Biomedical subjects

A Gentil

Publications and source records attributed to A Gentil.

At least 19 recordsLinked to original sources

PubMed2000

Mutation spectra induced by replication of two vicinal oxidative DNA lesions in mammalian cells.

Ionizing radiations often induce multiple and clustered DNA lesions at the site of DNA interaction. As a model, we have studied the toxicity and the mutagenicity of two adjacent oxidative bases as clustered DNA lesions in mammalian cells using shuttle vectors. The chosen oxidative lesions were 8-oxo-7,8-dihydroguanine, the formylamine residue resulting from the oxidation of a pyrimidine base and the tandem lesion 8-oxo-7,8-dihydroguanine/formylamine where both modifications are located at a vicinal position. A single-stranded DNA shuttle vector carrying a unique DNA lesion was constructed, transfected into simian COS7 cells and mutations induced after replication in mammalian cells were screened in bacteria. 8-oxo-7,8-dihydroguanine, as expected, does not affect greatly survival (70% bypass) whereas formylamine and the tandem lesions are blocking alterations, DNA polymerase bypass being of 45% and 17%, respectively. Base insertion opposite the lesion was studied. Under our experimental conditions, replication of 8-oxo-7, 8-dihydroguanine finally gives rise to guanine:cytosine pairing, rendering this lesion only slightly mutagenic. This is not the case for the formylamine that codes preferentially for adenine (71%). In addition, one-base deletions were observed targeted to the site to the lesion. Cytosine and thymine were inserted opposite the lesion with similar but low frequencies. Thus, coding properties of the formylamine render this residue very mutagenic when coming from the oxidative alteration of a cytosine. The coding properties of the tandem damage are a combination of the contribution of the two isolated lesions with a very high percentage of adenine insertion (94%) opposite the formylamine residue of the tandem lesion. The toxicity as well as the mutation spectrum of the tandem lesion allow us to speculate about the molecular mechanism with which the DNA polymerase replicates these two lesions.

Animals↗

PubMed2000

Transcription-coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome.

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

Cell Line↗

PubMed1998

Repair and mutagenic potency of 8-oxoG:A and 8-oxoG:C base pairs in mammalian cells.

Replication of the oxidative lesion 8-oxo-7,8-dihydroguanine (GO) leads to the formation of both 8-oxo-7,8-dihydroguanine:adenine (GO:A) and 8-oxo-7,8-di-hydroguanine:cytosine (GO:C) pairs. The repair and mutagenic potency of these two kinds of base pairs were studied in simian COS7 and human MRC5V1 cells using the shuttle vector technology. Shuttle vectors carrying a unique GO residue opposite either a C or an A were constructed, then transfected into recipient mammalian cells. DNA repair resulting in G:C pairs and mutation frequency, were determined using resistance to digestion by the Ngo MI restriction enzyme for screening and DNA sequencing of suspect mutants. Results showed that the GO:C mismatch was well repaired since almost no mutations were detected in the plasmid progeny obtained 72 h after cell transfection. The GO:A pair was poorly repaired since only 32-34% of the plasmid progeny contained G:C whereas two thirds contained A:T at the original site. Repair kinetics measured with a non-replicating vector deleted by 13 bp at the SV40 replication origin, showed that GO:A was slowly repaired. Only 30% of the mispairs were corrected in 12 h. During this time 100% of the plasmids containing GO:A pairs were replicated as seen by the replication kinetics in a vector with an intact SV40 replication origin. These results show that, under our experimental conditions, replication is occurring before completion of DNA repair which explains the high mutagenic potency of the GO:A mispair.

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↗

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↗

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↗

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↗

PubMed1994

Mutagenesis in monkey cells of a vector containing a single d(GPG) cis-diamminedichloroplatinum(II) adduct placed on codon 13 of the human H-ras proto-oncogene.

