Ethel Moustacchi

Chromatin Assembly Coupled to DNA Repair: A New Role for Chromatin Assembly Factor I

DNA repair in the eukaryotic cell disrupts local chromatin organization. To investigate whether the resetting of nucleosomal arrays can be linked to the repair process, we developed model systems, with both Xenopus egg extract and human cell extracts, to follow repair and chromatin assembly in parallel on circular DNA templates. Both systems were able to carry out nucleotide excision repair of DNA lesions. We observed that UV-dependent DNA synthesis occurs simultaneously with chromatin assembly, strongly indicating a mechanistic coupling between the two processes. A complementation assay established that chromatin assembly factor I (CAF1) is necessary for this repair associated chromatin formation.

Use of the alkaline comet assay to detect DNA repair deficiencies in human fibroblasts exposed to UVC, UVB, UVA and gamma-rays

The alkaline single cell gel electrophoresis (comet) assay applied to human fibroblasts allowed us to analyze the response to components of the solar spectrum (UVB and UVA) in comparison with the well-established response to UVC and gamma-rays. DNA strand breaks related to nucleotide excision repair of DNA photoproducts were produced 1 h after exposure to UVB or UVC in the normal cell line but not in the repair deficient XPD and TTD-2 cell lines. In contrast, the immediate production of DNA strand breaks observed in all cell lines after exposure to UVA or gamma-rays was followed by restitution of high molecular weight DNA upon post-exposure incubation. These results imply that (1) fibroblasts as well as lymphocytes can be analysed by the comet assay and (2) the comet assay clearly distinguishes cellular nucleotide excision repair capacity without the use of inhibitors of DNA synthesis.

An analysis of in vivo hprt mutant frequency in circulating T-lymphocytes in the normal human population: a comparison of four datasets☆

In this paper, we have compared mutant frequency data at the hprt locus in circulating T-lymphocytes from four large datasets obtained in the UK (Sussex), the USA (Vermont), France (Paris) and The Netherlands (Leiden). In total, data from > 500 non-exposed individuals ranging in age from newborns (cord blood samples) to > 80 years old have been included in the analysis. Based on raw data provided by the four laboratories, a model is presented for the analysis of mutant frequency estimations for population monitoring. For three of the laboratories, a considerable body of data was provided on replicate estimates of mutant frequency from single blood samples, as well as estimates from repeat blood samples obtained over a period of time from many of the individual subjects. This enabled us to analyse the sources of variation in the estimation of mutant frequency. Although some variation was apparent in the results from the four laboratories, overall the data were in general agreement. Thus, in all laboratories, cellular cloning efficiency of T-cells was generally high (> 30%), although in each laboratory considerable variation between experiments and subjects was seen. Mutant frequency per clonable T-cell was in general found to be inversely related to cloning efficiency. With the exception of a few outliers (which are to be expected), mutant frequencies at this locus were in the same range in each dataset; no effect of subject gender was found, but an overall clear age effect was apparent. When log mutant frequency was analysed vs log (age + 0.5) a consistent trend from birth to old age was seen. In contrast, the effect of the smoking habit did differ between the laboratories, there being an association of smoking with a significant increase in mutant frequency in the Sussex and Leiden datasets, but not in those from the Vermont or Paris datasets. Possible reasons for this are discussed. One of the objectives of population monitoring is an ability to detect the effect of accidental or environmental exposure to mutagens and carcinogens among exposed persons. The large body of data from non-exposed subjects we have analysed in this paper has enabled us to estimate the size of an effect that could be detected, and the number of individuals required to detect a significant effect, taking known sources of variation into account.(ABSTRACT TRUNCATED AT 400 WORDS)

Photochemotherapy (PUVA) of psoriasis using 3-carbethoxypsoralen, a non-carcinogenic compound in mice.

The carcinogenic risk of photochemotherapy (PUVA) with bi‐functional furocoumarins such as 8‐methoxypsoralen (8‐MOP) which form cross‐links in cellular DNA has initiated a search for active but less hazardous psoralens. A new compound, 3‐carbethoxypsoralen (3‐CPs), studied in the yeast Saccharomyces cerevisiae (eukaryote), has been shown to be very photoactive on DNA and to form only mono‐additions to DNA. These lesions appear to be more easily repaired than the cross‐links induced by 8‐MOP. 3‐CPs produces less nuclear genetic events such as nuclear mutations and mitotic crossovers, but more cytoplasmic ‘petite’ mutations (damage to mitochondrial DNA) than 8‐MOP. In mice it was demonstrated that after local or intra‐peritoneal administration, in contrast to 8‐MOP. 3‐CPs is non‐toxic, non‐erythematogenic, and non‐carcinogenic. A study of ten psoriatic patients has shown that local applications of 3‐CPs plus UV‐A exhibit about the same therapeutic activity for the clearing of psoriatic lesions as local treatment with 8‐MOP plus UV‐A, but without any localized hyperpigmentation.

Oxidation of guanine in cellular DNA by solar UV radiation: biological role.

Abstract. The formation of cyclobutane pyrimidine dimers (CPD) and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo) was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3‐2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8‐oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 102 and 102 more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that efficient induction of 8‐oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8‐oxodGuo were produced between 290–320 nm and 320–340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8‐oxodGuo greatly differed from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8‐oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8‐oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation

Analysis by alkaline comet assay of cancer patients with severe reactions to radiotherapy: defective rejoining of radioinduced DNA strand breaks in lymphocytes of breast cancer patients.

