Affect DNA Damage Research Articles

Roles of cytochromes P450 and ribosome inhibition in the interaction between two preoccupying mycotoxins, aflatoxin B1 and deoxynivalenol

Mycotoxins are a threat to human and animal health. Climate change increases their occurrence and our dietary exposure. Although humans and animals are concomitantly exposed to several mycotoxins, their combined effects are poorly characterised. This study investigated the interaction between aflatoxin B1 (AFB1), the most potent natural carcinogen, and deoxynivalenol (DON), which is among the most prevalent mycotoxins. AFB1 is associated with hepatocellular carcinoma through its bioactivation by cytochrome P450 (CYP450) enzymes; while DON induces ribotoxic stress leading to an alteration of intestinal, immune and hepatic functions. Analysis of DNA damage biomarkers γ-H2AX and 53BP1 revealed that DON reduces the genotoxicity of AFB1. This effect was mimicked with cycloheximide, another ribosome inhibitor; moreover DOM-1, a DON-derivative lacking ribosome inhibition, did not affect DNA damage. Exposure to DON, alone or in combination with AFB1, decreased the protein levels and/or activities of CYP1A2 and CYP3A4 in a time- and dose-dependent manner. Altogether, these results revealed an original interaction between DON and AFB1, DON inhibiting the genotoxicity of AFB1. The underlying mechanism involves ribosome inhibition by DON and the subsequent impairment of CYP450s, responsible for the bioactivation of AFB1. This work highlights the importance of studying mycotoxins not only individually but also in mixture and of considering food contaminants as part of the exposome.

Circ_0025373 inhibits carbon black nanoparticles-induced malignant transformation of human bronchial epithelial cells by affecting DNA damage through binding to MSH2

Carbon black nanoparticles (CBNPs) have been demonstrated to induce DNA damage in epithelial cells. However, the potential of the damage to initiate carcinogenesis and the underlying mechanism remain poorly understood. Therefore, we constructed an in vitro model of malignant transformation of human bronchial epithelial cells (16HBE-T) by treating 40 μg/mL CBNPs for 120 passages. We observed tumor-like transformation and sustained DNA damage. Using transcriptome sequencing and RIP-seq, we identified the overexpression of the critical DNA mismatch repair genes MutS homolog 2 (MSH2) and its related circular RNA, circ_0025373, in the 16HBE-T cells. Mechanistically, circ_0025373 was found to inhibit DNA damage by binding to MSH2, thereby modifying its expression and influencing its nuclear and cytoplasmic distribution, which lead to inhibition of CBNP-induced malignant transformation of human bronchial epithelial cells. Our findings provide novel evidence on the carcinogenicity of CBNPs, and offer biological insights into the potential epigenetic regulation and potential therapeutic targets for lung carcinogenesis.

NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

Olaparib has been approved as a therapeutic option for metastatic pancreatic ductal adenocarcinoma patients with BRCA1/2 mutations. However, a significant majority of pancreatic cancer patients have inherent resistance or develop tolerance to olaparib. It is crucial to comprehend the molecular mechanism underlying olaparib resistance to facilitate the development of targeted therapies for pancreatic cancer. In this study, we conducted an analysis of the DepMap database to investigate gene expression variations associated with olaparib sensitivity. Our findings revealed that NLRP4 upregulation contributes to increased resistance to olaparib in pancreatic cancer cells, both in vitro and in vivo. RNA sequencing and Co-IP MS analysis revealed that NLRP4 is involved in the DNA damage response and autophagy pathway. Our findings confirmed that NLRP4 enhances the capacity for DNA repair and induces the production of significant levels of reactive oxygen species (ROS) and autophagy in response to treatment with olaparib. Specifically, NLRP4-generated mitochondrial ROS promote autophagy in pancreatic cancer cells upon exposure to olaparib. However, NLRP4-induced ROS do not affect DNA damage. The inhibition of mitochondrial ROS using MitoQ and autophagy using chloroquine (CQ) may render cells more susceptible to the effects of olaparib. Taken together, our findings highlight the significant roles played by NLRP4 in the processes of autophagy and DNA repair when pancreatic cancer cells are treated with olaparib, thereby suggesting the potential therapeutic utility of olaparib in pancreatic cancer patients with low NLRP4 expression.

