Functional Nucleotide Excision Repair Research Articles

Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2

Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

NER enzymes maintain genome integrity and suppress homologous recombination in the absence of exogenously induced DNA damage in Pseudomonas putida.

In addition to its prominence in producing genetic diversity in bacterial species, homologous recombination (HR) plays a key role in DNA repair and damage tolerance. The frequency of HR depends on several factors, including the efficiency of DNA repair systems as HR is involved in recovery of replication forks perturbed by DNA damage. Nucleotide excision repair (NER) is one of the major DNA repair pathways involved in repair of a broad range of DNA lesions generally induced by exogenous chemicals or UV-irradiation and its functions in the cells not exposed to DNA-damaging agents have attracted less attention. In this study we have developed an assay that enables to investigate HR between chromosomal loci of the soil bacterium Pseudomonas putida both in growing and stationary-phase cells. The present assay detects HR events between two non-functional alleles of phenol degrading genes that produce a functional allele and allow the growth of bacteria on phenol as a sole carbon source. Our results indicate that HR between chromosomal loci takes place mainly in the growing cells and the frequency of HR is reduced during the following starvation in NER-proficient P. putida but not in the case when bacteria lack UvrA or UvrB enzymes. The absence of UvrA or UvrB resulted in a hyper-recombination phenotype in P. putida, the cells were filamented and their growth was impaired even in the absence of exogenous DNA damage. However, NER-deficient derivatives that overcame growth defects emerged rapidly. Such adaptation resulted in the decline of the frequency of HR. Although HR in actively replicating P. putida was still elevated in the adapted variants of the UvrA- and UvrB-deficient strains, the dynamics of emergence of the recombinants in these strains turned similar to NER-proficient bacteria. Additionally, we observed that HR was enhanced in the absence of the transcription repair coupling factor Mfd in growing cells but not during starvation. The frequency of HR was not affected by the UvrA homologue UvrA2 neither in NER-proficient bacteria nor in the absence of UvrA, suggesting a minor role of UvrA2 in NER. Thus, we conclude that NER functions are important also without exogenously induced DNA damage in P. putida and both transcription-coupled and global genome NER act to suppress HR in growing cells, whereas UvrA and UvrB are involved in the maintenance of the genome integrity also in stationary-phase cells.

Implication of SUMO E3 ligases in nucleotide excision repair.

Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.

α-N-Methylation of Damaged DNA-binding Protein 2 (DDB2) and Its Function in Nucleotide Excision Repair

DDB2 exhibits a high affinity toward UV-damaged DNA, and it is involved in the initial steps of global genome nucleotide excision repair. Mutations in the DDB2 gene cause the genetic complementation group E of xeroderma pigmentosum, an autosomal recessive disease manifested clinically by hypersensitivity to sunlight exposure and an increased predisposition to skin cancer. Here we found that, in human cells, the initiating methionine residue in DDB2 was removed and that the N-terminal alanine could be methylated on its α-amino group in human cells, with trimethylation being the major form. We also demonstrated that the α-N-methylation of DDB2 is catalyzed by the N-terminal RCC1 methyltransferase. In addition, a methylation-defective mutant of DDB2 displayed diminished nuclear localization and was recruited at a reduced efficiency to UV-induced cyclobutane pyrimidine dimer foci. Moreover, loss of this methylation conferred compromised ATM (ataxia telangiectasia mutated) activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity of cells toward UV light exposure. Our study provides new knowledge about the posttranslational regulation of DDB2 and expands the biological functions of protein α-N-methylation to DNA repair.

