Induction and repair of DNA damage in normal and ataxiatelangiectasia skin fibroblasts treated with neocarzinostatin

Cells from patients with the hereditary multisystem disorderataxia-telangiectasia (A-T) are hypersensitive to the cytotoxicaction of DNA-breaking agents, such as X-rays, bleomycin andneocarzinostalin (NCS). A defect in the repair of a certainDNA lesion induced by all three agents may underlie this hypersensitivity.This DNA lesion may be a certain type of DNA strand break. Mostof the previous experiments done with X-rays and bleomycin failedto show any retardation in the rejoining of DNA strand breaksin A-T cells. However, since both A-T homozygous and heterozygouscells are particularly hypersensitive to NCS, we studied thetime course of strand breakage induction and repair in A-T skinfibroblast strains treated with NCS, using the sensitive methodof alkaline or neutral elution. A linear dose response was obtainedfor the induction by NCS of single-strand breaks and double-strandbreaks. A-T cells did not respond with a higher initial extentof strand breakage compared with normal cells. NCS is an appropriateagent for studying the kinetics of rejoining strand breaks,due to its rapid action in the cells; this action, which iscompleted within 2–4 min, was studied by monitoring strandbreak induction, inhibition of DNA synthesis and decrease incellular survival. The time course of strand break rejoiningfound after NCS treatment was very similar to that found followingX-irradiation: with both single- and double-strand breaks, arapid phase of rejoining was first noticed (t 5 min for single-strandbreaks and 20–25 min for double-strand breaks). This wasfollowed by a second, slow phase that continued for severalhours. No difference could be detected between normal and A-Tcells either with regard to the time course of rejoining orthe fraction of non-rejoined breaks remaining several hoursafter treatment.     

Abnormal response of ataxia-telangiectasia cells to agents that break the deoxyribose moiety of DNA via a targeted free radical mechanism

A defect in DNA repair coupled to anomalous DNA synthesis afterinduction of certain radiogenic DNA damage is suspected to underliethe radiosensitivity of cells from patients with ataxia-telangiectasia(A-T). The response of cultured skin fibroblasts from A-T patientsand A-T heterozygotes to six agents inducing various levelsof DNA strand breakage by different mechanisms was studied toobtain further information on the nature of the ‘A-T criticalDNA lesion’. The A-T cells showed varying degrees of hypersensitivityto the cytotoxic action of the quinone-containing anti-tumorantibiotics streptonigrin and adriamycin and to hydrogen peroxide.This hypersensitivity was accompanied by reduced inhibitionof DNA synthesis compared to normal cells after treatment withthese agents. A limited degree of cellular hypersensitivitythat was not sufficient to allow for definition of a separatesensitivity range was shown by A-T heterozygous cells. On theother hand, the A-T cells showed a normal response to paraquat,saframycin A and ellipticine. Taken together with previous resultsshowing hypersensitivity of A-T cells to ionizing radiation,bleomycin and neocarzinostatin, these data indicate that thecritical DNA lesion in A-T cells is a strand break caused bydeoxyribose destruction following the action of free radicalstargeted into the DNA.     

Deficiency in the repair of UV-induced DNA damage in human skin fibroblasts compromised for the ATM gene

Ataxia-telangiectasia (A-T), is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. A-T cells exhibit a greatly decreased survival and a reduction in DNA synthesis inhibition as well as p53 induction in response to ionizing radiation. Occasionally, some strains of A-T cells have been reported to manifest a slightly enhanced sensitivity with no consistent observations of a deficiency in either cell cycle control or the repair of DNA damage after treatment with ultraviolet (UV) light. In the present study it is shown that skin fibroblasts from four A-T patients, compared with the control, display enhanced sensitivity to the killing effect of UV-light, moderate radioresistant DNA synthesis, and a reduction in viral recovery in the host cell reactivation (HCR) assay. PCR based analysis indicated that three of these UV-sensitive A-T cell strains bear a large deletion in the ATM gene, and no ATM polypeptide was detected in their cell free extracts. Moreover, it is shown that, in non-replicative conditions, these A-T cells are less efficient than normal cells in repairing the T4 endonuclease V sensitive sites. These results constitute the first clear evidence showing the deficiency of A-T cells in the repair of UV-induced DNA damage, and provide further information on the relationship between cell cycle control and DNA repair in human cells.     

