DNA Damage Sensor Research Articles

Upregulated DNA Damage-Linked Biomarkers in Parkinson's Disease Model Mice.

The present study examined expression of DNA damage markers in VMAT2 Lo PD model mice. The results demonstrate there is a significant increase in these DNA damage markers mostly in the brain regions of 18- and 23-month-old model mice, indicating oxidative stress-induced DNA lesion is an important pathologic feature of this mouse model.

Mec1 regulates PAS recruitment of Atg13 via direct binding with Atg13 during glucose starvation-induced autophagy

Mec1 is a DNA damage sensor, which performs an essential role in the DNA damage response pathway and glucose starvation-induced autophagy. However, the functions of Mec1 in autophagy remain unclear. In response to glucose starvation, Mec1 forms puncta, which are recruited to mitochondria through the adaptor protein Ggc1. Here, we show that Mec1 puncta also contact the phagophore assembly site (PAS) via direct binding with Atg13. Functional analysis of the Atg13-Mec1 interaction revealed two previously unrecognized protein regions, the Mec1-Binding Region (MBR) on Atg13 and the Atg13-Binding Region (ABR) on Mec1, which mediate their mutual association under glucose starvation conditions. Disruption of the MBR or ABR impairs the recruitment of Mec1 puncta and Atg13 to the PAS, consequently blocking glucose starvation-induced autophagy. Additionally, the MBR and ABR regions are also crucial for DNA damage-induced autophagy. We thus propose that Mec1 regulates glucose starvation-induced autophagy by controlling Atg13 recruitment to the PAS.

Cisplatin-induced cell death increases the degradation of the MRE11-RAD50-NBS1 complex through the autophagy/lysosomal pathway

Cisplatin and other platinum-based anticancer agents are among the most widely used chemotherapy drugs in the treatment of different types of cancer. However, it is common to find patients who respond well to treatment at first but later relapse due to the appearance of resistance to cisplatin. Among the mechanisms responsible for this phenomenon is the increase in DNA damage repair. Here, we elucidate the effect of cisplatin on the MRN (MRE11-RAD50-NBS1) DNA damage sensor complex. We found that the tumor suppressor FBXW7 is a key factor in controlling the turnover of the MRN complex by inducing its degradation through lysosomes. Inhibition of lysosomal enzymes allowed the detection of the association of FBXW7-dependent ubiquitylated MRN with LC3 and the autophagy adaptor p62/SQSTM1 and the localization of MRN in lysosomes. Furthermore, cisplatin-induced cell death increased MRN degradation, suggesting that this complex is one of the targets that favor cell death. These findings open the possibility of using the induction of the degradation of the MRN complex after genotoxic damage as a potential therapeutic strategy to eliminate tumor cells.

Engineered RecA Constructs Reveal the Minimal SOS Activation Complex.

The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacteria's ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key "SOS activation signal." Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that allow LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response.

The splicing regulators TIA1 and TIAL1 are required for the expression of the DNA damage repair machinery during B cell lymphopoiesis.

B cell lymphopoiesis requires dynamic modulation of the B cell transcriptome for timely coordination of somatic mutagenesis and DNA repair in progenitor B (pro-B) cells. Here, we show that, in pro-B cells, the RNA-binding proteins Tcell intracellular antigen 1 (TIA1) and TIA1-like protein (TIAL1) act redundantly to enable developmental progression. They are global splicing regulators that control the expression of hundreds of mRNAs, including those involved in DNA damage repair. Mechanistically, TIA1 and TIAL1 bind to 5' splice sites for exon definition, splicing, and expression of DNA damage sensors, such as Chek2 and Rif1. In their absence, pro-B cells show exacerbated DNA damage, altered P53 expression, and increased cell death. Our study uncovers the importance of tight regulation of RNA splicing by TIA1 and TIAL1 for the expression of integrative transcriptional programs that control DNA damage sensing and repair during B cell development.