Increased ultraviolet radiation sensitivity ofEscherichia coligrown at low temperature

The repair of DNA damage caused by ultraviolet radiation (UVR) is well understood in both lower and higher organisms. Genetic studies carried out at optimum temperature for growth, 37 °C in Escherichia coli, have revealed the major pathways of DNA repair. We show that E. coli cells grown at 20 °C are more sensitive to UVR than cells grown at 37 °C. The analysis of knockout mutants demonstrates that cells impaired in recombinational DNA repair pathways show increased UV sensitivity at 20 °C. Cells with mutations in the nucleotide excision repair (NER) pathway genes are highly sensitive to UVR when grown at 37 °C and retain that sensitivity when grown at 20 °C, whereas wild-type cells are not sensitive when grown at 37 °C but become more sensitive to UVR when grown at low temperatures. Our results taken along with reports from the literature suggest that the UVR sensitivity of E. coli cells at low temperature could be due to impaired NER function.

Post-treatment recovery of suboptimal DNA repair capacity and gene expression levels in colorectal cancer patients

DNA repair in blood cells was observed to be suboptimal in cancer patients at diagnosis, including colorectal cancer (CRC). To explore the causality of this phenomenon, we studied the dynamics of DNA repair from diagnosis to 1 yr follow-up, and with respect to CRC treatment. Systemic CRC therapy is targeted to DNA damage induction and DNA repair is thus of interest. CRC patients were blood-sampled three times in 6-mo intervals, starting at the diagnosis, and compared to healthy controls. DNA repair was characterized by mRNA levels of 40 repair genes, by capacity of nucleotide excision repair (NER), and by levels of DNA strand breaks (SBs). NER and base excision repair genes were significantly under-expressed (P < 0.016) in patients at diagnosis compared to controls, in accordance with reduced NER function (P = 0.008) and increased SBs (P = 0.015). Six months later, there was an increase of NER capacity, but not of gene expression levels, in treated patients only. A year from diagnosis, gene expression profiles and NER capacity were significantly modified in all patients and were no longer different from those measured in controls. All patients were free of relapse at the last sampling, so we were unable to clarify the impact of DNA repair parameters on treatment response. However, we identified a panel of blood DNA repair-related markers discerning acute stage of the disease from the remission period. In conclusion, our results support a model in which DNA repair is altered as a result of cancer.

A Selective Mitochondrial-Targeted Chlorambucil with Remarkable Cytotoxicity in Breast and Pancreatic Cancers

Nitrogen mustards, widely used as chemotherapeutics, have limited safety and efficacy. Mitochondria lack a functional nucleotide excision repair mechanism to repair DNA adducts and are sensitive to alkylating agents. Importantly, cancer cells have higher intrinsic mitochondrial membrane potential (Δψmt) than normal cells. Therefore, selectively targeting nitrogen mustards to cancer cell mitochondria based on Δψmt could overcome those limitations. Herein, we describe the design, synthesis, and evaluation of Mito-Chlor, a triphenylphosphonium derivative of the nitrogen mustard chlorambucil. We show that Mito-Chlor localizes to cancer cell mitochondria where it acts on mtDNA to arrest cell cycle and induce cell death, resulting in a 80-fold enhancement of cell kill in a panel of breast and pancreatic cancer cell lines that are insensitive to the parent drug. Significantly, Mito-Chlor delayed tumor progression in a mouse xenograft model of human pancreatic cancer. This is a first example of repurposing chlorambucil, a drug not used in breast and pancreatic cancer treatment, as a novel drug candidate for these diseases.

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps.

Functional Analysis of Rad14p, a DNA Damage Recognition Factor in Nucleotide Excision Repair, in Regulation of Transcription in Vivo

Rad14p is a DNA damage recognition factor in nucleotide excision repair. Intriguingly, we show here that Rad14p associates with the promoter of a galactose-inducible GAL1 gene after transcriptional induction in the absence of DNA lesion. Such an association of Rad14p facilitates the recruitment of TBP, TFIIH, and RNA polymerase II to the GAL1 promoter. Furthermore, the association of RNA polymerase II with the GAL1 promoter is significantly decreased in the absence of Rad14p, when the coding sequence was deleted. These results support the role of Rad14p in transcriptional initiation. Consistently, the level of GAL1 mRNA is significantly decreased in the absence of Rad14p. Similar results are also obtained at other galactose-inducible GAL genes such as GAL7 and GAL10. Likewise, Rad14p promotes transcription of other non-GAL genes such as CUP1, CTT1, and STL1 after transcriptional induction. However, the effect of Rad14p on the steady-state levels of transcription of GAL genes or constitutively active genes such as ADH1, PGK1, PYK1, and RPS5 is not observed. Thus, Rad14p promotes initial transcription but does not appear to regulate the steady-state level. Collectively, our results unveil a new role of Rad14p in stimulating transcription in addition to its well-known function in nucleotide excision repair.

