衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer

Breast cancer is the most common malignancy in women. Radiotherapy is frequently used in patients with breast cancer, but some patients may be more susceptible to ionizing radiation, and increased exposure to radiation sources may be associated to radiation adverse events. This susceptibility may be related to deficiencies in DNA repair mechanisms that are activated after cell-radiation, which causes DNA damage, particularly DNA double strand breaks. Some of these genetic susceptibilities in DNA-repair mechanisms are implicated in the etiology of hereditary breast/ovarian cancer (pathologic mutations in the BRCA 1 and 2 genes), but other less penetrant variants in genes involved in sporadic breast cancer have been described. These same genetic susceptibilities may be involved in negative radiotherapeutic outcomes. For these reasons, it is necessary to implement methods for detecting patients who are susceptible to radiotherapy-related adverse events. This review discusses mechanisms of DNA damage and repair, genes related to these functions, and the diagnosis methods designed and under research for detection of breast cancer patients with increased radiosensitivity.     

C --> T mutagenesis and gamma-radiation sensitivity due to deficiency in the Smug1 and Ung DNA glycosylases

The most common genetic change in aerobic organisms is a C:G to T:A mutation. C --> T transitions can arise through spontaneous hydrolytic deamination of cytosine to give a miscoding uracil residue. This is also a frequent DNA lesion induced by oxidative damage, through exposure to agents such as ionizing radiation, or from endogenous sources that are implicated in the aetiology of degenerative diseases, ageing and cancer. The Ung and Smug1 enzymes excise uracil from DNA to effect repair in mammalian cells, and gene-targeted Ung(-/-) mice exhibit a moderate increase in genome-wide spontaneous mutagenesis. Here, we report that stable siRNA-mediated silencing of Smug1 in mouse embryo fibroblasts also generates a mutator phenotype. However, an additive 10-fold increase in spontaneous C:G to T:A transitions in cells deficient in both Smug1 and Ung demonstrates that these enzymes have distinct and nonredundant roles in suppressing C --> T mutability at non-CpG sites. Such cells are also hypersensitive to ionizing radiation, and reveal a role of Smug1 in the repair of lesions generated by oxidation of cytosine.     

Repair Responses to DNA Damage: Enzymatic Pathways in E coli and Human Cells

Bacteria and eukaryotic cells employ a variety of enzymatic pathways to remove damage from DNA or to lessen its impact upon cellular functions. Most of these processes were discovered in Escherichia coli and have been most extensively analyzed in this organism because suitable mutants have been isolated and characterized. Analogous pathways have been inferred to exist in mammalian cells from the presence of enzyme activities similar to those known to be involved in repair in bacteria, from the analysis of events in cells treated with DNA damaging agents, and from the analysis of the few naturally occurring mutant cell types. Excision repair of pyrimidine dimers produced by UV in E coli is initiated by an incision event catalyzed by a complex composed of uvrA, uvrB, and uvrC gene products. Multiple exonuclease and polymerase activities are available for the subsequent excision and resynthesis steps. In addition to the constitutive pathway, which produces short patches of 20–30 nucleotides, an inducible excision repair process exists that produces much longer patches. This long patch pathway is controlled by the recA-lexA regulatory circuit and also requires the recF gene. It is apparently not responsible for UV-induced mutagenesis. However, the ability to perform inducible long patch repair correlates with enhanced bacterial survival and with a major component of the Weigle reactivation of bacteriophage with double-strand DNA genomes. Mammalian cells possess an excision repair pathway similar to the constitutive pathway in E coli. Although not as well understood, the incision event is at least as complex, and repair resynthesis produces patches of about the same size as the constitutive short patches. In mammalian cells, no patches comparable in size to those produced by the inducible pathway of E coli are observed. Repair in mammalian cells may be more complicated than in bacteria because of the structure of chromatin, which can affect both the distribution of DNA damage and its accessibility to repair enzymes. A coordinated alteration and reassembly of chromatin at sites of repair may be required. We have observed that the sensitivity of digestion by staphylococcal nuclease (SN) of newly synthesized repair patches resulting from excision of furocoumarin adducts changes with time in the same way as that of patches resulting from excision of pyrimidine dimers. Since furocoumarin adducts are formed only in the SN-sensitive linker DNA between nucleosome cores, this suggests that after repair resynthesis is completed, the nucleosome cores in the region of the repair event do not return exactly to their original positions. We have also studied excision repair of UV and chemical damage in the highly repeated 172 base pair α DNA sequence in African green monkey cells. In UV irradiated cells, the rate and extent of repair resynthesis in this sequence is similar to that in bulk DNA. However, in cells containing furocoumarin adducts, repair resynthesis in α DNA is only about 30% of that in bulk DNA. Since the frequency of adducts does not seem to be reduced in α DNA, it appears that certain adducts in this unique DNA may be less accessible to repair. Endonuclease V of bacteriophage T4 incises DNA at pyrimidine dimers by cleaving first the glycosylic bond between deoxyribose and the 5′ pyrimidine of the dimer and then the phosphodiester bond between the two pyrimidines. We have cloned the gene (denV) that codes for this enzyme and have demonstrated its expression in uvrA recA and uvrB recA cells of E coli. Because T4 endonuclease V can alleviate the excision repair deficiency of xeroderma pigmentosum when added to permeabilized cells or to isolated nuclei after UV irradiation, the cloned denV gene may ultimately be of value for analyzing DNA repair pathways in cultured human cells.     

