UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts

Rodent UV-sensitive mutant cell lines of complementation groups 6 and 8 are the genetic counterparts of human Cockayne syndrome CS-B and CS-A, respectively. The original mutant in this group, UV61, was described as defective in cyclobutane pyrimidine dimer removal after high doses of UV. We have examined the responses of several cell lines from group 6 to low doses of UV irradiation, and find that these mutants have wild-type capacity for DNA repair as indicated by incision, cyclobutane pyrimidine dimer, and (6-4) photoproduct removal. ERCC6, the product of the gene defective in CS-B and group 6 mutants, is implicated in the regulation of repair of actively transcribed genes in Cockaynesyndrome; however, this protein clearly is not required for the processing of low levels of damage in CHO cells, which occurs remarkably efficiently, 40–50% of dimers being removed in both wild-type and group 6 mutants in 5 hours following 0.1 Jm^-2 of UV. The group 8 mutant cell line US31, on the other hand, is very deficient in repair of UV damage, showing a more extreme phenotype than is seen in the corresponding human syndrome CS-A. In both complementation groups, expression of mutations in a gene involved in regulation of DNA repair takes very different forms in human and rodent cells. Environ. Mol. Mutagen. 29:152–160, 1997. © 1997 Wiley-Liss, Inc.     

Expression of DNA repair and apoptosis genes in mitochondrial mutant and normal cells following exposure to ionizing radiation

In double‐strand DNA damage repair, nonhomologous end joining (NHEJ) is more error‐prone than homologous recombination repair (HRR), indicating that the relative prevalence of NHEJ may lead to more incorrect repair and thus to increases in chromosome damage. If DNA damage is extensive and cells are unable to repair that damage they typically undergo apoptosis. The mechanism(s) by which cells decide to switch from DNA repair to apoptosis is unknown. Since DNA repair and apoptosis are both energy‐demanding processes, the answer may involve ATP utilization. We used human mitochondrial mutant cell lines obtained from people with phenotypic manifestations of compromised ATP generation. We hypothesized that these cells may not have adequate capacity for dealing with the additional demands for ATP required for repairing DNA damage after genotoxic exposure, perhaps making the cells more prone to undergo apoptosis instead of initiating repair. This study describes changes in the expression of genes involved in NHEJ or HRR, as well as genes involved in apoptosis, in one normal and two mitochondrial mutant human cell lines following ionizing radiation exposure. Compared to normal cells, both mutant cell lines showed reduced expression of genes involved in NHEJ and HRR. Analysis of expression changes in genes involved in apoptosis revealed marked increases in expression in the mutants compared to normal cells. These results indicate that following ionizing radiation exposure, mitochondrial mutant cells have decreased levels of mRNA expression of DNA repair genes and increased expression levels of genes involved in apoptosis compared to normal cells. This study provides information that might be useful in characterizing energy dependent processes following exposure to stress or genotoxic agents. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.     

Development and characterization of CHO repair-proficient cell lines for comparative mutagenicity and metabolism of heterocyclic amines from cooked food

In order to understand the role of repair and metabolism in the mutagenicity of heterocyclic amines from cooked foods, we previously developed the nucleotide excision repair-deficient CHO 5P3NAT2 cell line engineered to coexpress the mouse CYP1A2 and human N-acetyltransferase genes. In the present study, we have made a matched repair-competent cell line by mutagenizing 5P3NAT2 cells with ethyl methanesulfonate and selecting for resistance to cytotoxicity by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In the differential cytotoxicity (DC) assay, 4 out of 15 clones showed no cytotoxic effect with IQ at the highest dose (30 microg/ml) tested, in contrast to repair-deficient 5P3NAT2 cells, which showed approximately 100% cytotoxicity at 0.3 microg/ml. Subsequently, these IQ-resistant clones were examined for resistance to killing by UV irradiation. All four IQ-resistant clones, which show resistance to UV similar to that of repair-proficient AA8 cells, still express both the CYP1A2 and N-acetyltransferase genes. Sequence analysis of CXPD cDNA from the 5P3NAT2R9 clone revealed an A:T-->G:C reversion event at the site of the UV5 mutation. This base change results in reversion of the codon 116 tyrosine in UV5 cells back to the original cysteine in AA8 cells, thereby restoring wild-type CXPD activity and repair function. In contrast to 5P3NAT2 cells, the repair-proficient 5P3NAT2R9 revertant cell line shows little IQ-induced cell killing, and dramatically lower levels of induced mutation at the adenine phosphoribosyltransferase (Aprt) gene locus over the range of 2-40 microg/ml IQ. This matched pair of repair-proficient/deficient cell lines can provide insight not only into the genotoxicity of heterocyclic amine dietary carcinogens such as IQ and PhIP, but also into the effects of nucleotide excision repair on the ultimate mutagenicity of these compounds.     

