Search

ATM and DNA-PKcs coordinate the DNA damage response at multiple levels following the exposure to chemotherapy. The Topoisomerase II poison etoposide (ETO) is an effective chemotherapeutic agent that induces DNA double-strand breaks (DSB), but it is responsible from the chromosomal rearrangements frequently found in therapy-related secondary tumors. Targeted inhibition of DNA-PKcs in ATM-defective tumors combined with radio- or chemotherapy has been proposed as relevant therapies. Here, we explored the DNA repair mechanisms and the genetic consequences of targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumor cells after exposure to ETO. We demonstrated that chemical inhibition of DNA-PKcs followed by treatment with ETO resulted in the accumulation of chromatid breaks and decreased mitotic index in both A-T cells and ATM-knocked-down (ATMkd) tumor cells. The HR repair process in DNA-PKcs-inhibited ATMkd cells amplified the RAD51 foci number, with no correlated increase in sister chromatid exchanges. The analysis of post-mitotic DNA lesions presented an augmented number of persistent unresolved DSB, without alterations in the cell cycle progression. Long-term examination of chromosome aberrations revealed a strikingly high number of chromatid and chromosome exchanges. By using genetic and pharmacological abrogation of PARP-1, we demonstrated that alternative end-joining (alt-EJ) repair pathway is responsible for those chromosome abnormalities generated by limiting c-NHEJ activities during directed inhibition of DNA-PKcs in ATM-deficient cells. Targeting the non-oncogenic addiction to DNA-PKcs of ATM-defective tumors stimulates the DSB repair by alt-EJ, which is liable for the origin of cells carrying stable chromosome aberrations that may eventually restrict the therapeutic strategy. [ABSTRACT FROM AUTHOR]

Interstitial telomeric sequences (ITSs) in hamster cells are hot spots for spontaneous and induced chromosome aberrations (CAs). Most data on ITS instability to date have been obtained in DNA repair-proficient cells. The classical non-homologous end joining repair pathway (C-NHEJ), which is the principal double strand break (DSB) repair mechanism in mammalian cells, is thought to restore the morphologically correct chromosome structure. The production of CAs thus involves DNA-PKcs-independent repair pathways. In our current study, we investigated the participation of DNA-PKcs from the C-NHEJ pathway in the repair of spontaneous or radiation-induced DSBs in ITSs using wild-type and DNA-PKcs mutant Chinese hamster ovary cells. Our data demonstrate that DNA-PKcs stabilizes spontaneous DSBs within ITSs from the chromosome 9 long arm, leading to the formation of terminal deletions. In addition, we show that DNA-PKcs-dependent C-NHEJ is employed following radiation-induced DSBs in other ITSs and restores morphologically correct chromosomes, whereas DNA-PKcs independent mechanisms co-exist in DNA-PKcs proficient cells leading to an excess of CAs within ITSs. [ABSTRACT FROM AUTHOR]

Broken chromosomes arising from DNA double-strand breaks result from endogenous events such as the production of reactive oxygen species during cellular metabolism, as well as from exogenous sources such as ionizing radiation. Left unrepaired or incorrectly repaired they can lead to genomic changes that may result in cell death or cancer. DNA-dependent protein kinase (DNA-PK), a holoenzyme that comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer Ku70/Ku80, has a major role in non-homologous end joining—the main pathway in mammals used to repair double-strand breaks. DNA-PKcs is a serine/threonine protein kinase comprising a single polypeptide chain of 4,128 amino acids and belonging to the phosphatidylinositol-3-OH kinase (PI(3)K)-related protein family. DNA-PKcs is involved in the sensing and transmission of DNA damage signals to proteins such as p53, setting off events that lead to cell cycle arrest. It phosphorylates a wide range of substrates in vitro, including Ku70/Ku80, which is translocated along DNA. Here we present the crystal structure of human DNA-PKcs at 6.6 Å resolution, in which the overall fold is clearly visible, to our knowledge, for the first time. The many α-helical HEAT repeats (helix–turn–helix motifs) facilitate bending and allow the polypeptide chain to fold into a hollow circular structure. The carboxy-terminal kinase domain is located on top of this structure, and a small HEAT repeat domain that probably binds DNA is inside. The structure provides a flexible cradle to promote DNA double-strand-break repair. [ABSTRACT FROM AUTHOR]

We previously reported that cells exposed to low doses of ionizing radiation (IR) in the G2-phase of the cell cycle activate a checkpoint that is epistatically regulated by ATM and ATR operating as an integrated module. In this module, ATR interphases exclusively with the cell cycle to implement the checkpoint, mainly using CHK1. The ATM/ATR module similarly regulates DNA end-resection at low IR-doses. Strikingly, at high IR-doses, the ATM/ATR coupling relaxes and each kinase exerts independent contributions to resection and the G2-checkpoint. DNA-PKcs links to the ATM/ATR module and defects cause hyper-resection and hyperactivation of G2-checkpoint at all doses examined. Surprisingly, our present report reveals that cells irradiated in S-phase utilize a different form of wiring between DNA-PKcs/ATM/ATR: The checkpoint activated in G2-phase is regulated exclusively by ATR/CHK1; similarly at high and low IR-doses. DNA end-resection supports ATR-activation, but inhibition of ATR leaves resection unchanged. DNA-PKcs and ATM link now epistatically to resection and their inhibition causes hyper-resection and ATR-dependent G2-checkpoint hyperactivation at all IR-doses. We propose that DNA-PKcs, ATM and ATR form a modular unit to regulate DSB processing with their crosstalk distinctly organized in S- and G2- phase, with strong dependence on DSB load only in G2-phase. [ABSTRACT FROM AUTHOR]

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors. Mitotic errors promote chromosome fragmentation and rearrangements through chromothripsis. Here, the authors identify NHEJ as the primary DSB repair pathway underlying chromothripsis and investigate the kinetics of fragment reassembly across the cell cycle. [ABSTRACT FROM AUTHOR]

