Association of ERCC2 Gene Polymorphisms with Susceptibility to Diffuse Large B-Cell Lymphoma.

BACKGROUND The objective of this study was to detect the association between ERCC excision repair 2, TFIIH core complex helicase subunit (ERCC2) gene polymorphisms and diffuse large B-cell lymphoma (DLBCL) susceptibility. MATERIAL AND METHODS This study used a case-control design. ERCC2 gene rs1799793 (Asp312Asn) and rs13181 (Lys751Gln) polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) both in DLBCL patients and healthy controls. The association between ERCC2 gene polymorphisms and DLBCL risk was assessed by χ² test. Odds ratios (ORs) with corresponding 95% confidence intervals (95% CIs) were used to address the association strength. Subgroup analyses were also performed to investigate the genetic effects of ERCC2 polymorphisms on clinical characteristics of DLBCL patients. RESULTS A significant association was discovered between the rs1799793 A allele and increased DLBCL risk (P=0.031, OR=1.928, 95% CI=1.052-3.534). The C allele of rs13181 was obviously associated with elevated DLBCL susceptibility (P=0.047, OR=1.820, 95% CI=1.002-3.305). The subgroup analysis demonstrated that rs1799793 and rs13181 polymorphisms had no relationship with serum lactate dehydrogenase level, nidus number, B-symptoms, Ann Arbor stages, or immunological types in DLBCL cases (P>0.05 for all). CONCLUSIONS Minor allele carriers of ERCC2 gene rs1799793 (Asp312Asn) and rs13181 (Lys751Gln) polymorphisms had higher susceptibility to DLBCL.

XPD Functions as a Tumor Suppressor and Dysregulates Autophagy in Cultured HepG2 Cells.

BACKGROUND: Recent clinical studies have linked polymorphisms in the xeroderma pigmentosum group D (XPD) gene, a key repair gene involved in nucleotide excision repair, to increased risk of hepatocellular carcinoma (HCC). However, the cellular effects of XPD expression in cultured HCC cells remain largely uncharacterized. Therefore, the aim of this study was to characterize the in vitro cellular effects of XPD expression on the HCC cell line HepG2. MATERIAL AND METHODS: HepG2 cells were transfected as follows to create four experimental groups: pEGFP-N2/XPD plasmid (XPD) group, EGFP-N2 plasmid (N2) control group, lipofectamine™ 2000 (lipid) control group, and non-transfected (CON) control group. An MTT cell proliferation assay, Annexin V-APC apoptosis assay, colony formation assay, scratch wound migration assay, Transwell migration assay, and Western blotting of the autophagic proteins LC3 and p62 were conducted. RESULTS: XPD expression significantly inhibited HepG2 cell proliferation (p<0.05), significantly promoted HepG2 cell apoptosis (p<0.05), significantly inhibited HepG2 colony formation (p<0.05), significantly decreased HepG2 cells' migratory ability (p<0.05), and significantly lowered HepG2 cells' invasive capacity (p<0.05). Western blotting showed that XPD expression significantly increased LC3 expression (p<0.05) and significantly reduced p62 expression (p<0.05). CONCLUSIONS: XPD expression serves as a tumor suppressor and dysregulates autophagic protein degradation in HepG2 cells in vitro. Further in vivo pre-clinical studies and clinical trials are needed to validate XPD's potential as a tumor-suppressive gene therapy.

XPD-dependent activation of apoptosis in response to triplex-induced DNA damage.

DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation.

The effect of tobacco, XPC, ERCC2 and ERCC5 genetic variants in bladder cancer development.

BACKGROUND: In this work, we have conducted a case-control study in order to assess the effect of tobacco and three genetic polymorphisms in XPC, ERCC2 and ERCC5 genes (rs2228001, rs13181 and rs17655) in bladder cancer development in Tunisia. We have also tried to evaluate whether these variants affect the bladder tumor stage and grade. METHODS: The patients group was constituted of 193 newly diagnosed cases of bladder tumors. The controls group was constituted of non-related healthy subjects. The rs2228001, rs13181 and rs17655 polymorphisms were genotyped using a polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: Our data have reported that non smoker and light smoker patients (1-19PY) are protected against bladder cancer development. Moreover, light smokers have less risk for developing advanced tumors stage. When we investigated the effect of genetic polymorphisms in bladder cancer development we have found that ERCC2 and ERCC5 variants were not implicated in the bladder cancer occurrence. However, the mutated homozygous genotype for XPC gene was associated with 2.09-fold increased risk of developing bladder cancer compared to the control carrying the wild genotype (p = 0.03, OR = 2.09, CI 95% 1.09-3.99). Finally, we have found that the XPC, ERCC2 and ERCC5 variants don't affect the tumors stage and grade. CONCLUSION: These results suggest that the mutated homozygous genotype for XPC gene was associated with increased risk of developing bladder. However we have found no association between rs2228001, rs13181 and rs17655 polymorphisms and tumors stage and grade.

Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification.

We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.

The XPD subunit of TFIIH is required for transcription-associated but not DNA double-strand break-induced recombination in mammalian cells.