Imparied glutathione biosynthesis in cultured human ataxia-telangiectasia cells

Cell lines established from donors with the inherited disorder ataxia-telangiectasia (A-T) exhibit exceptional sensitivity to ionizing radiation and chemicals known to produce increased levels of intracellular H2O2, suggesting a deficiency in glutathione-dependent detoxication reactions. Glutathione (GSH) biosynthesis in fibroblast and lymphoblast cultures derived from individuals known to be clinically unaffected, homozygous, or heterozygous for A-T was assessed. Following GSH depletion by diethylmaleate, fibroblasts (GM 3492) from a clinically unaffected individual resynthesized GSH at a rate approximately twice that observed in fibroblasts from known heterozygotes (GM 3488 and GM 3489). Unrelated A-T homozygote fibroblast lines GM 3487B and GM 5823 resynthesized GSH only very slowly. GM 3492 cells repleted intracellular GSH by 6 h after depletion, the heterozygote lines by 18 h. The A-T homozygotes replaced only 30% of the intracellular GSH pool by 24 h. A lymphoblast cell line from the A-T homozygote (GM 3189) also exhibited slow resynthesis after depletion. However, if these cells were permeabilized by treatment with digitonin, GSH synthesis proceeded at a rate exceeding synthesis in permeabilized or untreated normal lymphoblasts (GM 3323). The first enzyme in GSH synthesis, gamma-glutamylcysteine synthetase, was found to be elevated about 2.7-fold in A-T homozygote fibroblasts, suggesting that a substrate for GSH synthesis may be rate limiting. A-T homozygote lymphoblasts contained about 2-fold more gamma-cystathionase activity over other cell lines tested suggesting increased flux through the transsulfuration pathway for cysteine production in response to reduced cysteine supply. Transport of cysteine and cystine was found to be 8- and 5-fold slower in A-T homozygotes that did not affect fibroblasts while glutamate and methionine transport Vmax did not differ among the cell lines tested. These experiments demonstrate that cells from A-T homozygotes are deficient in cysteine transport, thus limiting GSH resynthesis after a depleting challenge such as radiation or GSH-depleting xenobiotic compounds.     

Evidence of DNA repair/processing defects in cultured skin fibroblasts from breast cancer patients

Cultured skin fibroblasts from 14 breast cancer (BC) patients were compared with those from 8 healthy subjects and 4 ataxia-telangiectasia (A-T) cases for sensitivity to low dose-rate (0.007 Gy/min) gamma-irradiation assessed by a colony-forming assay and for postirradiation DNA synthesis inhibition determined by the method of [(3)H]thymidine incorporation. Fibroblasts from all but two BC patients exhibited moderately enhanced radiosensitivity in the colony-forming assay, occupying an intermediate position between the controls and the A-T cases. Fibroblasts from the radiosensitive BC patients also showed an intermediate response with respect to radio-induced DNA synthesis inhibition compared with those from controls and A-T cases. In a host cell reactivation assay using an irradiated herpes simplex virus for plaque-forming ability, the fibroblasts from 7 BC patients, used as host cells, resulted in a significantly reduced (P < 0.0001) recovery of the virus relative to the 8 control fibroblasts, suggesting a deficiency in DNA repair in the former. A number of the BC fibroblasts analyzed in an assay for potentially lethal damage repair confirmed the repair deficiency in the fibroblasts from the BC patients. Defects in DNA repair and/or DNA processing after exposure to genotoxic agents would lead to genomic instability and hence would be responsible for cancer predisposition. Our data suggest that most BC patients may carry various genes resulting in such defects, and additional studies on normal cells from a larger cohort of BC patients and their family members are warranted to establish a connection between mutations or polymorphisms in specific DNA repair genes and susceptibility to breast cancer.     