Purpose: TIPRL1 (target of rapamycin signaling pathway regulator-like 1) is a known interactor and inhibitor of protein phosphatases PP2A, PP4 and PP6 – all pleiotropic modulators of the DNA Damage Response (DDR). Here, we investigated the role of TIPRL1 in the radiotherapy (RT) response of Head and Neck Squamous Cell Carcinoma (HNSCC). Methods: TIPRL1 mRNA (cBioportal) and protein expression (immunohistochemistry) in HNSCC samples were linked with clinical patient data. TIPRL1-depleted HNSCC cells were generated by CRISPR/Cas9 editing, and effects on colony growth, micronuclei formation (microscopy), cell cycle (flow cytometry), DDR signaling (immunoblots) and proteome (mass spectrometry) following RT were assessed. Mass spectrometry was used for TIPRL1 phosphorylation and interactomics analysis in irradiated cells. Results: TIPRL1 expression was increased in tumor versus non-tumor tissue, with high tumoral TIPRL1 expression associating with lower locoregional control and decreased survival of RT-treated patients. TIPRL1 deletion in HNSCC cells resulted in increased RT sensitivity, a faster but prolonged cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Upon irradiation, ATM phosphorylates TIPRL1 at Ser265. A non-phospho Ser265Ala mutant could not rescue the increased radiosensitivity phenotype of TIPRL1-depleted cells. While binding to PP2A-like phosphatases was confirmed, DNA-dependent protein kinase (DNA-PKcs), RAD51 recombinase and nucleosomal histones were identified as novel TIPRL1 interactors. Histone binding, although stimulated by RT, was adversely affected by TIPRL1 Ser265 phosphorylation. Conclusions: Our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in RT resistance through modulation of the DDR, highlighting its potential as a new HNSCC predictive marker and therapeutic target. [ABSTRACT FROM AUTHOR]

Modeling a biological process equips us with more comprehensive insight into the process and a more advantageous experimental design. Non-homologous end joining (NHEJ) is a major double-strand break (DSB) repair pathway that occurs throughout the cell cycle. The objective of the current work is to model the fast and slow phases of NHEJ in G1 phase of the cell cycle following exposure to ionizing radiation (IR). The fast phase contains the major components of NHEJ; Ku70/80 complex, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and XLF/XRCC4/ligase IV complex (XXL). The slow phase in G1 phase of the cell cycle is associated with more complex lesions and involves ATM and Artemis proteins in addition to the major components. Parameters are mainly obtained from experimental data. The model is successful in predicting the kinetics of DSB foci in 13 normal, ATM-deficient, and Artemis-deficient mammalian fibroblast cell lines in G1 phase of the cell cycle after exposure to low doses of IR. The involvement of ATM provides the model with the potency to be connected to different signaling pathways. Ku70/80 concentration and DNA-binding rate as well as XXL concentration and enzymatic activity are introduced as the best targets for affecting NHEJ DSB repair process. On the basis of the current model, decreasing concentration and DNA binding rate of DNA-PKcs is more effective than inhibiting its activity towards the Artemis protein. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1(XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.Cell Research (2008) 18:134–147. doi: 10.1038/cr.2007.111; published online 24 December 2007 [ABSTRACT FROM AUTHOR]

Artemis is a multifunctional phospho-protein with roles in V(D)J recombination, repair of double-strand breaks by nonhomologous end-joining and regulation of cell-cycle checkpoints after DNA damage. Here, we describe a new function of Artemis as a negative regulator of p53 in response to oxidative stress in both primary cells and cancer cell lines. We show that depletion of Artemis under typical culture conditions (21% oxygen) leads to a spontaneous phosphorylation and stabilization of p53, and resulting cellular G1 arrest and apoptosis. These effects are suppressed by co-depletion of DNA-PKcs, but not ATM, indicating that Artemis is an inhibitor of DNA–PKcs-mediated stabilization of p53. Culturing of cellsat 3% oxygen or treatment with an antioxidant abrogated p53 stabilization, indicating that oxidative stress is the responsible cellular stimulus. Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation. In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion. These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.Oncogene (2009) 28, 2196–2204; doi:10.1038/onc.2009.100; published online 27 April 2009 [ABSTRACT FROM AUTHOR]

Non-homologous DNA end-joining (NHEJ) - the main pathway for repairing double-stranded DNA breaks - functions throughout the cell cycle to repair such lesions. Defects in NHEJ result in marked sensitivity to ionizing radiation and ablation of lymphocytes, which rely on NHEJ to complete the rearrangement of antigen-receptor genes. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes, but which might also contribute to some of the genetic changes that underlie cancer and ageing. [ABSTRACT FROM AUTHOR]

Background: Peptide receptor radionuclide therapy (PRRT) uses [177Lu]Lu-[DOTA0-Tyr3]octreotate ([177Lu]Lu-DOTA-TATE) to treat patients with neuroendocrine tumours (NETs) overexpressing the somatostatin receptor 2A (SSTR2A). It has shown significant short-term improvements in survival and symptom alleviation, but there remains room for improvement. Here, we investigated whether combining [177Lu]Lu-DOTA-TATE with chemotherapeutics enhanced the in vitro therapeutic efficacy of [177Lu]Lu-DOTA-TATE. Results: Transfected human osteosarcoma (U2OS + SSTR2A, high SSTR2A expression) and pancreatic NET (BON1 + STTR2A, medium SSTR2A expression) cells were subjected to hydroxyurea, gemcitabine or triapine for 24 h at 37oC and 5% CO2. Cells were then recovered for 4 h prior to a 24-hour incubation with 0.7–1.03 MBq [177Lu]Lu-DOTA-TATE (25 nM) for uptake and metabolic viability studies. Incubation of U2OS + SSTR2A cells with hydroxyurea, gemcitabine, and triapine enhanced uptake of [177Lu]Lu-DOTA-TATE from 0.2 ± 0.1 in untreated cells to 0.4 ± 0.1, 1.1 ± 0.2, and 0.9 ± 0.2 Bq/cell in U2OS + SSTR2A cells, respectively. Cell viability post treatment with [177Lu]Lu-DOTA-TATE in cells pre-treated with chemotherapeutics was decreased compared to cells treated with [177Lu]Lu-DOTA-TATE monotherapy. For example, the viability of U2OS + SSTR2A cells incubated with [177Lu]Lu-DOTA-TATE decreased from 59.5 ± 22.3% to 18.8 ± 5.2% when pre-treated with hydroxyurea. Control conditions showed no reduced metabolic viability. Cells were also harvested to assess cell cycle progression, SSTR2A expression, and cell size by flow cytometry. Chemotherapeutics increased SSTR2A expression and cell size in U2OS + SSTR2A and BON1 + STTR2A cells. The S-phase sub-population of asynchronous U2OS + SSTR2A cell cultures was increased from 45.5 ± 3.3% to 84.8 ± 2.5%, 85.9 ± 1.9%, and 86.6 ± 2.2% when treated with hydroxyurea, gemcitabine, and triapine, respectively. Conclusions: Hydroxyurea, gemcitabine and triapine all increased cell size, SSTR2A expression, and [177Lu]Lu-DOTA-TATE uptake, whilst reducing cell metabolic viability in U2OS + SSTR2A cells when compared to [177Lu]Lu-DOTA-TATE monotherapy. Further investigations could transform patient care and positively increase outcomes for patients treated with [177Lu]Lu-DOTA-TATE. [ABSTRACT FROM AUTHOR]

