XPA Levels Research Articles

Combining Phenotypes of Nucleotide Excision Repair Pathway to Predict the Risk of Head and Neck Squamous Cell Carcinomas in a Chinese Population

Background Nucleotide excision repair (NER) is pivotal in the development of smoking-related malignancies. Nine core genes (XPA, XPB, XPC, XPD, XPF, XPG, ERCC1, DDB1, and DDB2) are highly involved in the NER process. We combined two phenotypes of NER pathway (NER protein and NER gene mRNA expression) and evaluated their associations with the risks of the head and neck squamous cell carcinomas (HNSCCs) in a Chinese population. Methods We conducted a case-control study of 337 HNSCC patients and 285 cancer-free controls by measuring the expression levels of nine core NER proteins and NER gene mRNA in cultured peripheral lymphocytes. Results Compared with the controls, cases had statistically significantly lower protein expression levels of XPA (P < 0.001) and lower mRNA expression levels of XPA and XPB (P = 0.005 and 0.001, respectively). After dividing the subjects by controls' medians of expression levels, we found an association between increased risks of HNSCCs and low XPA protein level (Ptrend = 0.031), as well as low mRNA levels of XPA and XPB (Ptrend = 0.024 and 0.001, respectively). Subsequently, we correlated the two phenotypes and found associations between the NER mRNA and protein levels. Finally, the sensitivity of the expanded model with protein and mRNA expression levels, in addition to demographic variables, on HNSCCs risk was significantly improved. Conclusions Combining two phenotypes of NER pathway may be more effective than the model only including one single phenotype for the assessment of risks of HNSCCs.

Time-Dependent Induction of the Nucleotide Excision Repair Gene XPA and RAD51 in Homologous Recombination in Human Lymphocytes Exposed to Low Doses of Ionizing Radiation

Introduction: The aim of the present study was to understand the effect of low-doses of ionizing radiation (LDIR) on repair genes expression in blood samples that were taken from healthy donors. The next purpose was to examine the time-effect on the modified gene expression caused by low-doses of ionizing radiation. Material and method: The RNA of peripheral blood lymphocytes (PBLs) taken from four healthy donors was isolated at different time points after exposure included 4, 24, 48, 72, and 168 hours and then cDNA was synthesized. Modification of XPA and RAD51 expression levels due to LDIR (2, 5, 10 cGy) were evaluated by relative quantitative reverse transcription-polymerase chain reaction.Results: Significant up-regulation of both repair genes was observed at the 4 and 168 h following to 10 cGy. Also, this dose could increase expression levels of RAD51 at 48 and 72 h after radiation. For lower doses at 5 cGy, only XPA levels were significantly up-regulated after 168 h. A significant regression was found between the XPA levels and the dose, at 168 h after irradiation to PBLs that can represent a new potential biomarker for biological dosimetry purposes.Conclusion: The results of this study could support the hypothetical role of the different DNA repair pathways in response to LDIR. This led us to propose a molecular biodosimetry method for ionizing radiation in the range of LDIR.

Increased levels of XPA might be the basis of cisplatin resistance in germ cell tumours

BackgroundGerm cell tumours (GCTs) represent a highly curable malignity as they respond well to cisplatin (CDDP)-based chemotherapy. Nevertheless, a small proportion of GCT patients relapse or do not respond to therapy. As this might be caused by an increased capacity to repair CDDP-induced DNA damage, identification of DNA repair biomarkers predicting inadequate or aberrant response to CDDP, and thus poor prognosis for GCT patients, poses a challenge. The objective of this study is to examine the expression levels of the key nucleotide excision repair (NER) factors, XPA, ERCC1 and XPF, in GCT patients and cell lines.MethodsTwo hundred seven GCT patients’ specimens with sufficient follow-up clinical-pathological data and pairwise combinations of CDDP-resistant and -sensitive GCT cell lines were included. Immunohistochemistry was used to detect the ERCC1, XPF and XPA protein expression levels in GCT patients’ specimen and Western blot and qRT-PCR examined the protein and mRNA expression levels in GCT cell lines.ResultsGCT patients with low XPA expression had significantly better overall survival than patients with high expression (hazard ratio = 0.38, 95% confidence interval: 0.12–1.23, p = 0.0228). In addition, XPA expression was increased in the non-seminomatous histological subtype, IGCCCG poor prognosis group, increasing S stage, as well as the presence of lung, liver and non-pulmonary visceral metastases. Importantly, a correlation between inadequate or aberrant CDDP response and XPA expression found in GCT patients was also seen in GCT cell lines.ConclusionsXPA expression is an additional independent prognostic biomarker for stratifying GCT patients, allowing for improvements in decision-making on treatment for those at high risk of refractoriness or relapse. In addition, it could represent a novel therapeutic target in GCTs.