DNA Base Damage in Chromatin of γ-Irradiated Cultured Human Cells

We report on the chemical characterization of DNA base damage in chromatin of γ-irradiated cultured human cells. Chromatin was isolated from unirradiated and irradiated cells and analyzed by gas chroma-tography/mass spectrometry with selected-ion monitoring after acidic hydrolysis of chromatin and trimethylsilylation of hydrolysates. Prior to analysis of chromatin samples, experimental conditions for acidic hydrolysis were optimized by determining the relative molar response factors of modified bases under non-acidic and acidic conditions, and their release from DNA under various acidic conditions. A number of modified bases in chromatin isolated from irradiated cells were identified and quantitated. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, cytosine glycol, thymine glycol, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. Radiation doses ranging from 42 to 420 Gy (J . kg¹) were used. Background levels of all modified bases were observed in chromatin isolated from unirradiated cells. The radiation yields of a number of modified bases were increased significantly over their background levels at a dose as low as 42 Gy. In most cases, linear dose-yield relationships were obtained up to ≈200Gy. At radiation doses higher than 420 Gy, no additional increase in the yields of modified bases was observed. The yields of guanine-derived bases amounted to ≈ 45% of the total net yield of modified bases measured, followed by almost equal yields of adenine-, cytosine- and thymine-derived bases. Modified bases identified were typical products of hydroxyl radical attack on DNA bases, indicating the involvement of hydroxyl radical, although their induction in part by the direct effect of ionizing radiation through ionization of DNA bases cannot be excluded. The yields of modified bases were lower than those previously measured after γ-irradiation of fully expanded chromatin in aqueous buffer solutions.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

DNA repair of clustered uracils in HeLa cells

Two or more base damages, abasic sites or single-strand breaks (SSBs) within two helical turns of the DNA form a multiply damaged site (MDS) or clustered lesion. Studies in vitro and in bacteria indicate that attempts to repair two closely opposed base lesions can potentially form a lethal double-strand break (DSB). Ionizing radiation and chemotherapeutic agents introduce complex lesions, and the inability of a cell to repair MDSs is believed to contribute to the lethality of these treatments. The goal of this work was to extend the in vitro studies by examining MDS repair in mammalian cells under physiological conditions. Here, two opposing uracil residues separated by 3, 5, 7, 13 or 29 base-pairs were chosen as model DNA lesions. Double-stranded oligonucleotides containing no damage, a single uracil residue or the MDS were introduced into a non-replicating mammalian construct within the firefly luciferase open reading frame, or at the 5' or 3' end of the luciferase expression cassette. Following transient transfection into HeLa cells, luciferase activity was measured or plasmid DNA was re-isolated from the cells. Formation of a DSB was expected to decrease luciferase expression. However, certain single uracil residues as well as the MDSs decreased luciferase activity, which suggested that the reduction in activity was not due to DSB formation. In fact, Southern analysis of the re-isolated plasmid did not show the presence of linear DNA and demonstrated that none of the constructs was destroyed during repair. Further analysis of the re-isolated DNA demonstrated that only a small percentage of molecules originally carrying a single lesion or an MDS contained deletions. This work indicates that the majority of the clustered lesions were not converted to DSBs and that repair systems in mammalian cells may have established mechanisms to avoid the accumulation of SSB-repair intermediates.     

