Nucleotide Excision Repair Capacity Research Articles

Thallium(I) induces a prolonged inhibition of (6-4)photoproduct binding and UV damage excision repair activities in zebrafish (Danio rerio) embryos via protein inactivation

Cyclobutane pyrimidine dimer (CPD) and (6-4)photoproduct (6-4 PP) are two major types of UV-induced DNA lesion and 6-4 PP is more mutagenic than CPD. Activated by lesion detection, nucleotide excision repair (NER) eliminates CPDs and 6-4 PPs. Thallium (Tl) is a toxic metal existing primarily as Tl+ in the aquatic environment. Ingestion of Tl+-contaminated foods and water is a major route of human poisoning. As Tl+ may inhibit enzyme activities via binding to sulfhydryl groups, this study explored if Tl+ could intensify UV mutagenicity by inactivating NER-linked damage recognition factors using zebrafish (Danio rerio) embryo as a model system. Incubation of Tl+ (as thallium nitrate) at 0.1–0.4 μg/mL with zebrafish extracts for 20 min caused a concentration-dependent inhibition of 6-4 PP binding activities as shown by a photolesion-specific band shift assay, while CPD binding activities were insensitive to Tl+. The ability of Tl+ to suppress 6-4 PP detection was stronger than that of Hg2+. Exposure of zebrafish embryos at 1 h post fertilization (hpf) to Tl+ at 0.4–1 μg/mL for 9 or 71 h also specifically inhibited 6-4 PP detection, indicating that Tl+ induced a prolonged inhibition of 6-4 PP sensing ability primarily via its direct interaction with damage recognition molecules. Tl+-mediated inhibition of 6-4 PP binding in embryos at distinct stages resulted in a suppression of NER capacity monitored by a transcription-based DNA repair assay. Our results revealed the potential of Tl+ to enhance UV mutagenicity by disturbing the removal of 6-4 PP through repressing the lesion detection step of NER.

TGFβ signaling links early life endocrine-disrupting chemicals exposure to suppression of nucleotide excision repair in rat myometrial stem cells

Environmental exposure to endocrine-disrupting chemicals (EDCs) is linked to the development of uterine fibroids (UFs) in women. UFs, non-cancerous tumors, are thought to originate from abnormal myometrial stem cells (MMSCs). Defective DNA repair capacity may contribute to the emergence of mutations that promote tumor growth. The multifunctional cytokine TGFβ1 is associated with UF progression and DNA damage repair pathways. To investigate the impact of EDC exposure on TGFβ1 and nucleotide excision repair (NER) pathways, we isolated MMSCs from 5-month-old Eker rats exposed neonatally to diethylstilbestrol (DES), an EDC, or to vehicle (VEH). EDC-MMSCs exhibited overactivated TGFβ1 signaling and reduced mRNA and protein levels of NER pathway components compared to VEH-MMSCs. EDC-MMSCs also demonstrated impaired NER capacity. Exposing VEH-MMSCs to TGFβ1 decreased NER capacity while inhibiting TGFβ signaling in EDC-MMSCs restored it. RNA-seq analysis and further validation revealed decreased expression of Uvrag, a tumor suppressor gene involved in DNA damage recognition, in VEH-MMSCs treated with TGFβ1, but increased expression in EDC-MMSCs after TGFβ signaling inhibition. Overall, we demonstrated that the overactivation of the TGFβ pathway links early life exposure to EDCs with impaired NER capacity, which would lead to increased genetic instability, arise of mutations, and fibroid tumorigenesis. We demonstrated that the overactivation of the TGFβ pathway links early life exposure to EDCs with impaired NER capacity, which would lead to increased fibroid incidence.

Susceptibility of DNA damage recognition activities linked to nucleotide excision and mismatch repair in zebrafish (Danio rerio) early and mid-early embryos to 2.5 to 4.5°C heat stress.

