Biomedical subjects

E Moustacchi

Publications and source records attributed to E Moustacchi.

At least 37 records · Page 2Linked to original sources

PubMed1996

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. The incision reaction was performed with HeLa cell-free extracts on angelicin or 8-methoxypsoralen (8-MOP)-modified plasmid DNA substrates carrying known amounts of mono- and biadducts, within various relative ratios. In the case of 8-MOP modified plasmids consisting in a mixture of mono- and biadducts on the same DNA molecule, the incision signal was mainly due to the presence of interstrand cross-links. The extent of incision was linear with the number of cross-links up to about 4 cross-links per plasmid and then reached a plateau. The sensitivity of incision defined as the increase of incision by 2-fold over the background level corresponded to about 1 cross-link per plasmid molecule, and about 7% of the total cross-links were repaired under our assay conditions. The incision activity on angelicin monoadducts yielded only 27% when compared to that on 8-MOP cross-links. Furthermore, 8-MOP cross-links lowered the incision extent of angelicin monoadducts when the two photoadducts were present on distinct plasmid DNA molecules. These data are in line with the more rapid excision of psoralen interstrand cross-links vs monoadducts observed in vivo.

Cell-Free System↗

PubMed1996

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.

Adaptation, Physiological↗

PubMed1996

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.

Animals↗

PubMed1996

Deregulated apoptosis in ataxia telangiectasia: association with clinical stigmata and radiosensitivity.

Ataxia telangiectasia (AT) is a recessive genetic disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation hypersensitivity, and increased predisposition to cancer. Reduced or delayed induction of the tumor suppressor protein p53 after gamma -irradiation was reported. These characteristics may be compatible with an inability to correctly regulate apoptosis. We show here that AT lymphocytes and EBV-transformed lymphoblasts demonstrate a significantly higher level of spontaneous apoptosis, whereas ionizing radiation-induced apoptosis is reduced compared to normal cells. However, neither AT nor normal primary fibroblasts undergo apoptosis after irradiation. Consequently, we conclude that the radiosensitivity of the AT cells is not related to an increased apoptotic response. Finally, we show that SV40-transformed AT fibroblasts undergo gamma- ray-induced apoptosis, while SV40-transformed normal cells do not. This result raises the question of the physiological relevance of the latter cellular model with respect to the AT phenotype.

Apoptosis↗

PubMed1996

Mutagenic specificity of solar UV light in nucleotide excision repair-deficient rodent cells.

To investigate the role of nucleotide excision repair (NER) in the cellular processing of carcinogenic DNA photoproducts induced by defined, environmentally relevant portions of the solar wavelength spectrum, we have determined the mutagenic specificity of simulated sunlight (310-1100 nm), UVA (350-400 nm), and UVB (290-320 nm), as well as of the "nonsolar" model mutagen 254-nm UVC, at the adenine phosphoribosyltransferase (aprt) locus in NER-deficient (ERCC1) Chinese hamster ovary (CHO) cells. The frequency distributions of mutational classes induced by UVB and by simulated sunlight in repair-deficient CHO cells were virtually identical, each showing a marked increase in tandem CC-->TT transitions relative to NER-proficient cells. A striking increase in CC-->TT events was also previously documented for mutated p53 tumor-suppressor genes from nonmelanoma tumors of NER-deficient, skin cancer-prone xeroderma pigmentosum patients, compared to normal individuals. The data therefore indicate that the aprt gene in NER-deficient cultured rodent cells irradiated with artificial solar light generates the same distinctive "fingerprint" for sunlight mutagenesis as the p53 locus in NER-deficient humans exposed to natural sunlight in vivo. Moreover, in strong contrast to the situation for repair-component CHO cells, where a significant role for UVA was previously noted, the mutagenic specificity of simulated sunlight in NER-deficient CHO cells and of natural sunlight in humans afflicted with xeroderma pigmentosum can be entirely accounted for by the UVB portion of the solar wavelength spectrum.

Adenine Phosphoribosyltransferase↗

PubMed1996

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.

