Inhibition Of Nucleotide Excision Repair Research Articles

A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

Abstract 6155: Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression

Abstract Lurbinectedin (Zepzelca) is a marine-derived anti-tumor agent currently approved for metastatic small cell lung cancer (SCLC). Lurbinectedin has been previously shown to exert its anti-tumor effect by binding to the minor groove of DNA and inhibition of nucleotide excision repair. In addition, lurbinectedin inhibits transcription by inducing degradation of RNA polymerase II. While these activities hint towards a general mechanism, specific molecular targets for lurbinectedin remain obscure. We demonstrate that in vitro, Lurbinectedin specifically induced downregulation of MCL1 protein, a member of the BCL2 family of pro-survival proteins, in a dose- (0-30 nM) and time-dependent manner (0-24 h) by Western analysis. Expression of other pro-survival proteins BCL2 and BCL2L1 were not affected. The downregulation of MCL1 was consistent in all nine tumor cell lines tested regardless of indication. Increased activity or expression of MCL1 is a known compensatory mechanism to offset loss of BCL2 or BCL2L1. We therefore hypothesized that lurbinectedin induced MCL1 loss, along with inhibition of the pro-survival proteins BCL2 and BCL2L1 would lead to an enhancement in cell killing. Consistent with the dominant pro-survival role of BCL2 in SCLC, the combination of lurbinectedin with a specific BCL2 inhibitor (venetoclax) in vitro induced synergistic cytotoxicity (measured by Bliss Sum analysis) in a panel of small cell lung cancer lines. In solid tumor lines other than SCLC, the combination of lurbinectedin with a specific BCL2L1 inhibitor (A-1331852) was also synergistic. Correspondingly in vivo, enhancement in the efficacy of lurbinectedin was observed in combination with venetoclax in SCLC models, while in solid tumor models other than SCLC the combination with a specific BCL2L1 inhibitor enhanced efficacy. Collectively, these data identify MCL1 as a specific lurbinectedin target and suggest that combinations that target other pro-survival proteins may represent a viable strategy to enhance the anti-tumor activity of lurbinectedin. Citation Format: Kedar S. Vaidya, Aparna Gupta, Robert Hauptschein, Robin C. Humphreys. Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6155.

Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

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.

Protective role of electrophile-reactive glutathione for DNA damage repair inhibitory effect of dibromoacetonitrile

Dibromoacetonitrile (DBAN) is a disinfection byproduct (DBP) and linked with cancer in rodents, but the mechanism of its carcinogenicity has not been fully elucidated. We recently reported that DBAN induced inhibition of nucleotide excision repair (NER). In this study, we investigated if glutathione (GSH) is involved in the DBAN-induced inhibition of NER. Human keratinocytes HaCaT were pretreated with L-buthionine-(S,R)-sulfoximine (BSO) to deplete intracellular GSH. BSO treatment markedly potentiated the DBAN-induced NER inhibition as well as intracellular oxidation. The recruitment of NER proteins (transcription factor IIH, and xeroderma pigmentosum complementation group G) to DNA damage sites was inhibited by DBAN, which was further exacerbated by BSO treatment. Our results suggest that intracellular GSH protects cells from DBAN-induced genotoxicity including inhibition of DNA damage repair.

Modulation of ERCC1-XPF Heterodimerization Inhibition via Structural Modification of Small Molecule Inhibitor Side-Chains.

Inhibition of DNA repair enzymes is an attractive target for increasing the efficacy of DNA damaging chemotherapies. The ERCC1-XPF heterodimer is a key endonuclease in numerous single and double strand break repair processes, and inhibition of the heterodimerization has previously been shown to sensitize cancer cells to DNA damage. In this work, the previously reported ERCC1-XPF inhibitor 4 was used as the starting point for an in silico study of further modifications of the piperazine side-chain. A selection of the best scoring hits from the in silico screen were synthesized using a late stage functionalization strategy which should allow for further iterations of this class of inhibitors to be readily synthesized. Of the synthesized compounds, compound 6 performed the best in the in vitro fluorescence based endonuclease assay. The success of compound 6 in inhibiting ERCC1-XPF endonuclease activity in vitro translated well to cell-based assays investigating the inhibition of nucleotide excision repair and disruption of heterodimerization. Subsequently compound 6 was shown to sensitize HCT-116 cancer cells to treatment with UVC, cyclophosphamide, and ionizing radiation. This work serves as an important step towards the synergistic use of DNA repair inhibitors with chemotherapeutic drugs.

