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Abstract The Saccharomyces cerevisiae silencing information regulator (SIR) complex contains up to four proteins, namely Sir1, Sir2, Sir3, and Sir4. While Sir2 encodes a NAD‐dependent histone deacetylase, other SIR proteins mainly function as structural and scaffold components through physical interaction with various proteins. The SIR complex displays different conformation and composition, including Sir2 homotrimer, Sir1‐4 heterotetramer, Sir2‐4 heterotrimer, and their derivatives, which recycle and relocate to different chromosomal regions. Major activities of the SIR complex are transcriptional silencing through chromosomal remodeling and modulation of DNA double‐strand‐break repair pathways. These activities allow the SIR complex to be involved in mating‐type maintenance and switching, telomere and subtelomere gene silencing, promotion of nonhomologous end joining, and inhibition of homologous recombination, as well as control of cell aging. This review explores the potential link between epigenetic regulation and DNA damage response conferred by the SIR complex under various conditions aiming at understanding its roles in balancing cell survival and genomic stability in response to internal and environmental stresses. As core activities of the SIR complex are highly conserved in eukaryotes from yeast to humans, knowledge obtained in the yeast may apply to mammalian Sirtuin homologs and related diseases.

AIM: This study aims to examine the short-term and long-term effects of aerobic exercise on the cell division cycle protein 42（ Cdc42） and DNA-damage response（ DDR）-related factors in the hippocampus of adolescent mice. METHODS: A cohort of 40 C57BL/6 male mice, aged 8 weeks, was randomly divided into two groups: a sedentary group and an exercise group, each consisting of twenty mice, respectively. Both groups were provided with a standard diet. The exercise group underwent a 12-week treadmill exercise regimen at a moderate intensity level of 75% VO2 max. The sedentary group, serving as the control, was exposed to treadmill noise and vibration without engaging in any physical activity. Ten mice from the sedentary group and 10 mice from the exercise group, all aged 5 months, were randomly assigned to the sedentary young （Sed-young） group and the exercise young （Exe-young） group. The remaining 10 mice from each group were designated as the sedentary old （Sed-old） group and the exercise old （Exe-old） group. These mice were allowed to continue their respective diets fed until they reached 18 months of age. At the end of the study, all mice underwent Y-maze testing, body composition analysis, and had their hippocampal tissue collected for further analysis. The protein expression and activity of hippocampal Cdc42 were detected by Western blot and pull-down assays. The relative expression levels of hippocampal Cdc42, Pak4, Pak5, Cofilin, Chk2, Atm and P53 in the mice were determined using RTqPCR. RESULTS: Adolescent mice that engaged in aerobic exercise exhibited reduced body fat percentages （P<0. 05） and demonstrated enhanced spatial awareness and memory abilities （P<0. 05） that could persisted throughout their lifespan（ P<0. 05）. In both young and old age, the exercise group displayed significantly diminished expression and activity of the Cdc42 protein in the hippocampus compared to the sedentary group. Furthermore, the hippocampus of adolescent mice exhibited significantly decreased transcriptional levels of Cdc42, Pak4, Pak5, Cofilin, Atm, Chk2 and P53 following aerobic activity （P<0. 05）, and these reduced transcriptional levels （Cdc42, Pak4, Pak5, Cofilin, Chk2 and P53） endured until old age （P<0. 05）. CONCLUSION: The expression of the Cdc42 and DDR-related genes can be consistently reduced through long-term aerobic exercise training starting from adolescence, thereby providing protection to hippocampus neurons against DNA damage and cell death or senescence throughout the aging process. [ABSTRACT FROM AUTHOR]

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the "chronic inflammation-dysplasia-cancer" carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations. [ABSTRACT FROM AUTHOR]

Mitochondrial outer membrane permeabilization (MOMP) is crucial for the cytosolic accumulation of mitochondrial DNA (mtDNA) species that are required to jumpstart innate and adaptive immunity. Recent data reported by Ghosh et al. suggest that tumor protein p53 regulates MOMP-dependent type I interferon (IFN) production, not only via MOMP-promoting effects, but also by directing mtDNA-degrading exonucleases to proteasomal processing. [ABSTRACT FROM AUTHOR]

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the “chronic inflammation-dysplasia-cancer” carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations.

