Host Cell Reactivation Research Articles

The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells

Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome.

Xeroderma pigmentosum complementation group G patient with a novel homozygous missense mutation and no neurological abnormalities

We describe an unusual xeroderma pigmentosum (XP) patient with a mutation in XP complementation group G, representing only the third reported Japanese XP-G patient. A 40-year-old men (XP3HM), born from consanguineous parents experienced sun sensitivity and pigmentary changes of sun-exposed skin since childhood. He developed a squamous cell carcinoma on his lower lip at the age of 40. He has neither neurological abnormalities nor Cockayne syndrome. The primary fibroblasts of the patient were hypersensitive to killing by UV (D(0) = 0.6 J/m(2)) and the post-UV unscheduled DNA synthesis was 8% of normal. Host cell reactivation complementation analysis implicated XP complementation group G. We identified a novel homozygous mutation (c.194T>C) in a conserved portion of the XPG(ERCC5) gene, resulting in a predicted amino acid change; p.L65P. We confirmed that this genetic change reduced DNA repair thus linking this mutation to increased skin cancer.

Decreased repair of singlet oxygen-induced DNA damage in xeroderma pigmentosum group A cells determined by plasmid host cell reactivation

Decreased repair of singlet oxygen-induced DNA damage in xeroderma pigmentosum group A cells determined by plasmid host cell reactivation

Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea

Nucleus pulposus intervertebral disc cells are routinely confronted with high osmolality in their microenvironment and respond to this stress in vitro by regulating cell cycle progression and by activating a DNA repair machinery in order to counteract its genotoxic effect. In the present study, we attempted to identify the origin of this osmo-regulatory response, by using an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea. High salt and sorbitol were found to activate similar molecular pathways, including the p38 MAPK and the p53-p21(WAF1)-pRb axis, that were not stimulated by high urea. On the other hand, only high urea led to the phosphorylation of ERKs and JNKs. Furthermore, salt- and sorbitol-treated cells were able to phosphorylate histone H2A.X on Ser139, in contrast to cells exposed to urea, indicating a common mechanism for DNA repair, which was achieved by a p53-dependent activation of the G1 checkpoint by both solutes. DNA repair, as directly measured by a host cell reactivation assay, occurred under conditions of hyperosmolar salt and sorbitol, although to a lesser extent in sorbitol-treated cells than in cells exposed to high salinity. Taken as a whole, our findings suggest that the hyperosmolality-provoked DNA damage and the responses of nucleus pulposus cells induced by this genotoxic stress most probably originate from cell volume alterations mediated by hypertonicity and not from increased intracellular ionic concentration.

DNA Repair Capacity in Peripheral Lymphocytes Predicts Survival of Patients With Non–Small-Cell Lung Cancer Treated With First-Line Platinum-Based Chemotherapy

Platinum-based regimens are the standard chemotherapy for patients with advanced non-small-cell lung cancer (NSCLC). DNA repair capacity (DRC) in tumor cells plays an important role in resistance to platinum-based drugs. We have previously reported that efficient DRC, as assessed by an in vitro lymphocyte-based assay, was a determinant of poor survival in patients with NSCLC in a relatively small data set. In this larger independent study of 591 patients with NSCLC, we further evaluated whether DRC in peripheral lymphocytes predicts survival of patients with NSCLC who receive platinum-based chemotherapy. All patients were recruited at The University of Texas MD Anderson Cancer Center and donated blood samples before the start of any chemotherapy. We measured DRC in cultured T lymphocytes by using the host-cell reactivation assay, and we assessed associations between DRC in peripheral lymphocytes and survival of patients with NSCLC who were treated with first-line platinum-based chemotherapy. We found an inverse association between DRC in peripheral lymphocytes and patient survival. Compared with patients in the low tertile of DRC, patients with NSCLC in the high tertile of DRC had significantly worse overall and 3-year survival (adjusted hazard ratio [HR], 1.33; 95% CI, 1.04 to 1.71; P = .023; and HR, 1.35; 95% CI, 1.04 to 1.76; P = .025, respectively). This trend was more pronounced in patients with early-stage tumors, adenocarcinoma, or squamous cell carcinoma. We confirmed that DRC in peripheral lymphocytes is an independent predictor of survival for patients with NSCLC treated with platinum-based chemotherapy.