DNA damage is a constant event in every cell caused by exogenous factors such as ultraviolet and ionizing radiation (UVR/IR) and intercalating drugs, or endogenous metabolic and replicative stress. Proteins of the DNA damage response (DDR) network sense DNA lesions and induce cell cycle arrest, DNA repair, and apoptosis. Genetic defects of DDR or DNA repair proteins can be associated with immunodeficiency, bone marrow failure syndromes, and cancer susceptibility. Although various diagnostic tools are available to evaluate DNA damage, their quality to identify DNA repair deficiencies differs enormously and depends on affected pathways. In this study, we investigated the DDR biomarkers γH2AX (Ser139), p-ATM (Ser1981), and p-CHK2 (Thr68) using flow cytometry on peripheral blood cells obtained from patients with combined immunodeficiencies due to non-homologous end-joining (NHEJ) defects and ataxia telangiectasia (AT) in response to low-dose IR. Significantly reduced induction of all three markers was observed in AT patients compared to controls. However, delayed downregulation of γH2AX was found in patients with NHEJ defects. In contrast to previous reports of DDR in cellular models, these biomarkers were not sensitive enough to identify ARTEMIS deficiency with sufficient reliability. In summary, DDR biomarkers are suitable for diagnosing NHEJ defects and AT, which can be useful in neonates with abnormal TREC levels (T cell receptor excision circles) identified by newborn screening. We conclude that DDR biomarkers have benefits and some limitations depending on the underlying DNA repair deficiency. [ABSTRACT FROM AUTHOR]

Carnosol, a rosemary polyphenol, displays anticancer properties and is suggested as a safer alternative to conventional surgery, radiotherapy, and chemotherapy. Given that its effects on gingiva carcinoma have not yet been investigated, the aim of this study was to explore its anti-tumor selectivity and to unravel its underlying mechanisms of action. Hence, oral tongue and gingiva carcinoma cell lines exposed to carnosol were analyzed to estimate cytotoxicity, cell viability, cell proliferation, and colony formation potential as compared with those of normal cells. Key cell cycle and apoptotic markers were also measured. Finally, cell migration, oxidative stress, and crucial cell signaling pathways were assessed. Selective anti-gingiva carcinoma activity was disclosed. Overall, carnosol mediated colony formation and proliferation suppression in addition to cytotoxicity induction. Cell cycle arrest was highlighted by the disruption of the c-myc oncogene/p53 tumor suppressor balance. Carnosol also increased apoptosis, oxidative stress, and antioxidant activity. On a larger scale, the alteration of cell cycle and apoptotic profiles was also demonstrated by QPCR array. This was most likely achieved by controlling the STAT5, ERK1/2, p38, and NF-ĸB signaling pathways. Lastly, carnosol reduced inflammation and invasion ability by modulating IL-6 and MMP9/TIMP-1 axes. This study establishes a robust foundation, urging extensive inquiry both in vivo and in clinical settings, to substantiate the efficacy of carnosol in managing gingiva carcinoma. [ABSTRACT FROM AUTHOR]

Using a semiquantitative PCR-based approach, a breakpoint cluster region of AML1 was associated with the nuclear matrix. Inhibition of topoisomerase II (topoII) by etoposide stimulated the appearance of histone γH2AX foci, indicative of DNA double-strand breaks (DSBs). The majority of these foci were associated with the nuclear matrix. Nuclear matrix-associated multiprotein complexes involved in topoII-induced DNA DSB repair were visualized. Colocalization studies demonstrated that these complexes included the main components of the nonhomologous end joining repair system (Ku80, DNA-PKcs, and DNA ligase IV). Thus, it was suggested that nonhomologous DNA end joining is a possible mechanism of topoII-induced chromosomal rear-rangements. [ABSTRACT FROM AUTHOR]

The human tumor suppressor gene ataxia telangiectasia mutated (ATM) encodes a 3056 amino-acid protein kinase that regulates cell cycle checkpoints. ATM is defective in the neurodegenerative and cancer predisposition syndrome ataxia-telangiectasia. ATM protein kinase is activated by DNA damage and responds by phosphorylating downstream effectors involved in cell cycle arrest and DNA repair, such as p53, MDM2, CHEK2, BRCA1 and H2AX. ATM is probably a component of, or in close proximity to, the double-stranded DNA break-sensing machinery. We have observed purified human ATM protein, ATM-DNA and ATM-DNA-avidin bound complexes by single-particle electron microscopy and obtained three-dimensional reconstructions which show that ATM is composed of two main domains comprising a head and an arm. DNA binding to ATM induces a large conformational movement of the arm-like domain. Taken together, these three structures suggest that ATM is capable of interacting with DNA, using its arm to clamp around the double helix.Oncogene (2003) 22, 3867-3874. doi:10.1038/sj.onc.1206649 [ABSTRACT FROM AUTHOR]

Tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) enzymes belong to the poly (ADP-ribose) polymerase (PARP) family participates in process of poly-ADP-ribosylation of different target proteins which leads to ubiquitin-mediated proteasomal degradation. Tankyrases are also involved in the pathophysiology of many diseases, especially cancer. Their functions include cell cycle homeostasis (primarily in mitosis), telomere maintenance, Wnt signaling pathway regulation, and insulin signaling (particularly GLUT4 translocation). Studies have implicated that genetic changes, mutations in the tankyrase coding sequence, or up regulation and down regulation of tankyrase are reflected in the numerous disease conditions. Investigations are pursued to develop putative molecules that target tankyrase in various diseases such as cancer, obesity, osteoarthritis, fibrosis, cherubism, and diabetes, thereby providing a new therapeutic treatment option. In the present review, we described the structure and function of tankyrase along with its role in different disease conditions. Furthermore, we also presented cumulative experimental evidences of different drugs acting on tankyrase. [ABSTRACT FROM AUTHOR]

