inhibited IGF-I-stimulated DNA synthesis. When both the MAPK and PI3 kinase pathways were inhibited using a combination of these compounds, the IGF-I-stimulated DNA synthesis was completely negated. These results indicate that both IGF-I and -II are potent mitogens for zebrafish embryonic cells and that activation of both the MAPK and PI3 kinase-signaling pathways is required for the mitogenic action of IGFs in zebrafish embryonic cells.     

Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors

Cells from organisms with renewable tissues can permanently withdraw from the cell cycle in response to diverse stress, including dysfunctional telomeres, DNA damage, strong mitogenic signals, and disrupted chromatin. This response, termed cellular senescence, is controlled by the p53 and RB tumor suppressor proteins and constitutes a potent anticancer mechanism. Nonetheless, senescent cells acquire phenotypic changes that may contribute to aging and certain age-related diseases, including late-life cancer. Thus, the senescence response may be antagonistically pleiotropic, promoting early-life survival by curtailing the development of cancer but eventually limiting longevity as dysfunctional senescent cells accumulate.     

The molecular control of DNA damage-induced cell death

Because of the singular importance of DNA for genetic inheritance, all organisms have evolved mechanisms to recognize and respond to DNA damage. In metazoans, cells can respond to DNA damage either by undergoing cell cycle arrest, to facilitate DNA repair, or by undergoing cell suicide. Cell death can either occur by activation of the apoptotic machinery or simply be a consequence of irreparable damage that prevents further cell division. In germ cells, mechanisms for limiting alterations to the genome are required for faithful propagation of the species whereas in somatic cells, responses to DNA damage prevent the accumulation of mutations that might lead to aberrant cell proliferation or behavior. Several of the genes that regulate cellular responses to DNA damage function as tumor suppressors. The clinical use of DNA damaging agents in the treatment of cancer can activate these tumor suppressors and exploits the cellular suicide and growth arrest mechanisms that they regulate. It appears that in some but not all types of tumors the propensity to undergo apoptosis is a critical determinant of their sensitivity to anti-cancer therapy. This review describes current understanding of the molecular control of DNA damage-induced apoptosis with particular attention to its role in tumor suppression and cancer therapy.     

Apoptosome dysfunction in human cancer

Apoptosis is a cell suicide mechanism that enables organisms to control cell number and eliminate cells that threaten survival. The apoptotic cascade can be triggered through two major pathways. Extracellular signals such as members of the tumor necrosis factor (TNF) family can activate the receptor-mediated extrinsic pathway. Alternatively, stress signals such as DNA damage, hypoxia, and loss of survival signals may trigger the mitochondrial intrinsic pathway. In the latter, mitochondrial damage results in cytochrome c release and formation of the apoptosome, a multimeric protein complex containing Apaf-1, cytochrome c , and caspase-9. Once bound to the apoptosome, caspase-9 is activated, and subsequently triggers a cascade of effector caspase activation and proteolysis, leading to apoptotic cell death. Recent efforts have led to the identification of multiple factors that modulate apoptosome formation and function. Alterations in the expression and/or function of these factors may contribute to the pathogenesis of cancer and resistance of tumor cells to chemotherapy or radiation. In this review we discuss how disruption of normal apoptosome formation and function may lead or contribute to tumor development and progression.