Fish at early life stages are sensitive to temperature change because of their narrower temperature tolerance ranges. Initiated by damage detection, DNA mismatch repair (MMR) and nucleotide excision repair (NER) maintain genome integrity respectively by eliminating mismatched nucleotides and helix-distorting DNA lesions. As discharge of heated effluent from power plants may elevate water temperatures to only 2 to 6°C higher than ambient, this study explored if temperatures within this range affected MMR and NER-linked damage detection activities in fish embryos using zebrafish (Danio rerio) embryo as a model organism. Exposure of early embryos at 10h post fertilization (hpf) to a warmer temperature at + 4.5°C for 30min enhanced damage recognition activities targeting UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) that distorted helical structures. Conversely, photolesions sensing activities were inhibited in 24 hpf mid-early embryos under the same stress conditions. A much higher temperature at + 8.5°C imposed similar effects on UV damage detection. A mild heat stress at + 2.5°C for 30min, however, repressed both CPD and 6-4PP binding activities in 10 and 24 hpf embryos. Inhibition of damage recognition under mild heat stress impeded the overall NER capacity evidenced by a transcription-based repair assay. Warmer water temperatures at + 2.5 and + 4.5°C also inhibited G-T mismatch binding activities in 10 and 24 hpf embryos, but G-T recognition was more sensitive to + 4.5°C stress. Inhibition of G-T binding partially correlated with a downregulation of Sp1 transcription factor activity. Our results showed the potential of water temperature elevation within 2 to 4.5°C to disturb DNA damage repair in fish at embryonic stages.

Deregulated DNA damage response network in Behcet's disease

Behcet's disease (BD) is a chronic, relapsing systemic vasculitis of unknown etiology. Since the DNA repair enzyme NEIL1 has been identified as one of the two genetic risk factors for BD by whole exome study, we examined the potential involvement of the DNA damage response (DDR) network in BD. Peripheral blood mononuclear cells from 26 patients and 26 age−/sex-matched healthy controls were studied. Endogenous DNA damage levels were increased in active BD patients compared to controls or patients in remission. In parallel, BD patients had defective nucleotide excision repair capacity. RNA-sequencing revealed reduced expression of NEIL1 that negatively correlated with DNA damage accumulation. On the other hand, expression of genes involved in senescence and senescence-associated secretory phenotype positively correlated with individual endogenous DNA damage levels. We conclude that deregulated DDR contributes to the proinflammatory environment in BD.

TGF-Β AND EARLY-LIFE EXPOSURE TO ENDOCRINE-DISRUPTING CHEMICALS IMPAIR NUCLEOTIDE EXCISION REPAIR IN EKER RAT MYOMETRIAL STEM CELLS

Uterine fibroids (UFs) are the most common noncancerous tumors in women of reproductive age, and their prevalence is associated with environmental exposure to endocrine-disrupting chemicals (EDCs). Each UFs is thought to arise from a single transformed myometrial stem cell (MMSCs) that acquires a driver mutation in pivotal genes such as Tsc-2 in Eker rats. The Eker rat is a unique animal model of spontaneous development of UFs in adulthood, that are characteristically similar to human UFs. There is evidence that a deficient DNA repair capacity could be involved in the emergence of such mutations. We have previously shown that the TGF-β1 pathway is more active in Eker rat MMSCs exposed to EDCs compared to VEH. There is evidence that TGF-β links to the DNA damage response with implications for tumor origin and development. The objective of this work was to evaluate the role of TGF-β1 and EDCs exposure on nucleotide excision repair (NER, a DNA repair mechanism) capacity in MMSCs.

Aluminum (Al) causes a delayed suppression of nucleotide excision repair (NER) capacity in zebrafish (Danio rerio) embryos via disturbance of DNA lesion detection

