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  • EXPRESSION  (17)
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  • 1
    Keywords: treatment ; ASSOCIATION ; polymorphism ; POLYMORPHISMS ; single nucleotide polymorphism ; SUSCEPTIBILITY ; VARIANTS ; SKIN ; mechanisms ; prevention ; HEALTH ; PROMOTER ; BREAST ; breast cancer ; BREAST-CANCER ; cancer prevention ; smoking ; SNP ; REPAIR ; WOMEN ; LYMPHOCYTES ; DAMAGE ; GENOTYPES ; cancer risk ; CANCER-PATIENTS ; INDIVIDUALS ; case-control studies ; DNA-DAMAGE ; CANCER PATIENTS ; SUSCEPTIBILITY GENE ; BODY ; RISK ; GENE ; ENZYMES ; DISEASE ; lung cancer ; LUNG-CANCER ; PATIENT ; MECHANISM ; DNA ; TUMORS ; validation ; DRUG ; RNA ; GENES ; THERAPY ; VITRO ; LUNG ; COMBINATION ; CANCER ; EXPRESSION ; IN-VITRO ; CELLS ; CELL ; tumor ; AGENTS ; radiotherapy ; NSCLC ; CANCER-RISK ; cancer research ; RNA EXPRESSION ; ENZYME ; case control studies ; analysis ; GENOTYPE ; PROFILES ; single-nucleotide ; development ; PROMOTER POLYMORPHISM ; XRCC1 ; VARIANT ; WEIGHT ; SINGLE NUCLEOTIDE POLYMORPHISMS ; SNPs ; case-control study ; GEMCITABINE ; CAPACITY ; DEFICIENCY ; small cell lung cancer ; AGENT ; SINGLE ; DNA repair ; MPO ; APE1
    Abstract: Cells in the body are permanently attacked by DNA-reactive species, both from intracellular and environmental sources. Inherited and acquired deficiencies in host defense mechanisms against DNA damage (metabolic and DNA repair enzymes) can modify cancer susceptibility as well as therapy response. Genetic profiles should help to identify high-risk individuals who subsequently can be enrolled in preventive measures or treated by tailored therapy regimens. Some of our attempts to define such risk profiles are presented. Cancer susceptibility: Single nucleotide polymorphisms (SNPs) in metabolic and repair genes were investigated in a hospital-based lung cancer case-control study. When evaluating the risk associated with different genotypes for N-acetyltransferases (Wikman et al. 2001) and glutathione-S-transferases (Risch et al. 2001), it is mandatory to distinguish between the three major histological subtypes of lung tumors. A promoter polymorphism of the myeloperoxidase gene MPO was shown to decrease lung cancer susceptibility mainly in small cell lung cancer (SCLC) (Dally et al. 2002). The CYP3A4*1B allele was also linked to an increased SCLC risk and in smoking women increased the risk of lung cancer eightfold (Dally et al. 2003b). Polymorphisms in DNA repair genes were shown to modulate lung cancer risk in smokers, and reduced DNA repair capacity elevated the disease risk (Rajaee-Behbahani et al. 2001). Investigations of several DNA repair gene variants revealed that lung cancer risk was only moderately affected by a single variant but was enhanced up to approximately threefold by specific risk allele combinations (Popanda et al. 2004). Therapy response: Inter-individual differences in therapy response are consistently observed with cancer chemotherapeutic agents. Initial results from ongoing studies showed that certain polymorphisms in drug transporter genes (ABCB1) differentially affect response outcome in histological subgroups of lung cancer. Stronger beneficial effects were seen in non-small cell lung cancer (NSCLC) patients following gemcitabine and in SCLC patients following etoposide-based treatment. Several DNA repair parameters (polymorphisms, RNA expression, and DNA repair capacity) were measured in vitro in lymphocytes of patients before radiotherapy and correlated with the occurrence of acute side effects (radio-hypersensitivity). Our initial analysis of several repair gene variants in breast cancer patients (n = 446) who received radiotherapy revealed no association of single polymorphisms and the development of side effects (moist desquamation of the irradiated normal skin). The risk for this side effect was, however, strongly reduced in normal weight women carrying a combination of XRCC1 399Gln and APE1 148Glu alleles, indicating that these variants afford some protection against radio-hypersensitivity (Chang-Claude et al. 2005). Based on these data we conclude that specific metabolic and DNA repair gene variants can affect cancer risk and therapy outcome. Predisposition to hereditary cancer syndromes is dominated by the strong effects of some high-penetrance tumor susceptibility genes, while predisposition to sporadic cancer is influenced by the combination of multiple low-penetrance genes, of which as a major challenge, many disease-relevant combinations remain to be identified. Before translating these findings into clinical use and application for public health measures, large population-based studies and validation of the results will be required.