• Berzosertib plus gemcitabine was well tolerated in 38 patients with advanced NSCLC. • Preliminary signs of clinical efficacy were observed. • Response rate was numerically increased in patients with high TMB and LOH scores. • Potential DDRi biomarkers should be investigated in genomically selected patients. • Berzosertib plus chemotherapy is being investigated in other treatment settings. Berzosertib (formerly M6620, VX-970) is an intravenous, highly potent and selective, first-in-class ataxia telangiectasia and Rad3-related (ATR) protein kinase inhibitor. We assessed the safety, tolerability, preliminary efficacy, and pharmacokinetics (PK) of berzosertib plus gemcitabine in an expansion cohort of patients with advanced non-small cell lung cancer (NSCLC). The association of efficacy with TP53 status and other tumor markers was also explored. Adult patients with advanced histologically confirmed NSCLC received berzosertib 210 mg/m2 (days 2 and 9) and gemcitabine 1000 mg/m2 (days 1 and 8) at the recommended phase 2 dose established in the dose escalation part of the study. Thirty-eight patients received at least one dose of study treatment. The most common treatment-emergent adverse events were fatigue (55.3%), anemia (52.6%), and nausea (39.5%). Gemcitabine had no apparent effect on the PK of berzosertib. The objective response rate (ORR) was 10.5% (4/38, 90% confidence interval [CI]: 3.7–22.5%). In the exploratory analysis, the ORR was 30.0% (3/10, 90% CI: 9.0–61.0%) in patients with high loss of heterozygosity (LOH) and 11.0% (1/9, 90% CI: 1.0–43.0%) in patients with low LOH. The ORR was 33.0% (2/6, 90% CI: 6.0–73.0%) in patients with high tumor mutational burden (TMB), 12.5% (2/16, 90% CI: 2.0–34.0%) in patients with intermediate TMB, and 0% (0/3, 90% CI: 0.0–53.6%) in patients with low TMB. Berzosertib plus gemcitabine was well tolerated in patients with advanced, pre-treated NSCLC. Based on the observed clinical efficacy, future clinical trials should involve genomically selected patients. [ABSTRACT FROM AUTHOR]

Radiation therapy has been used worldwide for many decades as a therapeutic regimen for the treatment of different types of cancer. Just over 50% of cancer patients are treated with radiotherapy alone or with other types of antitumor therapy. Radiation can induce different types of cell damage: directly, it can induce DNA single- and double-strand breaks; indirectly, it can induce the formation of free radicals, which can interact with different components of cells, including the genome, promoting structural alterations. During treatment, radiosensitive tumor cells decrease their rate of cell proliferation through cell cycle arrest stimulated by DNA damage. Then, DNA repair mechanisms are turned on to alleviate the damage, but cell death mechanisms are activated if damage persists and cannot be repaired. Interestingly, some cells can evade apoptosis because genome damage triggers the cellular overactivation of some DNA repair pathways. Additionally, some surviving cells exposed to radiation may have alterations in the expression of tumor suppressor genes and oncogenes, enhancing different hallmarks of cancer, such as migration, invasion, and metastasis. The activation of these genetic pathways and other epigenetic and structural cellular changes in the irradiated cells and extracellular factors, such as the tumor microenvironment, is crucial in developing tumor radioresistance. The tumor microenvironment is largely responsible for the poor efficacy of antitumor therapy, tumor relapse, and poor prognosis observed in some patients. In this review, we describe strategies that tumor cells use to respond to radiation stress, adapt, and proliferate after radiotherapy, promoting the appearance of tumor radioresistance. Also, we discuss the clinical impact of radioresistance in patient outcomes. Knowledge of such cellular strategies could help the development of new clinical interventions, increasing the radiosensitization of tumor cells, improving the effectiveness of these therapies, and increasing the survival of patients. [ABSTRACT FROM AUTHOR]