PEITC Induces DNA Damage and Inhibits DNA Repair-Associated Proteins in Human Retinoblastoma Cells In Vitro.

Phenethyl isothiocyanate (PEITC), a natural product, exists in biological activities, including anticancer activity in many human cancer cells. No information shows that PEITC affects DNA damage in human retinoblastoma (RB) cells in vitro. In this study, the aim of experiments was to determine whether PEITC decreased total viable cell number or not by inducing protein expressions involved in DNA damage and repair in Y79 RB cells in vitro. Total cell viability was measured by PI exclusion assay, and PEITC reduced the total Y79 viable cell numbers in a dose-dependent manner. DNA condensation and DNA impairment were conducted by DAPI staining and comet assays, respectively, in Y79 cells. The findings show that PEITC induced DNA condensation dose-dependently based on the brighter fluorescence of cell nuclei stained by DAPI staining. PEITC-induced DNA damage showed a more extended DNA migration smears than that of the control, which was performed by a comet assay. Western blotting was performed to measure the protein expressions involved in DNA damage and repair, which showed that PEITC at 2.5-10 μM increased NRF2, HO-1, SOD (Mn), and catalase; however, it decreased SOD (Cu/Zn) except 10 μM PEITC treatment, and decreased glutathione, which were associated with oxidativestress. Furthermore, PEITC increased DNA-PK, MDC1, H2A.XpSer139, ATMpSer1981, p53, p53pSer15, PARP, HSP70, and HSP90, but decreased TOPIIα, TOPIIβ, and MDM2pSer166 that were associated with DNA damage and repair mechanism in Y79 cells. The examination from confocal laser microscopy shows that PEITC increased H2A.XpSer139 and p53pSer15, and decreased glutathione and TOPIIα in Y79 cells. In conclusion, the cytotoxic effects of PEITC on reducing the number of viable cells may be due to the induction of DNA damage and the alteration of DNA repair proteins in Y79 cells in vitro.

Obesity-associated non-oxidative genotoxic stress alters trophoblast turnover in human first-trimester placentas.

Placental growth is most rapid during the first trimester (FT) of pregnancy, making it vulnerable to metabolic and endocrine influences. Obesity, with its inflammatory and oxidative stress, can cause cellular damage. We hypothesized that maternal obesity increases DNA damage in the FT placenta, affecting DNA damage response and trophoblast turnover. Examining placental tissue from lean and obese non-smoking women (4-12 gestational weeks), we observed higher overall DNA damage in obesity (COMET assay). Specifically, DNA double-strand breaks were found in villous cytotrophoblasts (vCTB; semi-quantitative γH2AX immunostaining), while oxidative DNA modifications (8-hydroxydeoxyguanosine; FPG-COMET assay) were absent. Increased DNA damage in obese FT placentas did not correlate with enhanced DNA damage sensing and repair. Indeed, obesity led to reduced expression of multiple DNA repair genes (mRNA array), which were further shown to be influenced by inflammation through in vitro experiments using tumor necrosis factor-α treatment on FT chorionic villous explants. Tissue changes included elevated vCTB apoptosis (TUNEL assay; caspase-cleaved cytokeratin 18), but unchanged senescence (p16) and reduced proliferation (Ki67) of vCTB, the main driver of FT placental growth. Overall, obesity is linked to heightened non-oxidative DNA damage in FT placentas, negatively affecting trophoblast growth and potentially leading to temporary reduction in early fetal growth.

High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.

Ccq1 restrains Mre11-mediated degradation to distinguish short telomeres from double-strand breaks.

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.

Exploring the effects of topoisomerase II inhibitor XK469 on anthracycline cardiotoxicity and DNA damage.

Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIβ has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIβ selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and β) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.