    Type of Publication: Book chapter
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  • 2
    Keywords: CANCER ; EXPRESSION ; carcinoma ; CELL ; Germany ; human ; LUNG ; lung cancer ; LUNG-CANCER ; EXPOSURE ; RISK ; GENE ; PROTEIN ; METABOLISM ; TISSUE ; PATIENT ; RISK-FACTORS ; FREQUENCY ; polymorphism ; SUSCEPTIBILITY ; PROMOTER ; OVARIAN-CANCER ; WOMEN ; MEN ; risk factors ; smoking ; PROSTATE-CANCER ; cancer risk ; RISK FACTOR ; CYP3A4 ; LINKAGE DISEQUILIBRIUM ; CANCER-PATIENTS ; CARCINOMAS ; POLYMERASE-CHAIN-REACTION ; adenocarcinoma ; ADENOCARCINOMAS ; CARRIERS ; case-control studies ; CLINICAL PRESENTATION ; CYP3A,genetic polymorphism,lung cancer susceptibility,small cell lung cancer,LightCycler ; EXPRESSED HUMAN CYTOCHROME-P450S ; GENETIC VARIANT ; HUMAN LIVER-MICROSOMES ; PROSTATE TUMORS ; PROTEIN LEVELS ; squamous cell carcinoma ; TOBACCO
    Abstract: CYP3A isozymes are involved in tobacco carcinogen- and steroid-metabolism, and are expressed in human lung tissue showing interindividual variation in expression and activity. The CYP3A4* 1 B allele has been associated with a two-fold higher promoter activity and with high-grade prostate cancers. The very frequent intron 3 polymorphism in the CYP3A5 gene (CYP3A5*3) results in decreased CYP3A5 protein levels. A case-control study was conducted in 801 Caucasian lung cancer patients that included 330 adenocarcinomas, 260 squamous cell carcinomas, 171 small cell lung cancers (SCLC) and 432 Caucasian hospital-based controls. CYP3A-genotyping was performed by capillary polymerase chain reaction followed by fluorescence-based melting curve analysis. A significantly increased SCLC risk for CYP3A4* 1B allele carriers [odds ratio (OR) 2.25, 95% confidence interval (CI) 1.11-4.55, P = 0.02] was found. After dividing cases and controls by gender, an increased lung cancer risk for CYP3A4* 1B carriers (OR 3.04, 95% CI 0.94-9.90, P= 0.06) for women but not for men (OR 1.00, 95% CI 0.56-1.81) was revealed. Heavier smoking men (greater than or equal to 20 pack-years) with the CYP3A4* 1 B allele had a significant OR for lung cancer of 3.42 (95% CI 1.65-7.14, P= 0.001) compared to * 1A/1* 1A carriers with lower tobacco exposure (〈 20 pack-years). For women, the respective OR was 8.00 (95% CI 2.12-30.30, P = 0.005). Genotype frequencies were generally in Hardy-Weinberg equilibrium, except for CYP3A5 where a greater than expected number of CYP3A5* 1 homozygotes was observed among cases (P = 0.006). In addition, we observed linkage disequilibrium of CYP3A4 and CYP3A5 (P 〈 0.00001), but a nonsignificantly increased lung cancer risk was only found for homozygous CYP3A5* 1 allele carriers (OR 5.24,95% CI 0.85-102.28, P = 0.14) but not for heterozygotes. To confirm our observation that the CYP3A4* 1B allele increases SCLC risk and modifies the smoking-related lung cancer risk in a gender-specific manner, further studies, including CYP3A haplotype analysis, will be necessary. Pharmacogenetics 13:607-618 (C) 2003 Lippincott Williams Wilkins
    Type of Publication: Journal article published
    PubMed ID: 14515059
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  • 3
    Keywords: CANCER ; EXPRESSION ; carcinoma ; CELL ; Germany ; LUNG ; COMMON ; lung cancer ; LUNG-CANCER ; EXPOSURE ; RISK ; GENE ; GENES ; HYBRIDIZATION ; DNA ; MECHANISM ; primary ; RISK-FACTORS ; mechanisms ; ASSOCIATION ; polymorphism ; POLYMORPHISMS ; SUSCEPTIBILITY ; NO ; AMPLIFICATION ; AGE ; DNA-REPAIR ; REPAIR ; CIGARETTE-SMOKING ; risk factors ; smoking ; PCR ; cancer risk ; DAMAGE ; RISK FACTOR ; REGION ; CARCINOGENS ; adenocarcinoma ; case-control studies ; squamous cell carcinoma ; INDIVIDUALS ; CANCER-RESEARCH ; SMOKERS ; NUCLEOTIDE EXCISION-REPAIR ; CELL CARCINOMA ; case control study ; case-control study ; REGRESSION ; OCCUPATIONAL-EXPOSURE ; CARCINOGEN ; HEAVY ; LUNG ADENOCARCINOMA ; PIGMENTOSUM GROUP-A