Radiation therapy has been used worldwide for many decades as a therapeutic regimen for the treatment of different types of cancer. Just over 50% of cancer patients are treated with radiotherapy alone or with other types of antitumor therapy. Radiation can induce different types of cell damage: directly, it can induce DNA single- and double-strand breaks; indirectly, it can induce the formation of free radicals, which can interact with different components of cells, including the genome, promoting structural alterations. During treatment, radiosensitive tumor cells decrease their rate of cell proliferation through cell cycle arrest stimulated by DNA damage. Then, DNA repair mechanisms are turned on to alleviate the damage, but cell death mechanisms are activated if damage persists and cannot be repaired. Interestingly, some cells can evade apoptosis because genome damage triggers the cellular overactivation of some DNA repair pathways. Additionally, some surviving cells exposed to radiation may have alterations in the expression of tumor suppressor genes and oncogenes, enhancing different hallmarks of cancer, such as migration, invasion, and metastasis. The activation of these genetic pathways and other epigenetic and structural cellular changes in the irradiated cells and extracellular factors, such as the tumor microenvironment, is crucial in developing tumor radioresistance. The tumor microenvironment is largely responsible for the poor efficacy of antitumor therapy, tumor relapse, and poor prognosis observed in some patients. In this review, we describe strategies that tumor cells use to respond to radiation stress, adapt, and proliferate after radiotherapy, promoting the appearance of tumor radioresistance. Also, we discuss the clinical impact of radioresistance in patient outcomes. Knowledge of such cellular strategies could help the development of new clinical interventions, increasing the radiosensitization of tumor cells, improving the effectiveness of these therapies, and increasing the survival of patients.

The Saccharomyces cerevisiae silencing information regulator (SIR) complex contains up to four proteins, namely Sir1, Sir2, Sir3, and Sir4. While Sir2 encodes a NAD-dependent histone deacetylase, other SIR proteins mainly function as structural and scaffold components through physical interaction with various proteins. The SIR complex displays different conformation and composition, including Sir2 homotrimer, Sir1-4 heterotetramer, Sir2-4 heterotrimer, and their derivatives, which recycle and relocate to different chromosomal regions. Major activities of the SIR complex are transcriptional silencing through chromosomal remodeling and modulation of DNA double-strand-break repair pathways. These activities allow the SIR complex to be involved in mating-type maintenance and switching, telomere and subtelomere gene silencing, promotion of nonhomologous end joining, and inhibition of homologous recombination, as well as control of cell aging. This review explores the potential link between epigenetic regulation and DNA damage response conferred by the SIR complex under various conditions aiming at understanding its roles in balancing cell survival and genomic stability in response to internal and environmental stresses. As core activities of the SIR complex are highly conserved in eukaryotes from yeast to humans, knowledge obtained in the yeast may apply to mammalian Sirtuin homologs and related diseases., (© 2024 The Authors. mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.)

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) encodes a transcriptional repressor involved in the DNA-damage response. A SUMOylation increase on HIC1 Lysine314 favors the direct transcriptional repression of SIRT1 and thus the P53-dependent apoptotic response to irreparable DNA double strand breaks (DSBs). HIC1 is also essential for DSBs repair but in a SUMOylation-independent manner. Here, we show that repairable DSBs induced by a 1 h Etoposide treatment results in three specific posttranslational modifications (PTMs) of HIC1. Two of these PTMs, phosphorylation of Serine 694 and Acetylation of Lysine 623 are located in the conserved HIC1 C-terminal region located downstream of the Zinc Finger DNA-binding domain. By contrast, phosphorylation of Serine 285 found in the poorly conserved central region is unique to the human protein. We showed that Ser694 phosphorylation is mediated mainly by the PIKK kinase ATM and is essential for the DNA repair activity of HIC1 as demonstrated by the lack of efficiency of the S694A point mutant in Comet assays. Thus, our results provide the first evidence for a functional role of the conserved HIC1 C-terminal region as a novel ATM substrate that plays an essential role in the cellular HIC1-mediated cellular response to repairable DSBs. • Upon induction of repairable DSBs, HIC1 is phosphorylated on Ser285 and Ser694 and acetylated of Lys623. • ATM-dependent phosphorylation of HIC1 on Ser694 is required for DNA repair activity in functional Comet assays. • The highly conserved C-terminus is an essential player in the DNA repair function exhibited by HIC1. • Here, we identify HIC1 as a previously unrecognized substrate of ATM during the DNA damage response. [ABSTRACT FROM AUTHOR]

