Search PubMed⌕ Search

Biomedical subjects

A Sarasin

Publications and source records attributed to A Sarasin.

At least 91 records · Page 5Linked to original sources
PubMed1997

Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes.

Trichothiodistrophy (TTD), xeroderma pigmentosum (XP), and Cockayne's syndrome (CS) are three distinct human diseases with sensitivity to ultraviolet (UV) radiation affected by mutations in genes involved in nucleotide excision repair (NER). Among the many responses of human cells to UV irradiation, both nuclear accumulation of p53, a tumor suppressor protein, and alterations in cell-cycle checkpoints play crucial roles. The purpose of this study was to define the signals transmitted after UV-C-induced DNA damage, which activates p53 accumulation in TTD/XP-D fibroblasts, and compare this with XP-D cell lines that carry different mutations in the same gene, XPD. Our results showed that p53 was rapidly induced in the nuclei of TTD/XP-D and XP-D fibroblasts in a dose-dependent manner after UV-C irradiation, as seen in XP-A and CS-A fibroblasts, much lower doses being required for the protein accumulation than in normal human fibroblasts, XP variant cells, and XP-C cells. The kinetics of accumulation of p53 and two effector proteins involved in cell-cycle arrest, WAF1 and GADD45, were also directly related to the repair potential of the cells, as in normal human fibroblasts their levels declined after 24 h, the time required for repair of UV-induced lesions, whereas NER-deficient TTD/XP-D cells showed p53, WAF1, and GADD45 accumulation for over 72 h after irradiation. Our results indicate that p53 accumulation followed by transcriptional activation of genes implicated in growth arrest is triggered in TTD/XP-D cells by the persistence of cyclobutane pyrimidine dimers, which are known to block transcription, on the transcribed strands of active genes.

Adolescent↗
PubMed1996

Long-term complementation of DNA repair deficient human primary fibroblasts by retroviral transduction of the XPD gene.

Due to their limited life time in culture and their relative resistance to DNA transfection, primary fibroblasts derived from UV-hypersensitive patients could not be used for cloning DNA repair gene and studying stable complementation with wild-type DNA repair genes. Primary cells were only used for complementation analysis after transient expression through cell fusion. DNA microinjection and transfection. We report the retroviral-mediated highly efficient transfer and stable expression of XPD/ERCC2 gene in fibroblast strains from eight different patients using the LXPDSN retroviral vector. Cells derived from skin biopsies of xeroderma pigmentosum and trichothiodystrophy patients were incubated with vector-containing suspension and selected with the neomycin-analog G418. LXPDSN vector specifically complemented cells belonging to the XP-D group. Long-term reversion of repair-deficient phenotype, monitored by UV survival and UDS analysis, has been achieved in these diploid fibroblasts. We demonstrate this methodology is a powerful tool to study phenotypic reversion of nucleotide excision repair-deficient cells such as cellular DNA repair properties and we suggest that it may be used to study other cellular parameters (cell cycle regulation, p53 stability or immunosurveillance-controlling factors) involved in UV-induced skin cancers and which reliability requires the use of untransformed cells.

Cell Line↗
PubMed1996

Use of an infectious Simian virus 40-based shuttle vector to analyse UV-induced mutagenesis in monkey cells.

SV40 based shuttle vectors able to be packaged as pseudovirions have been used either as naked DNA or as pseudovirus to analyse the mutation frequency and the UV-induced mutation spectra obtained after transfection or infection of COS7 monkey cells. The frequency of supF spontaneous mutants was similar whatever the state of the vector, indicating that the transfection step is not responsible for the high spontaneous mutation frequency when using shuttle vectors. Nevertheless the UV-induced mutation frequency of the supF gene was higher when transfected DNA was replicated into COS7 cells than when pseudovirus infection was performed. The UV induced mutation spectra was basically similar in both situations but a new hot-spot at nucleotide 110 was obtained after pseudovirus infection. UV-pretreated and control COS7 cells were infected with untreated or UV-damaged pi SVPC7 shuttle virus and the survival and the supF mutation frequency were analysed in the progeny. The survival of UV-damaged pseudovirus replicated in 10 J/m2 UV-pretreated cells was 2-fold higher than in untreated cells. This increase in the survival was accompanied by a slight enhancement in the number of supF mutants.

Animals↗
PubMed1996

Recovery of normal DNA repair and mutagenesis in trichothiodystrophy cells after transduction of the XPD human gene.