下调SMU1表达对细胞增殖和DNA双链断裂损伤反应的影响

SMU1是一个与细胞基因组复制和RNA剪切过程相关的新基因。该研究为进一步调查SMU1对细胞增殖及DNA双链断裂（DNAdouble—strand breaks,DNADSBs）损伤应答的影响,设计合成针对SMU1基因的小分子siRNA,并与对照siRNA（scramblel分别转染HEK293T或U2OS细胞。通过免疫印迹（Westernblot）检测证实,siSMU1转染细胞中SMU1的表达显著下降,采用台盼蓝染色细胞计数检测显示,SMU1表达下调显著降低细胞增殖能力。免疫荧光和免疫印迹法检测结果表明,SMU1表达下调显著增加细胞内源性DSBs损伤（7H2AXfoci和蛋白水平均升高）;而进一步用X-ray处理细胞造成外源性DSBs损伤后,SMUI沉默细胞显示出延长的DSBs损伤修复动力学（减缓的γH2AXfoci和蛋白水平消退）。以上结果提示,SMU1在细胞DSBs损伤修复反应中扮演重要角色,积极参与细胞基因纽完整性的维持。     

Oxidative Damage and Fragmentation of Mitochondrial DNA in Cellular Apoptosis

The molecular genetics and bioenergetics of oxidative damage, fragmentation, and fragility of mitochondrial DNA in cellular apoptosis is reviewed in connection with the redox mechanism of ageing.     

Methacryloxylethyl Cetyl Ammonium Chloride Induces DNA Damage and Apoptosis in Human Dental Pulp Cells via Generation of Oxidative Stress

The polymerizable antibacterial monomer methacryloxylethyl cetyl ammonium chloride (DMAE-CB) has provided an effective strategy to combat dental caries. However, the application of such material raises the question about the biological safety and the question remains open. The mechanism of this toxic action, however, is not yet clearly understood. The present study aims at providing novel insight into the possible causal link between cellular oxidative stress and DNA damage, as well as apoptosis in human dental pulp cells exposed to DMAE-CB. The enhanced formation of reactive oxygen species and depletion of glutathione, as well as differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase in DMAE-CB-treated cells indicated oxidative stress. By using substances that can alter GSH synthesis, we found that GSH was the key component in the regulation of cell response towards oxidative stress induced by DMAE-CB. The increase in oxidative stress-sensitive 8-Oxo-2''-deoxyguanosine (8-OHdG) content, formation of γ-H₂AX and cell cycle G1 phase arrest indicated that DNA damage occurred as a result of the interaction between DNA base and ROS beyond the capacities of antioxidant mechanisms in cells exposed to DMAE-CB. Such oxidative DNA damage thus triggers the activation of ataxia telangiectasia-mutated (ATM) signaling, the intrinsic apoptotic pathway, and destruction of mitochondrial morphology and function.     

Induction of Excision Repairable DNA Lesions in Lymphocytes Exposed to Lead and ALA In Vitro

Numbers of studies have been carried out on the potential of lead genotoxicity. The mechanisms of lead genotoxicity are not fully known but partly attributed to the formation of highly reactive oxygen metabolites (ROM). However, lead ions have no ability to generate ROM. In this study, we have investigated the ability of lead and ALA to induce excision repairable DNA lesions by using cytosine arabinoside or cytokinesis block micronucleus (ARA-C/CBMN) assay. N-methyl-N-nitrosourea was used as a positive control which is a mutagen and known to induce excision repair. The results of the ARA-C/CBMN assay show that ALA exposures have significantly (p < 0.01) increased the ratio of excision repairable DNA lesions in peripheral blood lymphocytes; however, lead have not. Accordingly, accumulation of ALA should be considered as an effective partner of lead induced DNA damage in lead exposure.     

DNA Damage and Radiosensitivity in Blood Cells from Subjects Undergoing 45 Days of Isolation and Confinement: An Explorative Study

The effect of confined and isolated experience on astronauts’ health is an important factor to consider for future space exploration missions. The more confined and isolated humans are, the more likely they are to develop negative behavioral or cognitive conditions such as a mood decline, sleep disorder, depression, fatigue and/or physiological problems associated with chronic stress. Molecular mediators of chronic stress, such as cytokines, stress hormones or reactive oxygen species (ROS) are known to induce cellular damage including damage to the DNA. In view of the growing evidence of chronic stress-induced DNA damage, we conducted an explorative study and measured DNA strand breaks in 20 healthy adults. The participants were grouped into five teams (missions). Each team was composed of four participants, who spent 45 days in isolation and confinement in NASA’s Human Exploration Research Analog (HERA). Endogenous DNA integrity, ex-vivo radiation-induced DNA damage and the rates of DNA repair were assessed every week. Our results show a high inter-individual variability as well as differences between the missions, which cannot be explained by inter-individual variability alone. The ages and sex of the participants did not appear to influence the results.     