Hypersensitivity to cell killing and faulty repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in human (ataxia-telangiectasia) fibroblasts treated with 4-nitroquinoline 1-oxide

Dermal fibroblast strains from ataxia-telangiectasia (A-T) patients and clinically normal subjects were exposed to 4-nitroquinoline 1-oxide (4NQO) or its 3-methyl derivative (3me4NQO), and their colony-forming abilities and DNA metabolic properties were compared. Three A-T strains, i.e., AT2BE and AT3BI representing genetic complementation group AB and AT4BI belonging to group C, displayed enhanced (2.4- to 2.8-fold) sensitivity to reproductive inactivation by 4NQO, but exhibited normal survival in response to 3me4NQO. The initial induction and subsequent enzymatic repair of alkali-labile lesions (i.e., damaged sites converted to single-strand breaks in alkali) were quantitated by conventional velocity sedimentation analysis of cellular DNA in alkaline sucrose gradients. On exposure to concentrations of each chemical that produced comparable amounts of DNA damage, A-T and normal cells removed alkali-labile lesions at similar rates. However, the three 4NQO-sensitive A-T strains appeared to be defective in acting on alkali-stable adducts (formed by the parent compound but not its derivative), as judged by strand-break accumulation during posttreatment incubation with 1-beta-D-arabinofuranosylcytosine (araC). Specifically, the number of araC-detectable sites repaired in these A-T strains during the critical 2-h period immediately following 4NQO treatment ranged from 40 to 60% of that processed by normal controls. AT5BI, a fourth A-T strain assigned to complementation group D, responded normally to 4NQO-induced cytotoxicity and removed both alkali-labile and alkali-stable (araC-detectable) lesions normally. We thus conclude that the hypersensitivity of AT2BE, AT3BI, and AT4BI strains to 4NQO may be attributed, at least in part, to faulty execution of the excision-repair process operative on alkali-stable 4NQO adducts.     

ATM-dependent activation of the gene encoding MAP kinase phosphatase 5 by radiomimetic DNA damage

Cellular responses to DNA damage are mediated by an extensive network of signaling pathways. The ATM protein kinase is a master regulator of the response to double-strand breaks (DSBs), the most cytotoxic DNA lesion caused by ionizing radiation. ATM is the protein missing or inactive in patients with the pleiotropic genetic disorder ataxia-telangiectasia (A-T). A major response to DNA damage is altered expression of numerous genes. While studying gene expression in control and A-T cells following treatment with the radiomimetic chemical neocarzinostatin (NCS), we identified an expressed sequence tag that represented a gene that was induced by DSBs in an ATM-dependent manner. The corresponding cDNA encoded a dual specificity phosphatase of the MAP kinase phosphatase family, MKP-5. MKP-5 dephosphorylates and inactivates the stress-activated MAP kinases JNK and p38. The phosphorylation-dephosphorylation cycle of JNK and p38 by NCS was attenuated in A-T cells. Thus, ATM modulates this cycle in response to DSBs. These results further highlight ATM as a link between the DNA damage response and major signaling pathways involved in proliferative and apoptotic processes.     

Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells

The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.     

Hyperthermia enhances X ray cell killing in normal and homozygous and heterozygous ataxia telangiectasia human cells

Two homozygous and heterozygous ataxia telangiectasia (AT) cell strains were compared to normal human cells for heat and radiation responses. The thermal sensitivities to one hour heating at 42.0 degrees C were comparable in the five cell strains. Thermal enhancement of radiosensitivity occurred in both homo- and hetero-zygous AT cell strains and was comparable to the thermal enhancement observed in the normal cell strain. Normal cells recovered during incubation at 37.0 degrees C between the two treatments of heat and X rays while homozygous AT cells showed no recovery. The heterozygous cell strains showed only a slight sequence dependence for heat and radiation sensitivity.     