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku's role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability. [ABSTRACT FROM AUTHOR]

Discovering synthetic lethal (SL) gene partners of cancer genes is an important step in developing cancer therapies. However, identification of SL interactions is challenging, due to a large number of possible gene pairs, inherent noise and confounding factors in the observed signal. To discover robust SL interactions, we devised SLIDE-VIP, a novel framework combining eight statistical tests, including a new patient data-based test iSurvLRT. SLIDE-VIP leverages multi-omics data from four different sources: gene inactivation cell line screens, cancer patient data, drug screens and gene pathways. We applied SLIDE-VIP to discover SL interactions between genes involved in DNA damage repair, chromatin remodeling and cell cycle, and their potentially druggable partners. The top 883 ranking SL candidates had strong evidence in cell line and patient data, 250-fold reducing the initial space of 200K pairs. Drug screen and pathway tests provided additional corroboration and insights into these interactions. We rediscovered well-known SL pairs such as RB1 and E2F3 or PRKDC and ATM, and in addition, proposed strong novel SL candidates such as PTEN and PIK3CB. In summary, SLIDE-VIP opens the door to the discovery of SL interactions with clinical potential. All analysis and visualizations are available via the online SLIDE-VIP WebApp. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) occur every cell cycle and must be efficiently repaired. Non-homologous end joining (NHEJ) is the dominant pathway for DSB repair in G1-phase. The first step of NHEJ is to bring the two DSB ends back into proximity (synapsis). Although synapsis is generally assumed to occur through passive diffusion, we show that passive diffusion is unlikely to produce the synapsis speed observed in cells. Instead, we hypothesize that DNA loop extrusion facilitates synapsis. By combining experimentally constrained simulations and theory, we show that a simple loop extrusion model constrained by previous live-cell imaging data only modestly accelerates synapsis. Instead, an expanded loop extrusion model with targeted loading of loop extruding factors (LEFs), a small portion of long-lived LEFs, and LEF stabilization by boundary elements and DSB ends achieves fast synapsis with near 100% efficiency. We propose that loop extrusion contributes to DSB repair by mediating fast synapsis. DNA double-strand breaks (DSBs) occur every cell cycle and must be repaired. Here the authors combine theory and simulations to establish a likely role for loop extrusion in bringing the DSB ends back into proximity for repair. [ABSTRACT FROM AUTHOR]

Dinaciclib is a selective cyclin-dependent kinase inhibitor, but its radiosensitizing effect remains unclear. The aim of this study is to investigate the radiosensitizing effect of Dinaciclib on cervical cancer cells. Two cervical cancer cell lines, Hela and Siha, were selected, and the IC50 was determined by CCK8. The radiosensitizing effect of Dinaciclib was verified by plate cloning assay, and the G2/M phase arrest and apoptosis of IR cells were verified by flow cytometry. Immunofluorescence assay was used to verify the formation of γH2AX foci following DNA damage. Western blot was performed to detect cell cycle, apoptosis, autophagy, and DNA damage-related pathways. Dinaciclib increased the cell sensitivity to IR. IR induced G2/M phase arrest and apoptosis, and Dinaciclib enhanced this effect. Further, Dinaciclib delayed DNA repair, including non-homologous end joining repair and homologous recombination repair, and reduced the expression of DNA repair proteins Ku80 (SiHa cells), Ku70, and RAD51, as well as the expression of apoptotic marker Bcl-2. The expression of autophagy marker Beclin1 induced tumor cell death and increased the formation of DNA damage marker γH2AX foci. Dinaciclib improves the sensitivity of cervical cancer cells to IR by inducing cell cycle arrest, delaying DNA repair, and increasing apoptosis. However, further research is needed to unravel the complexity of DNA repair pathways. [ABSTRACT FROM AUTHOR]

Although serine ADP-ribosylation (Ser-ADPr) by Poly(ADP-ribose)-polymerases is a cornerstone of the DNA damage response, how this regulates DNA repair and genome stability is unknown. Here, we exploit the ability to manipulate histone genes in Dictyostelium to identify that ADPr of the histone variant H3b at S10 and S28 maintains genome stability by integrating double strand break (DSB) repair with mitotic entry. Given the critical requirement for mitotic H3S10/28 phosphorylation, we develop separation of function mutations that maintain S10 phosphorylation whilst disrupting ADPr. Mechanistically, this reveals a requirement for H3bS10/28 ADPr in non-homologous end-joining by recruiting Ku to DSBs. Moreover, this also identifies H3bS10/S28 ADPr is critical to prevent premature mitotic entry with unresolved DNA damage, thus maintaining genome stability. Together, these data demonstrate how serine ADPr of histones coordinates DNA repair with cell cycle progression to maintain genome stability. Poly(ADP-ribose)-polymerases (PARPs) are a cornerstone of the DNA damage response that promote DNA repair by modifying target proteins with ADP-ribose. Here, the authors show serine ADP-ribosylation of the H3 variant H3b maintains genome stability by coupling DNA repair with mitotic entry in Dictyostelium by regulating double strand break repair by nonhomologous end-joining (NHEJ). [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6–1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G2/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was observed in the HEK293 by combining α-radiation and inhibitions. ATMi preferentially sensitized cancer cells and normal HEK293 cells to α-radiation. DNA-PKi and ATMi can sensitize cancer cells to X-ray, but the effectiveness was dependent on cancer cells itself. α-radiation reduced proliferation in primary fibroblast without G2/M arrest. [ABSTRACT FROM AUTHOR]