Aluminum (Al) is extensively used for making cooking utensils and its presence in the aquatic environment may occur through acid mine drainage and wastewater discharge. Al is known to induce genotoxicity in human cells, rodents, and fish. Nucleotide excision repair (NER) eliminates helix-twisting DNA lesions such as UV-induced dipyrimidine photoproducts. Because our earlier investigation revealed the operation of NER in zebrafish (Danio rerio) embryos, this study explored if inhibition of NER could be a mechanism of Al-induced genotoxicity using zebrafish embryo as a model system. An acute fish embryo toxicity test indicated that Al (as aluminum sulfate) at 2–15 mg/L were nonlethal to zebrafish embryos, yet exposure of embryos at 1 h post fertilization (hpf) to Al at 10–15 mg/L for 71 h significantly repressed their NER capacity monitored by a transcription-based DNA repair assay. Band shift analysis indicated a higher sensitivity of (6−4) photoproduct (6–4PP) than cyclobutane pyrimidine dimer (CPD) detecting activities to Al, reflecting the preferential influence of Al on the detection of strongly distorted DNA lesions. Time-course experiments showed a delayed response of NER to Al as repair machinery was unaffected by Al at 15 mg/L following a 35-h exposure, while Al treatment for the same period obviously inhibited 6–4PP binding activities although the gene expression of damage recognition factors remained active. Inhibition of 6–4PP detection blocked downstream lesion incision/excision detected by a terminal deoxy transferase-mediated end labeling assay. As the disturbance of damage sensing preceded that of the overall repair process, Al exposure was believed to downregulate NER capacity by inhibiting the activities of lesion detection proteins. Our results revealed the ability of Al to enhance its genotoxicity by suppressing NER capacity.

Oxidative Stress and Deregulated DNA Damage Response Network in Lung Cancer Patients.

The deregulated DNA damage response (DDR) network is associated with the onset and progression of cancer. Herein, we searched for DDR defects in peripheral blood mononuclear cells (PBMCs) from lung cancer patients, and we evaluated factors leading to the augmented formation of DNA damage and/or its delayed/decreased removal. In PBMCs from 20 lung cancer patients at diagnosis and 20 healthy controls (HC), we analyzed oxidative stress and DDR-related parameters, including critical DNA repair mechanisms and apoptosis rates. Cancer patients showed higher levels of endogenous DNA damage than HC (p < 0.001), indicating accumulation of DNA damage in the absence of known exogenous genotoxic insults. Higher levels of oxidative stress and apurinic/apyrimidinic sites were observed in patients rather than HC (all p < 0.001), suggesting that increased endogenous DNA damage may emerge, at least in part, from these intracellular factors. Lower nucleotide excision repair and double-strand break repair capacities were found in patients rather than HC (all p < 0.001), suggesting that the accumulation of DNA damage can also be mediated by defective DNA repair mechanisms. Interestingly, reduced apoptosis rates were obtained in cancer patients compared with HC (p < 0.001). Consequently, the expression of critical DDR-associated genes was found deregulated in cancer patients. Together, oxidative stress and DDR-related aberrations contribute to the accumulation of endogenous DNA damage in PBMCs from lung cancer patients and can potentially be exploited as novel therapeutic targets and non-invasive biomarkers.

A Preclinical Study to Repurpose Spironolactone for Enhancing Chemotherapy Response in Bladder Cancer.

Neoadjuvant chemotherapy (NAC) followed by radical cystectomy is the standard-of-care for patients with muscle-invasive bladder cancer (MIBC). Defects in nucleotide excision repair (NER) are associated with improved responses to NAC. Excision Repair Cross-Complementation group 3 (ERCC3) is a key component of NER process. No NER inhibitors are available for treating patients with bladder cancer. We have developed an ex vivo cell-based assay of 6-4 pyrimidine-pyrimidinone (6-4PP) removal as a surrogate measure of NER capacity in human bladder cancer cell lines. The protein expression of ERCC3 was examined in human MIBC specimens and cell lines. Small molecule inhibitors were screened for NER inhibition in bladder cancer cell lines. Spironolactone was identified as a potent NER inhibitor. Combined effects of spironolactone with chemo-drugs were evaluated in vitro and in vivo. The efficacy between platinum and spironolactone on cytotoxicity was determined by combination index. A correlation between NER capacity and cisplatin sensitivity was demonstrated in a series of bladder cancer cell lines. Further, siRNA-mediated knockdown of ERCC3 abrogated NER capacity and enhanced cisplatin cytotoxicity. Spironolactone inhibited ERCC3 protein expression, abrogated NER capacity, and increased platinum-induced cytotoxicity in bladder cancer cells in vivo and in patient-derived organoids. Moreover, spironolactone exhibited the potential synergism effects with other clinical chemotherapy regimens in bladder cancer cell lines. Our data support the notion of repurposing spironolactone for improving the chemotherapy response of NAC in patients with MIBC. Further clinical trials are warranted to determine the safety and efficacy of spironolactone in combination with chemotherapy.