DNA break clustering as a predictor of cell death across various radiation qualities: influence of cell size, cell asymmetry, and beam orientation

Abstract Cosmic radiation, composed of high charge and energy (HZE) particles, causes cellular DNA damage that can result in cell death or mutation that can evolve into cancer. In this work, a cell death model is applied to several cell lines exposed to HZE ions spanning a broad range of linear energy transfer (LET) values. We hypothesize that chromatin movement leads to the clustering of multiple double strand breaks (DSB) within one radiation-induced foci (RIF). The survival probability of a cell population is determined by averaging the survival probabilities of individual cells, which is function of the number of pairwise DSB interactions within RIF. The simulation code RITCARD was used to compute DSB. Two clustering approaches were applied to determine the number of RIF per cell. RITCARD outputs were combined with experimental data from four normal human cell lines to derive the model parameters and expand its predictions in response to ions with LET ranging from ~0.2 keV/μm to ~3000 keV/μm. Spherical and ellipsoidal nuclear shapes and two ion beam orientations were modeled to assess the impact of geometrical properties on cell death. The calculated average number of RIF per cell reproduces the saturation trend for high doses and high-LET values that is usually experimentally observed. The cell survival model generates the recognizable bell shape of LET dependence for the relative biological effectiveness (RBE). At low LET, smaller nuclei have lower survival due to increased DNA density and DSB clustering. At high LET, nuclei with a smaller irradiation area—either because of a smaller size or a change in beam orientation—have a higher survival rate due to a change in the distribution of DSB/RIF per cell. If confirmed experimentally, the geometric characteristics of cells would become a significant factor in predicting radiation-induced biological effects. Insight Box: High-charge and energy (HZE) ions are characterized by dense linear energy transfer (LET) that induce unique spatial distributions of DNA damage in cell nuclei that result in a greater biological effect than sparsely ionizing radiation like X-rays. HZE ions are a prominent component of galactic cosmic ray exposure during human spaceflight and specific ions are being used for radiotherapy. Here, we model DNA damage clustering at sub-micrometer scale to predict cell survival. The model is in good agreement with experimental data for a broad range of LET. Notably, the model indicates that nuclear geometry and ion beam orientation affect DNA damage clustering, which reveals their possible role in mediating cell radiosensitivity.

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

MiR-302d Targeting of CDKN1A Regulates DNA Damage and Steroid Hormone Secretion in Bovine Cumulus Cells.

(1) Background: DNA damage in cumulus cells hinders oocyte maturation and affects steroid hormone secretion. It is crucial to identify the key factors that regulate cellular DNA damage and steroid hormone secretion. (2) Methods: Treatment of bovine cumulus cells with bleomycin to induce DNA damage. The effects of DNA damage on cell biology were determined by detecting changes in DNA damage degree, cell cycle, viability, apoptosis, and steroid hormones. It was verified that mir-302d targeted regulation of CDKN1A expression, and then affected DNA damage and steroid hormone secretion in cumulus cells. (3) Results: Bleomycin induced increased DNA damage, decreased G1-phase cells, increased S-phase cells, inhibited proliferation, promoted apoptosis, affected E2 and P4 secretion, increased CDKN1A expression, and decreased miR-302d expression. Knockdown of CDKN1A reduced DNA damage, increased G1-phase cells, decreased G2-phase cells, promoted proliferation, inhibited apoptosis, increased E2 and P4 secretion, and increased the expression of BRCA1, MRE11, ATM, CDK1, CDK2, CCNE2, STAR, CYP11A1, and HSD3B1. The expression of RAD51, CCND1, p53, and FAS was decreased. Overexpression of CDKN1A resulted in the opposite results. miR-302d targets CDKN1A expression to regulate DNA damage and then affects the cell cycle, proliferation, apoptosis, steroid hormone secretion, and the expression of related genes. (4) Conclusions: miR-302d and CDKN1A were candidate molecular markers for the diagnosis of DNA damage in bovine cumulus cells.