Histone deacetylase inhibitor sodium butyrate enhances cellular radiosensitivity by inhibiting both DNA nonhomologous end joining and homologous recombination

HDAC inhibitors have been proposed as radiosensitizers in cancer therapy. Their application would permit the use of lower radiation doses and would reduce the adverse effects of the treatment. However, the molecular mechanisms of their action remain unclear. In the present article, we have studied the radiosensitizing effect of sodium butyrate on HeLa cells. FACS analysis showed that it did not abrogate the γ-radiation imposed G2 cell cycle arrest. The dynamics of γ-H2AX foci disappearance in the presence and in the absence of butyrate, however, demonstrated that butyrate inhibited DSB repair. In an attempt to clarify which one of the two major DSBs repair pathways was affected, we synchronized HeLa cells in G1 phase and after γ-irradiation followed the repair of the DSBs by agarose gel electrophoresis. Since HR is not operational during G1 phase, by this approach we determined the rates of NHEJ only. The results showed that NHEJ decreased in the presence of butyrate. In another set of experiments, we followed the dynamics of disappearance of RAD51 foci in the presence and in the absence of butyrate after γ-radiation of HeLa cells. Since RAD51 takes part in HR only, this experiment allows the effect of butyrate on DSB repair by homologous recombination to be assessed. It showed that HR was also obstructed by butyrate. These results were confirmed by host cell reactivation assays in which the repair of plasmids containing a single DSB by NHEJ or HR was monitored. We suggest that after a DSB is formed, HDACs deacetylated core histones in the vicinity of the breaks in order to compact the chromatin structure and prevent the broken DNA ends from moving apart from each other, thus ensuring effective repair.

Effect of eIF3a on response of lung cancer patients to platinum-based chemotherapy by regulating DNA repair.

The purpose of this study is to test the hypothesis that eIF3a may regulate the expression of DNA repair proteins which, in turn, affects response of lung cancer patients to treatments by DNA-damaging anticancer drugs. Immunohistochemistry was used to determine the expression of eIF3a in 211 human lung cancer tissues followed by association analysis of eIF3a expression with patient's response to platinum-based chemotherapy. Ectopic overexpression and RNA interference knockdown of eIF3a were carried out in NIH3T3 and H1299 cell lines, respectively, to determine the effect of altered eIF3a expression on cellular response to cisplatin, doxorubicine, etoposide (VP-16), vincristine, and vinblastine by using MTT assay. The DNA repair capacity of these cells was evaluated by using host-cell reactivation assay. Real-time reverse transcriptase PCR and Western Blot analyses were carried out to determine the effect of eIF3a on the DNA repair genes by using cells with altered eIF3a expression. eIF3a expression associates with response of lung cancer patients to platinum-based chemotherapy. eIF3a knockdown or overexpression, respectively, increased and decreased the cellular resistance to cisplatin and anthrocycline anticancer drugs, DNA repair activity, and expression of DNA repair proteins. eIF3a plays an important role in regulating the expression of DNA repair proteins which, in turn, contributes to cellular response to DNA-damaging anticancer drugs and patients' response to platinum-based chemotherapy.