Quercetin protects radiation-induced DNA damage and apoptosis in kidney and bladder tissues of rats

Abstract

Ionizing radiation (IR) can induce cell damage and cell death through the reactive oxygen species generated by radiolytic hydrolysis. The present study was aimed to determine the possible protective effects of quercetin, a well-known antioxidant agent, against IR-induced bladder and kidney damage in rats. Sprague-Dawley rats were exposed to 8-Gy whole-abdominal IR and given either vehicle or quercetin (20 mg/kg, ip). Rats were decapitated at either 36 h or 10 days following IR, where quercetin or vehicle injections were repeated once daily, and kidney and bladder samples were obtained for the determination of myeloperoxidase and caspase-3 activities, an index of tissue neutrophil infiltration and apoptosis, respectively. Radiation-induced inflammation was evaluated through tissue cytokine, TNF-α levels. In order to examine oxidative DNA damage, tissue 8-hydroxydeoxyguanosine (8-OHdG) levels were measured. All tissues were also examined microscopically. In the saline-treated irradiation groups, myeloperoxidase and caspase-3 activities, 8-OHdG and TNF-α levels were found to be increased in both tissues (p < 0.05). In the quercetin-treated-IR groups, all these oxidant responses were prevented significantly (p < 0.05). The present data demonstrate that quercetin, through its free radical scavenging and antioxidant properties, attenuates irradiation-induced oxidative organ injury, suggesting that quercetin may have a potential benefit in radiotherapy by minimizing the adverse effects and will improve patient care.     

Effect of UV Radiation and Other Abiotic Stress Factors on DNA of Different Wild Plant Species Grown in Three Successive Seasons in Alpine and Subalpine Regions

Plants in natural ecosystems are exposed to a combination of UV radiation, ionizing radiation (IR) and other abiotic factors. These factors change with the altitude. We investigated DNA alterations of some wild plants of different plant families in natural ecosystems at three altitudes in Rila Mountain, Bulgaria (1500, 1782, and 2925 m above sea level (a.s.l.) exposed to UV radiation, IR and other abiotic stresses, to assess the tolerance of plant species to the changing environmental conditions in three successive growth seasons. For this purpose, physicochemical, cytogenetic, and molecular methods were applied. DNA damage was assessed by micronucleus test and molecular method comet assay adapted and applied by us to wild plant species from Onagraceae, Rosaceae, Boraginaceae, Saxifragaceae, Orobanchaceae, Asteraceae and Poaceae families, growing at three different altitudes. Variability in the DNA sensitivity and the level of tolerance was observed among the plant species in response to combined abiotic factors assessed by induced DNA damage and gross beta activity. The studied representatives of Poaceae were less susceptible than the other studied species at all three altitudes and showed close level of DNA injuries to that of unaffected control plant grown in laboratory conditions. The lower levels of DNA damage of these wild plant species corresponded to their lower ability to accumulate radionuclides. There was a particularly pronounced low level of DNA injuries in the plant species at the highest altitude. The level of DNA damage showed correlation with the values of some abiotic environmental factors. The results would contribute to the elucidation of the extent of adaptation of plant species to the continuously changing environment and would be useful in selecting sensitive herbaceous monitor species for environmental impact assessment at mountain and alpine sites.     

The role and mechanism of long non-coding RNAs in homologous recombination repair of radiation-induced DNA damage

DNA double-strand breaks can seriously damage the genetic information that organisms depend on for survival and reproduction. Therefore, cells require a robust DNA damage response mechanism to repair the damaged DNA. Homologous recombination (HR) allows error-free repair, which is key to maintaining genomic integrity. Long non-coding RNAs (lncRNAs) are RNA molecules that are longer than 200 nucleotides. In recent years, a number of studies have found that lncRNAs can act as regulators of gene expression and DNA damage response mechanisms, including HR repair. Moreover, they have significant effects on the occurrence, development, invasion and metastasis of tumor cells, as well as the sensitivity of tumors to radiotherapy and chemotherapy. These studies have therefore begun to expose the great potential of lncRNAs for clinical applications. In this review, we focus on the regulatory roles of lncRNAs in HR repair.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

The basal levels of 8-oxoG and other oxidative modifications in intact mitochondrial DNA are low even in repair-deficient (Ogg1(-/-)/Csb(-/-)) mice