Detrimental effects of UV‐B radiation in a xeroderma pigmentosum‐variant cell line

DNA polymerase η (pol η), of the Y‐family, is well known for its in vitro DNA lesion bypass ability. The most well‐characterized lesion bypassed by this polymerase is the cyclobutane pyrimidine dimer (CPD) caused by ultraviolet (UV) light. Historically, cellular and whole‐animal models for this area of research have been conducted using UV‐C (λ = 100–280 nm) owing to its ability to generate large quantities of CPDs and also the more structurally distorting 6‐4 photoproduct. Although UV‐C is useful as a laboratory tool, exposure to these wavelengths is generally very low owing to being filtered by stratospheric ozone. We are interested in the more environmentally relevant wavelength range of UV‐B (λ = 280–315 nm) for its role in causing cytotoxicity and mutagenesis. We evaluated these endpoints in both a normal human fibroblast control line and a Xeroderma pigmentosum variant cell line in which the POLH gene contains a truncating point mutation, leading to a nonfunctional polymerase. We demonstrate that UV‐B has similar but less striking effects compared to UV‐C in both its cytotoxic and its mutagenic effects. Analysis of the mutation spectra after a single dose of UV‐B shows that a majority of mutations can be attributed to mutagenic bypass of dipyrimidine sequences. However, we do note additional types of mutations with UV‐B that are not previously reported after UV‐C exposure. We speculate that these differences are attributed to a change in the spectra of photoproduct lesions rather than other lesions caused by oxidative stress. Environ. Mol. Mutagen. 55:375–384, 2014. © 2014 Wiley Periodicals, Inc.     

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.     

Comparison of the mutagenic responses of mismatch repair-proficient (TK6) and mismatch repair-deficient (MT1) human lymphoblast cells to the food-borne carcinogen PhIP.

Heterocyclic amines are ubiquitously present in cooked meats and fish. They represent an important class of food-borne carcinogens. We describe the cytotoxic, apoptotic, and mutagenic responses of mismatch repair-proficient (TK6) and mismatch repair-deficient (MT1) human lymphoblastoid cells to PhIP, the most abundant heterocyclic amine. Dose-dependent increases in cytotoxicity, in apoptosis, and in mutant fractions at the hprt locus were observed following PhIP treatment. We present a statistical method that is useful for comparing two populations. With this method, we show that the data fitted a model that assumes that the PhIP-induced mutation rate is dependent on the cell line. Estimated rates of increase of 22.8 x 10(-6) and 2.2 x 10(-6) mutation per cell per microg PhIP were found in MT1 and TK6, respectively, showing that MT1 is hypermutable to PhIP. MT1 also exhibited lower PhIP-induced apoptosis. We conclude from these results that mismatch repair-deficient cells are hypermutable to the food-borne carcinogen PhIP and that the PhIP-DNA adducts, when not eliminated by apoptosis, can be transformed into mutations.     

Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts.

The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent. Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts. Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA. Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E. coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences. In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity. The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.     

Induction and repair of formaldehyde-induced DNA-protein crosslinks in repair-deficient human cell lines

We have previously shown that the alkaline Comet assay (single cell gel electrophoresis) in a modified version is a sensitive test for the detection of formaldehyde-induced DNA-protein crosslinks (DPC). Our results also indicated that formaldehyde-induced DPC are related to the formation of chromosomal effects such as micronuclei and sister chromatid exchanges. To better understand the genetic consequences of formaldehyde-induced DPC we have now investigated the induction and removal of DPC in relationship to the formation of micronuclei in normal and repair-deficient human cell lines. We did not find significant differences between normal cells, a xeroderma pigmentosum (XP) cell line and a Fanconi anaemia (FA) cell line with respect to the induction and removal of DPC. However, the induction of micronuclei was enhanced in both repair-deficient cell lines, particularly in XP cells, under the same treatment conditions. Comparative investigations with the DNA-DNA crosslinker mitomycin C (MMC) revealed a delayed removal of crosslinks and enhanced induction of micronuclei in both repair-deficient cell lines. FA cells were found to be particularly hypersensitive to micronucleus induction by MMC. In contrast to the results with formaldehyde, induction of micronuclei by MMC occurred at much lower concentrations than the effects in the Comet assay. Our results suggest that more than one repair pathway can be involved in the repair of crosslinks and that disturbed excision repair has more severe consequences with regard to the formation of chromosomal aberrations after formaldehyde treatment than has disturbed crosslink repair.     