Abstract 9727: Reduced Bmpr2 Causes a Decrease in Foxf1 That Links Unrepaired DNA Damage to Persistent Pulmonary Hypertension

Introduction: B MPR2 mutation is the most common genetic cause of pulmonary arterial hypertension (PAH). Loss of BMPR2 causes pulmonary arterial (PA) endothelial cell (EC) dysfunction and impairs EC-mediated suppression of smooth muscle cells proliferation. These are characteristic features of PAH vascular cells as is the presence of DNA damage that can be induced by oxidative stress. We previously showed that EC- Bmpr2 -/- mice have persistent pulmonary hypertension (PH) with reoxygenation after hypoxia. However, the role of DNA damage in causing persistent PH related to loss of BMPR2 and oxidative stress is unknown. Hypothesis: We hypothesize that loss of BMPR2 causes unrepaired DNA damage and persistent PH by altering the expression of genes that maintain EC homeostasis. Methods and Results: DNA damage assessed by γH2AX foci was observed in PAEC of EC- Bmpr2 -/- mice in room air, that was more extensive after reoxygenation (10% hypoxia for 3 weeks followed by recovery in room-air for 4 weeks). Deletion of the DNA damage sensor ataxia-telangiectasia mutated (ATM) in EC of mice (EC- Atm -/- ) also resulted in persistent DNA damage and PH. RNA sequencing of PAEC after reoxygenation in EC- Bmpr2 -/- and EC- Atm -/- mice vs. controls showed downregulated EC differentiation and development genes enriched in biding motifs for Foxf1, a transcription factor associated with DNA repair and angiogenesis. Foxf1 expression was reduced in PAEC with loss of BMPR2 or ATM. Loss of Foxf1 decreased angiogenic genes Cldn5 and Vegfr2 , and DNA damage response genes Atm and p53 , and impaired PAEC migration and tube formation. Foxf1 overexpression increased these genes in PAEC. Immunohistochemistry showed decreased FOXF1 in PAEC of human pulmonary vascular lesions. Delivery of Foxf1 to EC- Bmpr2 -/- mice with an AAV2-peptide targeting the PA endothelium attenuated DNA damage in PAEC, restored angiogenesis genes and completely reversed persistent PH returning right ventricular systolic pressure to normal levels. Conclusions: We link loss of BMPR2 and DNA damage sensing in PAEC to persistent DNA damage and PAH and show that both are mediated by reduced FOXF1 . In EC- Bmpr2 -/- mice, Foxf1 gene therapy attenuated DNA damage restored angiogenesis genes, and prevented persistent PH.

Nanobodies targeting LexA autocleavage disclose a novel suppression strategy of SOS-response pathway

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections.

SN-38 Sensitizes BRCA-Proficient Ovarian Cancers to PARP Inhibitors through Inhibiting Homologous Recombination Repair.

As a multifunctional protein posttranslational modification enzyme in eukaryotic cells, Poly-ADP-ribose polymerase (PARP) acts as a DNA damage sensor, which helps to repair DNA damage through recruiting repair proteins to the DNA break sites. PARP inhibitors offer a significant clinical benefit for ovarian cancer with BRCA1/2 mutations. However, the majority of ovarian cancer patients harbor wild-type (WT) BRCA1/2 status, which narrows its clinical application. Here, we identified a small compound, SN-38, a CPT analog, which sensitizes BRCA-proficient ovarian cancer cells to PARP inhibitor treatment by inhibiting homologous recombination (HR) repair. SN-38 treatment greatly enhanced PARP inhibitor olaparib induced DNA double-strand breaks (DSBs) and DNA replication stress. Meanwhile, the combination of SN-38 and olaparib synergistically induced apoptosis in ovarian cancer. Furthermore, combination administration of SN-38 and olaparib induced synergistic antitumor efficacy in an ovarian cancer xenograft model in vivo. Therefore, our study provides a novel therapeutic strategy to optimize PARP inhibitor therapy for patients with BRCA-proficient ovarian cancer.

Sensitization of Patient-Derived Colorectal Cancer Organoids to Photon and Proton Radiation by Targeting DNA Damage Response Mechanisms.