Associations between expression levels of nine core nucleotide excision repair genes in lymphocytes and risk of head and neck squamous cell carcinomas in a Chinese population.

Head and neck squamous cell carcinomas (HNSCCs) are one of the most common cancers in the world, and nucleotide excision repair (NER) is involved in HNSCCs susceptibility. We investigated whether mRNA expression levels of nine core NER genes were associated with risk of HNSCCs in a Chinese population. In this study of 251 HNSCC patients and 232 healthy controls, we quantified NER gene mRNA expression levels in cultured peripheral lymphocytes using a quantitative real-time PCR. Compared with the controls, HNSCC patients had statistically significantly lower expression levels of XPA and XPB (P = 0.029 and 0.001, respectively). After dividing the subjects by the controls' median values of expression levels, we found a dose-dependent association between an increased risk of HNSCCs and low expression levels of XPB (adjusted OR 1.56 and 95% CI 1.07-2.28; Ptrend = 0.001). We also identified a significant multiplicative interaction between smoking status as well as alcohol status and mRNA expression levels of XPB (P = 0.014 and 0.042, respectively). Finally, after integrating demographic variables, we found the addition of smoking status and XPB expression levels to the model significantly improved the sensitivity of the expanded model on HNSCC risk. Reduced mRNA expression levels of XPB were associated with an increased risk of HNSCCs in a Chinese population.

DNA damage and repair proteins in cellular response to sulfur mustard in Iranian veterans more than two decades after exposure

Delayed effects of sulfur mustard (SM) exposure on the levels of five important damage/repair proteins were investigated in 40 SM-exposed veterans of Iran-Iraq war and 35 unexposed controls. A major DNA damage biomarker protein – phosphorylated H2AX – along with four DNA repair proteins in cell response to the genome damage MRE11, NBS1, RAD51, and XPA were evaluated in blood lymphocytes from the veterans and controls using western blotting. Mean levels of XPA, MRE11, RAD51 and NBS1 were lower in SM-exposed patients and the decrease in NBS1 was significant. Even though the raised level of phosphor-H2AX in SM-poisoned group compared to the controls was not significant it was consistent with DNA damage findings confirming the severity of damage to the DNA after exposure to SM. There were correlations between the values of RAD51 and NBS1 proteins as well as XPA and MRE11 proteins. More than two decades after exposure to SM, there is still evidences of DNA damage as well as impaired repair mechanisms in cells of exposed individuals. Such disorders in cellular level may contribute to long term health problems of the SM veterans.

Downregulated XPA promotes carcinogenesis of bladder cancer via impairment of DNA repair.

Bladder cancer is the most common malignant tumor of urinary system, largely resulting from failure of repair of DNA damage to the environmental insults. The function of XPA in nucleotide excision repair pathway has been well documented. However, participation of XPA in the repair of DNA double-strand break remains unknown. Here, we reported that bladder cancer expressed low XPA levels compared to adjacent non-tumor bladder tissue, and this phenotype was closely associated with chromosomal aberrations. Moreover, downregulated XPA appeared to increase incidence of chromosome aberration. XPA reduction increased cell viability of a bladder cancer cell line RT4, while XPA re-expression decreased the cell viability of RT4 cells. Since high mutation frequency is the basis of mutations of oncogenes and anti-oncogenes, and may be the essence of bladder cancer susceptibility, our study suggests that downregulated XPA may promote carcinogenesis of bladder cancer via impairment of DNA repair.