Detection of ataxia telangiectasia heterozygous cell lines by postirradiation cumulative labeling index: measurements with coded samples

Coded skin fibroblast cell strains from ataxia telangiectasia (AT) families and apparently normal individuals were obtained from two different sources. AT homozygous strains were clearly identified on the basis of marked hypersensitivity to cell killing by X-irradiation. AT heterozygotes were intermediate in their cytotoxic response between AT homozygotes and five normal reference cell strains. When density-inhibited cultures were X-irradiated and immediately subcultured to low density, a large fraction of AT heterozygous cells were irreversibly blocked in G1 as determined by cumulative labeling indices following incubation with [3H]thymidine. No such block occurred in four reference normal or AT homozygous strains. Three coded cell strains from apparently normal individuals resembled AT heterozygotes in their response; two of these strains were heterozygous for lysosomal storage disease. Thus, although the phenotype associated with the cellular response of AT heterozygous cells to X-irradiation is not specific to this disorder, the cumulative labeling indices assay may be a useful method for the detection of AT heterozygotes in kindreds with known AT.     

Ubiquitination capabilities in response to neocarzinostatin and H(2)O(2) stress in cell lines from patients with ataxia-telangiectasia

The human genetic disorder ataxia-telangiectasia (A-T) is due to lack of functional ATM, a protein kinase which is involved in cellular responses to DNA double strand breaks (DSBs) and possibly other oxidative stresses, as well as in regulation of several fundamental cellular functions. Studies regarding responses in A-T cells to the induction of DSBs utilize ionizing radiation or radiomimetic chemicals, such as neocarzinostatin (NCS), which induce DNA DSBs. This critical DNA lesion activates many defense systems, such as the cell cycle checkpoints. The cell cycle is also regulated through a timed and coordinated degradation of regulatory proteins via the ubiquitin pathway. Our recent studies indicate that the ubiquitin pathway is influenced by the cellular redox status and that it is the major cellular pathway for removal of oxidized proteins. Accordingly, we hypothesized that the absence of a functional ATM protein might involve perturbations to the ubiquitin pathway as well. We show here that upon treatment with NCS, there was a transient 50-70% increase in endogenous ubiquitin conjugates in A-T and wt lymphoblastoid cells. Ubiquitin conjugation capabilities per se and levels of substrates for conjugation were also similarly enhanced in wt and A-T cells upon NCS treatment. We also compared the ubiquitination response in A-T and wt cells using H(2)O(2) as the stress, in view of preexisting evidence of the effects of H(2)O(2) on ubiquitination capabilities in other types of cells. As with NCS treatment, there was an approximately 45% increase in endogenous ubiquitin conjugates by 2-4 h after exposure to H(2)O(2). Both cell types showed a rapid 50-150% increase in de novo formed 125I-ubiquitin conjugates. As compared with wt cells, unexposed A-T cells had higher endogenous levels of conjugates and enhanced conjugation capability. However, A-T cells mounted a more muted ubiquitination response to the stress. The enhanced ubiquitin conjugation in unstressed A-T cells and attenuated ability of these cells to respond to stress are consistent with the A-T cells being under oxidative stress and with their having an 'aged' phenotype. The indication that ubiquitin conjugate levels and ubiquitin conjugation capabilities are enhanced upon oxidative stress without significant changes in GSSG/GSH ratios indicates that assays of ubiquitination provide a sensitive measure of cellular stress. The data also add support to the impression that potentiated ubiquitination response to mild oxidative stress is a generalizable phenomenon.     