The Impact of Clonal Hematopoiesis on Clinical Outcomes and Clonal Dynamics in Older Lymphoma Patients Receiving Chemotherapy

Background: CHIP is more commonly encountered in cancer patients, such as those with lymphoma, than in the general population likely attributable to age, cancer risk factors and treatment. While the adverse health associations with clonal hematopoiesis of indeterminate potential (CHIP) are established, its impact on the immediate tolerability of chemotherapy and longer-term blood count reconstitution are unknown. Also, while it is known that certain CHIP drivers, especially those affecting DNA damage responses, may be selected during cancer treatment, comprehensive studies of CHIP clonal dynamics along the course of treatment are lacking. We hypothesized that CHIP may be a risk factor for chemotherapy complications in older lymphoma patients related to dysfunctional bone marrow and blood cells. Our Aims were to ascertain CHIP status using targeted sequencing, investigate the influence of CHIP on adverse events during chemotherapy, and the changes in clonal dynamics. Methods: We evaluated the prevalence, associations, clonal dynamics and impact of CHIP on treatment toxicity and blood count recovery in lymphoma patients aged > 60 undergoing 1 st or second line cytotoxic chemotherapy. CHIP (Ion torrent NGS, 53 genes including coverage for DNA damage response (DDR) mutations: TP53, PPM1D, CHEK2 and SRCAP was performed on peripheral blood (PB) samples at baseline, after 3 chemotherapy cycles, at 6 and 12 months post chemotherapy. Ion Torrent VAF cutoff was 2%. Integrated genomics viewer (IGV) was used to detect known variants at < 2% VAF. We compared blood count parameters and treatment complications between those with and without CHIP overall or DDR mutations in particular. Results: 78 of planned 180 patients are available for analysis and 74 with non-lymphoma-related mutations are included. Median age was 70, 58% female. 61% had DLBCL and 19% follicular lymphoma. 88% received first line chemotherapy (68% anthracycline containing) and 15% had received radiation in the past. With a median follow up of 19 months, 82% remain alive. All CHIP (and DDR) mutations were detected in 27% (1.4%) patients at baseline, in 33.3% (5.3%) after cycle 3, in 47.2% (27.8%) at 6 months and 38.7% (22.6%) 12 months post chemotherapy respectively (Figure 1). IGV inspection and deep sequencing confirmed that most DDR variants were present at <2% VAF at baseline and expanded with treatment. Moreover, DDR CHIP peaked at the 6 months post-chemotherapy timepoint, with contraction in overall prevalence and VAF by 12 months. In contrast, most non-DDR mutations remained stable or increased slightly. The clonal dynamics of CHIP and DDR mutations are visible in Figure 2. Comparing those, with or without baseline or acquired CHIP or DDR mutations, there were no significant differences in most baseline clinical and laboratory characteristics, chemotherapy dose delays or reductions. In particular, MCV and RDW did not differ at baseline or increase preferentially in those with or without mutations. However, patients with DDR mutations had higher eosinophil counts at baseline (0.2 versus 0.1, p=.01), higher eosinophil counts over time (p=.0003), were more likely to visit an emergency department (86% versus 52%, p=.03), and sustain infections on treatment (43% versus 20% p=.07). A higher percentage of patients with DDR mutations had monocyte counts that exceeded normal reference ranges 6 months post (23% versus 10%, p=.01) and (trend) 12 months post (30% versus 12.9%, p=0.3). More patients with CHIP had marginal zone lymphoma (21% versus 2.5%, p=.04), experienced fewer neurologic toxicities (22% versus 52%, p=.006) but more respiratory infections (19.4% versus 2.3%, p=.02). The monocytes of patients with CHIP (compared without) surpassed baseline monocyte counts at 6 months (57.1% versus 16%, p=.004), and 12 months (52.3% versus 20%, p=0.03) and exceeded normal reference range (21% versus 4%, p=.04) at 6 months. Conclusion: The prevalence of CHIP is high in older lymphoma patients, with preferential expansion of clones harboring DDR mutations during treatment. Most of these DDR variants would fall below routine clinical NGS reporting thresholds if sampled at baseline, or after post-therapy contraction. DDR CHIP and CHIP overall associate with different complication rates on chemotherapy and both associate with post-treatment monocytosis. These novel DDR CHIP immune alterations warrant further mechanistic study.

