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Biomedical subjects

A Sarasin

Publications and source records attributed to A Sarasin.

At least 73 records · Page 4Linked to original sources
PubMed1997

Photocarcinogenesis and inhibition of intercellular adhesion molecule 1 expression in cells of DNA-repair-defective individuals.

Cells from patients with xeroderma pigmentosum complementation group D (XP-D) and most patients with trichothiodystrophy (TTD) are deficient in excision repair of ultraviolet (UV) radiation-induced DNA damage. Although in both syndromes this defect is based on mutations in the same gene, XPD, only XP-D, not TTD, individuals have an increased risk of skin cancer. Since the reduction in DNA repair capacity is similar in XP-D and TTD patients, it cannot account for the difference in skin cancer risk. The features of XP-D and TTD might therefore be attributable to differences in the immune response following UV-irradiation, a factor which is presumed to be important for photocarcinogenesis. We have measured the capacity of UVB radiation to inhibit expression of the immunological key molecule intercellular adhesion molecule 1 (ICAM-1) in cells from three healthy individuals in comparison to cells from three XP-D and three TTD patients. Cells from XP-D patients, but not from TTD patients, exhibited an increased susceptibility to UVB radiation-induced inhibition of ICAM-1 expression. Transfection of XP-D cells with the wild-type XPD cDNA, but not with XPC cDNA, corrected this abnormal phenotype. Thus, the skin cancer risk in DNA repair-defective individuals correlated with the susceptibility of their cells to UVB radiation-induced inhibition of ICAM-1 expression, rather than with their defect in DNA repair. The XPD protein has dual roles: in DNA repair and transcription. The transcriptional role might be important for the control of expression of immunologically relevant genes and thereby contribute to the skin cancer risk of a DNA-repair-deficient individual.

Cells, Cultured↗
PubMed1997

Differential replication of a single N-2-acetylaminofluorene lesion in the leading or lagging strand DNA in a human cell extract.

DNA replication in eucaryotic cells is a complex process involving a variety of proteins that synthesize the leading and lagging strand in an asymmetric, coordinated manner. To investigate the effect of this asymmetry on the translesion synthesis of bulky lesions, we have constructed SV40 origin-containing plasmids with site-specific N-2-acetylaminofluorene adduct on either leading or lagging strand templates. These plasmids have been incubated with DNA replication-competent extracts made from human HeLa cells. Two-dimensional agarose gel electrophoresis analyses reveal a strong blockage of fork progression only when the N-2-acetylaminofluorene adduct is located on the leading strand template. Morever, the analysis revealed that replication with HeLa cell extracts of SV40 origin-dependent plasmids functions in both directions from the origin with equal efficiency but, probably due to an important asynchrony at the formation of the two forks, proceeds unidirectionally for a large number of individual molecules. The validity of the in vitro replication approach to study the fidelity of both leading- and lagging strand synthesis is discussed with regard to these new data.

2-Acetylaminofluorene↗
PubMed1997

Oncogenic rearrangements of the ret proto-oncogene in thyroid tumors induced after exposure to ionizing radiation.

A high frequency (approximately 60%) of ret rearrangements in Chernobyl papillary thyroid carcinomas (PTC) has been reported recently. The data suggested that the radiation exposure may be a direct inducer of activating rearrangements in the ret gene. In our study, we have analyzed for the presence of RET/PTC oncogenes using the RT-PCR, XL-PCR, Southern blot and direct sequencing techniques, 39 human thyroid tumors from patients who had received external radiation for benign or malignant conditions. As controls, we studied 39 'spontaneous' tumors. Our results indicate that: 1) the overall frequency of ret rearrangements was 84% in papillary carcinomas (16/19) and 45% (9/20) in follicular adenomas; 2) in contrast with the results obtained in the Chernobyl tumors, the most frequently observed chimeric gene was RET/PTC1; and 3) all the tumors were negative for RET/PTC2. In the 'spontaneous' tumors, only the papillary carcinomas presented a ret rearrangement (15%: 3/20). Our data confirm the crucial role played by the ret proto-oncogene activating rearrangements in the development of radiation-associated thyroid tumors, and show, for the first time, the presence of RET/PTC genes in follicular adenomas appeared after external irradiation.

Adenoma↗
PubMed1997

Retroviral-mediated correction of DNA repair defect in xeroderma pigmentosum cells is associated with recovery of catalase activity.