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.

Nucleotide Excision Repair Pathway Activity Is Inhibited by Airborne Particulate Matter (PM10) through XPA Deregulation in Lung Epithelial Cells.

Airborne particulate matter with a diameter size of ≤10 µm (PM10) is a carcinogen that contains polycyclic aromatic hydrocarbons (PAH), which form PAH–DNA adducts. However, the way in which these adducts are managed by DNA repair pathways in cells exposed to PM10 has been partially described. We evaluated the effect of PM10 on nucleotide excision repair (NER) activity and on the levels of different proteins of this pathway that eliminate bulky DNA adducts. Our results showed that human lung epithelial cells (A549) exposed to 10 µg/cm2 of PM10 exhibited PAH–DNA adducts as well as an increase in RAD23 and XPD protein levels (first responders in NER). In addition, PM10 increased the levels of H4K20me2, a recruitment signal for XPA. However, we observed a decrease in total and phosphorylated XPA (Ser196) and an increase in phosphatase WIP1, aside from the absence of XPA–RPA complex, which participates in DNA-damage removal. Additionally, an NER activity assay demonstrated inhibition of the NER functionality in cells exposed to PM10, indicating that XPA alterations led to deficiencies in DNA repair. These results demonstrate that PM10 exposure induces an accumulation of DNA damage that is associated with NER inhibition, highlighting the role of PM10 as an important contributor to lung cancer.

MiRNA profiling in adult T-cell leukemia lymphoma (ATLL), a systems virology study

Due to a lack of adequate knowledge regarding the mechanism of action and signaling pathways, Adult T-Cell Lymphoma/Leukemia (ATLL) remained a poor prognostic cancer. Understanding the pathogenesis and signaling pathway can be helpful for introducing the possible treatment for ATLL patients. In this study, differentially expressed microRNAs (DEMs) were detected based on the value of log fold change. The gene targets of miRNAs were extracted using miRDB online tool. Protein-protein interaction networks (PPINs) were reconstructed using STRING database and the sub-networks were then determined based on the network centrality measures. Pathway analysis was performed, and proteins participating in the enriched pathways were selected to propose the HTLV1 implicated signaling network (HISN). Thirty-three DEMs consisted of 31 downregulated and 2 upregulated DEMs were detected. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, the proteins contributing in HISN were involved in processes resulting in proliferation, inflammation, anti-apoptosis effects, differentiation, microtubule organizing center (MTOC) relocation, cellular proliferation, cell cycle progression, inhibition of nucleotide excision repair, accumulation of mutations, cellular transformation, matrix proteolytic degradation, and DNA damages. Overexpression of anti-apoptotic genes, including NRAS, CCND1, and CCND2 or deletion/silencing of proapoptotic genes, including TP53 and CASP3, inactivates the intrinsic apoptotic pathways and may lead to developing ATLL.