Therapeutic exposure to ionising radiation reveals inter‐individual variations in normal tissue responses. To examine whether a defect in DNA repair capacity might be involved in such hypersensitive phenotypes, we analysed, using the alkaline comet assay, the response as a function of time to in vitro irradiation at 5 Gy of lymphocytes from 17 breast cancer and 9 Hodgkins disease patients who developed severe reactions to radiotherapy in comparison with 22 patients with “average” reactions and 24 healthy donors. A difference between breast cancer over‐reactors and both patients with normal reactions and healthy donors was observed 30 and 60 min after exposure. A subgroup of breast cancer over‐reactors (7/17) reproducibly demonstrated increased levels of residual damage. When the kinetic analyses were prolonged to 120 min, results were in favour of delayed kinetics of rejoining in these patients. Among Hodgkins disease over‐reactors, only one patient showed defective repair. Interestingly, all patients with the most severe complications (grade 4 RTOG/EORTC), i.e., 5 breast cancer and 1 Hodgkins disease, showed impaired rejoining. Our results suggest that impairment in DNA strand break processing may be associated, in specific subgroups of breast cancer patients, with an individual risk of major toxicity of radiation therapy. Thus, the alkaline comet assay appears to be useful for documenting the DNA repair phenotype in cancer patients. Int. J. Cancer 83:83–90, 1999.

Radioadaptation for gene mutation and the possible molecular mechanisms of the adaptive response

This paper reviews the experimental results showing that a prior exposure to a low dose of ionising radiation induces an adaptive response expressed as a reduction of gene mutation in various cell systems. The data show that the mutagenic adaptation shares common features with the clastogenic adaptation, i.e., priming dose level, kinds of conditioning agents, time interval between conditioning and challenging treatments, degree of induced protective effect (40-75%), transitory response and inhibition by 3-aminobenzamide, a DNA repair inhibitor. Moreover, the deletion-type mutations are predominantly reduced in adapted cells, suggesting that the mechanism underlying mutagenic adaptation preferentially facilitates the removal of the DNA lesions leading to deletion-type mutations. These lesions are thought to be double-strand breaks which are likely to be also involved in the production of chromosomal damage. Recent findings on the molecular processes implicated in the cellular response to radiation provide some clues for the mechanisms that could be triggered by low-dose exposure and ultimately contribute to the protective effect. There is some evidence that the protein kinase C-mediated signalling pathway is a key step for the transduction of the low-dose-induced signal. Several recent reports indicate that the low-dose triggers changes in the expression of several genes whose products, though most of them are still not identified, would be related to DNA repair and/or control of cell cycle progression.

Lethal and mutagenic effects of elevated temperature on haploid yeast. I. Variations in sensitivity during the cell cycle.

SummaryThe lethal and cytoplasmic mutagenic effects of 52°C incubation during the cell cycle of a haploid strain of Saccharomyces cerevisiae were examined. Both effects varied periodically in a rather parallel pattern: the maximum thermosensitivity was seen at budding time, corresponding to the S period (Williamson, 1965). The 52°C induction of a nuclear forward mutation was also examined: canavanine-resistant mutants were induced by this treatment. Exponentially growing cells were much more sensitive than resting cells to the different effects of heating which were studied. On the other hand, on comparing asynchronous cultures of 6 different radiosensitive mutants only one (xrs5) showed a greater thermosensitivity than the corresponding wild type.

DNA damage and repair: consequences on dose-responses.

Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and cancer. Single or double strand breaks, bases modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions formed in different proportions according to agents and to DNA sequence context. They can result in cell death or in mutational events which in turn may initiate malignant transformation. Normal cells are able to repair these lesions with fidelity or by introducing errors. Base excision (BER) and nucleotide excision (NER) repair are error-free processes acting on the simpler forms of DNA damage. A specialized form of BER involves the removal of mismatched DNA bases occurring as errors of DNA replication or from miscoding properties of damaged bases. Severe damage will be repaired according to several types of recombinational processes: homologous, illegitimate and site-specific recombination pathways. The loss of repair capacity as seen in a number of human genetic diseases and mutant cell lines leads to hypersensitivity to environmental agents. Repair-defective cells show qualitative (mutation spectrum) and quantitative alterations in dose-effect relationships. For such repair-deficient systems, direct measurements at low doses are possible and the extrapolation from large to low doses fits well with the linear or the linear-quadratic no-threshold models. Extensive debate still takes place as to the shape of the dose-response relationships in the region at which genetic effects are not directly detectable in repair-proficient normal cells. Comparison of repair mutants and wild-type organisms pragmatically suggests that, for many genotoxins and tissues, very low doses may have no effect at all in normal cells.

Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia

SummaryThe correction of chromosomal hypersensitivity to mitomycin C (MMC) in Fanconi anemia (FA) human lymphoblasts is observed by growth in a medium conditioned by normal human cells. Under the same conditions, the cytotoxic effect of MMC on FA cells is restored to an almost normal level. The addition of interleukin-6 (IL-6) to an unconditioned culture medium increased the resistance of FA cells to MMC cytotoxicity. This correcting effect is partially abolished by addition of an anti-IL-6 antibody to the conditioned medium. Both lymphoblasts and fibroblasts derived from FA patients demonstrate a reduction in IL-6 production. Moreover, this lymphokine is not induced by tumor necrosis factors α and β (TNFα and TNFβ) in FA cells, as is the case in normal cells. It is suggested that the observed deficiency in IL-6 production may account for one of the major characteristics of FA disease, i.e., the defect in differentiation of the hematopoietic system.