Several newly identified tumor suppressor genes including ATM, NBS1, BRCA1 and BRCA2 are involved in DNA double-strand break repair (DSBR) and DNA damage-induced checkpoint activation. Many of the gene products involved in checkpoint control and DSBR have been studied in great detail in yeast. In addition to evolutionarily conserved proteins such as Chk1 and Chk2, studies in mammalian cells have identified novel proteins such as p53 in executing checkpoint control. DSBR proteins including Mre11, Rad50, Rad51, Rad54, and Ku are present in yeast and in mammals. Many of the tumor suppressor gene products interact with these repair proteins as well as checkpoint regulators, thus providing a biochemical explanation for the pleiotropic phenotypes of mutant cells. This review focuses on the proteins mediating G1/S, S, and G2/M checkpoint control in mammalian cells. In addition, mammalian DSBR proteins and their activities are discussed. An intricate network among DNA damage signal transducers, cell cycle regulators and the DSBR pathways is illustrated. Mouse knockout models for genes involved in these processes have provided valuable insights into their function, establishing genomic instability as a major contributing factor in tumorigenesis. [ABSTRACT FROM AUTHOR]

We investigated the intracellular mechanisms of retinoic acid (9-cis-RA, 13-cis-RA or all-trans-RA) and a cyclic AMP analog 8-Cl-cAMP on growth-inhibition and apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. The cyclic AMP analog, 8-Cl-cAMP, acted synergistically with RA in inducing and activating retinoic acid receptor β (RARβ) which correlated with the growth inhibition, cell cycle arrest, and apoptosis in both cell types. In addition, combined treatment of cells with RA plus 8-Cl-cAMP resulted in the release of cytochrome c, loss in mitochondrial membrane potential and activation of caspase-3 followed by cleavage of anti-poly(ADP-ribose)polymerase and DNA-dependent protein kinase (catalytic subunit). Interestingly, inhibition of caspase-3 activation blocked RA plus 8-Cl-cAMP induced apoptosis. Furthermore, mutations in a CRE-related motif within the RARβ promoter resulted in loss of both transcriptional activation of RARβ and synergy between RA and 8-Cl-cAMP. Thus, RARβ can mediate RA and/or cyclic AMP action in ovarian cancer cells by promoting apoptosis. Loss of RARβ expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion in inducing apoptosis via caspase-3 activation, and may have potential for combination biotherapy for the treatment of malignant disease such as ovarian cancer. [ABSTRACT FROM AUTHOR]

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a ∼ 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome. [ABSTRACT FROM AUTHOR]

The article reports on the status of p53 antioncogene together with ataxia telangiectasia mutated (ATM) showing the response of a tumor to therapy. The authors used short hairpin ribonucleic acids (RNAs) to cut down the formula of ATM in immortalized mouse embryonic fibroblasts (MEFs). They suggested that ATM is required for induction of apoptosis reacting to DNA damage but ATM is not needed for p53-mediated cell cycle arrest.

Background: Greater than half of cancer patients experience radiation therapy, for both radical and palliative objectives. It is well known that researches on radiation response mechanisms are conducive to improve the efficacy of cancer radiotherapy. p21 was initially identified as a widespread inhibitor of cyclin-dependent kinases, transcriptionally modulated by p53 and a marker of cellular senescence. It was once considered that p21 acts as a tumour suppressor mainly to restrain cell cycle progression, thereby resulting in growth suppression. With the deepening researches on p21, p21 has been found to regulate radiation responses via participating in multiple cellular processes, including cell cycle arrest, apoptosis, DNA repair, senescence and autophagy. Hence, a comprehensive summary of the p21's functions in radiation response will provide a new perspective for radiotherapy against cancer. Methods: We summarize the recent pertinent literature from various electronic databases, including PubMed and analyzed several datasets from Gene Expression Omnibus database. This review discusses how p21 influences the effect of cancer radiotherapy via involving in multiple signaling pathways and expounds the feasibility, barrier and risks of using p21 as a biomarker as well as a therapeutic target of radiotherapy. Conclusion: p21's complicated and important functions in cancer radiotherapy make it a promising therapeutic target. Besides, more thorough insights of p21 are needed to make it a safe therapeutic target. [ABSTRACT FROM AUTHOR]

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies. Depending on the cell cycle stage, cells can repair their genome via different pathways. Here the authors reveal mechanistic insights into repair of double strand breaks induced during G1 in an error-prone manner by Pol θ-dependent and PARP1-independent alt NHEJ during the SG2/M phases of the cell cycle [ABSTRACT FROM AUTHOR]

Purpose: Signaling by cancer stem cells (CSCs) is known to occur at least in part through conserved developmental pathways. Here, the role of one of these pathways, i.e., the hedgehog pathway, was evaluated in high-grade serous ovarian carcinoma (HGSOC). Methods and results: We found that in HGSOC, hedgehog inhibitors (HHIs) GANT61, LDE225 and GDC0449 reduced or inhibited the formation of spheroids enriched in CSCs. Primary malignant cells (PMCs) in ascites from HGSOC patients cultured in the presence of HHIs showed significant reduction in CSCs. Sonic hedgehog (SHH) significantly increased the expression of ALDH1A1, which was inhibited by GANT61. In the presence of a SHH neutralizing antibody (5E1), a significant reduction in the number of spheroids was observed in HGSOC-derived cell lines. Further, the motility, migration and clonogenic growth of the cells were significantly reduced by HHIs. In the presence of GANT61, a reduction of cells from PMCs in the G0 phase of the cell cycle was observed. The magnitude of difference in expression of Gli1 in tumors from the same HGSOC patients at presentation and at interval debulking surgery was greater in patients who had a recurrence on follow up. GANT61 also significantly inhibited the growth of CSCs in nude mice. Finally, RNA sequencing of HGSOC cells treated with GANT61 showed a significantly reduced expression of CSC markers. Conclusions: Our results indicate that the hedgehog pathway plays an important role in maintaining the integrity of CSCs in HGSOC and could be a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Precise gene editing aims at generating single-nucleotide modifications to correct or model human disease. However, precision editing with nucleases such as CRIPSR-Cas9 has seen limited success due to poor efficiency and limited practicality. Here, we establish a fluorescent DNA repair assay in human induced pluripotent stem (iPS) cells to visualize and quantify the frequency of DNA repair outcomes during monoallelic and biallelic targeting. We found that modulating both DNA repair and cell cycle phase via defined culture conditions and small molecules synergistically enhanced the frequency of homology-directed repair (HDR). Notably, targeting in homozygous reporter cells results in high levels of editing with a vast majority of biallelic HDR outcomes. We then leverage efficient biallelic HDR with mixed ssODN repair templates to generate heterozygous mutations. Synergistic gene editing represents an effective strategy to generate precise genetic modifications in human iPS cells. Precision editing with CRISPR-Cas9 often suffers from poor efficiency. Here the authors identify culture conditions and small molecules that synergize to promote homology-directed repair (HDR) in induced pluripotent stem (iPS) cells. [ABSTRACT FROM AUTHOR]