Inhibition of nucleotide excision repair and damage response signaling by dibromoacetonitrile: A novel genotoxicity mechanism of a water disinfection byproduct

Dibromoacetonitrile (DBAN) is a carcinogenic disinfection byproduct (DBP) but how it precipitates cancer is unknown. Nucleotide excision repair (NER) is a versatile repair mechanism for removing bulky DNA lesions to maintain genome stability, and impairment of this process is associated with cancer development. In this study, we found that DBAN inhibited NER and investigated its mechanism with other DNA damage responses. Human keratinocytes HaCaT were treated with DBAN followed by ultraviolet (UV) as a model inducer of DNA damage, pyrimidine dimers, which require NER for the removal. DBAN pretreatment exacerbated UV-cytotoxicity, and inhibited the repair of pyrimidine dimers. DBAN treatment delayed the recruitment of NER proteins, transcription factor IIH (TFIIH) and xeroderma pigmentosum complementation group G (XPG), to DNA damaged sites, and subsequent gap filling process. Moreover, DBAN suppressed the UV-induced double strand breaks (DSBs) formation, as well as phosphorylated histone H2AX (γ-H2AX), a widely used DNA damage marker. Altogether, DBAN could negatively impact the NER process and phosphorylation pathway responding to DNA damage. This study was the first to identify the inhibition of NER and damage response signaling as a genotoxicity mechanism of a class of DBPs and it may serve as a foundation for DBP carcinogenesis.

Low extracellular pH inhibits nucleotide excision repair

Nucleotide excision repair (NER) is the main pathway to repair bulky DNA damages including pyrimidine dimers, and the genetic dysregulation of NER associated proteins is well known to cause diseases such as cancer and neurological disorder. Other than the genetic defects, ‘external factors’ such as oxidative stress and environmental chemicals also affect NER. In this study, we examined the impact of extracellular pH on NER. We prepared the culture media, whose pH values are 8.4 (normal condition), 7.6, 6.6 and 6.2 under atmospheric CO2 conditions. Human keratinocytes, HaCaT, slightly died after 48 h incubation in DMEM at pH 8.4, 7.6 and 6.6, while in pH 6.2 condition, marked cell death was induced. UV-induced pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs), were effectively repaired at 60 min and 24 h, respectively, which were remarkably inhibited at pH 6.6 and 6.2. The associated repair molecule, TFIIH, was accumulated to the damaged sites 5 min after UVC irradiation in all pH conditions, but the release was delayed as the pH got lower. Furthermore, accumulation of XPG at 5 min was delayed at pH 6.2 and 6.6, and the release at 60 min was completely suppressed. At the low pH, the DNA synthesis at the gaps created by incision of oligonucleotides containing pyrimidine dimers was significantly delayed. In this study, we found that the low extracellular pH inhibited NER pathway. This might partially contribute to carcinogenesis in inflamed tissues, which exhibit acidic pH.

Cell line-dependent difference in glutathione levels affects the cigarette sidestream smoke-induced inhibition of nucleotide excision repair

We recently reported that cigarette sidestream smoke (CSS) induced inhibition of nucleotide excision repair (NER) and the cause was NER molecule degradation by aldehydes contained in CSS [Carcinogenesis39, 56–65, 2018; Mutat. Res.834, 42–50, 2018]. In this study, we examined the relationship between intracellular glutathione (GSH) levels and CSS-induced NER inhibition. CSS treatment decreased the intracellular GSH level in human keratinocytes HaCaT, in which the repair of pyrimidine (6−4) pyrimidone photoproducts (6-4PPs) after UVB irradiation was suppressed. We used l-buthionine-(S,R)-sulfoximine (BSO) to artificially deplete intracellular GSH level. BSO treatment remarkably accelerated the CSS-induced NER inhibition. The NER inhibition by CSS was attributed to the delay of accumulation of NER molecules (TFIIH and XPG) to DNA damaged sites, which was further enhanced by BSO treatment. CSS degraded TFIIH, and BSO promoted it as expected. Formaldehyde (FA), a major constituent of CSS, showed similar intracellular GSH reduction and NER inhibition, and BSO promoted its inhibitory effect. Five cultured cell lines showed considerable variability in intrinsic GSH levels, and CSS-induced NER inhibitory effect was significantly correlated with the GSH levels. Chemicals like aldehydes are known to react not only with proteins but also with DNA, causing DNA lesions targeted by NER. Our results suggest that the tissues and cells with low intrinsic GSH levels are susceptible to treatment with CSS and electrophilic compounds like aldehydes through NER inhibition, thus leading to higher genotoxicity and carcinogenicity.