VRK1 is a nuclear Ser-Thr chromatin kinase that does not mutate in cancer, and is overexpressed in many types of tumors and associated with a poor prognosis. Chromatin VRK1 phosphorylates several transcription factors, including p53, histones and proteins implicated in DNA damage response pathways. In the context of cell proliferation, VRK1 regulates entry in cell cycle, chromatin condensation in G2/M, Golgi fragmentation, Cajal body dynamics and nuclear envelope assembly in mitosis. This kinase also controls the initial chromatin relaxation associated with histone acetylation, and the non-homologous-end joining (NHEJ) DNA repair pathway, which involves sequential steps such as γH2AX, NBS1 and 53BP1 foci formation, all phosphorylated by VRK1, in response to ionizing radiation or chemotherapy. In addition, VRK1 can be an alternative target for therapies based on synthetic lethality strategies. Therefore, VRK1 roles on proliferation have a pro-tumorigenic effect. Functions regulating chromatin stability and DNA damage responses have a protective anti-tumor role in normal cells, but in tumor cells can also facilitate resistance to genotoxic treatments. [ABSTRACT FROM AUTHOR]

In recent years, roles of the immune system in immune surveillance of cancer have been explored using a variety of approaches. The roles of the adaptive immune system have been a major emphasis, but increasing evidence supports a role for innate immune effector cells such as natural killer (NK) cells in tumor surveillance. Here, we discuss some of the evidence for roles in tumor surveillance of innate immune cells. In particular, we focus on NK cells and other immune cells that express germline-encoded receptors, often labeled NK receptors. The impact of these receptors and the cells that express them on tumor suppression is summarized. We discuss in detail some of the pathways and events in tumor cells that induce or upregulate cell-surface expression of the ligands for these receptors, and the logic of how those pathways serve to identify malignant, or potentially malignant cells. How tumors often evade tumor suppression mediated by innate killer cells is another major subject of the review. We end with a discussion on some of the implications of the various findings with respect to possible therapeutic approaches.

In recent years, roles of the immune system in immune surveillance of cancer have been explored using a variety of approaches. The roles of the adaptive immune system have been a major emphasis, but increasing evidence supports a role for innate immune effector cells such as natural killer (NK) cells in tumor surveillance. Here, we discuss some of the evidence for roles in tumor surveillance of innate immune cells. In particular, we focus on NK cells and other immune cells that express germline-encoded receptors, often labeled NK receptors. The impact of these receptors and the cells that express them on tumor suppression is summarized. We discuss in detail some of the pathways and events in tumor cells that induce or upregulate cell-surface expression of the ligands for these receptors, and the logic of how those pathways serve to identify malignant, or potentially malignant cells. How tumors often evade tumor suppression mediated by innate killer cells is another major subject of the review. We end with a discussion on some of the implications of the various findings with respect to possible therapeutic approaches.

Abstract Background S-phase kinase-associated protein 2 (SKP2) is an oncogene and cell cycle regulator that specifically recognizes phosphorylated cell cycle regulator proteins and mediates their ubiquitination. Programmed cell death protein 4 (PDCD4) is a tumor suppressor gene that plays a role in cell apoptosis and DNA-damage response via interacting with eukaryotic initiation factor-4A (eIF4A) and P53. Previous research showed SKP2 may interact with PDCD4, however the relationship between SKP2 and PDCD4 is unclear. Methods To validate the interaction between SKP2 and PDCD4, mass spectrometric analysis and reciprocal co-immunoprecipitation (Co-IP) experiments were performed. SKP2 stably overexpressed or knockdown breast cancer cell lines were established and western blot was used to detect proteins changes before and after radiation. In vitro and in vivo experiments were performed to verify whether SKP2 inhibits cell apoptosis and promotes DNA-damage response via PDCD4 suppression. SMIP004 was used to test the effect of radiotherapy combined with SKP2 inhibitor. Results We found that SKP2 remarkably promoted PDCD4 phosphorylation, ubiquitination and degradation. SKP2 promoted cell proliferation, inhibited cell apoptosis and enhanced the response to DNA-damage via PDCD4 suppression in breast cancer. SKP2 and PDCD4 showed negative correlation in human breast cancer tissues. Radiotherapy combine with SKP2 inhibitor SMIP004 showed significant inhibitory effects on breast cancer cells in vitro and in vivo. Conclusions We identify PDCD4 as an important ubiquitination substrate of SKP2. SKP2 promotes breast cancer tumorigenesis and radiation tolerance via PDCD4 degradation. Radiotherapy combine with SKP2-targeted adjuvant therapy may improve breast cancer patient survival in clinical medicine.