XPA–A Biomarker With Potential Prognostic Value in Patients With Oropharyngeal Head and Neck Squamous Cell Carcinoma

Platinum-based chemotherapy resistance has been under investigation for a long time, looking at the nucleotide excision repair (NER) pathway, which is responsible for DNA adduct repair. One of the participating proteins in that pathway is XPA. There is little information about this protein regarding its prognostic value in patients with head and neck squamous cell carcinoma (HNSCC). Therefore, we investigated XPA expression as a prognostic factor by retrospectively looking at overall survival, time to recurrence, and correlation with clinical parameters. Tissue microarrays were constructed from 453 cases of HNSCC including 222 oral (49%), 126 pharyngeal (27.8%), and 105 laryngeal (23.2%) tumors. Two hundred ninety-three tumor blocks were evaluable for XPA immunohistochemistry. Expression levels were dichotomized into a high and low XPA expressing group followed by a comparison of age, gender, TNM status, grading, and UICC stage. Outcomes for overall survival and time to recurrence were analyzed by using the Kaplan-Meier method and performed for different subsites of the head and neck. Analysis of overall survival and time to recurrence showed no difference between both expression levels of XPA in the overall patient cohort. However, superior overall survival in patients with oropharyngeal SCC and a high XPA expression could be observed (P=.0386). Looking at SCCs of the oral cavity, a trend toward an inferior overall survival in patients with a high XPA expression was seen, whereas investigations in the hypopharynx and larynx showed no significant differences between high and low XPA expressing tumors. Looking generally at gender, M stage, and grading, no statistical correlation was found. Analyzing T and N stage in all tumors, a trend toward a lower XPA expression in advanced tumors (>pT4 P=.0543 and >pN1 P=.0546) could be seen. This trend was confirmed by statistical lower expression of XPA in patients with UICC stage IV looking at the overall patient cohort (P=.035). The shown results suggest that XPA might be a novel predictive marker for overall survival in patients with oropharyngeal SCC with a superior survival in tumors with a high XPA expression. Furthermore, this study shows that subsites in the head and neck will have to be looked at separately in the future to determine the predictive value of biomarkers for therapy outcome and pretherapeutic risk stratification of patients. To increase statistical power of this study and to evaluate the effect on platinum-based chemotherapy resistance, further studies with even larger patient cohorts will be needed and are ongoing.

Role of Ubiquitin Protein Ligase Ring2 in DNA Damage of Human Bronchial Epithelial Cells Exposed to Benzo[a]pyrene

Ubiquitylation of histones plays a pivotal role in DNA repair. The ubiquitin ligase Ring2 was recently shown to be the dominant ubiquitin ligase of histone H2A. In a series of experiments using the human bronchial epithelia cells (16HBE) and small interfering RNA (siRNA)-Ring2 cells exposed to benzo(a)pyrene (BaP), we measured dynamic changes in the levels of DNA damage, expressions of ubiquitinated histone H2A, and nucleotide excision repair (NER) subunit xeroderma pigmentosum (XP) groups A, C, and F (XPA, XPC, XPF). We found that in vitro exposure to BaP increased DNA damage in a time- and dose-dependent manner in 16HBE and siRNA-Ring2 cells. The results show that although decrease of Ring2 causes DNA hypersensitivity to BaP, the levels of XPA, XPC, and XPF were not affected. These results indicated that Ring2 may effect the DNA repair through other pathways but not through the expressions of NER protein.

A functional analysis of G23A polymorphism and the alternative splicing in the expression of the XPA gene

The XPA gene has a commonly occurring polymorphism (G23A) associated with cancer risk. This study assessed the functional significance of this polymorphism, which is localised near the translation start codon. Lymphoblastoid cell lines with alternative homozygous genotypes showed no significant differences in their XPA levels. The luciferase reporter assay detected no functional difference between the two sequences. Unexpectedly, we found that the alternatively spliced form of XPA mRNA lacked a part of exon 1. Only the reading frame downstream of codon Met59 was preserved. The alternative mRNA is expressed in various human tissues. The analysis of the 5’cDNA ends showed similar transcription start sites for the two forms. The in vitro expression of the alternative XPA labelled with the red fluorescent protein (mRFP) showed a lack of preferential nuclear accumulation of the XPA isoform. The biological role of the alternative XPA mRNA form remains to be elucidated.