Downregulation of the DNA Repair Gene DDB2 by Arecoline Is through p53's DNA-Binding Domain and Is Correlated with Poor Outcome of Head and Neck Cancer Patients with Betel Quid Consumption.

Arecoline is the principal alkaloid in the areca nut, a component of betel quids (BQs), which are carcinogenic to humans. Epidemiological studies indicate that BQ-chewing contributes to the occurrence of head and neck cancer (HNC). Previously, we have reported that arecoline (0.3 mM) is able to inhibit DNA repair in a p53-dependent pathway, but the underlying mechanism is unclear. Here we demonstrated that arecoline suppressed the expression of DDB2, which is transcriptionally regulated by p53 and is required for nucleotide excision repair (NER). Ectopic expression of DDB2 restored NER activity in arecoline-treated cells, suggesting that DDB2 downregulation was critical for arecoline-mediated NER inhibition. Mechanistically, arecoline inhibited p53-induced DDB2 promoter activity through the DNA-binding but not the transactivation domain of p53. Both NER and DDB2 promoter activities declined in the chronic arecoline-exposed cells, which were consistent with the downregulated DDB2 mRNA in BQ-associated HNC specimens, but not in those of The Cancer Genome Atlas (TCGA) cohort (no BQ exposure). Lower DDB2 mRNA expression was correlated with a poor outcome in HNC patients. These data uncover one of mechanisms underlying arecoline-mediated carcinogenicity through inhibiting p53-regulated DDB2 expression and DNA repair.

Asymmetric repair of UV damage in nucleosomes imposes a DNA strand polarity on somatic mutations in skin cancer.

Nucleosomes inhibit excision repair of DNA damage caused by ultraviolet (UV) light, and it has been generally assumed that repair inhibition is equivalent on both sides of the nucleosome dyad. Here, we use genome-wide repair data to show that repair of UV damage in nucleosomes is asymmetric. In yeast, nucleosomes inhibit nucleotide excision repair (NER) of the nontranscribed strand (NTS) of genes in an asymmetric manner, with faster repair of UV damage occurring on the 5′ side of the nucleosomal DNA. Analysis of genomic repair data from UV-irradiated human cells indicates that NER activity along the NTS is also elevated on the 5′ side of nucleosomes, consistent with the repair asymmetry observed in yeast nucleosomes. Among intergenic nucleosomes, repair activity is elevated on the 5′ side of both DNA strands. The distribution of somatic mutations in nucleosomes shows the opposite asymmetry in NER-proficient skin cancers, but not in NER-deficient cancers, indicating that asymmetric repair of nucleosomal DNA imposes a strand polarity on UV mutagenesis. Somatic mutations are enriched on the relatively slow-repairing 3′ side of the nucleosomal DNA, particularly at positions where the DNA minor groove faces away from the histone octamer. Asymmetric repair and mutagenesis are likely caused by differential accessibility of the nucleosomal DNA, a consequence of its left-handed wrapping around the histone octamer.

SIRT2 Prevents Cisplatin-Induced Neuron Injury By Promoting Nucleotide Excision Repair