A combination of MSH2 DNA mismatch repair deficiency and expression of the SV40 large T antigen results in cisplatin resistance of mouse embryonic fibroblasts

Mutations in the human mismatch repair (MMR) genes are associated with hereditary non-polyposis colorectal cancer as well as other sporadic cancers. MMR gene mutations have been implicated in the resistance of human tumours to cisplatin and several tumour-derived MMR-deficient cells show cisplatin resistance in vitro. In addition, hypoxia, a common feature of the tumour microenvironment, has been shown to influence tumour responses to conventional cancer treatments. We have examined the role of the mMSH2 MMR protein on repair of cisplatin-damaged DNA and cisplatin sensitivity in mMSH2-deficient murine fibroblasts and mMSH2-proficient controls under conditions of normoxia and hypoxia. Sensitivity to cisplatin was measured using the MTT assay and clonogenic survival. Repair of cisplatin-damaged DNA was measured using a host cell reactivation (HCR) assay employing a non-replicating recombinant virus expressing the β-galactosidase reporter gene. Sensitivity to cisplatin was significantly less and HCR of the cisplatin-damaged reporter gene was significantly greater in SV40-transformed mMSH2-deficient cells (MS5-7) compared to mMSH2-proficient controls (BC1-6) under both normoxic and hypoxic conditions. In contrast, sensitivity to cisplatin was significantly greater and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts under both normoxic and hypoxic conditions. Sensitivity to cisplatin was also significantly greater and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts transfected with a control plasmid under both normoxic and hypoxic conditions. In contrast, sensitivity to cisplatin was less and HCR was similar in primary mMSH2-deficient compared to mMSH2-proficient murine fibroblasts transfected with a plasmid expressing SV40 large T antigen under both normoxic and hypoxic conditions. These results suggest that loss of MMR alone does not result in increased resistance to cisplatin in murine fibroblasts and that additional concomitant alterations in cells expressing the SV40 large T antigen are responsible for cisplatin resistance through a modulation of DNA repair capacity and/or apoptosis.

Delay of Gap Filling during Nucleotide Excision Repair by Base Excision Repair: The Concept of Competition Exemplified by the Effect of Propolis

Nucleotide excision repair (NER) consists of a sequence of events including DNA damage recognition, excision of the damage containing oligonucleotide, gap filling, and ligation. We found that gap filling during the repair of ultraviolet (UV)C-induced DNA lesions was inhibited by various compounds, e.g., amoxicillin, and mixtures, e.g., propolis, the materials that could induce oxidative DNA damage in serum-supplemented cell cultures. Such inhibitory effect was also demonstrated by the immunostaining experiment and host cell reactivation assay. In this study, we link the repair of oxidative DNA damage with the inhibition of gap filling. Our experimental evidence includes the following: (1) induction of oxidative DNA damage and inhibition of gap filling were quantitatively correlated; (2) although the repair of UV-induced DNA damage was delayed in the presence of propolis, the repair of propolis-induced oxidative DNA damage proceeded regardless of preexposure to UV radiation; (3) inhibition of gap filling by propolis was absent in base excision repair (BER)-deficient cells; (4) suppression of propolis-induced oxidative DNA damage by β-carotene abolished the inhibition of gap filling; and (5) inhibition of gap filling was also found with typical BER-inducing agents such as hydrogen peroxide, menadione, and methyl methanesulfonate. We propose that competition may occur between NER and BER, which results in delay of gap filling. Our study reveals the dominancy of BER over NER.

A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes

The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20–24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose–response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the relative DRC measurement in this cohort of patients. The results indicate that our method is highly sensitive, reliable and reproducible, and thus, it suitable for population-based studies to quantify in vitro DNA-repair deficiencies.

Abstract 1206: HER2/neu positive receptor status is associated with a low DNA repair capacity in women with breast cancer