DNA damage has been reported to induce cell cycle–related neuronal death. This is significant as aberrant cell cycle re-entry of mature, post-mitotic neurons contributes to neurodegeneration. In this study, we investigate how DNA damage elicited by exposure to the topoisomerase I inhibitor camptothecin (CPT) leads to cycle-related death of cultured cortical neurons and examine the function of E2F1 in this process. CPT treatment induced cell cycle initiation of cortical neurons and elevated the expression of certain cell cycle components (e.g., cyclin D1, CDK4, E2F1) but failed to drive S phase entry or DNA synthesis. The arrest in the cell cycle is explained by the elevated expression of the CDK inhibitor p21Cip1. Though its level was increased after CPT treatment, E2F1 did not drive treated neurons into the G1-S phase transition. E2F1 overexpression led to cell cycle activation and acute neuronal apoptosis without detectable entry of the neurons into S phase. ChIPseq analysis demonstrated that E2F1 predominantly occupies positions on or near the promoters of cell cycle related genes. Instead, in CPT-treated neurons, E2F1 preferentially regulated DNA repair related genes. Our study reveals that the functions of E2F1 in postmitotic neurons are context-dependent and offers novel insights into the role of E2F1 in DNA damage induced cycle-related neuronal death. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade (ICB)-based or natural cancer immune responses largely eliminate tumours. Yet, they require additional mechanisms to arrest those cancer cells that are not rejected. Cytokine-induced senescence (CIS) can stably arrest cancer cells, suggesting that interferon-dependent induction of senescence-inducing cell cycle regulators is needed to control those cancer cells that escape from killing. Here we report in two different cancers sensitive to T cell-mediated rejection, that deletion of the senescence-inducing cell cycle regulators p16Ink4a/p19Arf (Cdkn2a) or p21Cip1 (Cdkn1a) in the tumour cells abrogates both the natural and the ICB-induced cancer immune control. Also in humans, melanoma metastases that progressed rapidly during ICB have losses of senescence-inducing genes and amplifications of senescence inhibitors. Metastatic cells also resist CIS. Such genetic and functional alterations are infrequent in metastatic melanomas regressing during ICB. Thus, activation of tumour-intrinsic, senescence-inducing cell cycle regulators is required to stably arrest cancer cells that escape from eradication. The growth of cancer cells can be stably arrested by cytokine-induced senescence. Here, the authors show that cancers with defects in senescence-inducing cell cycle regulator pathways are resistant to immune checkpoint blockade. [ABSTRACT FROM AUTHOR]

DNA damage occurs abundantly during normal cellular proliferation. This necessitates that cellular DNA damage response and checkpoint pathways monitor the cellular DNA damage load and that DNA damage signaling is quantitative. Yet, how DNA lesions are counted and converted into a quantitative response remains poorly understood. We have recently obtained insights into this question investigating DNA damage signaling elicited by single-stranded DNA (ssDNA). Intriguingly, our findings suggest that local and global DNA damage signaling react differentially to increasing amounts of DNA damage. In this mini-review, we will discuss these findings and put them into perspective of current knowledge on the DNA damage response. [ABSTRACT FROM AUTHOR]

The Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine–threonine kinases that have been implicated in DNA replication, DNA repair, transcription, chromatin structure, viral latency, cell cycle checkpoint control and chromosomal stability in various organisms. The functions of the TLKs appear to depend largely on their ability to regulate the H3/H4 histone chaperone ASF1, although numerous TLK substrates have been proposed. Over the last few years, a clearer picture of TLK function has emerged through the identification of new partners, the definition of specific roles in development and the elucidation of their structural and biochemical properties. In addition, the TLKs have been clearly linked to human disease; both TLK1 and TLK2 are frequently amplified in human cancers and TLK2 mutations have been identified in patients with neurodevelopmental disorders characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. A better understanding of the substrates, regulation and diverse roles of the TLKs is needed to understand their functions in neurodevelopment and determine if they are viable targets for cancer therapy. In this review, we will summarize current knowledge of TLK biology and its potential implications in development and disease. [ABSTRACT FROM AUTHOR]

Ewing sarcoma (ES) are aggressive pediatric bone and soft tissue tumors driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. Treatment of ES patients consists of up to 9 months of alternating courses of 2 chemotherapeutic regimens. Furthermore, EWS-ETS-targeted therapies have yet to demonstrate clinical benefit, thereby emphasizing a clinical responsibility to search for new therapeutic approaches. Our previous in silico drug screening identified entinostat as a drug hit that was predicted to reverse the ES disease signatures and EWS-FLI1-mediated gene signatures. Here, we establish preclinical proof of principle by investigating the in vitro and in vivo efficacy of entinostat in preclinical ES models, as well as characterizing the mechanisms of action and in vivo pharmacokinetics of entinostat. ES cells are preferentially sensitive to entinostat in an EWS-FLI1 or EWS-ERG-dependent manner. Entinostat induces apoptosis of ES cells through G0/G1 cell cycle arrest, intracellular reactive oxygen species (ROS) elevation, DNA damage, homologous recombination (HR) repair impairment, and caspase activation. Mechanistically, we demonstrate for the first time that HDAC3 is a transcriptional target of EWS-FLI1 and that entinostat inhibits growth of ES cells through suppressing a previously unexplored EWS-FLI1/HDAC3/HSP90 signaling axis. Importantly, entinostat significantly reduces tumor burden by 97.4% (89.5 vs. 3397.3 mm3 of vehicle, p < 0.001) and prolongs the median survival of mice (15.5 vs. 8.5 days of vehicle, p < 0.001), in two independent ES xenograft mouse models, respectively. Overall, our studies demonstrate promising activity of entinostat against ES, and support the clinical development of the entinostat-based therapies for children and young adults with metastatic/relapsed ES. Key messages: • Entinostat potently inhibits ES both in vitro and in vivo. • EWS-FLI1 and EWS-ERG confer sensitivity to entinostat treatment. • Entinostat suppresses the EWS-FLI1/HDAC3/HSP90 signaling. • HDAC3 is a transcriptional target of EWS-FLI1. • HDAC3 is essential for ES cell viability and genomic stability maintenance. [ABSTRACT FROM AUTHOR]