Allyl isothiocyanate induces DNA damage and inhibits DNA repair-associated proteins in a human gastric cancer cells in vitro.

Allyl isothiocyanate (AITC) is abundant in cruciferous vegetables and it present pharmacological activity including anticancer activity in many types of human cancer cells in vitro and in vivo. Currently, no available information to show AITC affecting DNA damage and repair-associated protein expression in human gastric cancer cells. Therefore, in the present studies, we investigated AITC-induced cytotoxic effects on human gastric cancer in AGS and SNU-1 cells whether or not via the induction of DNA damage and affected DNA damage and repair associated poteins expressions in vitro. Cell viability and morphological changes were assayed by flow cytometer and phase contrast microscopy, respectively, the results indicated AITC induced cell morphological changes and decreased total viable cells in AGS and SNU-1 cells in a dose-dependently. AITC induced DNA condensation and damage in a dose-dependently which based on the cell nuclei was stained by 4', 6-diamidino-2-phenylindole present in AGS and SNU-1 cells. DNA damage and repair associated proteins expression in AGS and SNU-1 cells were measured by Western blotting. The results indicated AITC decreased nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), glutathione, and catalase, but increased superoxide dismutase (SOD (Cu/Zn)), and nitric oxide synthase (iNOS) in AGS cells, however, in SNU-1 cells are increased HO-1. AITC increased DNA-dependent protein kinase (DNA-PK), phosphorylation of gamma H2A histone family member X on Ser139 (γH2AXpSer139 ), and heat shock protein 90 (HSP90) in AGS cells. AITC increased DNA-PK, mediator of DNA damage checkpoint protein 1 (MDC1), γH2AXpSer139 , topoisomerase II alpha (TOPIIα), topoisomerase II beta (TOPIIβ), HSP90, and heat shock protein 70 (HSP70) in SNU-1 cells. AITC increased p53, p53pSer15 , and p21 but decreased murine double minute 2 (MDM2)pSer166 and O6 -methylguanine-DNA methyltransferase (MGMT) in AGS cells; however, it has a similar effect of AITC except increased ataxia telangiectasia and Rad3 -related protein (ATR)pSer428 , checkpoint kinase 1 (CHK1), and checkpoint kinase 2 (CHK2) in SNU-1 cells. Apparently, both cell responses to AITC are different, nonetheless, all of these observations suggest that AITC inhibits the growth of gastric cancer cells may through induction off DNA damage in vitro.

SRSF1 is essential for primary follicle development by regulating granulosa cell survival via mRNA alternative splicing.

Granulosa cell abnormalities are characteristics of premature ovarian insufficiency (POI). Abnormal expression of serine/arginine-rich splicing factor 1 (SRSF1) can cause various diseases, but the role of SRSF1 in mouse granulosa cells remains largely unclear. In this study, we found that SRSF1 was expressed in the nuclei of both mouse oocytes and granulosa cells. The specific knockout of Srsf1 in granulosa cells led to follicular development inhibition, decreased granulosa cell proliferation, and increased apoptosis. Gene Ontology (GO) analysis of RNA-seq results revealed abnormal expression of genes involved in DNA repair, cell killing and other signalling pathways. Alternative splicing (AS) analysis showed that SRSF1 affected DNA damage in granulosa cells by regulating genes related to DNA repair. In summary, SRSF1 in granulosa cells controls follicular development by regulating AS of genes associated with DNA repair, thereby affecting female reproduction.

Androgen receptor contributes to repairing DNA damage induced by inflammation and oxidative stress in prostate cancer.