DNA repair deficiencies and cellular senescence are unrelated in xeroderma pigmentosum cell lines

Senescence of skin fibroblast cultures from normal individuals occurred after 23.9 +/- 6.3 (S.D.) passages; senescence in DNA repair-deficient cell lines from xeroderma pigmentosum patients occurred at 22.9 +/- 5.5 passages. Cells from xeroderma pigmentosum variant and Cockayne syndrome patients reached senescence at similar passage numbers. Xeroderma pigmentosum patients contract skin cancer as a consequence of their repair deficiencies but show no symptoms of premature ageing; neither do their cells age prematurely in vitro. The clinical spectrum and the life-span of fibroblasts in culture therefore lend no support for a correlation between ageing and the DNA repair or DNA replication deficiencies found in xeroderma pigmentosum and Cockayne syndrome cells.     

Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines

The purpose of this study was to determine the radiosensitivities of 31 human oesophageal squamous cell carcinoma cell lines with a colony-formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. Ataxia-telangiectasia-mutated (ATM) gene in these cells had five missense mutations, and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non-hereditary diseased patients must be a good target for radiotherapy.     

Oxidative DNA damage and cytogenetic effects in flight engineers exposed to cosmic radiation

This study set out to analyze biomarkers for genotoxic events, e.g., oxidative DNA damage, chromosomal damage and hprt mutations, among flight personnel, who are known to be occupationally exposed to ionizing radiation of cosmic origin. Twenty-three flight engineers were recruited while ground personnel served as a matched control group. Cumulative radiation doses during flight were calculated on the basis of subjects' flight records assuming an exposure rate of 6 μSv per hour of flight. Oxidative DNA damage in peripheral lymphocytes from flight engineers appeared significantly increased in comparison with controls and was associated with cumulative exposure to cosmic radiation. Frequencies of peripheral lymphocyte chromosome aberrations, micronuclei and hprt mutations appeared also to be increased in flight engineers, but not significantly. It was also observed that DNA damage was higher in flight engineers with a relatively shorter flight history in comparison with flight engineers with higher cumulative exposures to radiation, suggesting adaptation to DNA damage caused by ionizing radiation. DNA repair activities measured as unscheduled DNA synthesis were clearly increased in the higher-exposed subgroup of flight engineers, and appeared significantly correlated with cumulative radiation dose, as well as inversely with oxidative DNA damage. The implications for cancer risk assessment in relation to exposure to cosmic radiation are discussed. Environ. Mol. Mutagen. 32:121–129, 1998 © Wiley-Liss, Inc.     

Isolation and characterization of yeast mutants with thermoconditional sensitivity to the bifunctional alkylating agent nitrogen mustard

Summary Selection of mutants of Saccharomyces cerevisiae sensitive to the DNA cross-linking agent nitrogen mustard (HN2) at two temperatures (23 °C and 36 °C) yielded two isolates with thermoconditionally enhanced (ts) sensitivity to the mutagen. Both were due to single recessive nuclear genes. Mutant allele snm1–2 ^ts showed mainly ts-sensitivity to HN2, whereas mutant allele snm2-1 ^ts conferred ts-sensitivity to HN2, half mustard (HN1) and UV. In temperature-shift experiments it was determined that the functions of SNM1 and SNM2 are needed for recovery within 6 to 7 h. after mutagen exposure during incubation at 23 °C on YEPD when HN2 and UV are applied. After HN1 treatment the SNM2 coded function is required for recovery for about 14 hrs. This possibly indicates a handling of UV- and HN2-induced lesions different from that of HN1-induced lesions.     

Interactions among genes controlling sensitivity to radiation (RAD) and to alkylation by nitrogen mustard (SNM) in yeast

Summary Three haploid yeast mutants (snm) sensitive or thermoconditionally sensitive to the DNA cross-linking agent nitrogen mustard (HN2) were crossed with four rad strains representing mutations in the three pathways of DNA dark repair. The resulting haploid double and triple mutant strains were tested for their sensitivity to UV, HN2 and HN1. From the observed epistatic or synergistic interactions of the combinations of mutant alleles we could derive the relation of the SNM1 and SNM2 genes to the postulated repair pathways. Alleles snm1-1 and snml-2 ^ts were found epistatic to genes of the rad3 group, whereas snm2-1 ^ts was epistatic to rad6. The snm1 and snm2 mutant alleles interacted synergistically. From these data it is concluded that the SNM1 gene product plays a cross-link specific role in excision repair while the SNM2 gene product may be involved in a system of error-prone repair.     