Approximately half of high-grade serous epithelial ovarian cancers incur alterations in genes of homologous recombination ( BRCA1, BRCA2, RAD51C, Fanconi anemia genes), and the rest incur alterations in other DNA repair pathways at high frequencies. Such cancer-specific gene alterations can confer selective sensitivity to DNA damaging agents such as cisplatin and carboplatin, topotecan, etoposide, doxorubicin, and gemcitabine. Originally presumed to inhibit DNA repair, PARP inhibitors that have recently been approved by the FDA for the treatment of advanced ovarian cancer also act as DNA damaging agents by inducing PARP-DNA complexes. These DNA damaging agents induce different types of DNA lesions that require various DNA repair genes for the repair, but commonly induce replication fork slowing or stalling, also referred to as replication stress. Replication stress activates DNA repair checkpoint proteins (ATR, CHK1), which prevent further DNA damage. Hence, targeting DNA repair genes or DNA repair checkpoint genes augments the anti-tumor activity of DNA damaging agents. This review describes the rational basis for using DNA repair and DNA repair checkpoint inhibitors as single agents. The review also presents the strategies combining these inhibitors with DNA damaging agents for ovarian cancer therapy based on specific gene alterations. [ABSTRACT FROM AUTHOR]

ATM is a master regulator of the cellular response to DNA damage. The classical mechanism of ATM activation involves its monomerization in response to DNA double-strand breaks, resulting in ATM-dependent phosphorylation of more than a thousand substrates required for cell cycle progression, DNA repair, and apoptosis. Here, new experimental evidence for non-canonical mechanisms of ATM activation in response to stimuli distinct from DNA double-strand breaks is discussed. It includes cytoskeletal changes, chromatin modifications, RNA-DNA hybrids, and DNA single-strand breaks. Noncanonical ATM activation may be important for the pathology of the multisystemic disease Ataxia Telangiectasia. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3′-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast. [ABSTRACT FROM AUTHOR]

Purpose: The study mainly aimed to determine the biological function of a novelly synthesized phenanthroimidazole derivative, named L233, and to explore its potential mechanisms.Methods: Cell survival was examined using the MTT assays, and the DNA-damaging role of L233 was explored using the comet assay. Moreover, the western blotting assays and immunofluorescence assays were used to detect DNA damage biomarkers. Afterward, the flow cytometry was used to assess the effects of L233 on cell cycle distribution. As for the detection of cell apoptosis upon L233 treatment, the Hoechst 33342 staining, flow cytometry, and western blotting assays were all put into practice.Results: We find that L233 inhibits tumor cell growth more efficiently and safely than cisplatin. Moreover, it is a DNA-damaging agent, interrupting the cell cycle G1/S checkpoint transition and inducing cell apoptosis by not only activating ATM/CHK1 signaling pathway, but also targeting CHK1 to reduce the expression of RAP80 and PARP-1 to compromise the DNA damage repair in tumor cells.Conclusions: In summary, L233 is a promising anticancer drug for the development of novel chemotherapies in the future. [ABSTRACT FROM AUTHOR]

DNA damage is correlated with and may drive the ageing process. Neurons in the brain are postmitotic and are excluded from many forms of DNA repair; therefore, neurons are vulnerable to various neurodegenerative diseases. The challenges facing the field are to understand how and when neuronal DNA damage accumulates, how this loss of genomic integrity might serve as a 'time keeper' of nerve cell ageing and why this process manifests itself as different diseases in different individuals. [ABSTRACT FROM AUTHOR]

Ser/Thr protein phosphatase 5 (PPP5C) has been reported to participate in tumor progression. However, its functional role in hepatocellular carcinoma (HCC) remains unknown yet. In this study, we firstly evaluated the expression levels of PPP5C in six HCC cell lines by real-time PCR and found that PPP5C was widely expressed in HCC cells. To explore the role of PPP5C in HCC cell growth, lentivirus-mediated short hairpin RNA (shRNA) was employed to silence PPP5C expression in HepG2 and Bel-7404 cells. The expression of PPP5C was significantly downregulated in PPP5C knockdown cells. Knockdown of PPP5C markedly suppressed the proliferation and colony formation ability of HCC cells. Moreover, cell cycle analysis showed that PPP5C depletion in HepG2 cells led to G/G phase and G/M phase arrest. We demonstrate for the first time that PPP5C is essential for growth of HCC cells, which suggests that inhibition of PPP5C by RNAi may be a potential therapeutic strategy for the treatment of HCC. [ABSTRACT FROM AUTHOR]

To study the function of CaPpt1, we deleted PPT1 gene from the Candida albicans genome by sequentially replacing the entire coding region with the selectable markers ARG4 and HIS1. The results showed that the deletion of Ppt1 did not affect the hyphal formation of C. albicans under serum induction and caused enhanced sensitivity to DNA damage, Calcofluor white and salt-induced stress. We also found that Ppt1 was not required for the phenotypic response of cells treated with the genotoxins, methylmethane sulfonate and hydroxyurea. Flow cytometric analyses indicated that ppt1Δ cells and wild-type cells showed similar G/M arrest profiles when exposed to DNA damage stress. Ppt1 was not required for the activation of the DNA damage response pathway, as indicated by normal phosphorylation of Rad53 and Rfa2 in ppt1Δ cells under DNA damage stress. We suggest that Ppt1 plays important roles in response to various stress conditions in C. albicans. [ABSTRACT FROM AUTHOR]