Virtual Screening and Biological Evaluation of Inhibitors Targeting the XPA-ERCC1 Interaction

BackgroundNucleotide excision repair (NER) removes many types of DNA lesions including those induced by UV radiation and platinum-based therapy. Resistance to platinum-based therapy correlates with high expression of ERCC1, a major element of the NER machinery. The interaction between ERCC1 and XPA is essential for a successful NER function. Therefore, one way to regulate NER is by inhibiting the activity of ERCC1 and XPA.Methodology/Principal FindingsHere we continued our earlier efforts aimed at the identification and characterization of novel inhibitors of the ERCC1-XPA interaction. We used a refined virtual screening approach combined with a biochemical and biological evaluation of the compounds for their ability to interact with ERCC1 and to sensitize cells to UV radiation. Our findings reveal a new validated ERCC1-XPA inhibitor that significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction.ConclusionsNER is a major factor in acquiring resistance to platinum-based therapy. Regulating the NER pathway has the potential of improving the efficacy of platinum treatments. One approach that we followed is to inhibit the essential interaction between the two NER elements, ERCC1 and XPA. Here, we performed virtual screening against the ERCC1-XPA interaction and identified novel inhibitors that block the XPA-ERCC1 binding. The identified inhibitors significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction.

Important Role for Mycobacterium tuberculosis UvrD1 in Pathogenesis and Persistence apart from Its Function in Nucleotide Excision Repair

Mycobacterium tuberculosis survives and replicates in macrophages, where it is exposed to reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the roles of UvrA and UvrD1, thought to be parts of the nucleotide excision repair pathway of M. tuberculosis. Strains in which uvrD1 was inactivated either alone or in conjunction with uvrA were constructed. Inactivation of uvrD1 resulted in a small colony phenotype, although growth in liquid culture was not significantly affected. The sensitivity of the mutant strains to UV irradiation and to mitomycin C highlighted the importance of the targeted genes for nucleotide excision repair. The mutant strains all exhibited heightened susceptibility to representatives of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). The uvrD1 and the uvrA uvrD1 mutants showed decreased intracellular multiplication following infection of macrophages. Most importantly, the uvrA uvrD1 mutant was markedly attenuated following infection of mice by either the aerosol or the intravenous route.

Nucleotide Excision Repair by Mutant Xeroderma Pigmentosum Group A (XPA) Proteins with Deficiency in Interaction with RPA

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.

Association studies of excision repair cross-complementation group 1 ( ERCC1) haplotypes with lung and head and neck cancer risk in a Caucasian population

Background: The formation of bulky DNA adducts caused by diol epoxide derivatives of polycyclic aromatic hydrocarbons has been associated with tobacco-induced cancers, and inefficient repair of such adducts by the nucleotide excision repair (NER) system has been linked to increased risk of tobacco-induced lung and head and neck (H&N) cancers. The human excision repair cross-complementation group 1 (ERCC1) protein is essential for a functional NER system and genetic variation in ERCC1 may contribute to impaired DNA repair capacity and increased lung and H&N cancer risk. Methods: In order to comprehensively capture common genetic variation in the ERCC1 gene, Caucasian data from the International HapMap project was used to assess linkage disequilibrium and choose four tagSNPs (rs1319052, rs3212955, rs3212948, and rs735482) in the ERCC1 gene to genotype 452 lung cancer cases, 175 H&N cancer cases, and 790 healthy controls. Haplotypes were estimated using expectation maximization (EM) algorithm, and haplotype association with cancer was investigated using Haplo.stats software adjusting for known covariates. Results: The genotype and haplotype frequencies matched previous estimates from Caucasians. There was no significant difference in the prevalence of rs1319052, rs3212955, rs3212948, and rs735482 when comparing lung or H&N cancer cases with controls ( p-values > 0.05). Similarly, there was no association between ERCC1 haplotypes and lung or H&N cancer susceptibility in this Caucasian population ( p-values > 0.05). No associations were found when stratifying lung cancer cases by histology, sex, smoking status, or smoking intensity. Conclusions: This study suggests that ERCC1 polymorphisms and haplotypes do not play a role in lung and H&N cancer susceptibility in Caucasians.