DNA Repair Capacity for Personalizing Risk and Treatment Response − Assay Development and Optimization in Human Peripheral Blood Mononuclear Cells (PBMCs)

DNA repair capacity (DRC) is the ability of a cell to repair DNA damage. Differential DRC plays an important role in human disease, including lung and other cancers. Measuring DRC could aid in translational disease research and in personalizing treatment. We developed and optimized a flow cytometry-based assay to measure individual DRC using GFP-expressing plasmids modified by ultraviolet (UV) light for nucleotide excision repair (NER) and restriction enzyme digestion to induce a blunt double-strand cut between promoter and GFP expression regions for nonhomologous end joining (NHEJ). Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy volunteers were used to measure DRC and optimize the assay. Pathway specificity of the NHEJ DRC assay was confirmed using Ku80-/- MEF cells, which showed a 6-fold reduction in NHEJ compared to Ku80+/+. Using a cell mixing assay, we show a linear correlation between NHEJ DRC and the expected concentration of Ku80. NHEJ DRC measurements in cryopreserved PBMCs are quantifiable with low interindividual and inter-assay variability, and a titratable decrease in NHEJ activity was observed in PBMCs treated with the DNA-PK inhibitor NU7441. Pathway specificity of the NER DRC assay was confirmed by a decrease in measured NER activity in human XPC deficient compared to XPC proficient fibroblasts, with a linear correlation measured between NER DRC and expected XPC concentration by cell mixing assay. NER DRC is quantifiable, reproducible, and titratable in PBMCs from healthy volunteers. We measured both NER and NHEJ DRC in PBMCs obtained from newly diagnosed, untreated lung cancer patients; measured DRC differed in these PBMCs compared to healthy volunteers. With further investigation, measurement of NER and NHEJ DNA repair capacity may be useful in personalizing disease risk and response to DNA damaging therapies and small molecular inhibitors of DNA repair pathways using readily available human PBMCs.

Abstract 2106: Combined deacetylase and bromodomain inhibition downregulates ERCC2 and suppresses growth of metastatic colon cancer cells

Abstract DNA repair genes have potential clinical value in predicting cancer prognosis and treatment outcomes. Nucleotide excision repair (NER) proteins like ERCC2 play a critical role in maintaining genome integrity by recognizing and unwinding DNA at sites of damage1.Aberrant expression of ERCC2 alters NER capacity, influencing treatment outcomes2. The current investigation examined the expression of ERCC2 following epigenetic combination treatment in colorectal cancer (CRC) cells and preclinical models. Attention was drawn to ERCC2 based on three observations. First, from online databases, when ERCC2 was overexpressed in colon tumors the corresponding CRC patients had reduced overall survival3. Second, ERCC2 was the most highly downregulated gene when dietary histone deacetylase 3 (HDAC3) inhibitor sulforaphane (SFN) was combined withJQ1, an inhibitor of the bromodomain and extra terminal domain family, in human colon cancer cells and in colon polyps from the polyposis in rat colon (Pirc) model4. Third, as reported here for the first time, RNA-seq analyses of Pirc colon polyps from rats treated withJQ1 and the SFN analog 6-SFN5 identified Ercc2 as the most highly downregulated gene. RNA-seq data were corroborated by RT-qPCR and immunoblotting experiments. There is much interest in combination approaches that target epigenetic ‘readers, writers, and erasers' that are deregulated in cancer and other pathologies. The current work identified promising second-generation inhibitors with enhanced synergy and antitumor efficacy, especially in metastatic cells cultured in three-dimensions. Drug combinations decreased HDAC3, BRD4 and ERCC2 expression, while DNA damage and apoptosis markers were increased both in spheroids and in a mouse xenograft model. This investigation has potential clinical relevance for the identification of robust biomarkers that predict enhanced antitumor outcomes in CRC patients.1. Gillet LC, Schärer OD. Molecular mechanisms of mammalian global genome nucleotide excision repair. Chem Rev 2006;106:253-76.2. Shuck SC, Short EA, Turchi JJ. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Cell Res 2008;18:64-72.3. Liu J et al., The Differential Expression of Core Genes in Nucleotide Excision Repair Pathway Indicates Colorectal Carcinogenesis and Prognosis. Biomed Res Int 2018;2018:9651320.4. Rajendran P et al., Acetylation of CCAR2 establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition. Cancer Res 2019;79:918-27.5. Rajendran Pet al., HDAC turnover, CtIP acetylation and dysregulated DNA damage signaling in colon cancer cell treated with sulforaphane and related dietary isothiocyanates. Epigenetics 2013;8:612-23. Citation Format: Sabeeta Kapoor, Trace Gustafson, Mutian Zhang, Ying-Shiuan Chen, Jia Li, Nhung Nguyen, Jorge Enrique Perez, Wan Mohaiza Dashwood, Praveen Rajendran, Roderick Dashwood. Combined deacetylase and bromodomain inhibition downregulates ERCC2 and suppresses growth of metastatic colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2106.