ATM as a target for novel radiosensitizers

DNA damage checkpoints are complex signal transduction pathways that are critical for normal cellular recovery following potentially lethal genotoxic insults. The ataxia-telangiectasia mutated (ATM) protein kinase is a critical component in these pathways and integrates the cellular response to damage by phosphorylating key proteins involved in cell cycle regulation and DNA repair. Lack of normal ATM function in the inherited ataxia-telangiectasia (A-T) syndrome results in a pleiotropic clinical syndrome characterized by a marked increased risk of cancer and profound hypersensitivity to ionizing radiation. Cells derived from patients with A-T share some of these attributes with genomic instability, loss of normal cell cycle arrest pathways, defects in DNA repair and increased radiation sensitivity. The radiosensitivity of A-T cells suggests that pharmacological inhibitors of the ATM kinase should be effective radiosensitizing agents. In fact, caffeine inhibits ATM kinase activity at concentrations that result in an A-T-like phenotype with loss of cell cycle checkpoints and hypersensitivity to ionizing radiation. Although the clinical use of caffeine as a radiosensitizer is limited by potentially lethal systemic toxicities, more potent methyl xanthines may selectively inhibit the ATM pathway at clinically achievable levels. Interestingly, caffeine and other methyl xanthines preferentially radiosensitize cells that lack normal p53 function. Because p53 is commonly inactivated in epithelial malignancies, this suggests that small molecule inhibitors of ATM might selectively sensitize the majority of tumors to the lethal effects of ionizing radiation while sparing normal tissues.     

DNA damage repair and genetic polymorphisms: assessment of individual sensitivity and repair capacity

PURPOSE: To study the repair capacity after X-ray irradiation in human peripheral blood cells of healthy subjects, in relation to their genotypes. METHODS AND MATERIALS: The peripheral blood of 50 healthy subjects was irradiated in vitro with 2 Gy of X rays and the induced DNA damage was measured by Comet assay immediately after irradiation. DNA repair was detected by analyzing the cells at defined time intervals after the exposure. Furthermore, all subjects were genotyped for XRCC1, OGG1, and XPC genes. RESULTS: After X-ray irradiation, persons bearing XRCC1 homozygous variant (codon 399) genotype exhibited significantly lower Tail DNA values than those bearing wild-type and heterozygous genotypes. These results are also confirmed at 30 and 60 min after irradiation. Furthermore, XPC heterozygous subjects (variant codon 939) showed lower residual DNA damage 60 min after irradiation compared with wild-type and homozygous genotypes. CONCLUSION: The results of the present study show that polymorphisms in DNA repair genes could influence individual DNA repair capacity.     

Abnormal response of xeroderma pigmentosum cells to bleomycin

The repair of bleomycin-damaged DNA was examined in human fibroblasts isolated from patients having the disease xeroderma pigmentosum (XP). In normal fibroblasts, the appearance of low-molecular-weight DNA was observed in the presence of increasing amounts of the drug. The studies in XP fibroblasts produced results which differed from those obtained in normal cells in two ways. (a) Prelabeled XP cells from most complementation groups contained more low-molecular-weight DNA than observed in the other human fibroblasts examined. (b) When XP cells were exposed to low doses of bleomycin, the low-molecular-weight DNA disappeared, suggesting induction of a repair process. If the XP cells were exposed to bleomycin in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, the disappearance of low-molecular-weight DNA was not observed; instead, a normal dose response to the drug was observed. Our results suggest that XP cells show an "induced" repair response following bleomycin treatment and that blocking DNA chain elongation uncovers normal incisions in bleomycin-treated DNA.     

Enhanced transformation of xeroderma pigmentosum variant cells by ultraviolet light-irradiated simian virus 40.

The role of DNA repair in transformation was investigated by infecting repair-deficient xeroderma pigmentosum (XP) variant cells, XP variant heterozygous cells, and normal human fibroblasts with simian virus 40 which had been irradiated by ultraviolet light. The transformation frequencies obtained were compared to those observed for unirradiated virus. While normal and heterozygous cells showed no differences between transformation frequencies using either irradiated or untreated virus, two XP variant cell lines were transformed 2- to 7-fold more readily with irradiated virus than with unirradiated virus. XP variant cells were also found to produce lower than normal quantities of virus following infection with either damages or undamaged virus, suggesting that increased viral production was not contributing to the increased transformation seen for these cells. Finally, the proportion of cells which repair ultraviolet light-irradiated simian virus 40 was found to be similar for wild-type and XP variant cells, suggesting that enhanced transformation in the mutant cells was not associated with a reduction in the numbers of cells which repair damaged virus. Several possible mechanisms to account for the increased transformation of XP variant cells by ultraviolet light-irradiated simian virus 40 are proposed.     