Cell Line↗

PubMed1995

The mutagenic processing of psoralen photolesions leaves a highly specific signature at an endogenous human locus.

To assess the role of a given genotoxic agent in the etiology of human cancers, it is useful to establish the mutational specificity of this agent. The aim of this study was to investigate whether the processing of psoralen photolesions, interstrand cross-links (CL) and monoadducts (MA), leaves a specific molecular signature in the mutational events produced at an endogenous locus, HPRT. Human lymphoblasts were treated by 4,5',8-trimethylpsoralen (Me3Pso) in association with a double irradiation protocol (365 plus 365 nm) which allows us to increase the proportion of CL for a given constant number of total photoadducts. The molecular spectrum of mutations at the HPRT locus induced in these conditions was compared to the previously reported spectra of mutations induced by the same psoralen in combination with a single irradiation of either 365 nm (induction of MA and a low proportion of CL) or 405 nm (producing almost exclusively MA). In all treatment conditions, base substitutions constitute the major type of Me3Pso photoinduced mutations. The majority of base substitutions involve a T residue preferably within a 5'-TpA sequence which corresponds to the favoured sites of psoralen photoadducts. In other words, the Me3Pso photolesions induce at the endogenous HPRT locus a high specific signature. Moreover, base substitutions have been essentially found in the non-transcribed strand of the HPRT gene suggesting that the psoralen photolesions are preferentially removed from the transcribed strand. In spite of the considerable difference between the proportion of lesions of both types (CL or MA) induced in different treatment conditions, the kind of mutations and their sequence distribution are similar suggesting that the mutagenic processing of psoralen CL and MA is similar at least for the steps resulting in base substitutions.

Base Sequence↗

PubMed1995

p53 involvement in control of G2 exit of the cell cycle: role in DNA damage-induced apoptosis.

DNA damage in proliferating mammalian cells induces a complex cellular response comprising perturbation of the cell cycle and programmed cell death. The relationship between p53-dependent and p53-independent apoptotic cell death, as well as the cell cycle checkpoints induced by DNA damaging agents were explored in hematopoietic cells, using M1 myeloblastic leukemia cells, which are null for p53 expression, genetically engineered M1 variants, expressing p53ts and bcl-2 transgenes, as well as myeloblast enriched bone-marrow cells obtained from wild type p53 (wt p53) and p53-deficient mice. It is shown that gamma-irradiation of M1p53ts cells activated a function of the temperature sensitive mutant transgene p53 (p53ts), promoting increased apoptosis relative to parental, null p53 M1 cells. It is also shown that the kinetics of apoptotic cell death induced by gamma-irradiation correlated with the rapidity of exit from gamma-ray-induced G2 arrest for all the different hematopoietic cell types indicated above. Finally, data has been obtained to demonstrate that, in addition to a role in apoptosis and G1 arrest, wild-type p53 positively modulated the exit from the gamma-ray-induced G2 checkpoint. Taken together, these findings indicate that this new function for p53 is a component of the physiological pathway by which p53 exerts its role in apoptosis.

Animals↗

PubMed1995

The molecular mechanism underlying formation of deletions in Fanconi anemia cells may involve a site-specific recombination.

Spontaneous and induced chromosomal breakage is an important cellular feature of Fanconi anemia (FA), an inherited DNA repair disorder characterized by progressive bone marrow failure, developmental abnormalities, and predisposition to leukemia. We have previously reported that in comparison to normal cells, there is a substantial increase in frequency of intragenic deletions at an endogenous locus (HPRT) in FA lymphoblasts. Taken together with the increased chromosomal instability, these observations indicated that the wild-type FA gene(s) plays an important role in the maintenance of the genomic integrity. To obtain information on the mechanism(s) underlying the genomic rearrangements in FA, the breakpoint sites of deletions in 11 FA-derived HPRT- mutants were analyzed. The results indicate that a significant proportion of deletions involving a loss of a given exon are identical and that two deletions of different size have the same 3' breakpoint. Interestingly, it appears that in most of the mutants there is a common deletion signal sequence, which suggests that the mutations in the FA gene(s) may lead to an aberrant site-specific cleavage activity that might be responsible for the deletion proneness and the chromosomal instability characteristic of the FA pathology. From the similarity or even identity of the signal sequence at some of the breakpoints with the consensus heptamer which directs cleavage and joining in the assembly of immunoglobulin and T-cell receptor genes, we speculate that steps in common with the V(D)J recombinational process may be illegitimately involved in FA cells.