To determine whether expression of the XPD/ERCC2 repair gene in trichothiodystrophy (TTD) group D cells could restore mutagenesis characteristics of repair-proficient cells, we compared the UV mutagenesis of normal cells, TTD group D cells, and TTD group D cells retrovirally transduced by the wild-type XPD/ERCC2 gene (TTD + ERCC2 cells). We first verified the expression of the XPD protein, correction of UV cell survival, and DNA repair ability of TTD + ERCC2 cells. UV-induced mutations were studied using the pR2 shuttle vector. The addition of the XPD/ERCC2 gene in TTD cells led to a significant but partial decrease of mutation frequency compared with the parental TTD cells. Types of mutations of TTD + ERCC2 cells get closer to those observed in normal cells (ie., a reduction of multiple mutations). New hotspots appeared and some disappeared in the complemented line, suggesting that hotspot distribution is particular to each cell line and cannot be correlated with the repair status of the cells. In conclusion, the expression of the XPD/ERCC2 repair gene completely corrected UV hypersensitivity and almost all types of mutations of TTD group D cells, whereas hypermutagenesis was partially corrected.

Cell Line, Transformed↗
PubMed1996

Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells.

The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form. A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells. After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced. Our results show clearly that both lesions are mutagenic, but at different levels. Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T. With the T(6-4)T a large variety of mutations were observed. A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.

Animals↗
PubMed1996

Cell survival and shuttle vector mutagenesis induced by ultraviolet A and ultraviolet B radiation in a human cell line.

Although it is known that sunlight is carcinogenic,few molecular data are available concerning the mutagenic effects of ultraviolet (UV) B (290-320 nm) and UVA (320-400 nm) radiation in human cells. To analyze the biologic effects of UVA and UVB, we irradiated the 293 human cell line, derived from adenovirus-transformed human embryonic kidney cells, in which we had stably introduced a shuttle vector harboring the lacZ' bacterial gene as the mutagenesis target. Identical cell survival occurred after UVA doses 700-fold higher than UVB. This comparable to the UVA/UVB ratio that reaches the basal cell layer of the skin after sunlight exposure with UVB sunscreen. The frequency of UVA- and UVB- induced mutations increased with the UV dose as cell survival decreased. At cell survival levels greater than 10%, UVA and UVB induced similar frequencies of mutations in the episomal lacZ gene, whereas for cell survival lower than 10%, UVA induced twice as many mutations as UVB. Sequence analysis of 81 independent lacZ mutants (36 UVA- and 45 UVB-induced) revealed specific characteristics for some UV-induced-mutations, particularly for UVB. Mutations at A/T base pairs were induced more frequently by UVA than by UVB. The UVA-induced mutation spectrum that we have observed in human cells may help help to elucidate the mechanism of skin carcinogenesis.

Amino Acid Sequence↗
PubMed1996

Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and growth retardation. Clinical photosensitivity is present in approximately 50% of TTD patients but is not associated with an elevated frequency of cancers. Previous complementation studies show that the photosensitivity in nearly all of the studied patients is due to a defect in the same genetic locus that underlies the cancer-prone genetic disorder xeroderma pigmentosum group D (XP-D). Nucleotide-sequence analysis of the ERCC2 cDNA from three TTD cell strains (TTD1V1, TTD3VI, and TTD1RO) revealed mutations within the region from amino acid 713-730 and within previously identified helicase functional domains. The various clinical presentations and DNA repair characteristics of the cell strains can be correlated with the particular mutations found in the ERCC2 locus. Mutations of Arg658 to either His or Cys correlate with TTD cell strains with intermediate UV-sensitivity, mutation of Arg722 to Trp correlates with highly UV-sensitive TTD cell strains, and mutation of Arg683 to Trp correlates with XP-D. Alleles with mutation of Arg616 to Pro or with the combined mutation of Leu461 to Val and deletion of 716-730 are found in both XP-D and TTD cell strains.

Alleles↗
PubMed1996

[Xeroderma pigmentosum. A study in 40 Algerian patients].