Xeroderma pigmentosum (XP) is a rare inherited disease associated with photosensitivity, a very high susceptibility to develop neoplasm on sun-exposed skin and neurological abnormalities for some patients. We previously reported that diploid cell lines established from XP skin biopsies present an abnormal low level of catalase activity, which is involved in the defense against oxygen free radicals. This biochemical dysfunction, probably involved in the skin cancer formation, has been difficult to be directly related to the nucleotide excision repair (NER) defect in XP. In this paper we report that the retroviral-mediated transduction of XP diploid cells by the XPC and XPD/ERCC2 cDNAs fully and stably corrects the NER defect in terms of survival and unscheduled DNA synthesis (UDS) after ultraviolet (UV) irradiation. The catalase activity in transduced cells was recovered up to normal levels only in cells transduced with repair genes correcting the repair defect. These results imply that: (i) the reduced catalase activity in XP, which might result from cellular depletion of its NADPH cofactor, is directly related to impaired DNA repair, and (ii) this depletion might be one of the multiple cellular consequences of XP inborn defect.

Catalase↗
PubMed1997

Retrovirus-mediated gene transfer corrects DNA repair defect of xeroderma pigmentosum cells of complementation groups A, B and C.

With the aim to devise a long-term gene therapy protocol for skin cancers in individuals affected by the inherited autosomal recessive xeroderma pigmentosum we transferred the human DNA repair XPA, XPB/ERCC3 and XPC cDNAs, by using the recombinant retroviral vector LXSN, into primary and immortalized fibroblasts obtained from two XP-A, one XP-B (associated with Cockayne's syndrome) and two XP-C patients. After transduction, the complete correction of DNA repair deficiency and functional expression of the transgenes were monitored by UV survival, unscheduled DNA synthesis and recovery of RNA synthesis, and Western blots. The results show that the recombinant retroviruses are highly efficient vectors to transfer and stably express the human DNA repair genes in XP cells and correct the defect of DNA repair of group A, B and C. With our previous results with XPD/ERCC2, the present work extends further promising issues for the gene therapy strategy for most patients suffering from this cancer-prone syndrome.

Blotting, Western↗
PubMed1997

High prevalence of activating ret proto-oncogene rearrangements, in thyroid tumors from patients who had received external radiation.

A high frequency (about 60%) of ret rearrangements in papillary thyroid carcinomas of children exposed to radioactive fallout in Belarus after the Chernobyl accident, has been reported by three recent studies (Fugazzola et al., 1995; Ito et al., 1994; Klugbauer et al., 1995). These studies suggested that the radiation exposure may be a direct inducer of activating rearrangements in the ret gene. In order to confirm the postulated link between irradiation and the role of the ret proto-oncogene in thyroid tumorigenesis, we analysed for the presence of ret activating rearrangements using RT-PCR, XL-PCR, Southern blot and direct sequencing techniques, 39 human thyroid tumors (19 papillary carcinomas and 20 follicular adenomas), from patients who had received external radiation for benign or malignant conditions. As controls, we studied 39 'spontaneous' tumors (20 papillary carcinomas and 19 follicular adenomas). Our data concerning the radiation-associated tumors, showed that: (1) the overall frequency of ret rearrangements was 84% in papillary carcinomas (16/19) and 45% (9/20) in follicular adenomas; (2) in contrast with the results obtained in the Chernobyl tumors, the most frequently observed chimeric gene was RET/PTC1 instead of the RET/PTC3 and (3) all the tumors were negative for RET/PTC2. In the 'spontaneous' tumors, only the papillary carcinomas presented a ret rearrangement (15%:3/20): 1 RET/PTC1, 1 RET/ PTC3 and 1 uncharacterized. In conclusion, our results confirm the crucial role played by the ret proto-oncogene activating rearrangements in the development of radiation-associated thyroid tumors appearing after therapeutic or accidental ionizing irradiation, and show, for the first time, the presence of RET/PTC genes in follicular adenomas appeared after external irradiation.

Adenoma↗
PubMed1997

Activation of the NADPH oxidase in human fibroblasts by mechanical intrusion of a single cell with an ultramicroelectrode.

We present here a real-time and single cell study of an oxidative stress mechanism in human fibroblasts. Hydrogen peroxide released by a single normal or SV40-transformed human fibroblast was detected at the surface of an ultramicroelectrode while puncturing the cell membrane with the ultramicroelectrode tip itself or with a micropipette. This mechanical intrusion induced the emission of large quantities (10(-15)-10(-14) mol) of H2O2 by the cell with a very short time delay (<0.5 s). We show that this H2O2 production was an active neo-production by fibroblasts when the membrane was stressed by the cellular puncture and is a model which could mimic similar effects as particle (virus, bacteria, etc.) intrusion into the cell. Cell incubations in the presence of some inhibitors of the different NADPH oxidase enzymes, using ultramicroelectrode measurements of the short time effects (<20 min) let us believe that an NADPH oxidase-like enzyme may be implicated in this induced-H2O2 generation. Phenylarsine oxide (PAO), a specific NADPH oxidase inhibitor, at concentrations between 0.5-50 microM seemed to quickly kill the transformed cells preferentially to the normal cells, pointing out for the future a possible anti-cancerous chemotherapic use.