Radiation and chemotherapy-induced neuropathy are some of the most common causes of dose reduction and discontinuation of cancer treatment, and often cause a major permanent impairment of quality of life in cancer patients. Furthermore, chemotherapy-induced neuropathy acts as a risk factor for development of radiation-induced neuropathy in patients receiving concomitant or adjuvant radiotherapy. The lack of effective treatments for or prevention of this debilitating neuronal toxicity not only compromises optimal treatment, but also leaves cancer survivors with significant disability. Here, we sought to investigate the biological role and underline molecular mechanisms of sirtuin 2 (SIRT2) in cisplatin neurotoxicity. Genetic murine models were utilized to compare the development of cisplatin-induced peripheral neuropathy in transgenic Sirt2 knock-in (Sirt2-KI) mice versus wild-type (WT) mice. The hyperalgesia seen in neuropathy was measured in terms of mechanical and thermal thresholds. Immunostaining and western blot were used to monitor SIRT2 expression in dorsal root ganglion (DRG) sensory neurons. The efficiency of DNA damage repair via nucleotide excision repair (NER) was examined using a dual-luciferase reporter assay, while neuronal cytotoxicity was determined using a trypan blue staining assay. In addition to the genetic approach, the function of SIRT2, an NAD+deacetylase, was pharmacologically modified to validate its role in prevention and treatment of cisplatin-induced neuropathy. High expression of SIRT2 in Sirt2-KI mice produces significant resistance to cisplatin-induced neuropathy compared to WT mice. Cisplatin provoked a nuclear translocation of SIRT2 in DRG sensory neurons, protecting mice against neuropathy. Overexpression of SIRT2 resulted in significantly improved cultured neuronal cell survival through the promotion of nucleotide excision repair of cisplatin-induced DNA crosslinks. In vivo inhibition of NER abolished SIRT2-mediated neuroprotection as evidenced by reductions in mechanical and thermal hyperalgesia thresholds. Finally, administration of nicotinamide riboside activated SIRT2 and prevented cisplatin-induced neuropathy. SIRT2 prevents cisplatin-induced neuron injury by regulating the NER pathway. Identification of this novel function warrants future investigation of nicotinamide riboside-mediated neuroprotection during platinum-based cancer treatment.

Aldehyde-mediated protein degradation is responsible for the inhibition of nucleotide excision repair by cigarette sidestream smoke

We recently reported that cigarette sidestream smoke (CSS) can delay nucleotide excision repair (NER), which was due to the inhibition of repair protein accumulation to DNA damage sites. However, the mechanisms how the protein recruitment was inhibited remains unclear. We hypothesized that aldehydes in CSS could be a candidate taking a role for the inhibition, and tested our hypothesis by removing aldehydes from CSS using cigarette-filters. The NER inhibition potency of CSS or filtered CSS (F-CSS) was compared using human keratinocyte cell line, HaCaT. Cigarette-filters were able to reduce total aldehydes in CSS by half. Pretreating cells with CSS and F-CSS enhanced UVB-induced cell death, with the effect of CSS weakened by filtration.CSS strongly inhibited the repair of UVB-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts (6-4PPs), where the recruitments of repair molecules, TFIIH and XPG, were slowed down. F-CSS showed similar inhibition of NER and accumulation of related proteins, but the effect was weaker than CSS. Semicarbazide (SEM), an aldehyde-trapping agent, alleviated the NER delay induced by both CSS and F-CSS, further confirming that aldehydes in CSS were the main cause for the inhibition of NER and that the different amounts of aldehydes in CSS and F-CSS were responsible for the different inhibition efficiency. Furthermore, TFIIH level was decreased by treatment with CSS and restored in the presence of proteasome inhibitor, indicating that the degradation of NER proteins might be the cause of the inhibition of NER-protein recruitment. These results supported our hypothesis that aldehydes in CSS are the main contributor for the NER inhibition via protein degradation, and reconfirmed that exposure to CSS without filtration could be a severe threat to human health.

Cigarette sidestream smoke delays nucleotide excision repair: inhibited accumulation of repair proteins at DNA lesions

