[Fanconi anemia: genes and function(s) revisited].

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes.

Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts.

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e.

Mitochondrial alterations in fanconi anemia fibroblasts following ultraviolet A or psoralen photoactivation.

The genetic disease Fanconi anemia (FA), generally considered to be a DNA repair defect, has also been related to a deficiency in cellular defense against reactive oxygen species (ROS). Results show that mitochondrial matrix densification occurs rapidly and transiently in FA fibroblasts following 8-methoxypsoralen (8-MOP) photoreaction or ultraviolet A (320 to 380 nm) (UVA) irradiation. This effect is oxygen dependent because it is more important under 20 than under 5% oxygen tension.

Contribution of antioxidant enzymes to the adaptive response to ionizing radiation of human lymphoblasts.

Purpose: To investigate whether the adaptive response to ionizing radiation triggered by a low-dose pre-exposure could be due to the activation of the antioxidant defence system.

Materials And Methods: Human lymphoblastoid AHH-1 cells were irradiated with a 0.02 Gy gamma-radiation and 6 h later were exposed to a 3 Gy challenge dose according to a protocol allowing mutagenic adaptation.

Arrest of S-phase progression is impaired in Fanconi anemia cells.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis.

ATM protein is required for radiation-induced apoptosis and acts before mitochondrial collapse.

Purpose: To define the role of the ataxia telangiectasia (A-T) mutated gene (ATM) in activation and progress of apoptosis.

Material And Methods: Three normal and three A-T EBV-transformed cell lines were studied. Following irradiation (IR), Fas activation or ceramide exposure, viability and apoptosis were measured by trypan blue dye exclusion assay and as sub-G1 cell fraction by flow cytometric analysis of propidium iodide stained cultures, respectively.

Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells. Possible role of Dewar photoproducts in solar mutagenesis.

In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum.

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.

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

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.

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 Hodgkin's 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.

Fanconi anemia C protein acts at a switch between apoptosis and necrosis in mitomycin C-induced cell death.

Deregulation of apoptosis seems to be a hallmark of the Fanconi anemia (FA) syndrome. In order to further define the role of the FA protein from complementation group C (FAC) in apoptosis, we characterized parameters modified during the mitomycin-C (MMC)-induced apoptotic program. It is shown that despite a higher level of cell death for FA compared to normal lymphoblasts after MMC treatment, FA cells do not display a marked DNA fragmentation.

[Tumor and individual radiosensitivity. An introduction].

This text is intended to introduce the following articles, which correspond to the lectures given at the "Radiosensitivity" session of the 1998 SFRO Meeting. We first underline the somewhat disappointing results obtained so far for evaluating tumoral radiosensitivity. However, a few new tests could bring some hope in a not-too distant future.

Is Fanconi anemia caused by a defect in the processing of DNA damage?

Fanconi anemia (FA) is an autosomal genetic disease characterized by a complex array of developmental disorders, a high predisposition to bone marrow failure and to acute myelogenous leukemia. The chromosomal instability and the hypersensitivity to DNA cross-linking agents led to its classification with the DNA repair disorders. This review aimed at establishing whether it is still appropriate to consider 1/approximately FA within a DNA repair framework taking into account the recently discovered genetic heterogeneity characteristics of the defect (eight complementation groups).

The fidelity of double strand breaks processing is impaired in complementation groups B and D of Fanconi anemia, a genetic instability syndrome.

In mammalian cells, nonhomologous end-joining is the predominant mechanism to eliminate DNA double strand breaks. Such events are at the origin of deletion mutagenesis and chromosomal rearrangements. The hallmark of Fanconi anemia, an inherited cancer prone disorder, is increased chromosomal breakage associated to over-production of deletions.

Fanconi anemia C gene product plays a role in the fidelity of blunt DNA end-joining.