Androgen deprivation therapy remains the first-line therapy option for prostate cancer, mostly resulting in the transition of the disease to a castration-resistant state. The lack of androgen signaling during therapy affects various cellular processes, which sometimes paradoxically contributes to cancer progression. As androgen receptor (AR) signaling is known to contribute to oxidative stress regulation, loss of AR may also affect DNA damage level and the response mechanism in oxidant and inflammatory conditions of the prostate tumor microenvironment. Therefore, this study aimed to investigate the role of AR and AR-regulated tumor suppressor NKX3.1 upon oxidative stress-induced DNA damage response (DDR) in the inflammatory tumor microenvironment of the prostate. Intracellular reactive oxygen species (ROS) level was induced by either inflammatory conditioned media obtained from lipopolysaccharide-induced macrophages or oxidants and measured by dichlorodihydrofluorescein diacetate. In addition to this, DNA damage was subsequently quantified by counting gH2AX foci using an immunofluorescence-based Aklides platform. Altered expression of proteins function in DDR detected by western blotting. Cellular levels of ROS and ROS-induced DNA double-strand break damage were analyzed in the absence and presence of AR signaling upon treatment of prostate cancer cells by either oxidants or inflammatory microenvironment exposure. The results showed that AR suppresses intracellular ROS and contributes to DNA damage recognition under oxidant conditions. Besides, increased DNA damage due to loss of NKX3.1 under inflammatory conditions was alleviated by its overexpression. Moreover, the activation of the DDR mediators caused by AR and NKX3.1 activation in androgen-responsive and castration-resistant prostate cancer cells indicated that the androgen receptor function is essential both in controlling oxidative stress and in activating the ROS-induced DDR. Taken together, it is concluded that the regulatory function of androgen receptor signaling has a vital function in the balance between antioxidant response and DDR activation.

Activation of UBEC2 by transcription factor MYBL2 affects DNA damage and promotes gastric cancer progression and cisplatin resistance.

Ubiquitin-conjugating enzyme E2 C (UBE2C) plays a carcinogenic role in gastric cancer (GC); yet, its role in cisplatin (DDP) resistance in GC is enigmatic. This study sought to probe into the impact of UBE2C on DDP resistance in GC and its concrete molecular mechanism in GC progression. Bioinformatics analysis was used to analyze differentially expressed mRNAs and predict upstream regulatory molecules in GC. Real-time quantitative reverse transcriptase polymerase chain reaction and western blot were used to detect the expression of UBE2C and MYB proto-oncogene like 2 (MYBL2). Dual luciferase and chromatin immunoprecipitation (ChIP) assays were used to verify the binding relationship. Cell counting kit-8 was used to detect cell viability and calculate IC50 values. Flow cytometry was used to detect the cell cycle. Comet assay was used to detect DNA damage. Western blot was used to detect the expression of DNA loss-related proteins (γ-H2AX, ATM/p-ATM). The knockdown of highly expressed UBE2C in GC cell lines could reduce cell viability, induce G2/M arrest, induce apoptosis, and promote DNA damage and DDP sensitivity. Bioinformatics analysis predicted that the substantially upregulated MYBL2 was an upstream transcription factor in UBE2C. The binding relationship between the UBE2C promoter region and MYBL2 was verified by dual luciferase and ChIP. Overexpression of UBE2C in the rescue experiment was found to reverse the inhibited GC progression and promoted DDP sensitivity brought by the knockdown of MYBL2. In conclusion, the MYBL2/UBE2C regulatory axis may be a potential way to overcome DDP resistance in GC.

Targeting Tri-Methyl-Histone H3 (Lys27) in Rectal Cancer: A Novel Strategy to Enhance Radiosensitivity and Predict Response to Neoadjuvant Therapy