A Gene Expression Signature to Predict Nucleotide Excision Repair Defects and Novel Therapeutic Approaches.

Nucleotide excision repair (NER) resolves DNA adducts, such as those caused by ultraviolet light. Deficient NER (dNER) results in a higher mutation rate that can predispose to cancer development and premature ageing phenotypes. Here, we used isogenic dNER model cell lines to establish a gene expression signature that can accurately predict functional NER capacity in both cell lines and patient samples. Critically, none of the identified NER deficient cell lines harbored mutations in any NER genes, suggesting that the prevalence of NER defects may currently be underestimated. Identification of compounds that induce the dNER gene expression signature led to the discovery that NER can be functionally impaired by GSK3 inhibition, leading to synergy when combined with cisplatin treatment. Furthermore, we predicted and validated multiple novel drugs that are synthetically lethal with NER defects using the dNER gene signature as a drug discovery platform. Taken together, our work provides a dynamic predictor of NER function that may be applied for therapeutic stratification as well as development of novel biological insights in human tumors.

Role of Nucleotide Excision Repair in Cisplatin Resistance.

Cisplatin is a chemotherapeutic drug used for the treatment of a number of cancers. The efficacy of cisplatin relies on its binding to DNA and the induction of cytotoxic DNA damage to kill cancer cells. Cisplatin-based therapy is best known for curing testicular cancer; however, treatment of other solid tumors with cisplatin has not been as successful. Pre-clinical and clinical studies have revealed nucleotide excision repair (NER) as a major resistance mechanism against cisplatin in tumor cells. NER is a versatile DNA repair system targeting a wide range of helix-distorting DNA damage. The NER pathway consists of multiple steps, including damage recognition, pre-incision complex assembly, dual incision, and repair synthesis. NER proteins can recognize cisplatin-induced DNA damage and remove the damage from the genome, thereby neutralizing the cytotoxicity of cisplatin and causing drug resistance. Here, we review the molecular mechanism by which NER repairs cisplatin damage, focusing on the recent development of genome-wide cisplatin damage mapping methods. We also discuss how the expression and somatic mutations of key NER genes affect the response of cancer cells to cisplatin. Finally, small molecules targeting NER factors provide important tools to manipulate NER capacity in cancer cells. The status of research on these inhibitors and their implications in cancer treatment will be discussed.

Association Between DNA Damage Response, Fibrosis and Type I Interferon Signature in Systemic Sclerosis.

