Infrared laser and amber LED exposure does not affect base excision repair gene expression in human breast cancer cells

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

Microgravity alters the expressions of DNA repair genes and their regulatory miRNAs in space-flown Caenorhabditis elegans

We studied the effect of ionizing radiation on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in drug-sensitive human breast carcinoma (MCF-7) cells and its drug-resistant variant (MCF-7/ADR) cells. Northern blot and gel mobility shift assays showed that 135 cGy of ionizing radiation induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression in MCF-7/ADR cells. In MCF-7 cells, however, we observed little/no induction of bFGF gene expression and AP-1 binding activity after the stress. Nevertheless, MCF-7 cells transfected with plasmids containing c-jun gene contain high levels of bFGF protein. H-7 (60 micrograms/ml), a potent protein kinase C (PKC) inhibitor, inhibited the stress-induced AP-1 binding activity and bFGF gene expression in MCF-7/ADR cells. Corroborating this observation, overexpression of PKC alpha induced bFGF gene expression in MCF-7 cells. Taken together, these results suggest that stress-induced bFGF gene expression is mediated through the activation of PKC and AP-1 transcription factors. Differences in the levels of PKC activity and AP-1 binding factors may be responsible for differential expression of bFGF among breast cancer cell lines. Although there are large differences in response to ionizing radiation between MCF-7 and MCF-7/ADR cell lines, we observed no significant differences in radiocytotoxicity between them.

Activating mutations in KRAS proto-oncogene, a signature event driving the development, progression and therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC), remains undruggable. The occurrence of guanine oxidation (8-oxoguanine) in the KRAS promoter and up-regulation of KRAS gene transcription under oxidative stress has been shown to be associated with KRAS expression and cancer development and progression. However, the molecular mechanism by which 8-oxoguanine damage regulates KRAS expression is largely unknown. Here, we show that the base excision repair (BER) pathway, a fundamental DNA damage repair pathway that processes most of the endogenous damages including oxidative base damage is involved in regulation of KRAS expression and survival of PDAC. We show that Apurinic/apyrimidinic endonuclease (APE1), a key enzyme of the BER pathway, is highly elevated in pancreatic cancer tissue samples. To elucidate the role of active BER pathway in the regulation of KRAS expression, we used real-time PCR (RT-PCR) analysis. Inflicting cells with oxidative damage using glucose oxidase increased KRAS gene expression in control cells but not in APE1 downregulated cells. ChIP assay showed enhanced occupancy of APE1 in KRAS promoter upon oxidative stress. Consistent with this, using synthetic oligonucleotide containing the KRAS promoter region, we showed that APE1 could bind and cleave AP site in KRAS promoter. Further, ChIP-qPCR analysis showed decreased occupancy of MAZ, a transcription factor, to the KRAS promoter in APE1 downregulated cells. Down-regulation of APE1 also resulted in decreased KRAS expression and increased sensitivity of pancreatic cancer cells to routinely used chemotherapeutic agents such as Gemcitabine, 5-Fluorouracil, Oxaliplatin, etc., suggesting that targeting APE1 and in turn, BER can sensitize pancreatic cancer cells. Our study suggests that BER pathway or APE1 plays a significant role in the tumor-selective regulation of gene expression and sensitization of cancer cells to chemotherapy, and supports the further investigation of novel treatments that target this pathway for cancer therapy. Citation Format: Suravi Pramanik, Shrabasti Roychoudhury, Hannah Harris, Heyu Song, Kishor K. Bhakat. Role of base excision repair (BER) pathway in regulation of KRAS expression in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4495.

Photobiomodulation (PBM) induced by non-ionizing radiations emitted from low-power lasers and light-emitting diodes (LEDs) has been used for various therapeutic purposes due to its molecular, cellular, and systemic effects. At the molecular level, experimental data have suggested that PBM modulates base excision repair (BER), which is responsible for restoring DNA damage. There is a relationship between the misfunction of the BER DNA repair pathway and the development of tumors, including breast cancer. However, the effects of PBM on cancer cells have been controversial. Breast cancer (BC) is the main public health problem in the world and is the most diagnosed type of cancer among women worldwide. Therefore, the evaluation of new strategies, such as PBM, could increase knowledge about BC and improve therapies against BC. Thus, this work aims to evaluate the effects of low-power red laser (658nm) and blue LED (470nm) on the mRNA levels from BER genes in human breast cancer cells. MCF-7 and MDA-MB-231 cells were irradiated with a low-power red laser (69Jcm-2, 0.77 W cm-2) and blue LED (482Jcm-2, 5.35 W cm-2), alone or in combination, and the relative mRNA levels of theAPTX,PolB, andPCNAgenes were assessed by reverse transcription-quantitative polymerase chain reaction. The results suggested that exposure to low-power red laser and blue LED decreased the mRNA levels fromAPTX,PolB, andPCNAgenes in human breast cancer cells. Our research shows that photobiomodulation induced by low-power red laser and blue LED decreases the mRNA levels of repair genes from the base excision repair pathway in MCF-7 and MDA-MB-231 cells.