Tri-Methyl-Histone H3 (Lys27) is a modification of histone H3 involved in gene regulation and chromatin organization. It plays a crucial role in cell response to ionizing radiation and affects DNA damage repair, with altered levels being linked to decreased radiation sensitivity. Our hypothesis is that high levels of Tri-Methyl-Histone H3 (Lys27) in rectal tumors can predict response to neoadjuvant chemoradiation, and targeting SETD2, the histone methyltransferase responsible for adding the tri-methyl group to Lys27 of histone H3, may enhance radiosensitivity in rectal cancer cells. Biopsy samples from 25 patients with locally advanced rectal adenocarcinoma (Stage II-III) were obtained for analysis. Immunohistochemistry (IHC) was used to evaluate Tri-Methyl-Histone H3 (Lys27) expression levels in the tumor biopsies. The staining intensity was scored semi-quantitatively. Samples with 100% positive staining were grouped as "Tri-Methyl-High," and all others were grouped as "Tri-Methyl-Low." Fisher's exact test was used to analyze the data to determine the correlation between Tri-Methyl-Histone H3 (Lys27) levels and response to neoadjuvant chemoradiation. The primary endpoint was either a sustained clinical complete response (cCR) at one year or a pathological complete response (pCR). To further understand the role of Tri-Methyl-Histone H3 (Lys27) in response to ionizing radiation (IR), clonogenic assays, γH2aX foci staining, and western blot analysis of DNA damage response proteins and histones were performed on two established human rectal cancer cell lines following either RNA inhibition (RNAi) or drug targeting of SETD2. Twenty-five patients with rectal cancer had pre-treatment biopsies analyzed for Tri-Methyl-Histone H3 (Lys27) levels, 11 were classified as Tri-Methyl-High and 14 as Tri-Methyl-Low. The groups were well balanced in terms of age, sex, clinical T/N stage, and neoadjuvant treatment approach. The overall combined complete response (cCR/pCR) rate was 36%, while the rate for Tri-Methyl-High patients was 9% (1/11) compared to 57% (8/14) in Tri-Methyl-Low patients (p = 0.03). After treating two colorectal cancer cell lines (SW837 and HCT116) with IR (5 Gy) in vitro, Tri-Methyl-Histone H3 (Lys27) levels increased 3-fold. SETD2 RNAi reduced Tri-Methyl-Histone H3 (Lys27) and increased cell death when combined with IR. In vitro treatment with a novel SETD2 inhibitor, EZM0414, led to a 2-fold increase in DNA damage following IR (5 Gy) as measured by γH2aX foci staining. EZM0414 treatment showed a 37% improvement in the dose enhancement ratio. Our study uncovers a new biomarker, Tri-Methyl-Histone H3 (Lys27), that could be used to predict response to neoadjuvant chemoradiation in rectal cancer patients. Our preclinical data indicates that targeting SETD2 to reduce Tri-Methyl-Histone H3 (Lys27) mediated DNA repair could improve the efficacy of radiation therapy for rectal cancer patients.

The Molecular and Genetic Mechanisms of Inherited Bone Marrow Failure Syndromes: The Role of Inflammatory Cytokines in Their Pathogenesis.

Inherited bone marrow failure syndromes (IBMFSs) include Fanconi anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, dyskeratosis congenita, severe congenital neutropenia, and other rare entities such as GATA2 deficiency and SAMD9/9L mutations. The IBMFS monogenic disorders were first recognized by their phenotype. Exome sequencing has validated their classification, with clusters of gene mutations affecting DNA damage response (Fanconi anemia), ribosome structure (Diamond-Blackfan anemia), ribosome assembly (Shwachman-Diamond syndrome), or telomere maintenance/stability (dyskeratosis congenita). The pathogenetic mechanisms of IBMFSs remain to be characterized fully, but an overarching hypothesis states that different stresses elicit TP53-dependent growth arrest and apoptosis of hematopoietic stem, progenitor, and precursor cells. Here, we review the IBMFSs and propose a role for pro-inflammatory cytokines, such as TGF-β, IL-1β, and IFN-α, in mediating the cytopenias. We suggest a pathogenic role for cytokines in the transformation to myeloid neoplasia and hypothesize a role for anti-inflammatory therapies.

Retrospective evaluation of the value of DNA damage repair gene signature in response to platinum-based chemotherapy in advanced pancreatic ductal adenocarcinoma.