Mutations in the XPD gene can give rise to three phenotypically distinct disorders: xeroderma pigmentosum (XP), trichothiodystrophy (TTD) or combined XP and Cockayne syndrome (CS) (XP/CS). The role of Xeroderma Pigmentosum group D protein (XPD) in nucleotide excision repair explains the increased risk of skin cancer in XP patients but not all the clinical phenotypes found in XP/CS or TTD patients. Here, we describe that the XPD-defective UV5 cell line is impaired in transcription-associated recombination (TAR), which can be reverted by the introduction of the wild-type XPD gene expressed from a vector. UV5 cells are defective in TAR, despite having intact transcription and homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Interestingly, we find reduced spontaneous HR in XPD-defective cells, suggesting that transcription underlies a portion of spontaneous HR events. We also report that transcription-coupled repair (TCR)-defective cells, mutated in the Cockayne syndrome B (CSB) protein, have a defect in TAR, but not in DSB-induced HR. However, the TAR defect may be associated with a general transcription defect in CSB-deficient cells. In conclusion, we show a novel role for the XPD protein in TAR, linking TAR with TCR.

[The metabolic and molecular bases of Cockayne syndrome]. / Las bases metabólicas y moleculares del síndrome de Cockayne.

Cockayne is a segmental progeroid syndrome that has autosomal recessive inheritance pattern. It is mainly characterized by Intrauterine growth retardation, severe postnatal growth deficiency, cachectic dwarfism, microcephaly, wizened face, sensorineural hearing loss, cataracts, dental caries, cardiac arrhythmias, hypertension, atherosclerosis, proteinuria, micropenis, renal failure, skeletal abnormalities, skin photosensitivity, decreased subcutaneous adipose tissue, cerebral atrophy, dementia, basal ganglia calcifications, ataxia and apraxia. It has a complex phenotype given by genetic heterogeneity. There are five gene responsible for this syndrome: CSA, CSB, XPB, XPD and XPG, in which various mutations have been found. The biochemical effect of these mutations includes dysfunctional protein of the repair system for oxidative damage to DNA, the complex coupled to transcription and the nucleotide excision repair system. Considering the role played for these proteins and its effects on clinical phenotype when they are deficient, we suggest that these genes might be candidates for analyzing susceptibility to common chronic degenerative diseases related to oxidative stress and aging.

[Mechanism of regulation of hepatoma cell cycle by XPD/P44 subcomplex : an in vitro experiment].

OBJECTIVE: To explore the effects of xeroderma pigmentosum group D(XPD)/P44 subcomplex on the cell cycle of the hepatoma cells. METHODS: Human hematoma cells of the line SMMC-7721 were cultured and transfected with human XPD gene by Lipofectamine and 2 strains with stably transfected plasmid pEGFG-N2 and stably transfected recombinant plasmid pEGFG-N2/XPD were selected. After stably transfection,the antisense oligonucleotides of P44 were added to treat the stably transfected cells. The cells were divided into 6 groups: Group (1) (control group), Group (2) transfected with the blank plasmid pEGFP-N2, Group (3) transfected with the recombinant plasmid pEGFP-N2/XPD, Group (4) transfected with ASODN complementary to the translation initiation site of pEGFP-N2/XPD, Group (5) transfected with antisense oligodeoxynucleotides (ASODN) complementary to the translation terminal site of pEGFP-N2/XPD, and Group (6) transfected with ASODN complementary to the translation exon5 site of pEGFP-N2/XPD. The expression levels of wild-type P44, XPD, cdk7, cdk2, c-myc, and cdc25A were detected by RT-PCR and Western blotting. The cell growth and the cell cycle were examined by MTT and flow cytometry (FCM). RESULTS: The P44 and XPD mRNA expression levels of Group (4) were significantly higher than those of Groups (1) and (2) (both P < 0.01). Western blotting indicated that the changes of P44 and XPD protein expression levels were consistent with those of their mRNAs respectively; while the mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were all decreased. MTF method showed that the hepatoma cells grew slowly, FCM showed that the number of the cells arrested at the G1 stage of Group (3) were higher than those of Groups (1) and (2). After the blockage of P44 gene expression, the expression levels of XPD mRNA and protein were decreased. The XPD mRNA and protein expression levels of Groups (4), (5), and (6) were significantly higher than those of Group (3) (all P < 0.01). The mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were upregulated. MT method indicated that cells grew fast. FCM showed that the numbers of the cells arrested at the G1 stage of Group (4), (5), and (6) were all lower than that of Group ((3) The expression levels of cell cycle regulatory genes including cdk7, cdk2, c-myc, and cdc25A were markedly decreased,the hepatoma cells grew slowly; after the blockage of P44 gene expression the expression levels of XPD mRNA and protein were decreased, whereas the expression levels of the cell cycle regulatory genes mentioned above were enhanced, and the hepatoma cells grew faster. CONCLUSION: XPD gene inhibits the proliferation and promotes the apoptosis of hepatoma cells. The expression of XPD may be regulated by its molecular partner P44. XPD/P44 subcomplex is involved in the regulation of DNA damage checkpoint.