Abstract Hormone receptor status are now routinely used for therapy selection in breast cancer (BC). Estrogen receptor status is routinely use for the selection of tamoxifen adjuvant therapy after surgery. Substantial evidence now indicates that patients with BC tumors that have overexpression of the HER2/neu receptor have generally a poor prognosis. Approximately 15-20 percent of BC patients have an amplification of the HER2/neu gene. Overexpression of HER2/neu is associated with increased BC recurrence and worse prognosis. Because of its prognostic role as well as its ability to predict response to trastuzumab (Herceptin), breast tumors are routinely checked for overexpression of HER2/neu. Strategies targeting DNA repair defects for the treatment of patients with triple-negative and BRCA mutation-associated BC have received much attention recently, however the relationship between HER2/neu receptor status and the phenotypic expression of DNA repair capacity (DRC) measured in lymphocytes has never been previously examined. DRC is an important risk factor for several types of cancer including BC. In this study, we examined the association between BC and DRC and the status of estrogen, progesterone and HER2/neu receptors. Our main objective was to determine if levels of DRC measured in lymphocytes using a host cell reactivation assay with a luciferase reporter gene are associated with receptor status. The estrogen, progesterone and HER2/neu receptor status, DRC levels, and other selected covariates were compared among 125 Hispanic women with histopathologically confirmed BC. After variable transformation, the mean DRC was compared between positive and negative HER2/neu BC patients using the t-test and the Wilcoxon's test to assess the statistical significance of the mean DRC difference. Crude and multiple logistic regression adjusted odds ratios (OR) were used as measures of association when compared HER2/neu (+) with (−) BC patients. The two-sided Wald test was used to assess the adjusted OR statistical significance. BC women with a HER2/neu (+) (n=14) had 4.1 times more odds of having a low DRC (< 1.78) compared with those that were HER2/neu (−) (n=65): (OR = 4.3 (95% CI 1.1, 16.6) p = 0.047. The average DRC in controls without BC was 6.10% (n=560). Women with BC that were HER2/neu (+) had an average DRC of 1.6% while BC patients that were HER2/neu (−) had 2.4%, this difference was significant (p=0.008). No significant differences were evident in regards to the association between DRC and the status of estrogen and progesterone receptors (double negatives) nor with triple negative receptor status (estrogen, progesterone and HER2/neu).These results indicate that low DRC could, at least partially explain, why HER2/neu (+) tumors are more aggressive. Supported by the NCI Diversity Training Branch of the Center to Reduce Cancer Health Disparities through the MBRS SCORE Program (Grant SO6 GM008239-23). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1206. doi:10.1158/1538-7445.AM2011-1206

Abstract 4657: Identification and validation of genetic determinants of DNA repair phenotype in genome-wide association study of lung cancer

Abstract DNA repair is essential in protecting the integrity of the genome. There is considerable inter-individual variation in DNA repair capacity (DRC). We have reported that cellular DRC (measured in lymphocytes using the host-cell reactivation assay with a reporter gene chloramphenicol acetyltransferase [CAT] damaged by an activated tobacco carcinogen, benzo(a)pyrene diol epoxide [BPDE]) modulates susceptibility to lung cancer. To identify genetic polymorphisms predictive of the repair phenotype, we used phenotype and genotype data in a population of 1,474 European ancestry individuals from a Texas lung cancer genome-wide association case-control study using Illumina 317K platform (317498 SNPs). This discovery phase was followed by replication testing of the top 20 SNPs in an independent set of an additional 1,364 European ancestry cases and controls, using Illumina Bead Array Platform. In addition, we performed a replication analysis of all SNPs with P < 0.01 in 145 DNA repair-related genes in BER, NER, MMR, DSBR pathways from the discovery set, and applying a conservative Bonferroni-corrected P -value of 2.37E-5 for the joint P -value, based on the total number of analyzed SNPs in DNA repair genes. The DRC data were divided into quartiles by assigning the top quarter of participants (with highest values of DRC) as the DNA repair proficient group and the bottom quarter of participants with lowest values of DRC as the suboptimal DRC group to enhance the contrast. We used a generalized linear model with SNPs as predictors and DRC as the outcome. The covariates age, gender, smoking status, duration of smoking, number of cigarettes per day, DRC assay-related variables including the sample storage time, baseline reporter gene CAT expression level, blastogenic rate, and the case-control status of the study participants were adjusted in the model. We identified several suggestive loci contributing to the DRC phenotype: rs9390123 in PHACTR2 (phosphatase and actin regulator 2, P = 4.78E-05 in the discovery set and P = 1.08E-05 in the joint analysis); rs4756897 in USH1C (Usher syndrome 1C, P = 5.40E-05 in the discovery set and 8.71E-06 in the joint analysis); rs6785626 in CNTN4 (contactin 4, P = 0.000451 in the discovery set and P = 1.55E-05 in the joint analysis); rs13181 in ERCC2 (excision repair cross-complementing rodent repair deficiency-complementation group 2, joint P = 2.09E-05); rs7443927 in DUSP1 (dual specificity phosphatase 1, joint P =5.04E-05) and rs10878361 in HMGA2 (high mobility group AT-hook 2, joint P =2.54E-4). In conclusion, we identified and replicated a number of SNPs associated with DRC. These genetic determinants of DNA repair phenotype are easier and cheaper to measure, and subject to less misclassification than the phenotype and may be effective biomarkers for risk assessment. (This study was supported by NIH grants CA127219, CA055769, ES011740, CA131274 and CA016672) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4657. doi:10.1158/1538-7445.AM2011-4657