Contribution of proximal promoter elements to the regulation of basal and differential glutathione S-transferase P1 gene expression in human breast cancer cells

Expression of relaxin is increased in human breast cancer, and relaxin was shown to increase in vitro invasiveness through increased production and secretion of matrix metalloproteinases in human breast cancer cells. The role of estrogen in the promotion of breast cancer is well-known. The environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a known carcinogen but has been shown to have antiestrogenic effects in human breast cancer cells. In this study, we have employed real-time PCR and chromatin immunoprecipitation (ChIP) assays to investigate the influence of estrogen and TCDD on relaxin-1 (RLN1) and relaxin-2 (RLN2) gene expression in MCF-7 and T47D human breast cancer cells. Estrogen increased RLN2 transcripts in T47D and MCF-7 cells after just 4 h of exposure, whereas TCDD did not. RLN1 transcripts were only induced after 24 h of estrogen exposure. TCDD did have antiestrogenic activity and reduced the estrogen-mediated increase in RLN2 and RLN1 mRNA. The estrogen-mediated increase in RLN2 mRNA levels was not caused by changes in the mRNA stability. ChIP analysis revealed binding of estrogen receptor-alpha (ERalpha) to promoter sequences of the RLN2 gene. Thus, we provide evidence that RLN2 gene activity is directly regulated by activated ERalpha in human breast cancer cells and we show that activation of the arylhydrocarbon receptor by TCDD inhibits this regulation by estrogen.

Methionine restriction (MetR) is a dietary intervention that extends mean and maximum life span in rodents, at least in part, by reducing oxidative stress and promoting DNA stability in different tissues. Regarding DNA stability, DNA repair pathways play a critical role, both in the nuclear and mitochondrial fractions. Base excision repair (BER) is the main one involved in the repair of oxidative damage, as well as the main one in mitochondria. Despite the relevance of DNA repair in DNA maintenance, it is not known whether MetR regulates BER as a mechanism of preserving genomic stability. In this study we analyzed, for the first time, the effect of 40% MetR for 7 weeks on BER in rat brain cortex and liver, focusing on the expression of several key BER genes. In the brain cortex, MetR significantly increased the gene expression of the DNA glycosylase Ogg1 and the DNA endonuclease Ape1 while reducing DNA polymerase γ gene expression. Conversely, MetR led to a general reduction in the expression of BER genes in the liver. Our findings highlight a tissue-specific regulation of the BER gene expression in response to MetR. Different potential mechanisms underlying these changes in BER, such as DNA methylation or activation of signaling pathways, are discussed.

Breast cancer (BC) is a serious and widespread disease for which different treatments have been developed. In addition to the classic therapies, the treatment with retinoic acid (RA) is still being clinically investigated. RA reduces cancer cells proliferation and migration, but its molecular mechanism of action is not clear. In tumor development, autophagy promotes cancer cell survival and prevents apoptosis. Small heat shock protein B8 (HSPB8) acts together with its co-chaperone BCL-2 associated athanogene 3 (BAG3) stimulating BC proliferation and migration. We analyzed whether direct correlations exist between RA and HSPB8 or BAG3 and how this may play a role in BC. We measured HSPB8 and BAG3 gene expression in MCF-7 BC cells and we analyzed the potential correlation between the antiproliferative and antimigratory effect of RA with the expression level of HSPB8. We found that in MCF-7 cells RA reduces both HSPB8 and BAG3 gene expression and it alters the mitotic spindle organization. Notably, the effects of RA on HSPB8 levels are exerted at both transcriptional and translational levels. RA effects are possibly mediated by miR-574-5p that targets the HSPB8 transcript. Our results suggest that therapeutic doses of RA can efficiently counteract the adverse effects of HSPB8 in BC progression.