INTRODUCTION: Xeroderma pigmentosum (XP) is a rate autosomal recessive disorder related to DNA repair defects. Recently, modifications of oncogenes and mutations of the p53 suppressor gene have been reported in skin tumors of XP patients. The purpose is to study, through a series of 40 patients admitted to the Dermatologic Clinic of Algiers, the characteristics of XP in Algeria. PATIENTS AND METHODS: For each patient, familiarity, clinical and biological examinations and therapeutic results were studied. Biological studies have been axed mainly on analysis of DNA extracted from skin tumors of 18 patients to detect oncogene modifications by Southern blot and hybridization. A technic, based on single strand DNA conformation polymorphism (SSCP), has been carried out to detect rapidly mutations on the p53 gene. RESULTS: A consanguinity in the first degree is noted in 95 p. 100 of cases and a familiarity in 63 p. 100 of cases. The median age of patients is 10 years; sex ratio is close to one; 32 patients (80 p. 100) are classic XP and 8 (20 p. 100) are XP variant. In 18 tumors analysed, the Ha-ras gene is amplified and/or modified in 50 p. 100 of cases. Only 3 tumors (16.6 p. 100) show mutations of the p53 gene (transitions C-T). Surgical treatment isolated or associated to polychemotherapy permitted to resolve tumors in 75 p. 100 of cases. DISCUSSION: In Algeria, XP are mainly classic with a particularly high frequency of occular (62 p. 100) and neurological manifestations (62 p. 100). Genetic studies confirm modifications of the Haras gene in direct relation with unrepaired UV lesions in classic XP and mutations of the p53 tumor suppressor gene characteristic of mutation spectra induced by UV. Surgery is the treatment of choice for tumors; polychemotherapy is an alternative in advanced cases.

Adolescent↗
PubMed1995

Characteristics of UV-induced mutation spectra in human XP-D/ERCC2 gene-mutated xeroderma pigmentosum and trichothiodystrophy cells.

To study the relationships between mutagenesis and carcinogenesis, we compared the mutations and their frequency induced by ultraviolet irradiation at 254 nm (UV-C) in XP-D (GM-08207B/XP6BE), TTD/XP-D (TTD1VI-LAS-KMT11) and wild-type (MRC-5V1) human cells. XP-D and TTD/XP-D cells, mutated in the same XP-D/ERCC2 gene, are deficient in nucleotide excision repair. Whereas XP-D patients develop early skin tumors, TTD patients do not exhibit abnormal levels of cancers. After verification of UV hypersensitivity and DNA repair defect of the immortalized cell lines XP-D and TTD compared with a wild-type cell line, UV-induced mutagenesis was studied with a new shuttle vector pR2, carrying the target lacZ' gene. The UV-mutation frequencies in XP-D and TTD cells were similar and significantly increased compared with normal cells. Sequence analysis of 312 independent mutant plasmids revealed that more rearrangements were induced in TTD cells (16%) than in XP-D (5%) and normal cells (1%), while XP-D cells exhibited a twofold higher rate of tandem mutations compared with TTD and normal cells. In the three cell lines, a predominance of G:C to A:T transitions was found, especially in chiefly on the cytosine at 5'-TC-3' sites. The types of UV-induced point mutations in TTD cells were, however, more similar to those in normal cells than those found in XP-D cells. XP-D mutations were preferentially located in 5'-TCPur-3' sites, while mutations in normal and TTD cells were mostly at 5'-TCC-3' sites. Analysis of mutation spectra revealed differences in the location of the mutational hotspots between the three lines. Although the mutation frequency of the UV-irradiated pR2 vector is much higher in TTD and XP-D cells than in normal cells, the mutation spectrum is closer between TTD and normal cells as compared with XP-D cells. These dissimilarities could contribute to an explanation of some of the differences between the two syndromes.

Amino Acid Sequence↗
PubMed1995

[Genetic instability and cancer].

Multiple mutations are present in most human tumours. These genetic modifications appear to be necessary to produce and select pretumoral, tumoral and metastatic clones. These multiple mutations can be sequentially produced by genotoxic agents leading to cancers with a long latency period or due to mutations in genes implicated in the control of the genetic stability. Several human diseases are linked with a very high cancer incidence because they are hereditarily carrying a mutation on some DNA repair genes, mismatch repair genes or tumour suppressor genes. These abnormal functions will produced a mutator phenotype leading to a cascade of genetic modifications which will allow the rapid production of cancer cells.

Chromosome Fragility↗
PubMed1995

The specificity of p53 mutation spectra in sunlight induced human cancers.