Cisplatin (cis-[Pt(NH3)2Cl2]) is a widely used antitumor agent whose mutagenic activity raises the possibility of the induction of secondary cancer as a result of treatment. Mutation of the proto-oncogene H-ras is found in more than 30% of all human tumors, where it has been postulated to contribute to the initiation and progression of human cancers. Activating mutations in the H-ras gene are predominantly single-base substitutions, most frequently at codons 12, 13 and 61. In the present work we have studied the mutational spectra induced by a single cis-[Pt(NH3)2d(GpG)] adduct, the most frequent DNA crosslink formed by cisplatin. We have constructed a 25-mer-Pt oligonucleotide singly modified at codon 13 (GGT) within the human H-ras DNA sequence and we have inserted it into a single-stranded SV40-based shuttle vector able to replicate in simian COS7 cells. After replication in the mammalian host, vectors were extracted, amplified in bacteria and DNA from 124 randomly chosen colonies was sequenced. The observed mutation frequency was 21%. Base substitutions were the most frequent modification. 92% of the mutagenic events occurred at one or both of the platinated guanines of codon 13. The single G-->T transversion accounted for 65% of the total mutations scored. All single base substitutions were located at the G in the 3' position showing, for the first time, that the guanine at the 3' side of a cis-[Pt(NH3)2d(GpG)] adduct may be a preferential site for cisplatin induced mutations. The substitution G-->T at this position of the codon 13 of the H-ras proto-oncogene is known to induce the oncogenic properties of the p21ras protein.

Animals↗

PubMed1993

Implication of uracil in spontaneous mutagenesis on a single-stranded shuttle vector replicated in mammalian cells.

Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues. In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations. The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay. Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations. While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E. coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30%. The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed. This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.

Animals↗

PubMed1992

Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells.

Abasic sites are common DNA lesions produced either spontaneously or as a consequence of the action of some genotoxic agent. The mutagenic properties of a unique abasic site replicated in mammalian cells have been studied using a shuttle vector. A plasmid, able to replicate both in mammalian cells and in bacteria, carrying a unique abasic site chemically synthesized has been constructed. After replication in mammalian cells, plasmid DNA was recovered and used to transform bacteria. Mutants were screened without selection pressure by differential hybridization with a labelled oligonucleotide and their DNA was sequenced. A mutation frequency ranging from 1% to 3% was found, depending on the base originally inserted during the vector construction, opposite the abasic site. All the sequenced mutants correspond to single base-pair substitutions targeted at the abasic site. We observed a deficit in guanine incorporation opposite the abasic site, while the three other bases were incorporated with a similar efficiency. The mutational potency of abasic sites was observed without any voluntary preconditioning treatment of mammalian cells in order to induce "SOS" like conditions.

Animals↗

PubMed1992

Mutagenesis by O6 meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells.

The first or/and the second guanines of the human Ha-ras codon 12 (normally GGC) were substituted by O6 meG residues and the modified sequence was subsequently introduced into an SV40-based shuttle vector able to replicate in both simian cells and bacteria. After replication in simian COS7 cells (proficient in O6-alkyl-guanine transferase), plasmid DNA was extracted and mutations were screened in E. coli DH5 alpha cells. The vast majority of the mutations induced by O6 meG were G----A transitions. The mutation frequency observed at the second guanine of codon 12 (12G2 position: 3.75% +/- 0.4) was higher than the one observed at the first guanine (12G1 position: 1.09% +/- 0.6). This difference was confirmed by the results obtained when two adjacent O6 meG residues were positioned within codon 12. The higher mutation frequency observed for the 12G2 position could be attributed to differential repair or/and variation in polymerase fidelity. These results are in agreement with animal experiments where alkylating agents gave rise to mutation on G2 position of codon 12.

Animals↗

PubMed1992

Mutation spectrum of heat-induced abasic sites on a single-stranded shuttle vector replicated in mammalian cells.

The mutational potency of apurinic/apyrimidinic (AP) sites induced by heat-treatment under acidic conditions has been studied in mammalian cells. Abasic sites were induced on a single-stranded DNA shuttle vector carrying the supF tRNA gene, eliminating, therefore, any ambiguity concerning the damaged strand. This vector was able to replicate both in mammalian cells and in bacteria where the mutations induced in animal cells on the supF tRNA gene were screened by the white/blue beta-galactosidase assay in the presence of isopropyl-1-thio-beta-D-galactopyranoside and 5-bromo-4-chloro-3-indoyl-beta-D-galactoside. All white colonies contained plasmid with a mutation on the target gene which was directly sequenced. Our results show that one AP site was induced/22 min of heating as measured by sensitivity of DNA to alkali denaturation or treatment with the AP-endonuclease activity of the FPG protein (Fapy-DNA glycosylase). Putative AP sites decrease survival of the plasmid with a lethal hit of one AP site/single-stranded molecule. Mutation frequency was increased by a factor of approximately six after 2 h at 70 degrees C. Most of the induced mutations were point mutations not distributed at random and clustered in the gene region which will give rise to the mature tRNA. Mutations were abolished by treatments that eliminated AP sites such as alkali treatment or incubation with the Fapy-DNA glycosylase protein. Under our experimental conditions, when only single mutations were taken into account, the order of base insertion opposite AP sites was G greater than A greater than T greater than C.