Antioxidants↗
PubMed1997

The role of UV-B light in skin carcinogenesis through the analysis of p53 mutations in squamous cell carcinomas of hairless mice.

Mutation spectra of the p53 gene from human skin carcinomas have been connected to solar UV radiation. For comparison we have characterized the mutation spectrum of the p53 gene in a very large sample of squamous cell carcinomas from hairless mice induced with UV of wavelength 280-320 nm (UV-B), which have substantiated the mutagenic effects of UV-B radiation in vivo. Tumors from hairless mice, random bred SKH:HR1 as well as inbred SKH:HRA strains, which are analyzed for mutations in the conserved domains of the p53 protein present a very specific mutation spectrum. The observed mutation frequency after chronic UV-B radiation in the p53 gene ranged from 54% (SKH-HRA) to 73% (SKH-HR1) among the 160 tumors analyzed. Over 95% of the mutations were found at dipyrimidine sites located in the non-transcribed strand, the majority were C-->T transitions and 5% were CC-->TT tandem double mutations. Four distinct UV-B mutation hot spots have been identified for the first time: two major ones at codons 267 (33%) and 272 (19%) and two minor ones at codons 146 (10%) and 173 (4%). The codon 267 hot spot consists of a CpG preceded by a pyrimidine, which confirms in vivo an important role for this UV-B mutable site in UV-B-induced skin tumors that is not found in other types of mouse tumors. Comparison with mutation spectra from human skin carcinomas fully validates the merits of the hairless mouse model for studying the molecular mechanisms of skin carcinogenesis. For example, the murine hot spot at codon 272 does have a full equivalent in human skin carcinomas. In contrast, the human equivalent of the murine codon 267 lacks the dipyrimidine site and therefore fails to be a pronounced hot spot in human skin carcinomas; however, this site is one of the major hot spots in human internal cancers (evidently not induced by UV radiation but probably by deamination of the 5 methyl cytosine).

Animals↗
PubMed1997

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. The use of the alkaline comet assay (single cell gel electrophoresis assay) is proposed as a simple repair test for earlier prenatal diagnosis. Amniotic or chorionic villus cells in two pregnancies at risk for XP and one for TTD were examined in comparison with skin fibroblasts of family members or with repair-proficient or -deficient control cells. The comet assay and the UDS test were performed in parallel. In repair-proficient cells, DNA strand breaks due to the incision of UV-induced DNA damage result in increased migration of high molecular weight DNA in the comet assay. Fetal cells demonstrate repair capacity similar to that of fibroblasts. In incision repair-deficient XP and TTD cells, after post-UV incubation, migration does not occur and comet moments are reduced. Two fetuses belonging to two XP families responded normally and were diagnosed as unaffected. Fetal cells in a TTD family had reduced comet moments and a low UDS. This fetus was diagnosed and confirmed later as affected. Heterozygotes had normal responses with both assays. The comet assay offers discrimination similar to that of the UDS assay in identifying NER-deficient phenotypes. Practical advantages in view of prenatal diagnosis include the reduced number of cells required, a 24-h delay in obtaining results, and no need for radioactivity.

Cells, Cultured↗
PubMed1997

Transient sex differences of aromatase (CYP19) mRNA expression in the developing rat brain.

Sex differences in the activity of aromatase cytochrome P450 (CYP19) in the rat brain have been reported during pre- and postnatal development. It is unclear, however, whether these differences are reflected by corresponding differences in specific mRNA levels. To address this question, we have examined aromatase mRNA levels in specific regions of male and female rat brains by means of in situ hybridization (ISH). At prenatal stages of development, i.e. at gestational day 18 (GD18) and GD20, aromatase mRNA was detected in several preoptic, hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal sex differences in any of these regions. In contrast, clear-cut sex differences were determined at postnatal day (PN) 2; male animals expressed significantly more aromatase mRNA in the bed nucleus of stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN). Smaller but still significant differences (females > males) were obtained in the medial preoptic area (MPO). At PN6, sex differences of aromatase mRNA signals (males > females) were still present in the BST, but were no longer detectable in the SDN and the MPO. At PN15 and in adult animals, aromatase mRNA levels were similar in BST and medical amygdaloid nucleus of male and female rats. Since aromatase mRNA expression decreases during postnatal development, no ISH signals could be detected anymore in MPO, SDN and ventromedial hypothalamic nucleus. Our results are consistent with the concept that differential regulation of aromatase mRNA expression might be important for the establishment of different neuronal circuitry in male and female animals.