Ultraviolet (UV) irradiation emitted by the sun has been clearly implicated as a major carcinogen in the formation of skin cancers in man. Indeed, the high levels of cutaneous tumors in xeroderma pigmentosum patients (XP) who are deficient in repair of UV-induced lesions have confirmed that DNA damage produced by sunlight is directly involved in the cancer development. The tumor suppressor gene, p53, very frequently found modified in human cancers, has proved to be a perfect target gene for correlating mutation spectra with different cancer causing agents as there are nearly 300 potential mutation sites available for analysis. In a comparative analysis of p53 mutations found in internal cancers with those in skin tumours we show here that clear differences exist between the types of spectra obtained. The specificity of UV induced mutations in skin cancers is confirmed when single and tandem mutations are compared. Most of the p53 point mutations found are GC to AT transitions both in skin and internal tumors where in the latter they are located mainly at CpG sequences probably due to the deamination of the unstable 5-MeC. Moreover, mutations are targeted at py-py sequences in over 90% of skin tumors whereas in internal cancers the distribution is proportional to the frequency of bipyrimidine sequences in the p53 gene. Most significantly, all mutations found in XP skin tumors are targeted at py-py sites and more than 50% are tandem CC to TT transitions considered as veritable signatures of UV-induced lesions. Tandem mutations are also relatively common (14%) in skin tumors from normal individuals compared to their very rare occurrence in internal malignancies (0.8%). Finally, nearly all mutations observed in XP skin tumors are due to unrepaired lesions remaining on the coding strand whereas no strand bias is seen in mutation location of internal or skin tumors from normal individuals. In fact the mutation spectrum analysed in XP skin cancers has permitted the first demonstration of the existence of preferential repair in man. In conclusion, using the p53 gene as a probe it is obvious that the mutation spectra from skin tumors are very similar to those observed in UV-treated gene targets in model systems but statistically different from those described in other types of human cancer. This has allowed us to demonstrate, without ambiguity, the major role of UV-induced DNA lesions in sunlight related skin carcinogenesis.

Animals↗
PubMed1995

Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by an increased frequency of skin cancer following minimal sunlight exposure. Cells isolated from XP patients are also hypersensitive to UV rays and UV-like chemicals. This sensitivity is directly related to a defect in the early steps of nucleotide excision repair (NER) of damaged DNA. No efficient treatment is available for this disease and skin cancer prevention can only be achieved by strict avoidance of sunlight exposure. Thus, we are developing a model for gene therapy in XP, particularly for patients belonging to group D. We report here the construction of a retroviral vector (LXPDSN) containing the XPD (ERCC2) cDNA, which fully complements the DNA repair deficiency of primary skin fibroblasts. Efficient integration, mRNA synthesis, and protein expression of the XPD gene were obtained in all LXPDSN-transduced XP-D fibroblasts tested. Full correction of the DNA repair defect was observed with all DNA repair assays used, such as an increased survival after UV-radiation of the transduced cells, a normal level of DNA repair synthesis (UDS), and the reactivation of a UV-irradiated reporter vector. This retroviral vector will be used to modify keratinocytes genetically to produce repair proficient reconstituted skin for engraftment to XP patients.

Cell Survival↗
PubMed1995

Mutagenicity of a unique 8-oxoguanine in a human Ha-ras sequence in mammalian cells.

The processing of a unique 8-oxoguanine residue in DNA has been studied in mammalian cells using a single-stranded shuttle vector. A fragment of human Ha-ras carrying the lesion on the first (G1) or the second guanine (G2) of codon 12 was inserted in a shuttle plasmid. Extrachromosomal DNA is replicated in animal cells, extracted and used to transform bacteria to be amplified and individualized. DNA sequencing of bacterial clones showed the mutagenic potency of 8-oxoguanine in vivo to be approximately 4%. The presence of the 8-oxoguanine does not greatly affect survival of the progeny. No significant difference was observed between the mutation frequencies induced by 8-oxoguanine located either at the G1 or G2 position. The majority of the mutations, targeted at the lesion level, are G to T transversions. These base substitutions induced respectively glycine to cysteine (G1) or valine (G2) change in the P21ras protein. These mutations may contribute to activation of the protooncogene, leading to spontaneous tumorigenesis.

Animals↗
PubMed1995

Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes.

Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.

Biopsy↗
PubMed1994

Coding properties of a unique apurinic/apyrimidinic site replicated in mammalian cells.

The coding properties of abasic sites have been studied in mammalian cells using a single-stranded shuttle vector carrying a unique abasic site. The probe was produced by digestion with the uracil DNA glycosylase of a uracil-containing oligonucleotide which was inserted in the single-stranded vector. After replication in monkey COS7 cells able to support DNA replication of the vector, the plasmid progeny were isolated in bacteria. DNA sequencing of rescued plasmids showed that replication of abasic sites does not lead to preferential insertion of a given base opposite the non-coding site. The four bases were inserted with a frequency which was not statistically different from a random distribution. It appears therefore that the "A rule insertion" opposite a unique abasic site does not apply, at least with the sequence we used, for an exogenous single-stranded DNA replicated in mammalian cells. It was not necessary to induce SOS-like conditions by pretreatment of host cells, in order to replicate abasic sites.

Animals↗