Simple SummaryRadiotherapy plays an important role in the treatment of colorectal cancer (CRC). Approximately one-third of patients with rectal cancer show a pathological complete response upon total neoadjuvant treatment. Patient-derived CRC organoids were investigated regarding their radiotherapeutic treatment response—both in terms of conventional photon irradiation, the combination thereof with chemotherapy, as well as proton irradiation. By inhibition of an important sensor molecule for DNA damage, which has been shown to be activated upon irradiation, radioresistant organoids could be resensitized.Pathological complete response (pCR) has been correlated with overall survival in several cancer entities including colorectal cancer. Novel total neoadjuvant treatment (TNT) in rectal cancer has achieved pathological complete response in one-third of the patients. To define better treatment options for nonresponding patients, we used patient-derived organoids (PDOs) as avatars of the patient’s tumor to apply both photon- and proton-based irradiation as well as single and combined chemo(radio)therapeutic treatments. While response to photon and proton therapy was similar, PDOs revealed heterogeneous responses to irradiation and different chemotherapeutic drugs. Radiotherapeutic response of the PDOs was significantly correlated with their ability to repair irradiation-induced DNA damage. The classical combination of 5-FU and irradiation could not sensitize radioresistant tumor cells. Ataxia-telangiectasia mutated (ATM) kinase was activated upon radiation, and by inhibition of this central sensor of DNA damage, radioresistant PDOs were resensitized. The study underlined the capability of PDOs to define nonresponders to irradiation and could delineate therapeutic approaches for radioresistant patients.

PFOA-Induced Ovotoxicity Differs Between Lean and Obese Mice With Impacts on Ovarian Reproductive and DNA Damage Sensing and Repair Proteins.

Perfluorooctanoic acid (PFOA) is an environmentally persistent perfluoroalkyl substance that is widely used in consumer products. Exposure to PFOA is associated with reproductive and developmental effects including endocrine disruption, delayed puberty in girls, and decreased fetal growth. In the United States, obesity affects 40% of women and 20% of girls, with higher rates in minority females. Obesity causes infertility, poor oocyte quality, miscarriage, and offspring defects. This study proposed that PFOA exposure would impact estrous cyclicity, ovarian steroid hormones, and the ovarian proteome and further hypothesized that obesity would impact PFOA-induced ovotoxicity. Female wild type (KK.Cg-a/a; lean) or KK.Cg-Ay/J mice (obese) received saline (CT) or PFOA (2.5 mg/kg) per os for 15 days beginning at 7 weeks of age. There were no effects on food intake, body weight, estrous cyclicity, serum progesterone, and heart, spleen, kidney, or uterus weight (p > .05). Ovary weight was decreased (p < .05) by PFOA exposure relative to vehicle control-treated mice in lean but not obese mice. Liquid chromatography-tandem mass spectrometry was performed on isolated ovarian protein and PFOA exposure altered the ovarian abundance of proteins involved in DNA damage sensing and repair pathways and reproduction pathways (p < .05) differentially in lean and obese mice. The data suggest that PFOA exposure alters ovary weight and differentially targets ovarian proteins in lean and obese females in ways that might reduce female fecundity.

Exceptional behavior of breast cancer-associated type 1 gene in breast invasive carcinoma.

Cellular expression level of Breast Cancer-Associated Type 1 (BRCA1) encoded protein is the sign of genome integrity, stability, and surveillance. BRCA1 after sensing DNA damage activates repairing system and if mutated leaves genomic lesions unrepaired and triggers transformation of normal breast cells into cancerous ones. We conducted in silico study to have a holistic view of BRCA1's correlation with multiple variables of breast invasive carcinoma. We used user-friendly online GeneCardsSuite pathway-level enrichment analysis, UALCAN portal differential expression analysis, cBioPortal cancer genome platform for mutatome map construction, and cancer cell lines encyclopedia genomics of drug sensitivity toolkit to understand correlation of BRCA1 expression with the effectiveness of anti-cancer drugs. Contrary to general behavior of a tumor suppressor gene our study revealed BRCA1 overexpression under all circumstances. This novel finding needs to be explored further to understand functional impact of BRCA1 overexpression on the expression of many genes which are transcriptionally regulated by BRCA1 and promotion of tumriogenesis. Our study highlights the potential role of BRCA1-regulated genes in oncogenesis and recommends use of BRCA1-linked genes as future therapeutic targets for effective disease management.