4137 Background: Mutations affecting DNA Damage Repair (DDR) genes have been associated with clinical benefits from platinum-based chemotherapy (PBC) in ovarian and breast cancer. Mutations of DDR genes, including BRCA1/2, PALB2, ATM, ATR, CHK1, or RAD51, are found in around 15% of pancreatic ductal adenocarcinoma (PDAC). Nevertheless, whether advanced PDAC patients harboring DDR gene mutations could benefit more from PBC has not been well established. In this study, we aimed to compare the prevalence of DDR gene mutations between advanced PDAC patients who benefited from PBC and those who did not. Methods: We retrospectively analyzed samples from patients with advanced PDAC who received PBC at our institution from 2008 to 2022. Patients with partial response or progression-free survival (PFS) > 6 months were considered responders, and those with progression disease as the best response were considered non-responders. Tumor molecular profiling was performed on tumor samples by Next Generation Sequencing (NGS) of DNA. Fisher’s exact test was used to compare DDR genomic alterations in responders vs. non-responders. Overall survival (OS) was defined as the time from the start of PBC to the date of death. PFS was defined as the time between the start of PBC and the first date of documented progression or death, whichever occurs first. Survival outcomes were estimated using Kaplan-Meier curves, and differences were tested using the long-rank test. Results: A total of 132 patients with advanced PDAC treated with PBC were included in this study. The median age at diagnosis was 55 years (range 27-79 years), and 71 (53.79%) patients were male. In this cohort, 88 (66.67%) patients were identified as responders. Tumor molecular profiling showed that among responders, 42 patients (47.7%) had genomic alterations in BRCA1/2 or PALB2, and 10 patients (11.3%) had alterations in other DDR genes. In contrast, only 17 patients (38.64%) showed DDR gene alternations among non-responders. Importantly, mutations in DDR genes are significantly associated with the responder group (OR=2.28; 95% CI 1.03-5.17, p=0.04). In line with these findings, patients with DDR gene mutations showed significantly longer PFS (median PFS 9.95 months) compared with patients without DDR gene mutations (median PFS 6.51 months) (HR= 0.57; 95%CI 0.39-0.85; p=0.005). Moreover, significantly longer OS was found in patients with DDR gene mutations (median OS 20.5 months) compared with those without DDR gene mutations (median OS 16.8 months) (HR=0.67; 95%CI 0.45-1; p=0.05). Conclusions: Genomic alterations in DDR genes are significantly associated with the benefit of PBC treatment in advanced PDAC patients. Our results argue in favor of using DDR genes as a decision-making tool for advanced PDAC patient stratification even before the stand-of-care chemotherapies.

Hexavalent chromium induces γH2AX and RAD51 involved in DNA damage repair in BEAS-2B cells by modulating LNC-DHFR-4:1

Hexavalent chromium [Cr(VI)] is rarely found in nature. Its occurrence in the environment is mainly due to anthropogenic sources. Our previous studies have shown that Cr(VI) exposure could change the expression profile of long noncoding RNAs (lncRNAs). However, the relationship between lncRNAs and genetic damage induced by Cr(VI) remains unclear. In this study, RT–qPCR was used to verify the expression of genes and lncRNAs involved in DNA damage repair in BEAS-2B cells exposed to different Cr(VI) concentrations. After screening out LNC-DHFR-4:1, overexpression and knockdown models of BEAS-2B cells were used to further identify the relationship between the lncRNA and RAD51. RT–qPCR and indirect immunofluorescence were used to detect expression. Our results revealed that with increasing Cr(VI) concentration, γH2AX expression was increased, while the expression of RAD51 was decreased. Meanwhile, LNC-DHFR-4:1 acted as a competitive endogenous RNA to regulate the expression of γH2AX and RAD51, which further affected DNA damage repair. The overexpression of LNC-DHFR-4:1 induced a twofold decrease in γH2AX and a onefold increase in RAD51, and its knockdown showed the opposite results. These results suggested that LNC-DHFR-4:1 might be a potential biomarker of Cr(VI)-induced DNA damage repair in BEAS-2B cells.