Cyclosporin A, but not everolimus, inhibits DNA repair mediated by calcineurin: implications for tumorigenesis under immunosuppression

Unlike other immunosuppressive drugs including everolimus, cyclosporin A causes a dramatic increase of UV-induced skin cancer, a feature that is reminiscent of xeroderma pigmentosum (XP), where defective nucleotide excision repair (NER) of UV-induced DNA damage results in cutaneous carcinogenesis. The molecular basis of the clinically important differential activities of cyclosporin A and everolimus is still unclear. We measured post-UV cell survival of cyclosporin A- and everolimus-treated human fibroblasts and lymphoblasts using a cell proliferation assay (MTT). The cellular NER capacity was assessed by host cell reactivation. Using an ELISA and specific antibodies, cyclobutane pyrimidine and pyrimidine-6,4-pyrimidone photoproduct removal from the cellular genome was measured. The effect of calcineurin on NER was investigated using a calcineurin A expression vector and specific RNAi. Cyclosporin A led to a dose dependent decrease in post-UV cell survival, inhibited NER and blocked photoproduct removal. In contrast, none of these effects where seen in everolimus-treated cells. Overexpression of calcineurin A resulted in increased NER and complemented the Cyclosporin A-induced reduction of NER. Downregulation of calcineurin using RNAi inhibited NER comparable to cyclosporin A-treatment. We conclude that cyclosporin A, but not everolimus, leads to an increased skin cancer risk via a calcineurin signalling-dependent impairment of NER.

Early host cell reactivation of an oxidatively damaged adenovirus-encoded reporter gene requires the Cockayne syndrome proteins CSA and CSB.

Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for normal reactivation.

Prospective analysis of DNA damage and repair markers of lung cancer risk from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial

Mutagen challenge and DNA repair assays have been used in case-control studies for nearly three decades to assess human cancer risk. The findings still engender controversy because blood was drawn after cancer diagnosis so the results may be biased, a type called 'reverse causation'. We therefore used Epstein-Barr virus-transformed lymphoblastoid cell lines established from prospectively collected peripheral blood samples to evaluate lung cancer risk in relation to three DNA repair assays: alkaline Comet assay, host cell reactivation (HCR) assay with the mutagen benzo[a]pyrene diol epoxide and the bleomycin mutagen sensitivity assay. Cases (n = 117) were diagnosed with lung cancer between 0.3 and 6 years after blood collection and controls (n = 117) were frequency matched on calendar year and age at blood collection, gender and smoking history; all races were included. Case and control status was unknown to laboratory investigators. In unconditional logistic regression analyses, statistically significantly increased lung cancer odds ratios (OR(adjusted)) were observed for bleomycin mutagen sensitivity as quartiles of chromatid breaks/cell [relative to the lowest quartile, OR = 1.2, 95% confidence interval (CI): 0.5-2.5; OR = 1.4, 95% CI: 0.7-3.1; OR = 2.1, 95% CI: 1.0-4.4, respectively, P(trend) = 0.04]. The magnitude of the association between the bleomycin assay and lung cancer risk was modest compared with those reported in previous lung cancer studies but was strengthened when we included only incident cases diagnosed more than a year after blood collection (P(trend) = 0.02), supporting the notion the assay may be a measure of cancer susceptibility. The Comet and HCR assays were unrelated to lung cancer risk.