Cigarette sidestream smoke (CSS) contains many carcinogens that induce DNA damage. DNA damage plays an important role in the initiation of cancer and several diseases, and repair is the major defense mechanism; however, the relationship between CSS and the repair of DNA damage remains unclear. We herein investigated whether CSS influences nucleotide excision repair (NER) in vivo and in vitro. HR-1 hairless mouse skin treated with CSS was exposed to UVB, as a result of which pyrimidine dimers (cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs)) were formed and repaired via the NER pathway. The immunohistochemical staining of CPDs revealed that their repair was delayed by the CSS treatment. This delay in NER and the underlying mechanisms were examined in the human skin cell lines, HaCaT and HSC-1. Dot-blot assays, enzyme-linked immunosorbent assay and local ultraviolet irradiation assays demonstrated that CSS delayed the repair of CPDs and 6-4PPs. The recruitment of the repair molecules, TFIIH, XPA and XPG to pyrimidine dimers was markedly inhibited by CSS. Semicarbazide, which reacts with aldehydes, recovered the CSS-induced inhibition of NER, and formaldehyde exerted similar inhibitory effects to those of CSS. These results suggest that aldehydes in CSS interfere with the recruitment of NER molecules to damaged sites, leading to a delay in the repair of pyrimidine dimers.

Triptolide induces DNA breaks, activates caspase-3-dependent apoptosis and sensitizes B-cell lymphoma to poly(ADP-ribose) polymerase 1 and phosphoinositide 3-kinase inhibitors.

Triptolide is the primary compound isolated from Tripterygium wilfordii, which has been reported to inhibit nucleotide excision repair as well as exhibit anti-inflammatory and antitumor activities. However, the action of triptolide in DNA breaks remains unknown. The present study investigated the effects of triptolide in the induction of DNA breaks and apoptosis in a murine B-cell lymphoma cell line, CH12F3. An MTT assay revealed that X-ray repair cross-complementing protein 1 (XRCC1)-/- CH12F3 cells were more sensitive to 6 nM triptolide compared with the wild-type CH12F3 cells, which suggests that low levels of triptolide induce DNA breaks in a manner that is dependent on the XRCC1-mediated repair pathway. Flow cytometric analysis identified that 50 nM triptolide increased the phospho-histone H2AX level, demonstrating that triptolide induces double-strand breaks. Western blot analysis revealed that triptolide up-regulated Rad51 and nuclear proliferating cell nuclear antigen. Annexin V/propidium iodide staining identified that triptolide promoted apoptosis and western blot analysis confirmed that triptolide activated caspase-3-dependent apoptosis. The results of the present study also demonstrated that 5 nM triptolide sensitized CH12F3 lymphoma cells to poly(ADP-ribose) polymerase 1 and phosphoinositide 3-kinase inhibitors, suggesting that triptolide may be a potent antitumor drug for sensitizing lymphoma cells to chemotherapeutic agents.

Oxidative stress induced by UVA photoactivation of the tryptophan UVB photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) inhibits nucleotide excision repair in human cells

Potentially mutagenic DNA lesions induced by UVB (wavelengths 280–320 nm) are important risk factors for solar ultraviolet (UV) radiation-induced skin cancer. The carcinogenicity of the more abundant UVA (320–400 nm) is less well understood but is generally regarded to reflect its interaction with cellular chromophores that act as photosensitisers. The arylhydrocarbon receptor agonist 6-formylindolo[3,2-b] carbazole (FICZ), is a UVB photoproduct of tryptophan and a powerful UVA chromophore. Combined with UVA, FICZ generates reactive oxygen species (ROS) and induces oxidative DNA damage. Here we demonstrate that ROS generated by FICZ/UVA combinations also cause extensive protein damage in HaCaT human keratinocytes. We show that FICZ/UVA-induced oxidation significantly inhibits the removal of potentially mutagenic UVB-induced DNA photolesions by nucleotide excision repair (NER). DNA repair inhibition is due to FICZ/UVA-induced oxidation damage to the NER proteome and DNA excision repair is impaired in extracts prepared from FICZ/UVA-treated cells. NER protects against skin cancer. As a likely UVB photoproduct of intracellular tryptophan, FICZ represents a de facto endogenous UVA photosensitiser in sun-exposed skin. FICZ formation may increase the risk of solar UV-induced skin cancer by promoting photochemical damage to the NER proteome and thereby preventing the removal of UVB-induced DNA lesions.

Nucleotide excision repair is a potential therapeutic target in multiple myeloma

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.