Cellular and molecular characteristics of an immortalized ataxia-telangiectasia (group AB) cell line

Ataxia-telangiectasia (A-T) is a multisystem hereditary disease featuring neurodegeneration, immunodeficiency, extreme cancer proneness, chromosomal instability, and radiosensitivity. A-T is found in many ethnic groups, and is genetically heterogeneous: four complementation groups have been identified in A-T so far. Attempts to isolate the A-T gene are based in part on gene transfer experiments, using permanent A-T fibroblast lines, obtained by transformation with SV40. "Immortalization" of A-T primary diploid fibroblasts using SV40 is difficult, possibly because of the chromosomal instability of these cells. The number of currently available permanent A-T fibroblast lines is small, and not all of them have been assigned to specific complementation groups. Using the assay of X-ray induced inhibition of DNA synthesis, we have assigned the A-T strain AT22IJE to complementation group AB. Origin-defective SV40 was used to transfect these cells, and one transformant (AT22IJE-T), which survived crisis, was found to have the typical characteristics of permanent cell lines obtained in this way. "In-gel renaturation" analysis did not show any DNA amplification of high degree in AT22IJE-T. Cytogenetic analysis showed considerable chromosomal instability in the new cell line, and medium conditioned by these cells contained the clastogenic activity which is characteristic of the parental strain as well. Other parameters of the "cellular A-T phenotype" have also been retained in the immortalized cells: hypersensitivity to the lethal effects of X-rays and neocarzinostatin, as well as "radioresistant" DNA synthesis. However, the sensitivity of AT22IJE-T to both DNA-damaging agents is less pronounced than that of the parental cells. The capacity of the cells for uptake of foreign DNA was tested by introducing into them the plasmid pRSVneo, using three different transfection methods. Satisfactory frequency of G418-resistant transfectants (0.66%) was achieved using a protocol recently published by Chen and Okayama (Mol. Cell Biol., 7: 2745-2752, 1987), which was found to be superior to the traditional calcium phosphate transfection method and to the polybrene-based method.     

Induction and repair of DNA and chromosome damage by neocarzinostatin in quiescent normal human fibroblasts

In an attempt to understand better the molecular basis of chromosome aberration formation, neocarzinostatin (NCS)-induced damage and repair were compared at the chromosome and DNA levels. Chromosome damage and repair in quiescent normal human fibroblasts (PA2 and DET 550) were assayed by the premature chromosome condensation technique in which the G1 prematurely condensed chromosomes are condensed and easily enumerated. DNA damage was monitored by neutral DNA elution. NCS induced chromosome breaks directly in the G1 cells; thus, its clastogenic action does not require passage of cells through S phase. The dose-response curve for NCS-induced damage suggested a two-hit-type event in the formation of chromosome breaks. The half-time of chromosome repair was approximately 4.5 hr, and all but one of the chromosome breaks were repaired by 46 hr. Neither 1-beta-D-arabinofuranosylcytosine nor cycloheximide blocked the repair of either DNA or chromosome damage induced by NCS in quiescent human cells.     

Radiation-induced G1 arrest is not defective in fibroblasts from Li-Fraumeni families without TP53 mutations

Radiation-induced G1 arrest was studied in four classes of early passage skin fibroblasts comprising 12 normals, 12 heterozygous (mut/wt) TP53 mutation-carriers, two homozygous (mut/-) TP53 mutation-carriers and 16 strains from nine Li-Fraumeni syndrome or Li-Fraumeni-like families in which no TP53 mutation has been found, despite sequencing of all exons, exon-intron boundaries, 3' and 5' untranslated regions and promoter regions. In an assay of p53 allelic expression in yeast, cDNAs from these non-mutation strains behaved as wild-type p53. Using two different assays, we found G1 arrest was reduced in heterozygous strains with mis-sense mutations and one truncation mutation, when compared to the range established for the normal cells. Heterozygous strains with mutations at splice sites behaved like normal cells, whilst homozygous (mut/-) strains showed either extremely reduced, or no, arrest. Strains from all nine non-mutation families gave responses within the normal range. Exceptions to the previously reported inverse correlation between G1 arrest and clonogenic radiation resistance were observed, indicating that these phenotypes are not strictly interdependent.     