Abstract A90: Nonmelanoma skin cancer epidemiology and DNA repair in Puerto Ricans

Abstract Introduction: Nonmelanoma skin cancers (NMSC) including basal cell and squamous cell carcinomas are the most common human cancers. DNA damage can be caused by exposure to solar UV light and other types of radiation, dietary factors, and ageing. The relationship between DNA damage and oncogenesis suggests that diminished DNA repair capacity (DRC) is involved in tumorigenesis. Purpose: The purpose of this study was to identify and assess selected exposures and DRC as predictors for NMSC in Puerto Rico. Methods: An incident-cases case-control study was conducted. A total of 477 NMSC cases and 365 controls were compared regarding DRC levels and other selected factors. Participants with low and high DRC levels were also compared regarding the same factors as in NMSC. DRC was measured using a host-cell reactivation assay with a luciferase reporter gene irradiated with UV light and transfected into human peripheral lymphocytes from participant's blood samples. A standardized epidemiological questionnaire was used to collect data from the participants. The crude and multiple logistic regression adjusted odds ratio was used to assess the associations between NMSC or DRC levels with selected factors after adjusting for age, gender, skin, hair, and eye color, and other potential confounders simultaneously. Results: Exposures increasing the odds for NMSC were being male, aging, light skin, presence of freckles, light hair, light eye color, increased sun exposure, severe insolations, family history of NMSC, and low DRC (p < 0.05). Protective factors found in the study included using sun block, taking aspirin regularly, and multivitamin supplements intake (p < 0.05). Factors associated with low DRC level were aging, light skin, sun exposure, severe insolations and not using sun block regularly. Discussion: The present study identified major epidemiologic factors for NMSC in Puerto Rico that are consistent with similar investigations elsewhere. Low DRC was strongly associated with NMSC (Matta et al, 2003). New unpublished data include the finding of the protective effect of multivitamin supplementation. Light skin, increased sun exposure, severe insolations and not using sun block regularly were associated with low DRC. Conclusions: The protective effect of multivitamins deserves further consideration to confirm our findings. Persons with light skin, increased sun exposure and not using sun block regularly had low DRC levels and increased odds for NMSC. DRC measurements might be used to monitor the potential preventive impact of multivitamins and sun block when used as preventive therapy. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A90.

Differential repair of polycyclic aromatic hydrocarbon DNA adducts from an actively transcribed gene

Polycyclic aromatic hydrocarbons (PAHs) are carcinogens with varying potencies. These compounds are metabolized to diol epoxides that react to form DNA adducts. Nucleotide excision repair is a critical cellular defense against these bulky DNA adducts which, if not repaired, can lead to mutations and the initiation of cancer. The structural features of the PAH-adducts play a role in differential repair of these adducts by the global genomic repair subpathway of nucleotide excision repair. DNA adducts derived from the PAHs containing bay-regions are repaired more rapidly than adducts derived from PAHs containing fjord-regions. We have employed the host cell reactivation assay to examine the rate of repair of these adducts in an actively transcribing gene. The pGL3 plasmid containing a luciferase gene was damaged with diol epoxides of benzo[ a]pyrene (B[ a]P-DE), dibenzo[ a,l]pyrene (DB[ a,l]P-DE), benzo[g]chrysene (B[g]Ch-DE), and benzo[ c]phenanthrene (B[ c]Ph-DE). The plasmids were transfected into B-lymphocytes with normal repair capacity as well as lymphocytes derived from patients with the XP-A, XP-C and CS-B syndromes. We found that XPA cells were able to transcribe slowly past B[g]Ch-adducts but not the other PAHs. Using the amount of luciferase produced as a measure of DNA repair, we found that the relative rates of repair in the actively transcribing luciferase gene was B[ a]P-DE > DB[ a,l]P-DE, B[ g]Ch-DE, >B[ c]Ph-DE in repair proficient and XP-C cells. These results indicate that the abilities to transcribe past and to repair the PAH adducts are dependent on different structural features of the DNA adducts.