XPD structure reveals its secrets.

The XPD protein is central to our understanding of the relationship between NER deficiencies and human disorders. Three recent papers report the crystal structures of XPD from archaea. Apart from anticipated helicase domains the structures reveal a 4FeS cluster and novel "Arch" domain. The structures help our understanding of genotype-phenotype relationships in the XPD gene.

Cisplatin plus weekly CPT-11/docetaxel in advanced esophagogastric cancer: a phase I study with pharmacogenetic assessment of XPD, XRCC3 and UGT1A1 polymorphisms.

PURPOSE: A multicenter phase I trial to establish the recommended dose of CPT-11/docetaxel plus cisplatin in advanced esophagogastric cancer patients and to correlate the efficacy and toxicity with genetic polymorphisms in DNA repair genes (XPD and XRCC3) and the UGT1A1 gene. METHODS: Four dose levels with a fixed dose of cisplatin (60 mg/m(2)), day 1, and dose-escalation of CPT-11 (50-70 mg/m(2)) and docetaxel (25-30 mg/m(2)), days 1 and 8, every 3 weeks were planned. Polymorphisms of XPD (Asp312Asn and Lys751Gln), XRCC3 (Thr241Met) and UGT1A1*28 were examined in baseline peripheral blood. RESULTS: Twenty-eight patients were included at three different dose levels. Dose-limiting toxicities were febrile neutropenia and diarrhea; the recommended dose was established at CPT-11 60 mg/m(2) and docetaxel 25 mg/m(2) plus cisplatin 60 mg/m(2). Objective response was observed in 13 patients (50%). Median time to progression was 6.6 months, and median survival was 11.3 months. Median time to progression was 9.7 months for patients harboring the XRCC3 Met241Met genotype versus 8.4 months for patients with Thr241Met and 3.1 months for those with Thr241Thr (P = .04). CONCLUSIONS: CPT-11/docetaxel plus cisplatin is active in patients with advanced esophagogastric cancer. XRCC3 Met241Thr polymorphisms could be a useful marker to predict prognosis in patients treated with a cisplatin-based chemotherapy. However, these results are required to be confirmed with a great number of patients.

The XPD 751Gln allele is associated with an increased risk for esophageal adenocarcinoma: a population-based case-control study in Sweden.

Mechanisms behind the strong associations of esophageal adenocarcinoma risk with gastroesophageal reflux (GOR) and body mass remain to be defined. In a nationwide population-based case-control study, we examined associations of polymorphisms in the DNA repair genes XPD, XPC, XRCC1 and XRCC3 with risk of esophageal adenocarcinoma, squamous-cell carcinoma (SCC) and gastric cardia adenocarcinoma, and paid special attention to possible interactions with symptomatic reflux or body mass. We collected blood samples from 96, 81 and 126 interviewed incident cases of esophageal adenocarcinoma, esophageal SCC and gastric cardia adenocarcinoma, respectively, and 472 randomly selected controls, frequency-matched with regard to age and sex. DNA was extracted and polymorphisms in XPD codon 751 (Lys-->Gln), codon 312 (Asp-->Asn), C insertion in intron 10 of XPD, XPC codon 939 (Lys-->Gln), XRCC1 codon 399 (Arg-->Gln) and XRCC3 codon 241 (Thr-->Met) were examined using PCR-RFLP. Odds ratios (ORs) derived from multivariate logistic regression with adjustments for potential confounding factors estimated relative risks. XPD codon 751 Lys/Gln and Gln/Gln genotypes, compared with Lys/Lys genotype, were both associated with a more than doubled risk for esophageal adenocarcinoma (OR=2.4; 95% CI=1.4-4.4; OR=2.7, 95% CI=1.3-5.9). The combined effects of these genotypes and symptomatic GOR or body mass showed borderline significant deviation from additivity. Excess risks for esophageal SCC were also noted for XPD 751Gln variant genotypes. Other studied variants were not found to be related to the three tumors. Our study suggests that XPD 751Gln allele is a potential genetic marker for susceptibility to esophageal adenocarcinoma.

The DNA repair genes XPB and XPD defend cells from retroviral infection.

Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A host degradation system prevents a majority of the cDNA molecules from completing the obligatory genomic integration necessary for pathogenesis. We demonstrate that the human TFIIH complex proteins XPB (ERCC3) and XPD (ERCC2) play a principal role in the degradation of retroviral cDNA. DNA repair-deficient XPB and XPD mutant cell lines exhibited an increase in transduction efficiency by both HIV- and Moloney murine leukemia virus-based retroviral vectors. Replicating Moloney murine leukemia virus viral production was greater in XPB or XPD mutant cells but not XPA mutant cells. Quantitative PCR showed an increase in total cDNA molecules, integrated provirus, and 2LTR circles in XPB and XPD mutant cells. In the presence of a reverse transcription inhibitor, the HIV cDNA appeared more stable in mutant XPB or XPD cells. These studies implicate the nuclear DNA repair proteins XPB and XPD in a cellular defense against retroviral infection.