Trichothiodystrophy, a human DNA repair disorder with heterogeneity in the cellular response to ultraviolet light

Trichothiodystrophy (TTD) is an autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and physical retardation. Some patients are photosensitive. A previous study by Stefanini et al. (Hum. Genet., 74: 107-112, 1986) showed that cells from four photosensitive patients with TTD had a molecular defect in DNA repair, which was not complemented by cells from xeroderma pigmentosum, complementation group D. In a detailed molecular and cellular study of the effects of UV light on cells cultured from three further TTD patients who did not exhibit photosensitivity we have found an array of different responses. In cells from the first patient, survival, excision repair, and DNA and RNA synthesis following UV irradiation were all normal, whereas in cells from the second patient all these responses were similar to those of excision-defective xeroderma pigmentosum (group D) cells. With the third patient, cell survival measured by colony-forming ability was normal following UV irradiation, even though repair synthesis was only 50% of normal and RNA synthesis was severely reduced. The excision-repair defect in these cells was not complemented by other TTD cell strains. These cellular characteristics of patient 3 have not been described previously for any other cell line. The normal survival may be attributed to the finding that the deficiency in excision-repair is confined to early times after irradiation. Our results pose a number of questions about the relationship between the molecular defect in DNA repair and the clinical symptoms of xeroderma pigmentosum and TTD.     

The 399Gln polymorphism in the DNA repair gene XRCC1 modulates the genotoxic response induced in human lymphocytes by the tobacco-specific nitrosamine NNK

DNA repair plays a critical role in protecting the genome of the cell from the insults of cancer-causing agents such as those found in tobacco smoke. Reduced DNA repair capacity would, therefore, constitute a significant risk factor for smoking-related cancers. Recently, a number of polymorphisms in several DNA repair genes have been discovered, and it is possible that these polymorphisms may affect DNA repair capacity and thus modulate cancer susceptibility in exposed populations. In the current study, we explored the relationship between two polymorphisms in the DNA repair gene XRCC1 (polymorphisms in codons 194 and 399) and the genotoxic response induced by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The sister chromatid exchange (SCE) assay was used as a marker of genetic damage. Our results, using whole blood cultures from 47 volunteers, indicated that treatment of cells with 0.24, 0.72 and 1.44 mM of NNK induced a concentration-dependent increase in the mean number of SCE (P<0.001). There was a significant difference (P<0.05) in response to NNK treatment between cells from individuals with the 399Gln allele (either homozygous or heterozygous) and cells from individuals with the homozygous 399 Arg/Arg genotype. Treatment of cells that have the 399Gln allele with 0.24, 0.72 and 1.44 mM NNK resulted in 22.8, 35.8 and 52.8% increases in NNK-induced SCE, respectively. Treatment of cells with the 399 Arg/Arg genotype using the same NNK concentrations resulted in 16.0, 15.5 and 32.6% increases in NNK-induced SCE, respectively. In contrast, no significant difference in NNK-induced SCE was observed between cells with the codon 194 Arg/Arg genotype and cells with the codon 194 Arg/Trp genotype at all concentrations of NNK tested. These data suggest that the Arg399Gln amino acid change may alter the phenotype of the XRCC1 protein, resulting in deficient DNA repair. Our study underscores the important role of polymorphisms in DNA repair genes in influencing the genotoxic responses to environmental mutagens, and justifies additional studies to investigate their potential role in susceptibility to cancer.