    Abstract: Polymorphisms of genes coding for DNA repair can affect lung cancer risk. A common single nucleotide (-4) G-to-A polymorphism was identified previously in the 5' untranslated region of the XPA gene. In a case-control study in European Caucasians, the influence of this polymorphism on primary lung cancer risk overall and according to histologic subtypes was investigated. Four hundred sixty-three lung cancer cases (including 204 adenocarcinoma and 212 squamous cell carcinoma) and 460 tumor-free hospital controls were investigated using PCR amplification and melting point analysis of sequence-specific hybridization probes. Odds ratios (OR) were calculated by multiple logistic regression analysis adjusting for age, gender, smoking habits, and occupational exposure and showed a slightly enhanced risk for all lung cancer cases as well as for squamous cell carcinoma and adenocarcinoma cases. Gene-environment interactions were analyzed with respect to smoking and occupational exposure. A nearly 3-fold increased risk for adenocarcinoma associated with the XPA AA genotype was observed for occupationally exposed individuals (OR, 2.95; 95% confidence interval, 1.42-6.14) and for heavy smokers (OR, 2.52; 95% confidence interval, 1.17-5.42). No genotype-dependent increase in OR was found for nonexposed individuals or those smoking 〈20 pack-years. The significant effect of the XPA polymorphism in heavy smokers and occupationally exposed individuals suggests an important gene-environment interaction for the XPA gene. The underlying mechanisms as to why AA homozygotes are predisposed to lung adenocarcinoma and which specific carcinogens are involved remains to be determined
    Type of Publication: Journal article published
    PubMed ID: 15598786
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  • 4
    Keywords: CANCER ; CELLS ; EXPRESSION ; TUMORS ; COMPLEX ; prognosis ; RETINOIC ACID RECEPTORS
    Abstract: Impairment of endogenous differentiation pathways like retinoic acid (RA) signaling seems to be a central pathogenetic event in astrocytic gliomas. Among others, expression of the differentiation-promoting RA chaperon protein cellular retinoic acid binding protein 2 (CRABP2) is extenuated in high-grade gliomas. Against this background, we aimed at identifying potential pathomechanisms underlying reduced CRABP2 expression in these tumors. Employing MassARRAY methylation analysis we detected extensive CpG methylation upstream of the CRABP2 gene locus in a study sample comprising 100 astrocytic gliomas of WHO grade II to IV. Compared to non-tumorous control samples tumors revealed increased CpG methylation and methylation levels were inversely correlated to CRABP2 mRNA expression. Substantiating our in situ findings, CRABP2 mRNA levels increased in glioma cell lines after exposure to the demethylating agent 5-aza-2'-deoxycytidine. Finally, a distinct CpG methylation signature distinguished between primary glioblastoma on the one hand and the group of astrocytoma WHO II-III and secondary glioblastoma on the other hand. Altogether, our observations suggest that epigenetic silencing of CRABP2 might contribute to an immature phenotype in glioma cells.