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

BackgroundThe Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances.A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products) and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing).ResultsIn this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP) and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis.The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts.ConclusionsThe methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity of different cell types can be compared with each other. The described assay is also highly flexible, and the activity of other repair mechanisms can be determined using modifications of this method.

Helicase-like transcription factor confers radiation resistance in cervical cancer through enhancing the DNA damage repair capacity

Helicase-like transcription factor (HLTF) is a member of the SWI/SNF (mating type switching/sucrose non-fermenting) family of ATPases/helicases and also has a RING-finger motif characteristic of ubiquitin ligase proteins. These features have led to suggestions that HLTF functions like yeast Rad5, which promotes replication through DNA lesions via a post-replication repair pathway. However, the function of HLTF in higher eukaryotes is still unknown. Herein, we found the overexpression of HLTF in radiation recurrent human uterine cervical carcinoma tissues when compared to disease free survived patients tissues. In this study, we used RNA interference techniques to investigate the potential function of HLTF in cervical cancer cell line HeLa and found that the cell proliferation was reduced by knockdown (KD) of HLTF. A host-cell reactivation assay showed that the capacity for repair to DNA damage induced by X-ray irradiation was reduced in HLTF KD cells. X-rays also increased apoptosis in HLTF KD cells. These results suggest that HLTF is involved in DNA repair and apoptosis in cancer cells, which might represent a target for gene therapies of human cancer.

Feasibility of a prospective host cell reactivation assay (HCRA) in non-small cell lung cancer (NSCLC) patients.

10586 Background: DNA repair capacity (DRC)—the ability to repair BPDE-induced DNA adducts—measured in peripheral blood lymphocytes (PBLs) by the HCRA has been shown to correlate with survival in patients (p) treated with cisplatin-based chemotherapy. p with high DRC had significantly shorter survival (Bosken et al. JNCI 2002). However, the HCRA of DRC has not been tested prospectively. Methods: Cryopreserved PBLs from 41 NSCLC p were cultured for 96 hours under phytohaemagglutinin stimulation. The pmaxGFP plasmid was nucleoporated in the cells of the host under two different experimental conditions: (i) undamaged (GFP-UND) and (ii) 254nm UV light-damaged plasmid DNA (1,000 J/m2) (GFP-DAM). After transfection, the cells were incubated for 24 hours in culture media to allow, firstly, repair of UV-damaged plasmids, and secondly, reexpression of the GFP reporter protein. By flow cytometry, the dead cells (positively stained with propidium iodide) were excluded and DRC was calculated in alive cells as follows: percentage DRC = (pmaxGFP-DAM/pmaxGFP-UND) * 100, where pmaxGFP-DAM and pmaxGFP-UND correspond to the percentage of alive cells positive for GFP protein. Results: There were no differences in DRC according to gender, season of the year or histology. Median DRC was 8.77 for p with PS 0, 10.49 for p with PS 1, and 21.60 for p with PS 2 (p=0.009). p treated with chemotherapy, with or without surgery and/or radiotherapy, had a higher relative risk (RR) of death associated with increased DRC (p=0.07) (Table). Conclusions: HCRA measured in PBLs is feasible and can be useful in monitoring NSCLC p. Serial measurements to detect increments in DRC can provide a marker of acquired resistance to cisplatin-based chemotherapy. The correlation of DRC with the expression of DNA repair genes in the tumor should be investigated. Cox proportional hazards model of the RR of death associated with increased DRC Unadjusted RR (95%CI) 0.82 (0.66-1.02) p=0.07 Adjusted* RR (95%CI) (for age, sex, cigarettes/day, stage) 0.81 (0.65-1.02) p=0.07 No significant financial relationships to disclose.