Abstract 2986: Melanocytes are deficient in repair of oxidative DNA damage and UVC induced photoproducts

Abstract Melanomas occur mainly in sunlight-exposed skin, xeroderma pigmentosum complementation (XP) patients, defective in nucleotide excision repair function, have 1000-fold higher incidence of melanoma. These findings indicate that sunlight induced “bulky” photoproducts are responsible for melanomagenesis. On the other hand, sunlight also induces a high level of reactive oxygen species (ROS) in melanoctes (MC), oxidative DNA damage (ODD) therefore may contribute to melanomagenesis and that the XP protein may participate in ODD repair. To address these questions we determined: 1) the effect of melanin on UVA (320-400 nm) irradiation induced ODD and UV photoproducts using formamidopyrimidine glycosylase (Fpg) and UvrABC incision assays, 2) the repair capacity for ODD and UV-induced photoproducts in MC and XPA cells using host cell reactivation and in vitro DNA repair synthesis assays. We found: 1) UVA irradiation-inducing a significantly higher amount of Fpg sensitive ODD in MC than in normal human skin fibroblasts (NHSF). In contrast, UVA irradiation induces an insignificant amount of UvrABC sensitive sites in either of these two types of cells. 2) Compared to NHSF, MC have a reduced repair capacity for ODD and photoproducts. 3) H2O2 modified DNA induces a higher mutation frequency in MC than in NHSF. 4) XPA cells are deficient in ODD repair and ODD induces a higher mutation frequency in XPA cells than in NHSF. Based on these results, we propose: 1) UVA is a major etiological agent for melanomagenesis and the mechanism is through melanin sensitizes UVA in induction of ODD and reduced repair capacity in MC; 2) the reduced repair capacity for ODD contributes to the high melanoma incidence in XPA patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2986.

Abstract 5379: Mechanism of fludarabine and oxaliplatin combined activity depends on the activity of XPF endonuclease

Abstract We set out to determine the mechanism by which fludarabine and oxaliplatin exert more than additive cell killing in chronic lymphocytic leukemia (CLL). We postulate that enhanced activity of oxaliplatin and fludarabine combination is based on termination by fludarabine of DNA re-synthesis associated with nucleotide excision repair (NER) following removal of oxaliplatin adducts. The blocked repair would result in single-strand and double-strand DNA breaks which would activate cell DNA damage signaling and apoptosis. More than additive apoptotic killing was observed in 49% (±22) of CLL cells after exposure to oxaliplatin and fludarabine in vitro, although single agents showed minimal increase in cell death (3% ±9 for oxaliplatin, 13% ±14 for fludarabine), as demonstrated by Annexin V and PI staining at 24 hr (expected vs. observed p=0.0003; n=10). Oxaliplatin induced a 2-fold increase in DNA synthesis in CLL cells, which was inhibited by fludarabine, as determined by thymidine incorporation (n=5). Knockdown of XPF, the NER 5′ endonuclease, in CLL patient samples (n=3) eliminated oxaliplatin induced DNA synthesis (3-fold increase in thymidine incorporation with scramble siRNA compared to 1.4-fold increase with XPF siRNA). Wild type CHO (AA8) cells showed more than additive killing after combination treatment similar to the observation with CLL patient samples (predicted survival 50%; observed 21%). In contrast, only additive killing was observed in CHO cells lacking XPF (UV41) with equitoxic doses of oxaliplatin and fludarabine (predicted survival 56%; observed 54%) suggesting that XPF function is essential for the cooperative interaction of the combination. Alkaline assays demonstrated that inhibition of repair by fludarabine was accompanied by DNA strand break formation within 12 hr (n=4) in CLL patients samples. The increase in Olive tail moment ranged from 2- to 5-fold, confirming induction of single-strand breaks. The double-strand break repair proteins ATM, SMC1, H2AX and DNA-PKcs were phosphorylated as early as 2 hr post-treatment with the combination and persisted up to 24 hr (3-fold, 24-fold, 10-fold and 10-fold increase, respectively; n=4). This response suggests that DNA damage generated by fludarabine and oxaliplatin exposure was processed to double-strand DNA breaks in CLL cells. Our work provides evidence that the combination of oxaliplatin and fludarabine results in the formation of toxic DNA damage and that their combined activity depends on a functional NER pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5379.