    Type of Publication: Journal article published
    PubMed ID: 22275178
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  • 5
    Keywords: EXPRESSION ; IONIZING-RADIATION ; SURVIVAL ; tumor ; DIAGNOSIS ; FOLLOW-UP ; COHORT ; GLUTATHIONE ; METALLOTHIONEIN ; GENOME-WIDE ASSOCIATION
    Abstract: We assessed whether variants in 22 oxidative stress-related genes are associated with mortality of breast cancer patients and whether the associations differ according to radiotherapy. Using a prospective cohort of 1348 postmenopausal breast cancer patients, we estimated hazard ratios (HR) and 95% confidence intervals (CI) for 109 single nucleotide polymorphisms (SNPs) using Cox proportional hazards regression. Validation of results was attempted using two Scandinavian studies. Eleven SNPs in MT2A, NFE2L2, NQO1, PRDX1, and PRDX6 were significantly associated with overall mortality after a median follow-up of 5.7 years. Three SNPs in NQO1 (rs2917667) and in PRDX6 (rs7314, rs4916362) were consistently associated with increased risk of dying across all three study populations (pooled: HRNQO1_rs2917667 1.20, 95% CI 1.00-1.44, p = 0.051; HRPRDX6_rs7314 1.16, 95% CI 1.00-1.35, p = 0.056, HRPRDX6_rs4916362 1.14 95% CI 1.00-1.32, p = 0.062). Potential effect modification by radiotherapy was found for CAT_rs769218. In conclusion, genetic variants in NQO1 and PRDX6 may modify breast cancer prognosis.
    Type of Publication: Journal article published
    PubMed ID: 23489758
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  • 6
    Keywords: ANGIOGENESIS ; CELLS ; EXPRESSION ; ENDOTHELIAL GROWTH-FACTOR ; INHIBITION ; POPULATION ; PROTEIN ; mechanisms ; DOWN-REGULATION ; HYPERMETHYLATION ; MALIGNANT GLIOMAS ; astrocytoma ; SUBTYPES ; PROMOTER METHYLATION ; GLIOBLASTOMA ; AKAP12 ; Gravin ; SSeCKS ; SSECKS/GRAVIN/AKAP12
    Abstract: The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY (R) of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12 alpha promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12 alpha promoter methylation and markedly increased AKAP12 alpha mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined
    Type of Publication: Journal article published
    PubMed ID: 24042196
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  • 7
    Keywords: brain ; CELL ; DOWN-REGULATION ; REPRESSION ; ASTROCYTIC GLIOMAS
    Abstract: OBJECTIVES: Measuring concentrations of the differentiation-promoting hormone retinoic acid (RA) in glioblastoma tissues would help to understand the reason why RA treatment has been inefficient in clinical trials involving brain tumor patients. Here, we apply a recently established extraction and measurement protocol to screen glioblastoma tissues for the levels of the RA precursor retinol and biologically active RA. Combining this approach with mRNA analyses of 26 tumors and 8 normal brains, we identify a multifaceted disturbance of RA synthesis in glioblastoma, involving multiple aldehyde dehydrogenase 1 family and retinol dehydrogenase enzymes. Through database studies and methylation analyses, we narrow down chromosomal deletions and aberrant promoter hypermethylation as potential mechanisms accounting for these alterations. Employing chromatin immunoprecipitation analyses and cell-culture studies, we further show that chromatin at RA target genes is poised to RA substitution, but most glioblastoma cell cultures are completely resistant to RA treatment. This paradoxical RA response is unrelated to alternative RA signaling through the fatty acid-binding protein 5/peroxisome proliferator-activated receptor delta axis. Our data suggest a multifaceted disturbance of RA synthesis in glioblastoma and contribute to reconsider current RA treatment strategies. GLIA 2015;63:1850-1859.