The Role of Protein Arginine Methyltransferases in DNA Damage Response.

In response to DNA damage, cells have developed a sophisticated signaling pathway, consisting of DNA damage sensors, transducers, and effectors, to ensure efficient and proper repair of damaged DNA. During this process, posttranslational modifications (PTMs) are central events that modulate the recruitment, dissociation, and activation of DNA repair proteins at damage sites. Emerging evidence reveals that protein arginine methylation is one of the common PTMs and plays critical roles in DNA damage response. Protein arginine methyltransferases (PRMTs) either directly methylate DNA repair proteins or deposit methylation marks on histones to regulate their transcription, RNA splicing, protein stability, interaction with partners, enzymatic activities, and localization. In this review, we summarize the substrates and roles of each PRMTs in DNA damage response and discuss the synergistic anticancer effects of PRMTs and DNA damage pathway inhibitors, providing insight into the significance of arginine methylation in the maintenance of genome integrity and cancer therapies.

Structure of an Intranucleosomal DNA Loop That Senses DNA Damage during Transcription.

Transcription through chromatin by RNA polymerase II (Pol II) is accompanied by the formation of small intranucleosomal DNA loops containing the enzyme (i-loops) that are involved in survival of core histones on the DNA and arrest of Pol II during the transcription of damaged DNA. However, the structures of i-loops have not been determined. Here, the structures of the intermediates formed during transcription through a nucleosome containing intact or damaged DNA were studied using biochemical approaches and electron microscopy. After RNA polymerase reaches position +24 from the nucleosomal boundary, the enzyme can backtrack to position +20, where DNA behind the enzyme recoils on the surface of the histone octamer, forming an i-loop that locks Pol II in the arrested state. Since the i-loop is formed more efficiently in the presence of SSBs positioned behind the transcribing enzyme, the loop could play a role in the transcription-coupled repair of DNA damage hidden in the chromatin structure.

Prognostic Impact of TP53 Mutations and Tumor Mutational Load in Colorectal Cancer

The DNA damage response (DDR) is critical for maintaining genome stability, and abnormal DDR—resulting from mutations in DNA damage-sensing and repair proteins—is a hallmark of cancer. Here, we aimed to investigate the predictive power of DDR gene mutations and the tumor mutational load (TML) for survival outcomes in a cohort of 22 rectal cancer patients who received pre-operative neoadjuvant therapy. Univariate analysis revealed that TML-high and TP53 mutations were significantly associated with worse overall survival (OS) with TML-high retaining significance in multivariate analyses. Kaplan–Meier survival analyses further showed TML-high was associated with worse disease-free (p = 0.036) and OS (p = 0.024) results in our patient cohort. A total of 53 somatic mutations were identified in 22 samples with eight (36%) containing mutations in DDR genes, including ATM, ATR, CHEK2, MRE11A, RAD50, NBN, ERCC2 and TP53. TP53 was the most frequently mutated gene, and TP53 mutations were significantly associated with worse OS (p = 0.023) in Kaplan–Meier survival analyses. Thus, our data indicate that TML and TP53 mutations have prognostic value for rectal cancer patients and may be important independent biomarkers for patient management. This suggests that prognostic determination for rectal cancer patients receiving pre-operative neoadjuvant therapy should include consideration of the initial TML and tumor genetic status.

Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC

High-grade serous ovarian carcinoma (HGSOC) is responsible for the majority of ovarian cancer-associated deaths. PARP (poly-(ADP-ribose) polymerase) inhibitors (PARPi) (olaparib, rucaparib and niraparib) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (OC). Previous studies have reported an increased expression of PARP1, a sensor for DNA damage, in OC; however, its compartment (epithelial tumor vs stroma)-, anatomical site (fallopian tube, omentum, ovary and serous tubal in situ carcinoma)- and histotype-specific expression have not been studied. Here, we showed that PARP1 protein levels are higher in epithelial tumors compared to adjacent non-malignant stroma at four different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases. Furthermore, we found that PARP1 protein levels are elevated in tumors relative to normal tissue in the fallopian tube, omentum and ovary in patients with HGSOC, again with the most significant difference in the omentum. We also showed that PARP1 expression is increased in a malignant and invasive stage in mouse ovarian surface epithelium cells, and that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial OC. These findings indicate that a better understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed since PARP inhibitors are currently being used in the treatment of patients with OC.