Lung inflammation is associated with reduced pulmonary nucleotide excision repair in vivo

Chronic pulmonary inflammation is associated with increased lung cancer risk, but the underlying process remains unknown. Recently, we showed that activated neutrophils inhibit nucleotide excision repair (NER) in pulmonary epithelial cells in vitro via the release of myeloperoxidase (MPO). To evaluate the effect of neutrophils on NER in vivo, mice were intratracheally instilled with lipopolysaccharide (LPS) (20 microg), causing acute lung inflammation and associated neutrophil influx into the airways. Three days post-exposure, phenotypical NER capacity was assessed in lung tissue homogenate. LPS exposure inhibited pulmonary NER by approximately 50%. This finding was corroborated by down-regulation of the NER-associated genes Xpa and Xpf. To further elicit the role of neutrophils and MPO in this process, we utilized MPO-deficient mice as well as mice in which circulating neutrophils were depleted by antibody treatment. LPS-induced inhibition of pulmonary NER was not affected by either Mpo(-/-) or by depletion of circulating neutrophils. This contrasts with our previous in vitro observations, suggesting that inhibition of pulmonary NER following acute dosing with LPS is not fully mediated by neutrophils and/or MPO. In conclusion, these data show that LPS-induced pulmonary inflammation is associated with a reduction of NER function in the mouse lung.

Cisplatin pharmacogenetics, DNA repair polymorphisms, and esophageal cancer outcomes

Genetic variations or polymorphisms within genes of the nucleotide excision repair (NER) pathway alter DNA repair capacity. Reduced DNA repair (NER) capacity may result in tumors that are more susceptible to cisplatin chemotherapy, which functions by causing DNA damage. We investigated the potential predictive significance of functional NER single nucleotide polymorphisms in esophageal cancer patients treated with (n = 262) or without (n = 108) cisplatin. Four NER polymorphisms XPD Asp312Asn; XPD Lys751Gln, ERCC1 8092C/A, and ERCC1 codon 118C/T were each assessed in polymorphism-cisplatin treatment interactions for overall survival (OS), with progression-free survival (PFS) as a secondary endpoint. No associations with ERCC1 118 were found. Polymorphism-cisplatin interactions were highly significant in both OS (P = 0.002, P = 0.0001, and P < 0.0001) and PFS (P = 0.006, P = 0.008, and P = 0.0007) for XPD 312, XPD 751, and ERCC1 8092, respectively. In cisplatin-treated patients, variant alleles of XPD 312, XPD 751, and ERCC1 8092 were each associated with significantly improved OS (and PFS): adjusted hazard ratios of homozygous variants versus wild-type ranged from 0.22 [95% confidence interval (CI): 0.1-0.5] to 0.31 (95% CI: 0.1-0.7). In contrast, in patients who did not receive cisplatin, variant alleles of XPD 751 and ERCC1 8092 had significantly worse survival, with adjusted hazard ratios of homozygous variants ranging from 2.47 (95% CI: 1.1-5.5) to 3.73 (95% CI: 1.6-8.7). Haplotype analyses affirmed these results. DNA repair polymorphisms are associated with OS and PFS, and if validated may predict for benefit from cisplatin therapy in patients with esophageal cancer.