Mutations in genes controlling the correct functioning of the replicative, repair and recombination machineries may lead to genomic instability. A high level of spontaneous chromosomal aberrations amplified by treatment with DNA cross-linking agents is the hallmark of Fanconi anemia (FA), an inherited chromosomal instability syndrome associated with cancer proneness. Two of the eight FA genes have been cloned (FAA and FAC), but their function has not yet been defined.

Role of a mutant p53 protein in apoptosis: characterization of a function independent of transcriptional trans-activation.

Wild-type (wt) tumor suppressor p53 has been implicated in cellular radiosensitivity, mediated by its role in apoptosis and growth arrest. Intriguingly, it was observed that the temperature sensitive (ts) mutant p53val135 protein functions as a positive modulator of cellular radiosensitivity, as evident from acceleration of irradiation-induced apoptosis of M1p53ts (p53val135) cells at the non-permissive temperature; this effect was correlated with acceleration of exit from the G2 checkpoint of the cell cycle. In this work it is shown that the ability of mutant p53val135 to accelerate irradiation-induced apoptosis, at the non-permissive temperature, was devoid of transcriptional trans-activation of p53 target genes.

[Molecular mechanisms of carcinogenesis: the role of systems of DNA repair].

The initiation step of the carcinogenic process consists in an alteration of genes playing a central role in the cellular life. The next steps of promotion and progression result from anomalies in the response to growth factors, to hormones and/or from the action of tumor promotors leading to cellular hyperplasia. This process generally leads to genetic instability of the initiated cell which in turn allows selection of malignant and invasive clones.

Impaired DNA repair as assessed by the "comet" assay in patients with thyroid tumors after a history of radiation therapy: a preliminary study.

Purpose: Patients with a history of head and neck irradiation in childhood are at risk to develop thyroid tumors. The aim of this study was to determine if an impairement of DNA strand breaks repair could account for this observation.

Methods And Materials: Circulating unstimulated lymphocytes of a group of 13 patients who developed thyroid tumors after radiotherapy were submitted to the alkaline single-cell gel electrophoresis assay (SCGE or "comet" assay) after in vitro exposure to 2 and 5 Gy of gamma-rays.

Deregulated apoptosis is a hallmark of the Fanconi anemia syndrome.

Fanconi anemia (FA) is a genetic human disorder associated with bone marrow failure and predisposition to cancer. FA cells show poor growth capacity and spontaneous chromosomal anomalies as well as cellular and chromosomal hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). Because it is likely that disruption of the apoptotic control would lead to such a phenotype, we investigated the implication of apoptosis in the FA syndrome.

6,4,4'-Trimethylangelicin photoadduct immunodetection in DNA: induction and repair in Fanconi's anemia and normal human fibroblasts.

6,4,4'-Trimethylangelicin (TMA)-photoinduced monoadducts (MAs) were detected and quantified on DNA of normal human and Fanconi's anemia (FA) fibroblasts (complementation groups A and D) by immuno-electron microscopy. TMA-modified DNA was extracted from the cells just after photoreaction, or after a subsequent 24 h repair period, for analysis of the MA processing capabilities of the different cell lines. Unmodified DNA was extracted from the control cells in parallel.

The comet assay as a repair test for prenatal diagnosis of Xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are autosomal recessive diseases associated with extreme cutaneous photosensitivity, a defect in nucleotide excision repair (NER), and genetic complexity. Severe prognosis and lack of treatment led families at risk to request genetic counseling. Unscheduled DNA synthesis (UDS) is the classic method for diagnosis and requires 4 to 5 wk before conclusion.

Preferential repair incision of cross-links versus monoadducts in psoralen-damaged plasmid DNA by human cell-free extracts.

Upon UVA irradiation psoralens covalently bind to DNA as monoadduct and interstrand crosslink. Psoralen photoadducts are processed via an excision repair reaction that has been reproduced in vitro with transcriptionnally active cell-free extracts. A derived in vitro assay that allows direct quantification of the incised sites has been set up and used to compare the efficiency of the incision reaction on monoadducts and interstrand cross-links.

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.