    Type of Publication: Journal article published
    PubMed ID: 25944104
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  • 8
    Keywords: CANCER ; CELLS ; EXPRESSION ; IRRADIATION ; radiotherapy ; CELL ; Germany ; THERAPY ; TOXICITY ; COHORT ; RISK ; SURGERY ; radiation ; PATIENT ; DNA ; INDEX ; QUALITY ; SKIN ; treatment ; BREAST ; breast cancer ; BREAST-CANCER ; LESIONS ; RADIATION-THERAPY ; ASSAY ; WOMEN ; DNA-REPAIR ; REPAIR ; COMET ASSAY ; DAMAGE ; LYMPHOCYTES ; BEAM ; DNA-DAMAGE ; PARAMETERS ; CANCER-PATIENTS ; KINETICS ; body mass index ; DNA repair ; DNA repair capacity ; PERIPHERAL-BLOOD LYMPHOCYTES ; ATAXIA-TELANGIECTASIA ; HETEROZYGOTES ; INTRINSIC RADIOSENSITIVITY ; radiation tolerance,DNA repair capacity,breast neoplasms,body mass index
    Abstract: Background and purpose: Intrinsic and extrinsic factors can affect the occurrence of side effects of radiotherapy. The influence of therapy modalities, personal characteristics and individual DNA repair capacity on the risk of acute skin toxicity was thus evaluated.Materials and methods: In a prospective study of 478 female breast cancer patients receiving adjuvant radiotherapy of the breast after breast-conserving surgery, acute skin toxicity was documented systematically using a modified version of the common toxicity criteria. Prognostic personal and treatment characteristics were identified for the entire cohort. Individual DNA repair capacity was determined in a subgroup of 113 patients with alkaline comet assay using phytohemagglutinin stimulated lymphocytes. Using proportional hazards analysis to account for cumulative biologically effective radiation dose, the hazard for the development of acute skin reactions (moist desquamation) associated with DNA repair capacity was modeled.Results: Of the 478 participants, 84 presented with acute reactions by the end of treatment. Higher body mass index was significantly associated with an increased risk for acute reactions (hazard ratio = 1.09 per 1 kg/m(2)), adjusted for treating hospital and photon beam quality. The comet assay parameters examined, including background DNA damage in non-irradiated cells, DNA damage induced by 5 Gy, and DNA repair capacity, were not significantly associated with risk of acute skin toxicity.Conclusions: Higher BMI is predictive of acute skin toxicity, however, individual repair parameters as determined by the alkaline comet assay are not informative enough. More comprehensive analyses including late effects of radiotherapy and repair kinetics optimized for different radiation-induced DNA lesions are warranted. (C) 2003 Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 14643951
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  • 9
    Keywords: APOPTOSIS ; CANCER ; tumor ; carcinoma ; CELL ; Germany ; LUNG ; lung cancer ; LUNG-CANCER ; SYSTEM ; EXPOSURE ; RISK ; GENE ; GENES ; PATIENT ; RISK-FACTORS ; cell cycle ; CELL-CYCLE ; CYCLE ; ASSOCIATION ; polymorphism ; POLYMORPHISMS ; SUSCEPTIBILITY ; VARIANTS ; BREAST-CANCER ; risk factors ; smoking ; p53 ; cancer risk ; MUTATIONS ; TRANSFORMATION ; SQUAMOUS-CELL CARCINOMA ; adenocarcinoma ; case-control studies ; squamous cell carcinoma ; GASTRIC-CANCER ; DNA repair ; DNA-REPAIR GENES ; molecular epidemiology ; ONCOLOGY ; case-control study ; REGRESSION ; RE ; TUMOR-SUPPRESSOR ; VARIANT ; INCREASE ; CARCINOGEN ; case control studies ; analysis ; SUPPRESSOR ; GENOTYPE ; adenocarcinoma of the lung ; HISTOLOGY ; RISK-FACTOR ; CANCER-RISK ; PROLINE ; SQUAMOUS-CELL ; INCREASES ; cell cycle control ; CODON-72 ; P53 POLYMORPHISM
    Abstract: Alterations in cell cycle regulation and apoptosis leading to malignant transformation could be caused by common genetic variants in tumor suppressor genes. The effects of the TP53 polymorphism Arg72Pro on lung cancer risk have been investigated in numerous studies with, however, conflicting results. In many studies, important risk modifiers such as smoking or tumor histology were not taken into account. We therefore investigated the combined effects of polymorphisms in TP53 (Arg72Pro) and p21/CDKN1A (Ser31Arg) and smoking on lung cancer risk. Our case-control study consisted of 405 patients with lung cancer, mainly squamous-cell