Correlations and co-localizations of Hsp70 with XPA, XPG in human bronchial epithelia cells exposed to benzo[a]pyrene

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant known to cause DNA damage, which may be repaired through nucleotide excision repair (NER). The significantly negative correlation between Hsp70 levels and the level of DNA damage in workers exposed to coke oven emission had been found. However, little is known about how Hsp70 modulate the DNA repair process. In a series of experiments using the human bronchial epithelia cells (16HBE) exposed to different concentrations of BaP for 24 h, we measured expression of NER subunit xeroderma pigmentosum (XP) group A, C, F, G (XPA, XPC, XPF, XPG), excision repair cross-complementing 1 (ERCC1) and Hsp70, and analyzed their possible correlations. Co-localizations of Hsp70 with NER subunit were detected by confocal microscope. We found that in vitro exposure to BaP reduced cell viability in a dose-dependent manner ranging from 2 to 64 μM. Our results showed that levels of XPA, XPG and Hsp70 significantly increased at cells exposed to 1 or 2 μM BaP. In addition, curve estimation showed there was a significant correlation between relative ratios of Hsp70 and XPA, XPG in cells exposed to different concentrations of BaP. Moreover, confocal microscopy demonstrated increased co-localization of Hsp70 with XPA, XPG in nuclei of cells exposed to BaP. These results suggested that Hsp70 might play a role in nucleotide excision repair. However, the mechanisms underlying this observation need further investigation.

Elevation of XPA protein level in testis tumor cells without increasing resistance to cisplatin or UV radiation

Most testicular germ cell tumors are curable using cisplatin-based chemotherapy, and cell lines from these tumors are unusually sensitive to cisplatin and other DNA-damaging agents. It has been suggested that this might be caused by a lower-than normal nucleotide excision repair (NER) activity. Previous studies found that cell lines from testicular germ cell tumors have on average about one-third the level of the NER protein XPA in comparison to cell lines from other tumors. We asked whether over-expression of XPA protein would alleviate the cellular sensitivity and increase the DNA repair capacity of a testis tumor cell line. Increasing XPA levels in 833K cells by 10-fold did not increase resistance to UV irradiation. XPA was localized to the cell nucleus in all cell lines, before and after exposure to UV-radiation. 833K cells were proficient in removing UV radiation-induced photoproducts from the genome and increased XPA did not enhance the rate of removal. Further, over-expressing functional XPA protein did not correlate with increased resistance of 833K testis tumor cells to cisplatin. Thus, although the amount of XPA in this testis tumor cell line is lower than normal, it is sufficient for NER in vivo. The relative sensitivity of testis tumor cells to cisplatin, UV radiation, and other DNA damaging agents is likely related not to NER capacity, but to other factors such as the integrity of the p53 pathway in these cells.

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells

Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000–200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to ∼10,000 molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6–4) photoproducts from the genome with a half-life of 1 h. Cells in which XPA protein was reduced to about 10,000 molecules/cell removed (6–4) photoproducts more slowly, with a half-life of 3 h. A reduced rate of repair of (6–4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.

Development of New EBV-Based Vectors for Stable Expression of Small Interfering RNA to Mimick Human Syndromes: Application to NER Gene Silencing

We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPA(KD) and XPC(KD) (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced (HSA)KIN17. Several clones displaying undetectable protein levels of XPA, XPC, or (HSA)kin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, (HSA)KIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPA(KD) and XPC(KD) HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPC(KD) HeLa clones were more sensitive to UVC than their XPA(KD) or KIN17(KD) counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPA(KD) cells recovered a normal UVC sensitivity, XPC(KD) cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, (HSA)kin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.

Reduced levels of XPA, ERCC1 and XPF DNA repair proteins in testis tumor cell lines.

Over 80% of patients with advanced metastatic testis tumors can be cured using cisplatin-based combination chemotherapy. This is unusual as metastatic cancer in adults is usually incurable. Cell lines derived from testis tumors retain sensitivity to cisplatin in vitro. We previously investigated 2 testis tumor cell lines with a low capacity to remove cisplatin-induced DNA damage and found that they had low levels of the DNA nucleotide excision repair proteins XPA, ERCC1 and XPF. To determine whether low levels of XPA, ERCC1 and XPF proteins are characteristic of testis tumor cell lines, we investigated 35 cell lines derived from cancers to determine whether groups of cell lines from diverse tissue origins differ from one another in constitutive levels of these NER proteins. Quantitative immunoblotting was used to compare groups of cell lines representing prostate, bladder, breast, lung, cervical, ovarian and testis cancers. Only the 6 testis tumor cell lines showed significantly lower mean levels of XPA (p = 0.001), XPF (p = 0.001) and ERCC1 (p = 0.004) proteins from the other groups. Our results encourage further investigation of the possibility that low levels of these nucleotide excision repair proteins could be related to the favorable response of testis tumors to cisplatin-based chemotherapy.

