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.

XPA deficiency affects the ubiquitin-proteasome system function.

Xeroderma pigmentosum complementation group A (XPA), is defective in xeroderma pigmentosum patients, causing pre-disposition to skin cancer and neurological abnormalities, which is not well understood. Here, we analyzed the XPA-deficient cells transcriptional profile under oxidative stress. The imbalance in of ubiquitin-proteasome system (UPS) gene expression was observed in XPA-deficient cells and the involvement of nuclear factor erythroid 2-related factor-2 (NFE2L2) was indicated. Co-immunoprecipitation assays showed the interaction between XPA, apurinic-apyrimidinic endonuclease 1 (APE1) and NFE2L2 proteins. Decreased NFE2L2 protein expression and proteasome activity was also observed in XPA-deficient cells. The data suggest the involvement of the growth arrest and DNA-damage-inducible beta (GADD45ß) in NFE2L2 functions. Similar results were obtained in xpa-1 (RNAi) Caenorhabditis elegans suggesting the conservation of XPA and NFE2L2 interactions. In conclusion, stress response activation occurs in XPA-deficient cells under oxidative stress; however, these cells fail to activate the UPS cytoprotective response, which may contribute to XPA patient's phenotypes.

New polymorphisms of Xeroderma Pigmentosum DNA repair genes in myelodysplastic syndrome.

The association between Xeroderma Pigmentosum DNA repair genes (XPA rs1800975, XPC rs2228000, XPD rs1799793 and XPF rs1800067) polymorphisms and myelodysplastic syndrome (MDS) have not been reported. To assess the functional role between these polymorphisms and MDS, we evaluated 189 samples stratified in two groups: 95 bone marrow samples from MDS patients and 94 from healthy elderly volunteers used as controls. Genotypes for all polymorphisms were identified in DNA samples in an allelic discrimination experiment by real-time polymerase chain reaction (qPCR). We also studied the mRNA expression of XPA and XPC genes to evaluate if its polymorphisms were functional in 53 RNAm MDS patients by qPCR methodologies. To the rs2228000 polymorphism, the CT and TT polymorphic genotype were associated with increased odds ratio (OR) of more profound cytopenia (hemoglobin and neutrophils count). To the rs1799793 polymorphism, we found that the GG homozygous wild-type genotype was associated with a decreased chance of developing MDS. We observed low expression of XPA in younger patients, in hypoplastic MDS and patients with abnormal karyotype when presented AG or AA polymorphic genotypes. We also found that there was a statistically significant interaction between the presence of micromegakaryocyte on down regulation of XPC regarding the CT heterozygous genotype of the rs1800975 polymorphism. Our results suggest that new functional polymorphisms of Xeroderma Pigmentosum DNA repair genes in MDS are related to its pathogenesis and prognosis.

DNA repair gene expressions are related to bone marrow cellularity in myelodysplastic syndrome.

OBJECTIVE: To evaluate the expression of genes related to nuclear excision (ERCC8, XPA and XPC), homologous recombination and non-homologous end-joining (ATM, BRCA1, BRCA2 and LIG4) repair mechanisms, using quantitative PCR methodologies, and it relation with bone marrow cellularity in myelodysplastic syndrome (MDS). METHODS AND RESULTS: A total of 51 adult de novo patients with MDS (3 refractory anaemia (RA), 11 refractory anaemia with ringed sideroblasts (RARS), 28 refractory cytopenia with multilineage dysplasia (RCMD), 3 refractory anaemia with excess blasts type I (RAEB-I), 5 refractory anaemia with excess blasts type II (RAEB-II), and 1 chronic myelomonocytic leukaemia (CMML) were evaluated. For karyotype, 16.2% patients were defined as very low prognosis, 59.5% low risk, 8.1% intermediate risk, 5.4% high risk and 10.8% very high risk. For bone marrow cellularity, 17.6%, 17.6% and 64.7% presented as hypocellular, normocellular and hypercellular, respectively. Patients with hypocellular MDS had significantly decreased expression of ATM (p=0.000), BRCA1 (p=0.014), BRCA2 (p=0.003), LIG4 (p=0.004) and ERCC8 (p=0.000) than those with normocellular/hypercellular bone marrow, whereas XPA (p=0.049) and XPC (p=0.000) genes were increased. In patients with hypoplastic MDS, a low expression of ATM (p=0.0268), LIG4 (p=0.0199) and ERCC8 (p=0.0493) was significantly associated with the presence of chromosomal abnormalities. We detected positive correlations between BRCA1 and BRCA2 (r=0.416; p=0.007), ATM and LIG4 (r=0.472; p=0.001), LIG4 and BRCA1 (r=0.333; p=0.026), LIG4 and BRCA2 (r=0.334; p=0.025), ATM and XPA (r=0.377; p=0.008), ATM and XPC (r=0.287; p=0.046), LIG4 and XPC (r=0.371; p=0.007) and XPA and XPC genes (r=0.895; p=0.0000). We also found among all patients evaluated that correlation with LIG4 occurred most often. CONCLUSIONS: These correlations demonstrate the important intrinsic relations between single and double DNA strand breaks genes in MDS, emphasising that these genes are related to MDS pathogenesis.