Increased endogenous DNA damage and type I interferon pathway activation have been implicated in systemic sclerosis (SSc) pathogenesis. Because experimental evidence suggests an interplay between DNA damage response/repair (DDR/R) and immune response, we hypothesized that deregulated DDR/R is associated with a type I interferon signature and/or fibrosis extent in SSc. DNA damage levels, oxidative stress, induction of abasic sites and the efficiency of DNA double-strand break repair (DSB/R) and nucleotide excision repair (NER) were assessed in peripheral blood mononuclear cells (PBMCs) derived from 37 SSc patients and 55 healthy controls; expression of DDR/R-associated genes and type I interferon–induced genes was also quantified. Endogenous DNA damage was significantly higher in untreated diffuse or limited SSc (Olive tail moment; 14.7 ± 7.0 and 9.5 ± 4.1, respectively) as well as in patients under cytotoxic treatment (15.0 ± 5.4) but not in very early onset SSc (5.6 ± 1.2) compared with controls (4.9 ± 2.6). Moreover, patients with pulmonary fibrosis had significantly higher DNA damage levels than those without (12.6 ± 5.8 vs. 8.8 ± 4.8, respectively). SSc patients displayed increased oxidative stress and abasic sites, defective DSB/R but not NER capacity, downregulation of genes involved in DSB/R (MRE11A, PRKDC) and base excision repair (PARP1, XRCC1), and upregulation of apoptosis-related genes (BAX, BBC3). Individual levels of DNA damage in SSc PBMCs correlated significantly with the corresponding mRNA expression of type I interferon–induced genes (IFIT1, IFI44 and MX1, r=0.419-0.490) as well as with corresponding skin involvement extent by modified Rodnan skin score (r=0.481). In conclusion, defective DDR/R may exert a fuel-on-fire effect on type I interferon pathway activation and contribute to tissue fibrosis in SSc.

Heat stress modulates nucleotide excision repair capacity in zebrafish (Danio rerio) early and mid-early embryos via distinct mechanisms

Discharge of heated effluent at 8–12 °C above ambient into water areas is known to retard the growth of aquatic organisms due to heat stress. Nucleotide excision repair (NER) maintains genome integrity by removing helix-distorting adducts such as UV-induced DNA lesions. This study explored how NER in zebrafish (Danio rerio) embryos at different hours post fertilization (hpf) responded to + 8.5 °C heat shock for 30 min. Our transcription-based repair assay monitoring the ability of zebrafish extracts to upregulate a UV-suppressed gene expression detected a 2-fold increase of NER capacity in 10 hpf early embryos after heat stress. In contrast, heat stress caused a mild inhibition of NER capacity in 24 hpf mid-early embryos. Heat-treated and untreated 10 hpf zebrafish extracts displayed similar levels of UV-damaged-DNA binding activities, while an apparently weaker (6-4) photoproduct (6-4 PP) binding activity was present in heat-stressed 24 hpf zebrafish extracts. Heat stress enhanced UV-induced NER in 10 hpf embryos by increasing the efficiency of damage incision/excision based on both genomic DNA electrophoresis and terminal deoxytransferase (TdT)-mediated end labeling assay. UV-irradiated embryos preexposed to heat stress produced a significantly larger amount of NER-associated DNA fragments about 20–30 nucleotides in length than embryos only heat-treated or irradiated. Correlated with its inhibitory effect on 6-4 PP damage recognition, heat stress downregulated damage incision/excision activities in 24 hpf embryos. Hence, thermal stress may positively or negatively modulate NER capacity in zebrafish embryos at different stages by targeting at the step of DNA incision/excision or damage recognition.

Preliminary Evidence for a Hormetic Effect on DNA Nucleotide Excision Repair in Veterans with Gulf War Illness.