Effect of gamma rays on efficiency of gene transfer in DNA repair-proficient and -deficient cell lines

Ionizing radiation induces a number of molecular changes in cells, including DNA damage, mutation, genetic recombination, gene amplification, and chromosomal rearrangement. The studies described here make use of the process of DNA- mediated gene transfer to examine the molecular effects of ionizing radiation. Two Chinese hamster ovary cell lines, the wild-type, AA8-4, and a DNA repair-deficient line, EM9-1, that is sensitive to ionizing radiation, were transfected with the recombinant DNA plasmid, pSV2-GPT, either in the absence or presence of high- molecular-weight carrier DNA. Following transfection, cell populations were irradiated with graded doses of¹³⁷Cs - rays. Results demonstate that, on a per viable cell basis, ionizing radiation hinders the transfection of this plasmid when tested in the presence of carrier DNA. A similar dose response was seen for both the wild-type (AA8-4) and mutant (EM9-1) lines. However, in the absence of carrier DNA,¹³⁷Cs - rays clearly enhanced the gene transfer process. An enhancement factor of 3–5 was seen for AA8-4 cells and 2–3 for EM9-1 cells. This enhancement occurred at relatively low doses (e.g., 50 cGy) and was not substantially elevated by larger doses.     

Allelism between pso1-1 and rev3-1 mutants and between pso2-1 and snm1 mutants in Saccharomyces cerevisiaee

Summary In the yeast Saccharomyces cerevisiae, allelism between the psol-1 and the rev3-1 mutants on the one hand and the pso2-1 and snm1 mutants on the other, is demonstrated by the comparison of phenotypes, complementation tests and meiotic segregation analysis.     

Genotoxic effects of formaldehyde in the human lung cell line A549 and in primary human nasal epithelial cells

The alkaline comet assay was used to further characterize the induction of DNA-protein crosslinks (DPX) by formaldehyde (FA) and their removal in the human lung cell line A549 and in primary human nasal epithelial cells (HNEC). DPX were indirectly measured as the reduction of gamma ray-induced DNA migration. FA induced DPX in A549 cells in a concentration-related manner in the range of 100-300 microM. This result is in agreement with previous studies using different mammalian cell lines. The main new findings of the present study are: (i) Determination of cytotoxicity in relation to genotoxicity strongly depend on the method used. Cytotoxicity measured as the reduction in cell counts 48 hr after addition of FA to the cultures occurred parallel to the induction of DPX while colony forming ability was already reduced at 10 times lower FA concentrations; (ii) DPX induced by a 1-hr FA treatment were completely removed within 8 hr cultivation in fresh medium while in the presence of FA in the medium DPX levels remained unchanged for 24 hr; (iii) Induction and removal of DPX did not fundamentally differ between exponentially growing and confluent A549 cultures; (iv) Slowly proliferating HNEC showed the same sensitivity towards FA-induced DPX as A549 cells (i.e. the same FA concentrations induced DPX under the same experimental conditions) and removed DPX with a similar efficiency. In summary, these results contribute to a better understanding of the genotoxic activity of FA in vitro and indicate that the tested cultured primary and permanent human cells do not differ fundamentally with regard to the processing of FA-induced primary genotoxic effects.     

Intratumoral budding as a potential parameter of tumor progression in mismatch repair-proficient and mismatch repair-deficient colorectal cancer patients

In colorectal cancer, tumor budding at the invasive front (peritumoral budding) is an established prognostic parameter and decreased in mismatch repair–deficient tumors. In contrast, the clinical relevance of tumor budding within the tumor center (intratumoral budding) is not yet known. The aim of the study was to determine the correlation of intratumoral budding with peritumoral budding and mismatch repair status and the prognostic impact of intratumoral budding using 2 independent patient cohorts. Following pancytokeratin staining of whole-tissue sections and multiple-punch tissue microarrays, 2 independent cohorts (group 1: n = 289; group 2: n = 222) with known mismatch repair status were investigated for intratumoral budding and peritumoral budding. In group 1, intratumoral budding was strongly correlated to peritumoral budding (r = 0.64; P < .001) and less frequent in mismatch repair–deficient versus mismatch repair–proficient cases (P = .177). Sensitivity and specificity for lymph node positivity were 72.7% and 72.1%. In mismatch repair–proficient cancers, high-grade intratumoral budding was associated with right-sided location (P = .024), advanced T stage (P = .001) and N stage pN (P < .001), vascular invasion (P = .041), infiltrating tumor margin (P = .003), and shorter survival time (P = .014). In mismatch repair–deficient cancers, high intratumoral budding was linked to higher tumor grade (P = .004), vascular invasion (P = .009), infiltrating tumor margin (P = .005), and more unfavorable survival time (P = .09). These associations were confirmed in group 2. High-grade intratumoral budding was a poor prognostic factor in univariate (P < .001) and multivariable analyses (P = .019) adjusting for T stage, N stage distant metastasis, and adjuvant therapy. These preliminary results on 511 patients show that intratumoral budding is an independent prognostic factor, supporting the future investigation of intratumoral budding in larger series of both preoperative and postoperative rectal and colon cancer specimens.