Base Sequence↗

PubMed1995

p53-dependent pathway of radio-induced apoptosis is altered in Fanconi anemia.

Fanconi anemia belongs to a group of human genetic diseases characterized by chromosomal instability, sensitivity to genotoxic agents associated to impaired processing of DNA lesions, cell cycle anomalies and cancer predisposition. We recently added to this list of distinctive features reduced production of interleukin 6 and overproduction of tumor necrosis factor alpha. Since growth factor deprivation, TNF alpha treatment or DNA damage can trigger apoptosis, we monitored the apoptotic response of FA cell lines. We show here that, although the spontaneous rate of apoptosis is slightly more elevated in FA than in normal cell cultures, the apoptosis induced by gamma-irradiation is drastically reduced in FA. Since the induction of apoptosis by radiation is a p53-dependent mechanism, the induction of this protein in FA cells was also examined. We found that the p53 protein is not radio-induced in FA cells belonging to the two genetic complementation groups examined (C and D), in contrast to normal cells. Moreover, the same impairment in p53 induction is observed after exposure to mitomycin C, a chemical agent for which FA cells demonstrate a specific cellular and chromosomal hypersensitivity, as well as after u.v.-B irradiation, an agent known to cause oxidative stress. These observations are in line with recent reports showing that at least certain cell lines from other chromosome breakage syndromes, such as ataxia telangiectasia and Bloom syndrome, may be also defective for radiation-induced increase of p53 protein. As the p53 tumor suppressor gene encodes a transcriptional activator whose targets include genes that regulate genomic stability, cellular response to DNA damage and cell cycle progression, we suggest that altered expression of p53 may be relevant to the FA phenotype.

Cell Cycle↗

PubMed1995

Genotoxic potential of psoralen cross-links versus monoadducts in normal human lymphoblasts.

Using the 4,5',8-trimethylpsoralen in combination with the reirradiation protocol, we show that, in normal human lymphoblasts, the cytotoxic potential of photoinduced cross-links (CL) is higher than that of monoadducts (MA). In contrast to cytotoxicity, the significant increase in the proportion of CL, at a constant level of total adducts, had no effect on the induction of mutations at the HPRT locus. Comparison with the data obtained in yeast and rodent cells using the same double irradiation protocol shows that the mutagenic potential of CL versus MA varies between species. This suggests that the equilibrium between the excision, the recombinational and the mutagenic components of the repair pathways which probably determine the mutagenic efficiency of CL versus MA is likely to be species-dependent.

Cell Line↗

PubMed1995

DNA sequence analysis of HPRT- mutants induced in human lymphoblastoid cells adapted to ionizing radiation.

Radioadaptation to the mutagenic effect of ionizing radiation by pre-exposure of human cells to a low dose has been shown to decrease the proportion of HPRT- mutants of the deletion type. To determine whether point mutations would be affected by the adaptive treatment, the molecular nature of mutations induced after exposure to low, high or low plus high doses was established. DNA sequencing of 38 point mutants which still expressed mRNA was performed using reverse transcription/polymerase chain reaction amplification. Under all conditions, base substitutions were the most common mutational event (range 72-80%), the remainder being frameshift and small deletions. The types and proportions of base changes did not appear to be differentially modified. A clustering of mutations was observed in exon 8, independently of the radiation protocol. About 40% of the mutants exhibited incorrect splicing of mRNA. The lack of striking modifications between the different molecular spectra of point mutations suggests that the low-dose pre-exposure does not affect the production and/or the processing of lesions leading to point mutations. Thus the highly significant effect triggered by the low dose is the preferential reduction of deletion-type mutations. In view of the actual small data set, definitive conclusions will be drawn only when our observations are confirmed or can be generalized to human endogenous loci other than the HPRT locus, which is particularly prone to the recovery of deletion-type mutations.