IntroductionVeterans of the 1991 Gulf War were potentially exposed to a mixture of stress, chemicals and radiation that may have contributed to the persistent symptoms of Gulf War Illness (GWI). The genotoxic effects of some of these exposures are mediated by the DNA nucleotide excision repair (NER) pathway. We hypothesized that individuals with relatively low DNA repair capacity would suffer greater damage from cumulative genotoxic exposures, some of which would persist, causing ongoing problems.Materials and MethodsBlood samples were obtained from symptomatic Gulf War veterans and age-matched controls. The unscheduled DNA synthesis assay, a functional measurement of NER capacity, was performed on cultured lymphocytes, and lymphocyte mRNA was extracted and analyzed by sequencing.ResultsDespite our hypothesis that GWI would be associated with DNA repair deficiency, NER capacity in lymphocytes from affected GWI veterans actually exhibited a significantly elevated level of DNA repair (p = 0.016). Both total gene expression and NER gene expression successfully differentiated individuals with GWI from unaffected controls. The observed functional increase in DNA repair capacity was accompanied by an overexpression of genes in the NER pathway, as determined by RNA sequencing analysis.ConclusionWe suggest that the observed elevations in DNA repair capacity and NER gene expression are indicative of a “hormetic,” i.e., induced or adaptive protective response to battlefield exposures. Normally such effects are short-term, but in these individuals this response has resulted in a long-term metabolic shift that may also be responsible for the persistent symptoms of GWI.

Nucleotide excision repair capacity increases during differentiation of human embryonic carcinoma cells into neurons and muscle cells

Embryonic stem cells can self-renew and differentiate, holding great promise for regenerative medicine. They also employ multiple mechanisms to preserve the integrity of their genomes. Nucleotide excision repair, a versatile repair mechanism, removes bulky DNA adducts from the genome. However, the dynamics of the capacity of nucleotide excision repair during stem cell differentiation remain unclear. Here, using immunoslot blot assay, we measured repair rates of UV-induced DNA damage during differentiation of human embryonic carcinoma (NTERA-2) cells into neurons and muscle cells. Our results revealed that the capacity of nucleotide excision repair increases as cell differentiation progresses. We also found that inhibition of the apoptotic signaling pathway has no effect on nucleotide excision repair capacity. Furthermore, RNA-Seq-based transcriptomic analysis indicated that expression levels of four core repair factors, xeroderma pigmentosum (XP) complementation group A (XPA), XPC, XPG, and XPF-ERCC1, are progressively up-regulated during differentiation, but not those of replication protein A (RPA) and transcription factor IIH (TFIIH). Together, our findings reveal that increase of nucleotide excision repair capacity accompanies cell differentiation, supported by the up-regulated transcription of genes encoding DNA repair enzymes during differentiation of two distinct cell lineages.

ERCC2 Helicase Domain Mutations Confer Nucleotide Excision Repair Deficiency and Drive Cisplatin Sensitivity in Muscle-Invasive Bladder Cancer.

DNA-damaging agents comprise the backbone of systemic treatment for many tumor types; however, few reliable predictive biomarkers are available to guide use of these agents. In muscle-invasive bladder cancer (MIBC), cisplatin-based chemotherapy improves survival, yet response varies widely among patients. Here, we sought to define the role of the nucleotide excision repair (NER) gene ERCC2 as a biomarker predictive of response to cisplatin in MIBC. Somatic missense mutations in ERCC2 are associated with improved response to cisplatin-based chemotherapy; however, clinically identified ERCC2 mutations are distributed throughout the gene, and the impact of individual ERCC2 variants on NER capacity and cisplatin sensitivity is unknown. We developed a microscopy-based NER assay to profile ERCC2 mutations observed retrospectively in prior studies and prospectively within the context of an institution-wide tumor profiling initiative. In addition, we created the first ERCC2-deficient bladder cancer preclinical model for studying the impact of ERCC2 loss of function. We used our functional assay to test the NER capacity of clinically observed ERCC2 mutations and found that most ERCC2 helicase domain mutations cannot support NER. Furthermore, we show that introducing an ERCC2 mutation into a bladder cancer cell line abrogates NER activity and is sufficient to drive cisplatin sensitivity in an orthotopic xenograft model. Our data support a direct role for ERCC2 mutations in driving cisplatin response, define the functional landscape of ERCC2 mutations in bladder cancer, and provide an opportunity to apply combined genomic and functional approaches to prospectively guide therapy decisions in bladder cancer.See related commentary by Grivas, p. 907.