Expression of base excision DNA repair genes as a biomarker of oxidative DNA damage

Low-power lasers and LEDs (light-emitting diodes) have been used to induce photobiomodulation (PBM) based on therapeutic protocols to treat diseases and clinical conditions. PBM begins with the absorption of radiation by photoacceptors, generating trigger molecules, such as reactive oxygen species (ROS), which leads to molecular, cellular and systemic effects. Breast cancer (BC) is the main cause of cancer death among women, and treatment depends on BC subtype. Despite the PBM being considered safe, its applications on cancer patients are controversial, which could hinder the benefits of PBM on BC patients. Thus, this study aimed to evaluate the effects of a low-power infrared laser and an amber LED on cell viability and ROS levels in human BC triple-negative and luminal A cells. For that, MDA-MB-231 and MCF-7 cultures were exposed to infrared laser (830 nm; 150 mW; 1.19 W cm −2 ; 32, 64, and 97 J cm −2 ) and amber LED (617 nm; 1500 mW; 11.94 W cm −2 ; 358, 716, and 1074 J cm −2 ), alone and simultaneously, and cell viability was evaluated. Also, ROS levels were evaluated by flow cytometry. The results show that exposure to low-power infrared laser and amber LED does not alter the cell viability in MDA-MB-231 and MCF-7 cultures. However, exposure to simultaneous infrared laser and amber LED increases ROS generation in MCF-7 cells. These results suggest that the effects of PBM induced by simultaneous exposure to low-power radiations on redox state depend on the human BC cell subtype.

Development of novel approaches for quantitative analysis of gene expression in intact tumor cells should provide new means for cancer detection and for studying the response of cancer cells to biological and therapeutic reagents. We developed procedures for detecting the levels of expression of multiple genes in fixed as well as viable cells using molecular beacon imaging technology. We found that simultaneous delivery of molecular beacons targeting survivin and cyclin D1 mRNAs produced strong fluorescence in breast cancer but not in normal breast cells. Importantly, fluorescence intensity correlated well with the level of gene expression in the cells detected by real-time reverse transcription-PCR or Western blot analysis. We further show that molecular beacons can detect changes of survivin gene expression in viable cancer cells following epidermal growth factor stimulation, docetaxel treatment, and overexpression of p53 gene. Thus, molecular beacon imaging is a simple and specific method for detecting gene expression in cancer cells. It has great potential for cancer detection and drug development.

DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells

Dear Editor, Neuroblastoma is the most common non-central nerve system (CNS) solid tumor in pediatrics [1]. Neuroblastoma accounts for approximately 8% of all pediatric cancers but disproportionally causes a high cancer mortality (15%) in children [2]. Pediatric patients with low-risk neuroblastoma witness a 5-year overall survival rate > 90%, whereas the 5-year overall survival rate in high-risk neuroblastoma pediatric patients is < 40% [3]. Genetic susceptibility to neuroblastoma is a promising area of research and needs to be fully investigated. For sporadic neuroblastoma, genome-wide association studies (GWASs) have identified over a dozen causal genetic loci. Studies of candidate genes also reported a decent number of variants predisposing to neuroblastoma. However, the known genetic alternations still could not unveil the full genetic underpinnings of neuroblastoma. The base excision repair (BER) pathway, one of the DNA repair systems, is responsible for repairing numerous oxidized and alkylated bases by recognizing and excising damaged bases [4]. Many core proteins are involved in the BER pathway, including poly(ADP)ribose polymerase 1 (PARP1), human 8-oxoguanine DNA glycosylase (OGG1), flap endonuclease 1 (FEN1), apurinic/apyrimidinic endonuclease 1 (APEX1), DNA ligase III (LIG3), and x-ray repair cross-complementing group 1 (XRCC1). OGG1 is a bifunctional enzyme (DNA glycosylase and AP lyase) that incises at abasic sites via an AP lyase activity, leaving a single-strand DNA break intermediate. APEX1 initiates the repair of abasic sites in DNA by cleaving the phosphodiester backbone 5′ to an AP site, creating a nick in the DNA backbone. FEN1 participates in the penultimate steps of Okazaki fragment maturation and 5′-flap removal during long-patch BER. LIG3 catalyzes the last stage of BER by sealing the gap. XRCC1 and PARP1 serve as the scaffold protein. Intensive evidence suggests that aberrant BER pathway proteins result in a variety of diseases, especially cancers [4]. Single nucleotide polymorphisms (SNPs) of the BER pathway genes are associated with the risk of various cancer types. Functional analysis revealed that SNPs in the BER pathway genes may modify the kinetics of BER proteins and the DNA repair capacity of the BER system, ultimately affecting carcinogenesis [4]. However, evidence regarding the role of BER pathway gene SNPs in the risk of neuroblastoma waits to be added. To identify more neuroblastoma susceptibility variations in the BER pathway genes, we performed a case-control study in children at three center hospitals in East China. This study was conducted in Children's Hospital of Nanjing Medical University (Nanjing, Jiangsu), Anhui Provincial Children's Hospital (Hefei, Anhui), and Yuying Children's Hospital of Wenzhou Medical University (Wenzhou, Zhejiang) in East China. A total of 313 neuroblastoma pediatric patients and 762 cancer-free children were recruited in this study. The characteristics of the study subjects are summarized in Supplementary Table S1. Age (P = 0.823) and gender (P = 0.610) were distributed equivalently between the two groups. The study design and participant recruitment were described in our previous work [5]. We successfully genotyped 20 SNPs from 6 BER pathway genes in 313 neuroblastoma pediatric patients and 762 control children (Table 1). Specifically, 3 PARP1, 3 OGG1, 2 FEN1, 3 APEX1, 3 LIG3, and 6 XRCC1 SNPs were genotyped. The genotypic distributions of all candidate SNPs were in Hardy-Weinberg equilibrium (P ≥ 0.05) in the controls. The rs174538 of the FEN1 gene was associated with decreased neuroblastoma risk under the dominant model (adjusted odd ratio [OR] = 0.71, 95% confidence interval [CI] = 0.54-0.93, P = 0.012). However, no significant associations with neuroblastoma risk were found for the remaining SNPs in the single-locus analysis (all P ≥ 0.05; Supplementary Figure S1). We conducted the stratified analyses (Supplementary Table S2) to eliminate potential influences of FEN1 genotypes on neuroblastoma susceptibility by adjusting confounding factors (age, gender, and site of tumor origin). The protective role of rs174538 AG/GG in decreasing neuroblastoma risk was found in subgroups of age ≤18 months (adjusted OR = 0.60, 95% CI = 0.40-0.89, P = 0.011), females (adjusted OR = 0.59, 95% CI = 0.40-0.87, P = 0.009), and tumors arising from the mediastinum (adjusted OR = 0.53, 95% CI = 0.35-0.81, P = 0.003). Combined analysis stated that the 2 protective genotypes (rs174538 AG/GG and rs4246215 TG/GG genotypes) also decreased neuroblastoma risk in the following subgroups: age ≤ 18 months (adjusted OR = 0.62, 95% CI = 0.42-0.93, P = 0.019), females (adjusted OR = 0.61, 95% CI = 0.41-0.91, P = 0.015), and tumors originated from the mediastinum (adjusted OR = 0.54, 95% CI = 0.36-0.83, P = 0.005). We carried out false-positive report probability (FPRP) analysis to validate significant associations (Supplementary Table S3). The threshold for FPRP was preset as 0.2. At the prior probability level of 0.1, significant associations with FEN1 rs174538 A > G (GG/AG vs. AA) remained noteworthy in all subjects (FPRP = 0.121) as well as in the subgroups of females (FPRP = 0.185) and tumors originating from the mediastinum (FPRP = 0.160). In the combined analysis, significant findings for 2 vs. 0-1 protective genotypes (FPRP = 0.166) and its subgroup tumors originated from the mediastinum (FPRP = 0.183) could be called noteworthy. We further explored the biological effects of FEN1 rs174538 A > G on the neighboring