Adaptation, Physiological↗

PubMed1994

Identification and chromosomal localization of a DNA fragment implicated in the partial correction of the Fanconi anemia group D cellular defect.

Fanconi anemia (FA) cells, complementation group D, which had been transfected with mouse genomic DNA were partially corrected for their mitomycin C (MMC) hypersensitivity. A genomic DNA fragment which complements the resistance of FA(D) cells to MMC close to normal level has been cloned; it has no correcting activity in FA group A cells. It contains two highly conserved regions between the mouse and human genome, which flank mouse repeated DNA. This DNA fragment detects a 3.6-4-kb mRNA transcript in human cells. Moreover this fragment maps to chromosome 11q23, a region of particular interest since several genes involved in the control of major cellular functions are located in this area. This DNA fragment may belong to a gene directly or indirectly involved in FA(D) function.

Animals↗

PubMed1994

Radioadaptation to the mutagenic effect of ionizing radiation in human lymphoblasts: molecular analysis of HPRT mutants.

A 70% reduction of HPRT- mutant frequency in radioadapted human lymphoblastoid cells has been reported, as analyzed by the Southern blot method (O. Rigaud et al., Radiat. Res., 133: 94-101, 1993). The data reported here extend the previous molecular analysis to a collection of 118 mutants. Structural rearrangements of the HPRT gene were determined using the multiplex polymerase chain reaction assay. This allows us to define more precisely the deleted exons in mutants and to ensure the absence of small alterations in exons among mutants with no detectable change after Southern analysis. The phenotype of these latter mutants is likely to be due to point mutations. Overall results of both Southern and multiplex polymerase chain reaction analyses confirm that the proportion of deletion-type mutations is decreased in adapted cells (42%) compared to that in mutants treated with the high dose alone (77%). Mutants with no change at the HPRT gene level were further characterized with respect to their HPRT gene expression. The vast majority of adapted mutants (86%) were still expressing mRNA, whereas HPRT transcripts were detected in only 56% of the mutants induced by the high dose alone. From these data and those reported by others, possible mechanisms underlying the adaptive response are proposed.

Cell Line↗

PubMed1994

Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia. II. In vitro and in vivo spontaneous overproduction of tumor necrosis factor alpha.

We have previously shown an unbalanced cytokine production in Fanconi anemia (FA) cells, ie, an underproduction of interleukin 6 (IL-6) during growth. Among a number of cytokines analyzed, the only other anomalies detected concern tumor necrosis factor alpha (TNF alpha). In comparison to normal cells, this cytokine is overproduced by FA lymphoblasts from the four genetic complementation groups. Indeed, up to an eight-fold increase in TNF alpha is observed in the growth medium of FA cells. Moreover, addition of anti-TNF alpha antibodies partially corrects the FA hypersensitivity to treatment by mitomycin C (MMC). Treatment of FA cells with IL-6, which partially restored an almost normal sensitivity to MMC of FA cells also reduces the TNF alpha overproduction in FA lymphoblasts. No anomalies at the molecular level (Southern and Northern blot analyses) are detected for the TNF alpha gene and its mRNA. We have investigated the in vivo situation by assaying TNF alpha levels in the serum from FA homozygotes and obligate heterozygotes. In contrast to normal healthy donors or to aplastic anemia patients in whom serum TNF alpha is present only in trace amounts, all 36 FA patients and 21 FA parents monitored show a significantly (P < .001) higher level of serum TNF alpha activity. Consequently, abnormal TNF alpha production seems to be associated with the FA genetic background.

Base Sequence↗

PubMed1994

Comparison of the effects of DNA topoisomerase inhibitors on lymphoblasts from normal and Fanconi anemia donors.