Impairment of RAD17 Functions by miR-506-3p as a Novel Synthetic Lethal Approach Targeting DNA Repair Pathways in Ovarian Cancer.

Epithelial ovarian cancer (EOC) remains the most lethal gynecological cancer and development of chemo-resistance is a major factor in disease relapse. Homologous recombination (HR) is a critical pathway for DNA double strand break repair and its deficiency is associated to a better response to DNA damage-inducing agents. Strategies to inhibit HR-mediated DNA repair is a clinical need to improve patients’ outcome. MicroRNA (miRNAs) affect most of cellular processes including response to cancer treatment. We previously showed that miR-506-3p targets RAD51, an essential HR component. In this study we demonstrated that: i) another HR component, RAD17, is also a direct target of miR-506-3p and that it is involved in mediating miR-506-3p phenotypic effects; ii) the impairment of miR-506-3p binding to RAD17 3’ UTR reverted the miR-506-3p induced platinum sensitization; iii) miR-506-3p/RAD17 axis reduces the ability of EOC cell to sense DNA damage, abrogates the G2/M cell cycle checkpoint thus delaying the G2/M cell cycle arrest likely allowing the entry into mitosis of heavily DNA-damaged cells with a consequent mitotic catastrophe; iv) RAD17 expression, regulated by miR-506-3p, is synthetically lethal with inhibitors of cell cycle checkpoint kinases Chk1 and Wee1 in platinum resistant cell line. Overall miR-506-3p expression may recapitulate a BRCAness phenotype sensitizing EOC cells to chemotherapy and helping in selecting patients susceptible to DNA damaging drugs in combination with new small molecules targeting DNA-damage repair pathway.

The Role of PARP-1 and NF-κB in Bile-Induced DNA Damage and Oncogenic Profile in Hypopharyngeal Cells.

We recently documented that acidic bile, a gastroesophageal reflux content, can cause invasive hypopharyngeal squamous cell carcinoma, by inducing widespread DNA damage and promoting nuclear factor kappa B (NF-κB)-related oncogenic molecular events. Poly or adenosine diphosphate (ADP)-ribose polymerase-1 (PARP-1), a sensitive sensor of DNA damage, may interact with NF-κB. We hypothesized that PARP-1 is activated in hypopharyngeal cells (HCs) with marked DNA damage caused by acidic bile, hence there is an association between PARP-1 and NF-κB activation or its related oncogenic profile, in this process. In vitro study. We targeted PARP-1 and NF-κB(p65), using pharmacologic inhibitors, 1.0 μM Rucaparib (AG014699) and 10 μM BAY 11-7082 {3-[4=methylphenyl)sulfonyl]-(2E)-propenenitrile}, respectively, or silencing their gene expression (siRNAs) and used immunofluorescence, luciferase, cell viability, direct enzyme-linked immunosorbent assays, and qPCR analysis to detect the effect of targeting PARP-1 or NF-κB in acidic bile-induced DNA damage, PARP-1, p-NF-κB, and B-cell lymphoma 2 (Bcl-2) expression, as well as NF-κB transcriptional activity, cell survival, and mRNA oncogenic phenotype in HCs. We showed that (i) PARP-1 is overexpressed by acidic bile, (ii) targeting NF-κB adequately prevents the acidic bile-induced DNA double-strand breaks (DSBs) by gamma H2A histone family member X (γH2AX), oxidative DNA/RNA damage, PARP-1 overexpression, anti-apoptotic mRNA phenotype, and cell survival, whereas (iii) targeting PARP-1 preserves elevated DNA damage, NF-κB activation, and anti-apoptotic phenotype. We document for the first time that the activation of PARP-1 is an early event during bile reflux-related head and neck carcinogenesis and that NF-κB can mediate DNA damage and PARP-1 activation. Our data encourage further investigation into how acidic bile-induced activated NF-κB mediates DNA damage in hypopharyngeal carcinogenesis. NA Laryngoscope, 133:1146-1155, 2023.