To maintain genome stability, cells have evolved various DNA repair pathways to deal with oxidative DNA damage. DNA damage response (DDR) pathways, including ATM-Chk2 and ATR-Chk1 checkpoints, are also activated in oxidative stress to coordinate DNA repair, cell cycle progression, transcription, apoptosis, and senescence. Several studies demonstrate that DDR pathways can regulate DNA repair pathways. On the other hand, accumulating evidence suggests that DNA repair pathways may modulate DDR pathway activation as well. In this review, we summarize our current understanding of how various DNA repair and DDR pathways are activated in response to oxidative DNA damage primarily from studies in eukaryotes. In particular, we analyze the functional interplay between DNA repair and DDR pathways in oxidative stress. A better understanding of cellular response to oxidative stress may provide novel avenues of treating human diseases, such as cancer and neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Radioresistance is a frustrating obstacle for patients with colorectal cancers (CRCs) undergoing radiotherapy. There is an urgent need to find an effective agent to increase the sensitivity of CRCs to radiation. Honokiol, an active compound purified from magnolia, was found to radiosensitize CRC cells both in vitro and in vivo. However, the mechanism controlling important signaling that enhances radiosensitivity is currently unknown. In this study, we review important signaling pathways that are closely related to radiosensitization, such as cell cycle arrest, tumor angiogenesis, the Jak/STAT3 signaling pathway, and mismatch repair. Studies show that honokiol can interfere with these pathways at different levels. Overall analysis may shed light on the possible mechanism by which honokiol acts as a radiosensitizing agent for CRCs. [ABSTRACT FROM AUTHOR]

Darinaparsin (Dar) is a more potent cytotoxic arsenical than arsenic trioxide (ATO). We hypothesized that the increased cytotoxicity of Dar may be because of a decreased cytoprotective response. We observed that, unlike ATO, Dar does not induce heme oxygenase-1 (HO-1), even though it induces expression of other nuclear factor (erythroid-derived 2)-like 2 (NRF2)-dependent detoxifying enzymes to a greater extent than ATO, in both cancer cell lines and patient-derived leukemic cells. This strengthens the emerging evidence, showing that response to reactive oxygen species (ROS) is stimuli specific. Dar treatment prevents recruitment of the transcriptional coregulator Brahma-related gene 1 (BRG1) to the HMOX1 promoter, which is required for HMOX1 expression. The inability of Dar to induce HO-1 correlates with arrest in G2/M cell cycle phase and BRG1 phosphorylation. Inhibition of HO-1 increases the toxicity of ATO, but has no effect on Dar-induced apoptosis. Accordingly, the lack of HO-1 induction is involved in Dar's enhanced antileukemic properties. Our data highlight cytoprotective responses mediated by HO-1 and BRG1 as a novel target for enhancing the therapeutic range of arsenicals. [ABSTRACT FROM AUTHOR]

Viruses depend on host cell resources for replication and access to those resources may be limited to a particular phase of the cell cycle. Thus manipulation of cell cycle is a commonly employed strategy of viruses for achieving a favorable cellular environment. For example, viruses capable of infecting nondividing cells induce S phase in order to activate the host DNA replication machinery and provide the nucleotide triphosphates necessary for viral DNA replication (Flemington in J Virol 75:4475-4481, ; Sullivan and Pipas in Microbiol Mol Biol Rev 66:179-202, ). Viruses have developed several strategies to subvert the cell cycle by association with cyclin and cyclin-dependent kinase complexes and molecules that regulate their activity. Viruses tend to act on cellular proteins involved in a network of interactions in a way that minimal protein-protein interactions lead to a major effect. The complex and interactive nature of intracellular signaling pathways controlling cell division affords many opportunities for virus manipulation strategies. Taking the maxim 'Set a thief to catch a thief' as a counter strategy, however, provides us with the very same virus evasion strategies as 'ready-made tools' for the development of novel antivirus therapeutics. The most obvious are attenuated virus vaccines with critical evasion genes deleted. Similarly, vaccines against viruses causing cancer are now being successfully developed. Finally, as viruses have been playing chess with our cell biology and immune responses for millions of years, the study of their evasion strategies will also undoubtedly reveal new control mechanisms and their corresponding cellular intracellular signaling pathways. [ABSTRACT FROM AUTHOR]

Glioblastoma is a disease with poor outcomes despite standard therapy. Specific targeting of the DNA damage response is a strategy that is becoming increasingly employed in oncology and has intriguing potential for improving outcomes in glioblastoma. DNA damage targeting has implications for improving current therapy as well as the potential to leverage inherent differences in glioblastoma cells to widen the therapeutic window. [ABSTRACT FROM AUTHOR]

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term 'checkpoint' was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells. [ABSTRACT FROM AUTHOR]

In the presence of sustained DNA damage occurring in S-phase or G2, normal cells arrest before mitosis and eventually become senescent. The checkpoint kinases Chk1/Chk2 and the CDK inhibitor p21 are known to have important complementary roles in this process, in G2 arrest and cell cycle exit, respectively. However, additional checkpoint roles have been reported for these regulators and it is not clear to what extent their functions are redundant. Here we compared the respective roles of Chk1, Chk2 and p21 in DNA damage-induced G2 arrest in normal human fibroblasts, normal epithelial cells and frequently used p53 proficient cancer cells. We show that in normal cells, Chk1, but not Chk2, is involved in G2 arrest whereas neither are essential. In contrast, p21 is required. However, Chk1, but not Chk2, becomes necessary for arrest in U2OS osteosarcoma cells. We find that their ATM/p53/p21 response in G2 phase is defective, like in other cancer cells with wild-type p53, and conclude that cross-talk between the Chk1 and p21 pathways allows them to switch dependency for G2 arrest onto Chk1. Using the specific ATM inhibitor KU-55933 we confirm the essential role of ATM in the induction of p21 for G2 arrest of normal cells. Efficient p21 induction is required for nuclear sequestration of inactive cyclin B1-Cdk1 complexes preceding irreversible cell cycle exit in G2. Our results demonstrate that p21 is able to fulfill the Chk1 functions in G2 arrest under continuous genotoxic stress, which has important implications for cancer chemotherapy. [ABSTRACT FROM AUTHOR]

ATM is the most significant molecule involved in monitoring the genomic integrity of the cell. Any damage done to DNA relentlessly challenges the cellular machinery involved in recognition, processing and repair of these insults. ATM kinase is activated early to detect and signal lesions in DNA, arrest the cell cycle, establish DNA repair signaling and faithfully restore the damaged chromatin. ATM activation plays an important role as a barrier to tumorigenesis, metabolic syndrome and neurodegeneration. Therefore, studies of ATM-dependent DNA damage signaling pathways hold promise for treatment of a variety of debilitating diseases through the development of new therapeutics capable of modulating cellular responses to stress. In this review, we have tried to untangle the complex web of ATM signaling pathways with the purpose of pinpointing multiple roles of ATM underlying the complex phenotypes observed in AT patients. [ABSTRACT FROM AUTHOR]