Mitochondrial DNA (mtDNA) is assumed to be highly prone to damage by reactive oxygen species (ROS) because of its location in close proximity to the mitochondrial electron transport chain. Accordingly, mitochondrial oxidative DNA damage has been hypothesized to be responsible for various neurological diseases, ageing and cancer. Since 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most frequent oxidative base modifications, is removed from the mitochondrial genome by the glycosylase OGG1, the basal levels of this lesion are expected to be highly elevated in Ogg1^−/− mice. To investigate this hypothesis, we have used a mtDNA relaxation assay in combination with various repair enzymes (Fpg, MutY, endonuclease III, endonuclease IV) to determine the average steady-state number of oxidative DNA modifications within intact (supercoiled) mtDNA from the livers of wild-type mice and those deficient in OGG1 and/or the Cockayne syndrome B (CSB) protein for mice aged up to 23 months. The levels of all types of oxidative modifications were found to be less than 12 per million base pairs, and the difference between wild-type and repair-deficient (Ogg1^−/−/Csb^−/−) mice was not significant. Thus, the increase of 8-oxoG caused by the repair deficiency in intact mtDNA is not much higher than in the nuclear DNA, i.e., not more than a few modifications per million base pairs. Based on these data, it is hypothesized that the load of oxidative base modifications in mtDNA is efficiently reduced during replication even in the absence of excision repair.     

Unraveling DNA Repair in Human: Molecular Mechanisms and Consequences of Repair Defect

Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders.     

Inorganic arsenic compounds cause oxidative damage to DNA and protein by inducing ROS and RNS generation in human keratinocytes

Arsenic is a naturally occurring element that is present in food, soil, and water. Inorganic arsenic can accumulate in human skin and is associated with increased risk of skin cancer. Oxidative stress due to arsenic exposure is proposed as one potential mode of carcinogenic action. The purpose of this study is to investigate the specific reactive oxygen and nitrogen species that are responsible for the arsenic-induced oxidative damage to DNA and protein. Our results demonstrated that exposure of human keratinocytes to trivalent arsenite caused the generation of 8-hydroxyl-2′-deoxyguanine (8-OHdG) and 3-nitrotyrosine (3-NT) in a concentration- and time-dependent manner. Pentavalent arsenate had similar effects, but to a significantly less extent. The observed oxidative damage can be suppressed by pre-treating cells with specific antioxidants. Furthermore, we found that pre-treating cells with Nω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase (NOS), or with 5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphinato iron (III) chloride (FeTMPyP), a decomposition catalyst of peroxynitrite, suppressed the generation of both 8-OHdG and 3-NT, which indicated that peroxynitrite, a product of the reaction of nitric oxide and superoxide, played an important role in arsenic-induced oxidative damage to both DNA and protein. These findings highlight the involvement of peroxynitrite in the molecular mechanism underlying arsenic-induced human skin carcinogenesis.     

HNRNPC regulates RhoA to induce DNA damage repair and cancer‐associated fibroblast activation causing radiation resistance in pancreatic cancer

Pancreatic cancer (PC) is one of the most lethal types of cancer due to its asymptomatic nature in the early stages and consequent late diagnosis. Its mortality rate remains high despite advances in treatment strategies, which include a combination of surgical resection and adjuvant therapy. Although these approaches may have a positive effect on prognosis, the development of chemo‐ and radioresistance still poses a significant challenge for successful PC treatment. Heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) and RhoA have been implicated in the regulation of tumour cell proliferation and chemo‐ and radioresistance. Our study aims to investigate the mechanism for HNRNPC regulation of PC radiation resistance via the RhoA pathway. We found that HNRNPC and RhoA mRNA and protein expression levels were significantly higher in PC tissues compared to adjacent non‐tumour tissue. Furthermore, high HNRNPC expression was associated with poor patient prognosis. Using HNRNPC overexpression and siRNA interference, we demonstrated that HNRNPC overexpression promoted radiation resistance in PC cells, while HNRNPC knockdown increased radiosensitivity. However, silencing of RhoA expression was shown to attenuate radiation resistance caused by HNRNPC overexpression. Next, we identified RhoA as a downstream target of HNRNPC and showed that inhibition of the RhoA/ROCK2‐YAP/TAZ pathway led to a reduction in DNA damage repair and radiation resistance. Finally, using both in vitro assays and an in vivo subcutaneous tumour xenograft model, we demonstrated that RhoA inhibition can hinder the activity of cancer‐related fibroblasts and weaken PC radiation resistance. Our study describes a role for HNRNPC and the RhoA/ROCK2‐YAP/TAZ signalling pathways in mediating radiation resistance and provides a potential therapeutic target for improving the treatment of PC.