carcinoma (185) and adenocarcinoma (177) and 404 unmatched tumor-free hospital controls. Multivariate regression analysis showed a moderate but statistically significant risk of lung cancer overall. and especially of squamous-cell carcinoma (OR, 1.65; CI, 1.10-2.47) for TP53 72Pro allele carriers. The risk was markedly increased in heavy smokers (〉 20 pack-years) with squamous-cell carcinoma (OR, 2.80 in patients homozygous for 72Pro; CI, 1.19-6.58), but not in tight smokers (〈= 20 pack-years). The results for the p21 Ser31Arg polymorphism suggested that 31Ser is a moderate-risk allele for squamous-cell carcinoma. Analysis of the combined effects of the two polymorphisms revealed a higher OR for TP53 72Pro carriers homozygous for p21 31Ser than for 72Pro carriers in general; this effect being most pronounced in heavy smokers with squamous-cell carcinoma (OR, 3.84; CI, 1.46-10.1). Our data indicate that the TP53 Arg72Pro polymorphism increases the risk for squamous-cell carcinoma mainly in heavy smokers. The observed interaction with smoking is biologically plausible as, for the 72Pro p53 variant, decreased apoptosis and extended G1 cell cycle arrest is reported after carcinogen exposure. Nevertheless, confirmation by further molecular and epidemiological studies is warranted. (c) 2006 Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17059853
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  • 10
    Keywords: CELLS ; IN-VITRO ; IRRADIATION ; CELL ; evaluation ; Germany ; human ; GENE-EXPRESSION ; radiation ; TIME ; RESPONSES ; DNA ; CARCINOGENESIS ; INDUCTION ; KERATINOCYTES ; SKIN ; fibroblasts ; gene expression ; ASSAY ; REPAIR ; skin carcinogenesis ; genetics ; COMET ASSAY ; DAMAGE ; DNA-DAMAGE ; MAMMALIAN-CELLS ; Jun ; MORPHOLOGY ; NETHERLANDS ; ELECTROPHORESIS ; sensitivity ; heredity ; HaCaT ; PERIPHERAL-BLOOD LYMPHOCYTES ; RE ; PATTERN ; CELL-VIABILITY ; keratinocyte ; SKIN KERATINOCYTES ; INCREASE ; LEVEL ; DNA damage ; TESTS ; UV-RADIATION ; E ; STRAND BREAKS ; POLARITY ; TOXICOLOGY ; microbiology ; biotechnology ; German ; modification ; adherent HaCaT keratinocytes ; alkaline comet assay ; dose-dependence of DNA-damage induction ; HAMSTER V79 CELLS ; REPAIR CAPACITY ; trypsin effect
    Abstract: The comet assay is one of the well-accepted tests to measure radiation-induced DNA damage. The most commonly used protocols require single-cell suspensions that are embedded in agarose in order to perform electrophoresis. For adherently growing cells such as human HaCaT skin keratinocytes this method bears several problems. We show that trypsinization required for maintaining singlecell suspensions is prolonged after UV radiation and thereby reduces cell viability and allows partial repair, with the consequence of reduced damage detection after irradiation. Therefore, we here introduce a modified version of the comet assay where HaCaT cells are seeded onto comet slides 24 h before the assay and overlaid with agarose immediately after irradiation. Using this modification we are now able to reproducibly measure high DNA-damage levels (13-fold increase compared with controls) following irradiation with 60 J/cm 2 UVA as well as a dose-dependent increase of DNA damage after 10, 20 and 60 J/cm 2 UVA. Thus, by maintaining the cells in their natural configuration, i.e. adherently growing, we exclude several artefacts that are likely to influence the damage responses. These include: (i) trypsinization-dependent changes in cell morphology and polarity (clear lateral, i.e. adherent, and apical side of keratinocytes) which are likely of consequence for the gene-expression pattern, (h) trypsin- and dislodgement-induced damage reducing cell viability, and (iii) the time delay between damage induction and damage evaluation to unpredictable results due to partial repair. Since these advantages pertain to all adherently growing cells, this improved protocol is not restricted to HaCaT cells but offers great potential also with all non-haematopoietic cells for obtaining accurate results and for studying repair processes in a highly reproducible manner. (c) 2007 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17509930
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