Tricetin Reduces Inflammation and Acinar Cell Injury in Cerulein-Induced Acute Pancreatitis: The Role of Oxidative Stress-Induced DNA Damage Signaling.

Acute pancreatitis (AP) poses a worldwide challenge due to the growing incidence and its potentially life-threatening course and complications. Specific targeted therapies are not available, prompting the identification of new pathways and novel therapeutic approaches. Flavonoids comprise several groups of biologically active compounds with wide-ranging effects. The flavone compound, tricetin (TCT), has not yet been investigated in detail but sporadic reports indicate diverse biological activities. In the current study, we evaluated the potential protective effects of TCT in AP. TCT (30 μM) protected isolated primary murine acinar cells from the cytotoxic effects of cerulein, a cholecystokinin analog peptide. The protective effects of TCT were observed in a general viability assay (calcein ester hydrolysis), in an apoptosis assay (caspase activity), and in necrosis assays (propidium iodide uptake and lactate dehydrogenase release). The effects of TCT were not related to its potential antioxidant effects, as TCT did not protect against H2O2-induced acinar cell death despite possessing radical scavenging activity. Cerulein-induced expression of IL1β, IL6, and matrix metalloproteinase 2 and activation of nuclear factor-κB (NFκB) were reduced by 30 μM TCT. In vivo experiments confirmed the protective effect of TCT in a mouse model of cerulein-induced AP. TCT suppressed edema formation and apoptosis in the pancreas and reduced lipase and amylase levels in the serum. Moreover, TCT inhibited interleukin-1β (IL1β), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) expression in the pancreas and reduced the activation of the oxidative DNA damage sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Our data indicate that TCT can be a potential treatment option for AP.

KDM6B promotes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key regulator of PARthanatos. Loss of KDM6B protein or its demethylase activity conferred cancer cell resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second XRCC1-dependent repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

The interaction of oxidative stress with MAPK, PI3/AKT, NF-κB, and DNA damage kinases influences the fate of γ-radiation-induced bystander cells

Oxidative stress is associated with the induction of a plethora of effects on cellular macromolecules and signaling cascades. The onset of oxidative imbalance characterizes irradiated cells. The present study investigates the effects of ionizing radiation on oxidative stress induction in bystander cells and their interactions with critical cell signaling mediators. The effect of irradiated cell-conditioned medium (ICCM) from γ-irradiated hepatocellular carcinoma (HepG2) cells were studied in bystander HepG2 and normal liver (BRL-3A) cells at early (1 h, 2 h) and later (24 h) time points post-irradiation. Although ROS generation and lipid peroxidation showed the highest effects in both bystander cell groups at the early time points, antioxidant enzymes superoxide dismutase and catalase showed the lowest activity. Oxidative stress was persistent up to 24 h, but the highest level was seen in 1 h ICCM treated 8By cells. Although the levels of all pro-survival signaling factors (p-PI3K, p-Akt, p-p38MAPK, p-JNK, and p-NFκB) increased in bystander HepG2 cells, they showed a significant decrease in bystander BRL-3A cells. JAK2-STAT3 activation, however, was reduced only in BRL-3A cells, with no effect in HepG2 cells. However, in both bystander cell groups, activation of DNA damage sensors ATM, ATR, and cell cycle inhibitor p21 increased. Elevated ROS levels down-regulated the activation of PI3K, Akt, JNK, and NF-κB in BRL-3A cells but enhanced the activation of ATM and p21. In contrast, in HepG2 cells, increased ROS level elevated the activation of PI3K, JNK, p38MAPK, NF-κB with no effect on p-ATM or p21. ROS differentially influenced the interactions between the signaling mediators in the bystander cells. p-ATR levels, although increased in both bystander cell groups, showed no association with other factors. ICCM from the same HepG2 cells differently affected signaling factors in two groups of cells, highlighting the critical significance of the study in the field of radiation biology.