The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world. [ABSTRACT FROM AUTHOR]

Background: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage. Results: We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorageindependent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates. Conclusion: NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

The response to DNA damage is the mechanism that allows the interaction between stress signals, inflammatory secretions, DNA repair, and maintenance of cell and tissue homeostasis. Adipocyte dysfunction is the cellular trigger for various disease states such as insulin resistance, diabetes, and obesity, among many others. Previously, our group demonstrated that adipogenesis per se, from mesenchymal/stromal stem cells derived from human adipose tissue (hASCs), involves an accumulation of DNA damage and a gradual loss of the repair capacity of oxidative DNA damage. Therefore, our objective was to identify whether healthy adipocytes differentiated for the first time from hASCs, when receiving inflammatory signals induced with TNFα, were able to persistently activate the DNA Damage Response and thus trigger adipocyte dysfunction. We found that TNFα at similar levels circulating in obese humans induce a sustained response to DNA damage response as part of the Senescence-Associated Secretory Phenotype. This mechanism shows the impact of inflammatory environment early affect adipocyte function, independently of aging.

HOPS is a ubiquitin-like protein implicated in many aspects of cellular function including the regulation of mitotic activity, proliferation, and cellular stress responses. In this study, we focused on the complex relationship between HOPS and the tumor suppressor p53, investigating both transcriptional and non-transcriptional p53 responses. Here, we demonstrated that Hops heterozygous mice and mouse embryonic fibroblasts exhibit an impaired DNA-damage response to etoposide-induced double-strand breaks when compared to wild-type genes. Specifically, alterations in HOPS levels caused significant defects in the induction of apoptosis, including a reduction in p53 protein level and percentage of apoptotic cells. We also analyzed the effect of reduced HOPS levels on the DNA-damage response by examining the transcript profiles of p53-dependent genes, showing a suggestive deregulation of the mRNA levels for a number of p53-dependent genes. Taken together, these results show an interesting haploinsufficiency effect mediated by Hops monoallelic deletion, which appears to be enough to destabilize the p53 protein and its functions. Finally, these data indicate a novel role for Hops as a tumor-suppressor gene in DNA damage repair in mammalian cells.

Over recent years, several Cys2-His2 (C2H2) domain-containing proteins have emerged as critical players in repairing DNA-double strand breaks. Human FLYWCH1 is a newly characterised nuclear transcription factor with (C2H2)-type zinc-finger DNA-binding domains. Yet, our knowledge about FLYWCH1 is still in its infancy. This study explores the expression, role and regulation of FLYWCH1 in the context of DNA damage and repair. We provide evidence suggesting a potential contribution of FLYWCH1 in facilitating the recruitment of DNA-damage response proteins (DDRPs). We found that FLYWCH1 colocalises with γH2AX in normal fibroblasts and colorectal cancer (CRC) cell lines. Importantly, our results showed that enforced expression of FLYWCH1 induces the expression of γH2AX, ATM and P53 proteins. Using an ATM-knockout (ATMKO) model, we indicated that FLYWCH1 mediates the phosphorylation of H2AX (Ser139) independently to ATM expression. On the other hand, the induction of DNA damage using UV-light induces the endogenous expression of FLYWCH1. Conversely, cisplatin treatment reduces the endogenous level of FLYWCH1 in CRC cell lines. Together, our findings uncover a novel FLYWCH1/H2AX phosphorylation axis in steady-state conditions and during the induction of the DNA-damage response (DDR). Although the role of FLYWCH1 within the DDR machinery remains largely uncharacterised and poorly understood, we here report for the first-time findings that implicate FLYWCH1 as a potential participant in the DNA damage response signaling pathways.