Abstract 4155: USP11 regulates UV-induced DNA damage repair and cell survival associated with skin cancer

Abstract UV radiation is a major risk factor for skin cancer, the most common cancer in the United States. UV exposure leads to DNA damage by formation of dimers between adjacent pyrimidine bases. The nucleotide excision repair (NER) process is responsible for removal of UV-induced DNA damage. When the damage is left unrepaired due to inefficient NER in disorders like xeroderma pigmentosum, it leads to skin tumorigenesis. Although the main players of the NER pathway have been characterized, the molecular regulation of the pathway is less understood. Understanding the regulation, particularly post-translational regulation, of the NER pathway has the potential to yield modulators of NER efficiency to prevent skin cancer. We found that chronic UV irradiation led to down-regulation of ubiquitin specific peptidase 11 (USP11) in mouse skin tissue. We also found that USP11 is decreased in skin tumors in mice and humans. Moreover, high-throughput proteomics analysis had predicted that USP11 may interact with NER factor XPC. Hence, we undertook this study to determine the role of USP11 in NER and cellular response to UV. We found that USP11 promoted the NER pathway by regulating XPC activity. USP11 promoted XPC deubiquitination and retention at the DNA damage sites. We also found that USP11 interaction with ubiquitinated XPC was enhanced post-UV irradiation, and that USP11 was recruited to the chromatin post-UV damage. Further, squamous cell carcinoma cells showed reduced survival with inhibition of USP11 after UV exposure. Our findings indicate that USP11 plays an important role in maintaining NER capacity, and suggest that USP11 acts as a tumor suppressor via its role in DNA repair. Citation Format: Palak Shah, Lei Qiang, Seungwon Yang, Keyoumars Soltani, Yu-Ying He. USP11 regulates UV-induced DNA damage repair and cell survival associated with skin cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4155.

Neurodegeneration as the presenting symptom in 2 adults with xeroderma pigmentosum complementation group F.

ObjectiveTo describe the features of 2 unrelated adults with xeroderma pigmentosum complementation group F (XP-F) ascertained in a neurology care setting.MethodsWe report the clinical, imaging, molecular, and nucleotide excision repair (NER) capacity of 2 middle-aged women with progressive neurodegeneration ultimately diagnosed with XP-F.ResultsBoth patients presented with adult-onset progressive neurologic deterioration involving chorea, ataxia, hearing loss, cognitive deficits, profound brain atrophy, and a history of skin photosensitivity, skin freckling, and/or skin neoplasms. We identified compound heterozygous pathogenic mutations in ERCC4 and confirmed deficient NER capacity in skin fibroblasts from both patients.ConclusionsThese cases illustrate the role of NER dysfunction in neurodegeneration and how adult-onset neurodegeneration could be the major symptom bringing XP-F patients to clinical attention. XP-F should be considered by neurologists in the differential diagnosis of patients with adult-onset progressive neurodegeneration accompanied by global brain atrophy and a history of heightened sun sensitivity, excessive freckling, and skin malignancies.

Enhancement of UVB-induced DNA damage repair after a chronic low-dose UVB pre-stimulation

Absorption of solar ultraviolet (UV) radiation by DNA leads to the formation of the highly mutagenic cyclobutane pyrimidine dimer (CPD). The mutagenicity of CPD is caused, in part, by the fact that their recognition and repair by the nucleotide excision repair (NER) pathway is challenging and slow. It has been previously shown that a pre-stimulation with genotoxic agents improve NER efficiency of CPD, indicating a potential adaptive response of this repair pathway. We have pre-treated human dermal fibroblasts with repeated subletal low doses of UVB (chronic low-dose of UVB; CLUV) to determine whether it could enhance NER capacity to repair CPD. Our results show that CLUV pre-treatment greatly enhances CPD repair but have little effect on the repair of another UV-induced bypirimidine photoproduct, the pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). We have determined that the CLUV treatment activates p53 and we found an increase of DDB2 and XPC gene expression. This is consistent with an increasing level of NER recognition proteins, DDB2 and XPC, we found concentrated at the chromatin. This study represents the first demonstration that chronic UVB exposure can stimulate NER pathway. Altogether, these results shed light on the potential adaptability of the NER by chronic UVB irradiation and the mechanisms involved.