Xeroderma Pigmentosum Group A Protein and Chemotherapy Resistance in Human Germ Cell Tumors

The exceptional sensitivity of germ cell tumors (GCTs) of adolescents and adults to chemotherapy, in particular to cisplatin, has been attributed to low levels of xeroderma pigmentosum group A protein (XPA), a crucial component of the nucleotide excision repair DNA repair pathway. In different types of solid tumors, resistance to cisplatin has been associated with enhanced expression of XPA. To assess the role of XPA levels in clinical sensitivity and resistance of GCTs to chemotherapy, immunohistochemistry was performed on tumor samples of both unselected patients before therapy and patients with fully documented clinical course before and after therapy. In the case of high XPA levels, fluorescent in situ hybridization was applied to assess the possibility of gene amplification. XPA protein levels were investigated by Western blot analysis after repeated exposure to cisplatin in different GCT-derived cell lines. Finally, XPA levels of both sensitive and cisplatin-resistant GCT cell lines were compared with cell lines derived from other neoplasms. We found that the presence of XPA protein as assessed by immunohistochemistry differs among the various histologies of GCTs. It is found more frequently and with a more homogenous staining pattern in histologic subtypes showing a more differentiated phenotype. Overall, no differences in the presence of XPA was observed between samples of tumors refractory or sensitive to chemotherapy. No XPA gene amplification was found. Interestingly, all tumors resected in relapse after chemotherapy in the refractory group stained positive for XPA. However, XPA was not induced by repeated courses of sublethal doses of cisplatin in GCT-derived cell lines in vitro, and no correlation between XPA protein levels and sensitivity to cisplatin in three GCT-derived cell lines was observed. We therefore conclude that XPA does not play a critical role in overall treatment resistance of GCTs.

DNA repair capacity: inconsistency between effect of over-expression of five NER genes and the correlation to mRNA levels in primary lymphocytes

We have previously shown that high DNA repair capacity protects psoriasis patients against chemically induced basal cell carcinoma [Dybdahl et al. Mutat. Res. 433 (1999) 15–22]. We have used the same study persons to investigate the correlation between expression of eight genes involved in nucleotide excision repair and DNA repair capacity. mRNA levels of XPA, XPB, XPC, XPD, XPF, XPG, CSB and ERCC1 in primary lymphocytes from 33 individuals were quantified by dot-blots and normalized to β-actin. ERCC1 and XPD mRNA quantities were highly correlated (r=0.89; P<10−11) while XPA, XPB, XPC, XPG, XPFand CSB mRNAs were moderately correlated (r=0.2–0.7). Thus, the mRNA expressions seem to fall in at least two groups. There was a three to sevenfold variation in the expression levels of the mRNAs. This is in contrast to the more than a hundredfold variation in mRNA levels reported in cancer patients.DNA repair capacity was measured in a host cell reactivation assay, where primary lymphocytes were transfected with an UV-irradiated plasmid encoding firefly-luciferase. Only ERCC1 and XPD mRNA levels correlated with the DNA repair capacity (P<0.03). In order to see if ERCC1 or XPD activity was limiting for DNA repair, we cotransfected with plasmids encoding NER genes, thus over-expressing either XPB, XPC, XPD, CSB or ERCC1 in the host cell reactivation assay. Only XPB over-expression increased DNA repair capacity. Thus, there is no indication that neither XPD nor ERCC1 limits the DNA repair capacity. However, our results indicate that ERCC1 and XPD mRNA levels may be used as a proxy for DNA repair capacity in lymphocytes.

Mechanisms and implications of the age‐associated decrease in DNA repair capacity

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6–4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6–4) photoproducts (P< 0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.—Goukassian, D., Gad, F., Yaar, M., Eller, M. S., Nehal, U. S., Gilchrest, B. A. Mechanisms and implications of the age-associated decrease in DNA repair capacity. FASEB J. 14, 1325–1334 (2000)

Mechanisms and implications of the age-associated decrease in DNA repair capacity.

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.