Background: Perioperative cisplatin-based chemotherapy (CBC) can improve the outcome of patients with muscle-invasive bladder cancer (MIBC), but it is still to be defined which patients benefit. Mutations in DNA damage response genes (DDRG) can predict the response to CBC. The value of DDRG expression as a marker of CBC treatment effect remains unclear. Material and methods: RNA expression of the nine key DDRG (BCL2, BRCA1, BRCA2, ERCC2, ERCC6, FOXM1, RAD50, RAD51, and RAD52) was assessed by qRT-PCR in a cohort of 61 MICB patients (median age 66 y, 48 males, 13 females) who underwent radical cystectomy in a tertiary care center. The results were validated in the The Cancer Genome Atlas (TCGA) cohort of MIBC (n = 383). Gene expression was correlated with disease-free survival (DFS) and overall survival (OS). Subgroup analyses were performed in patients who received adjuvant cisplatin-based chemotherapy (ACBC) (Mannheim n = 20 and TCGA n = 75). Results: Low expression of RAD52 was associated with low DFS in both the Mannheim and the TCGA cohorts (Mannheim: p = 0.039; TCGA: p = 0.017). This was especially apparent in subgroups treated with ACBC (Mannheim: p = 0.0059; TCGA: p = 0.012). Several other genes showed an influence on DFS in the Mannheim cohort (BRCA2, ERCC2, FOXM1) where low expression was associated with poor DFS (p < 0.05 for all). This finding was not fully supported by the data in the TCGA cohort, where high expression of FOXM1 and BRCA2 correlated with poor DFS. Conclusion: Low expression of RAD52 correlated with decreased DFS in the Mannheim and the TCGA cohort. This effect was especially pronounced in the subset of patients who received ACBC, making it a promising indicator for response to ACBC on the level of gene expression.

Abstract Background The error-free branch of the DNA-damage tolerance (DDT) pathway is orchestrated by Lys63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA), and this polyubiquitination is mediated by a Ubc13-Uev complex in yeast. We have previously cloned OsUBC13 from rice, whose product functions as an E2 to promote Lys63-linked ubiquitin chain assembly in the presence of yeast or human Uev. Results Here we identify four highly conserved UEV1 genes in rice whose products are able to form stable heterodimers with OsUbc13 and mediate Lys63-linked ubiquitin chain assembly. Expression of OsUEV1s is able to rescue the yeast mms2 mutant from death caused by DNA-damaging agents. Interestingly, OsUev1A contains a unique C-terminal tail with a conserved prenylation site not found in the other three OsUev1s, and this post-translational modification appears to be required for its unique subcellular distribution and association with the membrane. The analysis of OsUEV1 expression profiles obtained from the Genevestigator database indicates that these genes are differentially regulated. Conclusions We speculate that different OsUev1s play distinct roles by serving as a regulatory subunit of the Ubc13-Uev1 complex to respond to diverse cellular, developmental and environmental signals.

• Upon DNA damage the epigenetic landscape orchestrates DNA repair • Sirtuins have a relevant role in DNA repair. • Sirtuins modulate repair pathways through deacetylation of repair factors or influencing chromatin accessibility. Cellular DNA is constantly challenged by damage-inducing factors derived from exogenous or endogenous sources. Thus, to protect against DNA damage, cells have evolved complex and finely regulated mechanisms collectively known as DNA-damage response (DDR). However, DNA repair in eukaryotes does not occur merely in naked DNA but also within a highly organized and compacted chromatin environment, which ultimately participates in regulating DDR pathways. Thus, remodelling of the chromatin surrounding areas containing damaged DNA is required to allow access to the DNA repair machinery, as well as post-translational modifications in many repair factors to recruit and activate them at the damaged site. Notably, proteins such as sirtuins, which are NAD+-dependent deacetylases, have evolved to modulate multiple repair pathways through deacetylation of some repair factors, influencing chromatin accessibility or indirectly modulating cell cycle and preventing oxidative stress. In this way, the purpose of this review is to summarize the recent knowledge that links sirtuins with DNA repair, with a particular emphasis on the molecular mechanisms associated with coordination and regulation of this vital process. [ABSTRACT FROM AUTHOR]

Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM−/− cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2 −/− Fancd2 −/− mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2 −/− Fancd2 −/− HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging. [Display omitted] • Genotoxic aldehydes in HSCs cause accelerated aging and myeloid bias • Aged HSCs show increased activation of p53 response • p53 activation is associated with enrichment of myeloid-biased HSCs • Deletion of p53 rescues HSCs from aging and myeloid bias Wang et al. show that DNA damage caused by genotoxic aldehydes in HSCs results in accelerated aging and differentiation bias to myeloid cells. This HSC aging is driven by the p53 pathway and indicates that aging in the blood system can arise as a consequence of the DNA-damage response. [ABSTRACT FROM AUTHOR]