gene expression by using released data from Genotype-Tissue Expression (GTEx) Portal (https://www.gtexportal.org/). We observed that rs174538 A allele was significantly associated with increased mRNA expression levels of fatty acid desaturase 2 (FADS2) and transmembrane protein 258 (TMEM258) in the whole blood, nerve-tibial, and cell-cultured fibroblasts (Figure 1A). The rs174538 A allele was also associated with increased expression of fatty acid desaturase 1 (FADS1) mRNA in the whole blood, but with decreased expression of FADS1 mRNA in the nerve-tibial (Figure 1B). eQTL analysis of the neuroblastoma risk factor FEN1 rs174538 A > G. A. FADS2 and TMEM258 levels in the whole blood, nerve-tibial, and cell-cultured fibroblasts; B. FADS1 level in the whole blood and nerve-tibial. Abbreviations: eQTL, expression quantitative loci; FEN1, flap endonuclease 1; FADS2, fatty acid desaturase 2; TMEM258, transmembrane protein 258 The implication of the BER pathway gene SNPs in cancer susceptibility has been highly documented. Plenty of SNPs within the BER pathway genes were found to predispose to various types of cancer. Our group previously carried out a study on BER gene polymorphisms and Wilms tumor susceptibility [6]. Significant associations with Wilms tumor susceptibility were shown for the OGG1 rs1052133, FEN1 rs174538, and FEN1 rs4246215 polymorphisms. Regarding the association of the BER pathway gene SNPs with neuroblastoma risk, only 3 studies were available by far; and all of them were performed by our research group. In these studies, we found that, none of the studied APEX1 polymorphisms were associated with neuroblastoma risk [5]. Such a negative association was also observed between neuroblastoma risk and polymorphisms in the OGG1 [7] and LIG3 genes [8]. However, all these studies were conducted to analyze a single gene in the BER pathway, and the results need to be validated in another independent study. Thus, here we attempted to validate the previous studies by adopting a systematical analysis of potentially functional SNPs in 6 core genes in the BER pathway. In the current study, no significant relationships were detected between neuroblastoma risk and the SNPs in PARP1, OGG1, APEX1, LIG3, and XRCC1 genes. Such results strengthen the previous findings that these variations may be too weak to impact neuroblastoma risk. To be noted, significant conferring roles of the same BER SNPs to the risk of other cancer types have been detected, such as PARP1 rs1136410 and thyroid cancer [9], OGG1 rs1052133 and Wilms tumor [6], FEN1 rs4246215 and Wilms tumor [6], APEX1 rs1130409 and renal cell carcinoma [10], LIG3 rs1052536 and lung cancer [11]. The different roles of these SNPs in specific cancer types indicated that specific cancer types should be set before interpreting the role of SNPs. Excitedly, we demonstrated that the rs174538 of the FEN1 gene could protect from neuroblastoma. FEN1 is a structure-specific nuclease involved in the removal of 5′-flap during long-patch BER and the maturation of Okazaki fragments in DNA replication. Moreover, FEN1 is also characterized as a 5′ exonuclease and a gap-dependent endonuclease, which mediates apoptotic DNA degradation during apoptosis. The FEN1 gene is mapped to chromosome 11 (11q12.2). Yang et al. [12] identified that the rs174538 A allele of the FEN1 gene decreased risk for lung cancer by decreasing FEN1 expression. Moreover, they detected that coke oven workers who carried the AA genotype have significantly lower DNA damage level than those with GG or GA genotypes. In a meta-analysis conducted for the overall cancer, the results suggested that the subjects with FEN1 rs174538 A allele have a decreased susceptibility to cancer in Chinese populations [13]. We further performed online expression quantitative trait loci (eQTL) analysis to interpret the possible mechanism of how rs174538 impacts neuroblastoma risk. eQTL evidence suggested that the A allele in rs174538 was significantly associated with the increased mRNA expression levels of FADS2 and TMEM258. Further functional experiments conducted in neuroblastoma cells are needed to show how the FEN1 rs174538 A allele can be associated with altered expressions of these genes. FADS2 was found to function as a potential oncogene in some types of cancer [14]. TMEM258 is a central mediator of endoplasmic reticulum quality control and intestinal homeostasis, yet its role in cancer remains unknown [15]. The exact relationship of FADS2 and TMEM258 with neuroblastoma risk waits to be elucidated. Taken together, the significant role of rs174538 A allele in cancer deserves more attention for further exploration. Although at the preliminary stage, our findings represent a novel mechanism by which rs174538 may modulate the expression of multiple nearby genes, thereby impacting the risk of neuroblastoma. Our study has several limitations. First, the sample sizes were small in some stratification analyses. Second, the number of analyzed SNPs was limited. Another limitation was the lack of incorporating analysis on environment factors and genetic-environmental factors. The fourth limitation was that the current study only focused on the subjects of the Han population. Replication of these findings in additional individuals of non-Chinese descent should be helpful to validate our findings. In conclusion, we showed a robust association of genetic variants in the FEN1 gene with neuroblastoma risk in a relatively large sample size of pediatric patients in East China. Intensive future research is warranted to extend the role of FEN1 gene loci in neuroblastoma susceptibility in individuals of non-Chinese ancestries. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Ethics Committees of Children's Hospital of Nanjing Medical University, Anhui Provincial Children's Hospital, and the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. Each participant signed an informed consent before participating to this study. Not applicable. All data generated or analyzed during this study are included in this published article and its additional files. The authors declare that they have no competing interests. This work was supported by the grants from Natural Science Foundation of Guangdong Province (2019A1515010360) and Guangdong Provincial Key Laboratory of Research in Structural Birth Defect Disease (2019B030301004). Z.Z., J.Z., H.L., J.H., and Y.W. designed the study, performed the experiments and wrote the manuscript. C.Z., Y.F., H.Z., H.W., and Y.W. collected the clinical samples and information. Z.Z. and J.H. analyzed the data and prepared all the tables and figures. Z.Z., J.H., and Y.W. coordinated the study. All authors reviewed and approved the final manuscript. Not applicable. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Is the DNA damage response (DDR) dysregulated in the eutopic endometrium of women with endometriosis? Endometrial expression of genes involved in DDR is modulated in women with endometriosis, compared to those without the disease. Ectopic endometriotic lesions are reported to harbour somatic mutations, thereby hinting at dysregulation of DDR and DNA repair pathways. However, it remains inconclusive whether the eutopic endometrium also manifests dysregulated DDR in endometriosis. For this case-control study conducted between 2015 and 2019, eutopic endometrial (E) samples (EE- from women with endometriosis, CE- from women without endometriosis) were collected in either mid-proliferative (EE-MP, n = 23; CE-MP, n = 17) or mid-secretory (EE-MS, n = 17; CE-MS, n = 9) phases of the menstrual cycle. This study compares: (i) DNA damage marker localization, (ii) expression of DDR genes and (iii) expression of DNA repair genes in eutopic endometrial samples from women with and without endometriosis. The study included (i) 40 women (aged 31.9 ± 0.81 years) with endometriosis and (ii) 26 control women (aged 31.4 ± 1.02 years) without endometriosis. Eutopic endometrial samples from the two groups were divided into different parts for histological analysis, immunohistochemistry, RNA extraction, protein extraction and comet assays. Eighty-four genes of relevance in the DNA damage signalling pathway were evaluated for their expression in eutopic endometrial samples, using RT2 Profiler PCR arrays. Validations of the expression of two GADD (Growth Arrest DNA Damage Inducible) proteins - GADD45A and GADD45G were carried out by immunoblotting. DNA damage was assessed by immunohistochemical localization of γ-H2AFX (a phosphorylated variant of histone H2AX) and 8-OHdG (8-hydroxy-2'-deoxyguanosine). RNA sequencing data from mid-proliferative (EE-MP, n = 4; CE-MP, n = 3) and mid-secretory phase (EE-MS and CE-MS, n = 4 each) endometrial samples were scanned to compare the expression status of all the genes implicated in human DNA repair. PCNA (Proliferating Cell Nuclear Antigen) expression was determined to assess endometrial proliferation. Residual DNA damage in primary endometrial cells was checked by comet assays. Public datasets were also scanned for the expression of DDR and DNA repair genes as our RNASeq data were limited by small sample size. All the comparisons were made between phase-matched endometrial samples from women with and without endometriosis. Endometrial expression of DDR genes and intensity of immunolocalized γ-H2AFX were significantly (P < 0.05) higher in EE, compared to CE samples. DDR proteins, especially those belonging to the GADD family, were found to be differentially abundant in EE, as compared to CE. These patterns were evident in both mid-proliferative and mid-secretory phases. Intriguingly, higher DDR was associated with increased cell proliferation in EE-MP, compared to CE-MP. Furthermore, among the differentially expressed transcripts (DETs) encoded by DNA repair genes, the majority showed up-regulation in EE-MP, compared to CE-MP. Interestingly, CE-MP and EE-MP had a comparable percentage (P > 0.05) of cells with residual DNA damage. However, unlike the mid-proliferative phase data, many DETs encoded by DNA repair genes were down-regulated in EE-MS, compared to CE-MS. An analysis of the phase-matched control and endometriosis samples included in the GSE51981 dataset available in the Gene Expression Omnibus database also revealed significant (P < 0.05) alterations in the expression of DDR and DNA repair genes in EE, compared to CE. N/A. The study was conducted on a limited number of endometrial samples. Also, the study does not reveal the causes underlying dysregulated DDR in the eutopic endometrium of women with endometriosis. Alterations in the expression of DDR and DNA repair genes indirectly suggest that eutopic endometrium, as compared to its healthy counterpart, encounters DNA damage-inducing stimuli, either of higher strength or for longer duration in endometriosis. It will be worthwhile to identify the nature of such stimuli and also explore the role of higher genomic insults and dysregulated DDR/DNA repair in the origin and/or progression of endometriosis. The study was supported by the Department of Biotechnology and Indian Council of Medical Research, Government of India. No conflict of interest is declared.