DNA topoisomerases modify supercoiled DNA through concerted breaking and rejoining of the DNA strands and consequently play a key role in DNA biosynthesis and processing. It has been suggested that topoisomerases may facilitate access to damaged sites of excision repair enzymes due to their property to relax supercoiled DNA. We show here that treatment with nalidixic acid and novobiocin, which affects topoisomerase II activity among other targets, impairs the incision of 8-methoxypsoralen photoinduced DNA interstrand cross-links in normal human fibroblasts. Since cells derived from Fanconi anemia (FA) demonstrate hypersensitivity to DNA cross-linking agents associated with a reduced repair efficiency of cross-links, we compared the effects of different topoisomerase I and II inhibitors on FA and normal lymphoblasts. No differences were found in growth inhibition or induction of chromosome aberrations between FA and normal cells. The specificity of inhibitors is questionable and even if topoisomerases are indeed inhibited alternative pathways may be involved. However, our observations provisionally suggested that topoisomerases activities are normal in FA cells.

Camptothecin↗

PubMed1994

Activated ras oncogene and specifically acquired resistance to cisplatin in human mammary epithelial cells: induction of DNA cross-links and their repair.

A human non-malignant mammary epithelial cell line, HBL100, and the ras-transformed HBL100/ras1 cell line were examined for their sensitivity to cis-diamminedichloroplatinum(II) (cisplatin). The clonogenic cell survival assay showed that HBL100/ras1 exhibited a 2.7-fold increased resistance compared to the parental HBL100 cell line. The responses to other agents interacting with DNA, such as mitomycin C, 8-methoxypsoralen plus UVA or doxorubicin, were very similar in both cell lines. The same is true for ionizing radiation (Alapetite et al., Int J. Radiat. Biol., 59, 385-396, 1991). In other words, the mechanism of acquired resistance in HBL100 appears to be limited to cisplatin. No difference was observed between the two cell lines in cisplatin uptake as determined by atomic absorption spectrometry. Alkaline elution showed that less interstrand cross-links were formed by this drug in the resistant HBL100/ras1 cells compared to HBL100 and, moreover, the removal of these adducts was clearly more efficient in the former cell line. This was confirmed by an in vitro excision repair assay which revealed a 2.2-fold increase in DNA repair activity in the extracts from HBL100/ras1 versus HBL100 cells. It is concluded that the transformation of human epithelial HBL100 cells by the ras gene resulted in an acquired resistance apparently limited to cisplatin, a feature associated with a reduced proportion of induced interstrand cross-links and a higher efficiency in their removal. The mechanism of involvement of the ras gene product in this process is still a matter of speculation.

Breast↗

PubMed1994

The mutational specificity of simulated sunlight at the aprt locus in rodent cells.

To elucidate the nature of sunlight mutagenesis in mammalian cells, the mutational specificity of simulated solar light (SSL) has been established at the Chinese hamster ovary adenine phosphoribosyltransferase (aprt) locus. Among a collection of 36 independent SSL-induced mutations, the majority were single or tandem double C-->T transitions at dipyrimidine sites. This is consistent with previous investigations of 254 nm UVC on the aprt gene, as well as on various other genetic targets from diverse species, and supports a pre-eminent role for cyclobutane dimers and/or (6-4) photoproducts in solar mutagenesis. However, some substantial differences were also noted in the frequency and distribution of mutational classes generated by SSL versus UVC at the aprt locus. In particular, a reduction in the proportion of SSL-induced C-->T transitions was accompanied by significant increases in the incidence of T-->G transversions (25% versus 6%, P < 0.05) and of tandem double CC-->TT events at mutational hotspots (25% versus 9%, P < 0.05). Furthermore, a much greater fraction of SSL-induced mutations could be attributed to photoproducts on the non-transcribed strand of the aprt gene than was observed following treatment with 254 nm UV (94% versus 64%, P < 0.002). The general significance of these disparities between SSL and UVC mutagenesis at the aprt locus remains to be established, underscoring the need for further investigations on the effects of solar light in mammalian cells.

Adenine Phosphoribosyltransferase↗