Xeroderma pigmentosum: low prevalence of germline XPA mutations in a Brazilian XP population.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by DNA repair defects that cause photophobia, sunlight-induced cancers, and neurodegeneration. Prevalence of germline mutations in the nucleotide excision repair gene XPA vary significantly in different populations. No Brazilian patients have been reported to carry a germline mutation in this gene. In this study, the germline mutational status of XPA was determined in Brazilian patients exhibiting major clinical features of XP syndrome. The study was conducted on 27 unrelated patients from select Brazilian families. A biallelic inactivating transition mutation c.619C>T (p.Arg207Ter) was identified in only one patient with a history of neurological impairment and mild skin abnormalities. These findings suggest that XP syndrome is rarely associated with inherited disease-causing XPA mutations in the Brazilian population. Additionally, this report demonstrates the effectiveness of genotype-phenotype correlation as a valuable tool to guide direct genetic screening.

Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions.

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.

DNA damage induced Pol eta recruitment takes place independently of the cell cycle phase.

When DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass. Intriguingly, recent evidence has linked TLS polymerases to processes that can also take place outside S phase such as nucleotide excision repair (NER). Here we show that Pol eta is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G(1). This observation was confirmed by different strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. The potential connection between Pol eta recruitment to DNA lesions outside S phase and NER was further evaluated. Interestingly, the recruitment of Pol eta to damage sites outside S phase did not depend on active NER, as UV-induced focus formation occurred normally in XPA, XPG and XPF deficient fibroblasts. Our data reveals that the re-localization of the TLS polymerase Pol eta to photo-lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.

Casos clinicos: xeroderma pigmentoso / Clinical cases: xeroderma pigmentosum

El Xeroderma pigmentoso es una patología poco frecuente con incapacidad de las células para reparar el daño causado en el ADN por las radiaciones ultravioleta evidenciándose quemaduras solares, ampollas, costras telangiectasias, y queratosis, además de alteraciones neurológicas, retardo en el crecimiento pondo-estatural y en la maduración sexual.

Gene transduction in skin cells: preventing cancer in xeroderma pigmentosum mice.

UV radiation is the most common risk factor for skin cancer. Patients with the autosomal recessive DNA repair disorder xeroderma pigmentosum (XP) suffer high incidence of skin cancer after sunlight exposure. XP-mutant mice are attractive models to study this syndrome, as they, too, develop UV radiation-induced skin tumors, mimicking the human phenotype. Recombinant adenovirus carrying the human XPA gene was used for in vivo gene therapy in UVB-irradiated skin of such mice. Virus s.c. injection led to the expression of the XPA protein in basal keratinocytes and prevented deleterious effects in the skin, including late development of squamous cell carcinoma. Thus, efficient adenovirus gene delivery to the skin is a promising tool for reconstitution of specific DNA repair defects in XP patients.

Complementation of the DNA repair deficiency in human xeroderma pigmentosum group a and C cells by recombinant adenovirus-mediated gene transfer.

Nucleotide excision repair (NER) is one of the most versatile DNA repair mechanisms, ensuring the proper functioning and trustworthy transmission of genetic information in all living cells. The phenotypic consequences caused by NER defects in humans are autosomal recessive diseases such as xeroderma pigmentosum (XP). This syndrome is the most sun-sensitive disorder leading to a high frequency of skin cancer. The majority of patients with XP carry mutations in the XPA or XPC genes that encode proteins involved in recognition of DNA damage induced by UV light at the beginning of the NER process. Cells cultured from XPA and XPC patients are hypersensitive to UV light, as a result of malfunctioning DNA repair. So far there is no effective long-term treatment for these patients. Skin cancer prevention can only be achieved by strict avoidance of sunlight exposure or by the use of sunscreen agents. We have constructed recombinant adenoviruses carrying the XPA and XPC genes that were used to infect XP-A and XP-C immortalized and primary fibroblast cell lines. UV survival curves and unscheduled DNA synthesis confirmed complete phenotypic reversion in XP DNA repair deficient cells with no trace of cytotoxicity. Moreover, transgene expression is stable for at least 60 days after infection. This efficient adenovirus gene delivery approach may be an important tool to better understand XP deficiency and the causes of DNA damage induced skin cancer.

Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance.

DNA integrity is threatened by the damaging effects of physical and chemical agents that can affect its function. Nucleotide excision repair (NER) is one of the most known and flexible mechanisms of DNA repair. This mechanism can recognize and remove damages causing DNA double-helix distortion, including the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (6-4) photoproducts, promoted by ultraviolet light (UV). The human syndrome xeroderma pigmentosum (XP) is clinically characterized chiefly by the early onset of severe photosensitivity of the exposed regions of the skin, a very high incidence of skin cancers and frequent neurological abnormalities. The xpa gene seems to be involved during UV damage recognition, in both global genome repair (GGR) and transcription-coupled repair (TCR). The modulation of xpa expression may modify the DNA repair rate in the cell genome, providing a valuable contribution to an understanding of the NER process. The controlled expression of the cDNA xpa in XP12RO deficient cells was achieved through the transfection of a muristerone-A inducible vector, pINXA. The INXA15 clone shows good induction of the XPA protein and total complementation of XP12RO cell deficiency. Overexpression of this protein resulted in UV cell survival comparable to normal control human cells. Moreover, low expression of the XPA protein in these cells is sufficient for total complementation in cellular UV sensitivity and DNA repair activity. These data demonstrate that XPA protein concentration is not a limiting factor for DNA repair.