Nuclear β-dystroglycan (β-DG) is involved in the maintenance of nuclear architecture and function. Nonetheless, its relevance in defined nuclear processes remains to be determined. In this study we generated a C2C12 cell-based DG-null model using CRISPR-Cas9 technology to provide insights into the role of β-DG on nuclear processes. Since DG-null cells exhibited decreased levels of lamin B1, we aimed to elucidate the contribution of DG to senescence, owing to the central role of lamin B1 in this pathway. Remarkably, the lack of DG enables C2C12 cells to acquire senescent features, including cell-cycle arrest, increased senescence-associated-β-galactosidase activity, heterochromatin loss, aberrant nuclear morphology and nucleolar disruption. We demonstrated that genomic instability is one driving cause of the senescent phenotype in DG-null cells via the activation of a DNA-damage response associated with mitotic failure, as shown by the presence of multipolar mitotic spindles, which in turn induced the formation of micronuclei and γH2AX foci (DNA-damage marker), telomere shortening and p53/p21 upregulation. Altogether, these events might ultimately lead to premature senescence, impeding the replication of the damaged genome. In summary, we present evidence supporting a role for DG in protecting against senescence, through the maintenance of proper lamin B1 expression/localization and proper mitotic spindle organization.

The MiT/TFE family of transcription factors (MITF, TFE3, and TFEB), which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents.

While the role of genus alpha human papillomaviruses in the tumorigenesis and tumor maintenance of anogenital and oropharyngeal cancers is well-established, the role of genus beta human papilloviruses (β-HPVs) in non-melanoma skin cancers (NMSCs) is less certain. Persistent β-HPV infections cause NMSCs in sun-exposed skin of people with a rare genetic disorder, epidermodysplasia verruciformis. However, β-HPV infections in people without epidermodysplasia verruciformis are typically transient. Further, β-HPV gene expression is not necessary for tumor maintenance in the general population as on average there is fewer than one copy of the β-HPV genome per cell in NMSC tumor biopsies. Cell culture, epidemiological, and mouse model experiments support a role for β-HPV infections in the initiation of NMSCs through a “hit and run” mechanism. The virus is hypothesized to act as a cofactor, augmenting the genome destabilizing effects of UV. Supporting this idea, two β-HPV proteins (β-HPV E6 and E7) disrupt the cellular response to UV exposure and other genome destabilizing events by abrogating DNA repair and deregulating cell cycle progression. The aberrant damage response increases the likelihood of oncogenic mutations capable of driving tumorigenesis independent of a sustained β-HPV infection or continued viral protein expression. This review summarizes what is currently known about the deleterious effects of β-HPV on genome maintenance in the context of the virus's putative role in NMSC initiation.

The protein phosphatase 2C family (PP2C), belonging the protein phosphatases, is a kind of importantly specific serine/threonine dephosphatase. Recent studies show that PP2C control a lot of critical cellular functions:proliferation, cell cycle arrest, senescence and programmed cell death, apoptosis and autophagy, showing potentially biological role in regulating immune response, aging, neurogenesis and tumorigenesis. Several dominant cellular signaling pathways controlled by PP2C gene subtypes were reviewed in this paper, such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase(PI3K)-AKT, transforming growth factor β(TGF-β)/Smads, transcription factor nuclear factor-κB (NF-κB) and DNA-damage response pathways, which offered new ways to clarify the molecular basis and regular mechanisms for those physiological and pathological processes. [ABSTRACT FROM AUTHOR]

The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies. [ABSTRACT FROM AUTHOR]

In this study, we report the identification and functional characterization of three Brachypodium distachyon UEV genes. All three BdUev1s form heterodimers with BdUbc13s, which are capable of catalyzing Lys63-linked polyubiquitination in vitro. The three BdUEV1 genes are also able to functionally complement the budding yeast mms2 mutant defective in DNA-damage tolerance. BdUev1A differs from the other two BdUev1s in that it contains an 18-amino acid tail, which appears to compromise its function in yeast, as deletion of this tail restores full function. BdUev1A is located in the cytoplasm and plasma membrane but not in the nucleus, whereas BdUev1B, BdUev1C and the C-terminally-truncated BdUev1A are mainly found in the nucleus. These and the BdUEV1 gene expression analysis allow us to speculate that although all three BdUev1s function by promoting Lys63-linked polyubiquitination, BdUev1B and BdUev1C are involved in DNA-damage response and possibly other nuclear functions, while BdUev1A is required for non-nuclear function(s).