The role of Bcl-x(L) protein in nucleotide excision repair-facilitated cell protection against cisplatin-induced apoptosis.

Many anticancer drugs target the genomic DNA of cancer cells by generating DNA damage and inducing apoptosis. DNA repair protects cells against DNA damage-induced apoptosis. Although the mechanisms of DNA repair and apoptosis have been extensively studied, the mechanism by which DNA repair prevents DNA damage-induced apoptosis is not fully understood. We studied the role of the antiapoptotic Bcl-x(L) protein in nucleotide excision repair (NER)-facilitated cell protection against cisplatin-induced apoptosis. Using both normal human fibroblasts (NF) and NER-defective xeroderma pigmentosum group A (XPA) and group G (XPG) fibroblasts, we demonstrated that a functional NER is required for cisplatin-induced transcription of the bcl-x(l) gene. The results obtained from our Western blots revealed that the cisplatin treatment led to an increase in the level of Bcl-x(L) protein in NF cells, but a decrease in the level of Bcl-x(L) protein in both XPA and XPG cells. The results of our immunofluorescence staining indicated that a functional NER pathway was required for cisplatin-induced translocation of NF-kappaB p65 from cytoplasm into nucleus, indicative of NF-kappaB activation. Given the important function of NF-kappaB in regulating transcription of the bcl-x(l) gene and the Bcl-x(L) protein in preventing apoptosis, these results suggest that NER may protect cells against cisplatin-induced apoptosis by activating NF-kappaB, which further induces transcription of the bcl-x(l) gene, resulting in an accumulation of Bcl-x(L) protein and activation of the cell survival pathway that leads to increased cell survival under cisplatin treatment.

The effects of HBx gene on the expression of DNA repair enzymes hOGG1 and hMYHα mRNA in HepG2 cells

To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHalpha and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the beta-actin as the interior control, real-time polymerase chain reaction (Real-time qPCR) was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHalpha in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector (HepG2/pDNA3.1). The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHalpha in the HepG2/HBx (0.021+/-0.007) was significantly lower than that of HepG2 (0.099+/-0.041) (P<0.05) and HepG2/pDNA3.1 (0.121+/-0.005) (P<0.05). However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains (P>0.05). The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 (P<0.05). It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHalpha mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.

Topical thymidine dinucleotide application protects against UVB-induced skin cancer in mice with DNA repair gene ( Ercc1)-deficient skin

Topical application of thymidine dinucleotides (pTpT) provides some protection against the effects of UV on the skin, however, many details of the protective mechanism have yet to be elucidated. We have used mice with an epidermis-specific knockout for the nucleotide excision repair gene, Ercc1, to investigate the mechanisms of protection. pTpT offered no protection against the pronounced UV-induced short-term erythema and skin thickening responses that are characteristic of DNA repair-deficient skin. It also had no effect on UV-induced apoptosis in Ercc1-deficient cultured keratinocytes. However, in these short-term experiments in both skin and keratinocyte culture pTpT did cause a slight reduction in proliferation. pTpT application during a chronic UV irradiation protocol provided some protection from UVB-induced skin carcinogenesis in epidermis-specific Ercc1 knockout mice. The median tumour free survival time was increased in the pTpT-treated group and treated animals had fewer tumours. In addition, pTpT-treated animals developed fewer large inwardly growing skin lesions than untreated animals. Furthermore, the proliferation response was reduced in chronically irradiated, non-lesional pTpT-treated skin. We conclude that cancer protection by pTpT in our mice is not modulated by an upregulation of DNA repair, as protection appears to be independent of a functional nucleotide excision repair pathway. We hypothesise instead that protection by pTpT is due to a reduction in epidermal proliferation.