Animals↗
PubMed1997

The consequence of translesional replication of unique UV-induced photoproducts.

The consequence of translesional replication of unique UV-induced photoproducts is reviewed here. Mutagenesis induced by unique UV-induced lesions, the thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4), [T(6-4)T] carried on single-stranded DNA vectors and replicated in bacteria, yeast and mammalian cells have been considered. It has been found that in all of the three species the (TT) dimers induce a low mutation frequency compared to the (6-4) photoproduct. The molecular analysis of the mutations induced is reported, showing specific differences depending on the species considered.

Animals↗
PubMed1997

A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in approximately 50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. Besides XPD and TTDA, the XPB gene product is also part of TFIIH. To date, three patients with the remarkable conjunction of XP and CS but not TTD have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein completely conserved in yeast, Drosophila, mouse, and man. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of "transcription syndromes."

Adolescent↗
PubMed1997

Quantitative detection of ultraviolet-specific p53 mutations in normal skin from Japanese patients.

We have previously developed sensitive methods to detect UV-specific p53 mutations (CC to TT tandem mutations) and have reported that such mutations could be found in the normal skin cell populations of sun-exposed body sites, but not in those of covered sites, in Australian cancer patients. We have now further refined our allele-specific PCR method for detecting CC to TT mutations at codons 247/248 of the p53 gene to allow quantitative measurements. Using DNA containing this mutation from a tumor as a standard for calibration and 5 micrograms of genomic DNA/PCR reaction, we could detect 1 mutant allele in about 10(6) wild-type alleles. It is essential to use purified primers and 64 degrees C as the annealing temperature for PCR. Our method has been applied in a study of the correlation of sun exposure and accumulation of CC to TT mutations in normal skin biopsies from Japanese patients. There were more p53 mutations in samples taken from sites that were chronically exposed to the sun than in those from covered sites. A significant trend of increased p53 mutation frequency with increase in age of subjects was found, suggesting the cumulative nature of the mutation. On the other hand, the p53 mutation frequency was higher in patients with premalignant tumors or nonmelanocytic skin cancer than in patients with only benign tumors. These results confirm the utility of PCR-based p53 gene mutation assays for the measurement of exposure to UV as well as for predicting the risk of UV-associated skin cancer.

Adult↗
PubMed1997

[DNA lesions: mechanisms of recognition and repair].

Numerous structural lesions or disorders accumulate in DNA either spontaneously or following genotoxic stresses. Living cells have developed various strategies to eliminate most of these lesions so as to preserve a life-compatible genetic information. Several human diseases are associated to some DNA-repair processes. Analysis of cells derived from these patients has allowed the isolation of repair genes and the molecular analysis of lesions proofreading mechanisms. Most of these diseases are extremely severe, pointing out to the biological extent of repair systems, and are associated to genetic instability which frequently lead to high cancer proneness.

Cockayne Syndrome↗
PubMed1997

Human cancer and DNA repair-deficient diseases.

Cancer development requires the accumulation of numerous genetic changes which are usually believed to occur through the presence of unrepaired DNA lesions. Exogenous or endogenous DNA-damaging agents can lead to mutations in the absence of efficient error-free repair, via replication of DNA damage. Several DNA repair pathways are present in living cells and well conserved from bacteria to human cells. Apart from mismatch repair, photolyases, base excision, and postreplication repair, the nucleotide excision repair (NER), the most versatile of these DNA repair systems, recognizes and eliminates a wide variety of DNA lesions and particularly those induced by ultraviolet (UV) light. The phenotypic consequences of an NER defect in humans are apparent in rare but dramatic diseases characterized by hypersensitivity to UV and a striking clinical and genetic heterogeneity. The xeroderma pigmentosum syndrome (XP), the Cockayne's syndrome (CS), and the photosensitive form of trichothiodystrophy (TTD) are three of these clinically distinct human disorders inherited as an autosomal recessive trait. Persistence of unrepaired DNA damage produced by exposure to UV light is associated, in the XP syndrome, with an extremely high level of skin tumors in sun-exposed sites. But the direct link of defective DNA repair to cancer seems to be complex, since, in contrast to patients with XP, those with TTD or CS do not have an increased frequency of skin cancers. The understanding of the absence of skin tumors in TTD and CS patients may offer a way to better protect normal individuals from the most rapidly increasing cancer: skin cancer.

Cockayne Syndrome↗
PubMed1997

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain.

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.

Animals↗