Herpesviruses are important pathogens that can cause significant morbidity and mortality in the human population. Herpesviruses have a double-stranded DNA genome, and viral genome replication takes place inside the nucleus. Upon entering the nucleus, herpesviruses have to overcome the obstacle of cellular proteins in order to enable viral gene expression and genome replication. In this review, we want to highlight cellular proteins that sense incoming viral genomes of the DNA-damage repair (DDR) pathway and of PML-nuclear bodies (PML-NBs) that all can act as antiviral restriction factors within the first hours after the viral genome is released into the nucleus. We show the function and significance of both nuclear DNA sensors, the DDR and PML-NBs, and demonstrate for three human herpesviruses of the alpha-, beta- and gamma-subfamilies, HSV-1, HCMV and KSHV respectively, how viral tegument proteins antagonize these pathways.

Many aspects of cellular physiology, including cellular response to genotoxic stress, are related to the circadian rhythmicity induced by the molecular clock. The current study investigated if the cellular response to DNA damage is in relation to endogenous expression levels of the PER2 protein, a key component of the molecular regulatory system that confers rhythmicity in mammalian cells. Human normal fibroblasts (CCD-34Lu) were subjected to serum shock to induce circadian oscillations of the PER2 protein and then irradiated with γ- rays at times corresponding to the trough and peak expression of the PER2 protein. To better examine cellular response to DNA damage, the experiments performed in this study were carried out in non-proliferating CCD-34Lu fibroblasts in order to maintain the cell and circadian cycles separated while they were being exposed to genotoxic stress. Study results demonstrated that clonogenic cell survival, double-strand break repair kinetics, and TP53 protein levels were affected in the cells irradiated at the trough than in those irradiated at peak expression of the PER2 protein. [ABSTRACT FROM AUTHOR]

The retinoblastoma protein (Rb), which typically functions as a transcriptional repressor of E2F-regulated genes, represents a major control hub of the cell cycle. Here, we show that loss of the Arabidopsis Rb homolog RETINOBLASTOMA- RELATED 1 ( RBR1) leads to cell death, especially upon exposure to genotoxic drugs such as the environmental toxin aluminum. While cell death can be suppressed by reduced cell-proliferation rates, rbr1 mutant cells exhibit elevated levels of DNA lesions, indicating a direct role of RBR1 in the DNA-damage response ( DDR). Consistent with its role as a transcriptional repressor, we find that RBR1 directly binds to and represses key DDR genes such as RADIATION SENSITIVE 51 ( RAD51), leaving it unclear why rbr1 mutants are hypersensitive to DNA damage. However, we find that RBR1 is also required for RAD51 localization to DNA lesions. We further show that RBR1 is itself targeted to DNA break sites in a CDKB1 activity-dependent manner and partially co-localizes with RAD51 at damage sites. Taken together, these results implicate RBR1 in the assembly of DNA-bound repair complexes, in addition to its canonical function as a transcriptional regulator. [ABSTRACT FROM AUTHOR]

Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR) and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi) machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs) and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports suggest that ncRNAs are involved in DDR signaling and homology-mediated DNA repair. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair.

Over the last decade, the treatment of tumor patients has been revolutionized by the highly successful introduction of novel targeted therapies, in particular small-molecule kinase inhibitors and monoclonal antibodies, as well as by immunotherapies. Depending on the mutational status, BRAF and MEK inhibitor combinations or immune checkpoint inhibitors are current first-line treatments for metastatic melanoma. However, despite great improvements of survival rates limitations due to tumor heterogeneity, primary and acquired therapy resistance, immune evasion, and economical considerations will need to be overcome. Accordingly, ongoing clinical trials explore the individualized use of small-molecule drugs in new targeted therapy combinations based on patient parameters and tumor biopsies. With focus on melanoma therapy this review aims at providing a comprehensive overview of such novel alternative and combinational therapy strategies currently emerging from basic research. The molecular principles and drug classes that may hold promise for improved tumor therapy combination regimens including kinase inhibition, induction of apoptosis, DNA-damage response inhibition, epigenetic reprogramming, telomerase inhibition, redox modulation, metabolic reprogramming, proteasome inhibition, cancer stem cell transdifferentiation, immune cell signaling modulation, and others, are explained in brief. In addition, relevant targeted therapy combinations in current clinical trials and individualized treatment strategies are highlighted.