Animals↗

PubMed1990

Mutagenic properties of a unique abasic site in mammalian cells.

The mutagenic properties of a true unique abasic site located opposite a guanine residue were studied. An oligonucleotide containing a chemically-produced abasic site was inserted into a shuttle vector able to replicate both in simian cells and in bacteria. Plasmid DNA was rescued from simian cells and screened in bacteria by differential hybridization with a labelled oligonucleotide probe. Mutations were easily detected and sequenced. Results showed that opposite a guanine the abasic site was error free repaired or replicated by mammalian cells with an efficiency of 99%. Point mutations occurred at a frequency of approximately 1% in control host cells and at more than 3% in UV-pre-irradiated host cells. Adenine, cytosine or thymine were found to have been inserted opposite the abasic site. No preferential insertion for a particular base was observed in contrast to that reported in bacteria.

Animals↗

PubMed1990

Mammalian cell processing of a unique uracil residue in simian virus 40 DNA.

The processing of a unique uracil in DNA has been studied in mammalian cells. A synthetic oligodeoxyribonucleotide carrying a potential Bgl II restriction site, where one base has been substituted with a uracil, was inserted in the early intron of SV40 genome. Various heteroduplexes were constructed in such a manner that the restitution of an active Bgl II restriction site corresponds in each case to the specific substitution of the uracil by one of the four bases normally present in the DNA. DNA cuts by this restriction enzyme in one or several constructed heteroduplexes immediately determine the type of base pair substitution produced at the site of the U residue. When the uracil is inserted opposite a purine it is fully repaired; when facing a guanine it is replaced by a cytosine and opposite an adenine it is replaced by a thymine. These results indicate the error-free repair of uracil when it appears in the cell with the usual mechanisms such as cytosine deamination or incorporation of dUTP in place of dTTP during replication. When the uracil is inserted opposite a pyrimidine no error free repair at all is detected for U:C or U:T mismatches. It appears, moreover, that in approximately 18% of the cases U:T mismatch leads to a C:G base pairing. In the majority of the U:pyrimidine mismatches, mutations occur in the vicinity of the uracil, including base substitutions and frameshifts by addition of one or several bases.

Animals↗

PubMed1990

Mechanisms and consequences of mutation induction in mammalian cells.

Mutations have been studied for several decades in order to understand biological processes of great significance and the selection of better-adapted species. Our knowledge both of mutation spectra induced by genotoxic agents and the mechanisms involved in DNA damage processing is more advanced in bacteria than in animal cells. However, the use of new technologies such as shuttle vectors or the polymerase chain reaction will undoubtedly allow rapid progress in the next few years. Shuttle vectors consist of target sequences for monitoring mutagenic activity and additional sequences permitting DNA replication and selection, both in bacteria and in mammalian cells. These plasmids are very efficient in allowing the production of mutation spectra of a particular genotoxin in animal cells. In most cases, base substitutions occur predominantly at the sites of base damage and the type of substitution depends on the kind of damage. This has been well characterized using ultraviolet (UV) light as a mutagen. UV-induced mutations are targeted opposite pyrimidine-pyrimidine sites, where the two major UV lesions are produced. The direct relationships existing between mutation and cancer are exemplified by some hereditary diseases where deficiency in an enzymatic repair system is linked to a high incidence of tumours. Similarly, activation of some cellular proto-oncogenes occurs via specific point mutations. A correlation does exist between the mutation spectra found in model systems and the specific mutation found in the activated oncogene in tumours induced by a given genotoxin. This is particularly well illustrated in the DNA repair deficiency syndrome, xeroderma pigmentosum. The specific mutations found in activated ras oncogenes isolated from UV-stimulated skin tumours correlate well with the mutagenic properties of unrepaired UV-induced DNA lesions.

Animals↗

PubMed1989

Carcinogen-induced mutagenesis in the simian virus 40 genome.

Here are reviewed the most interesting results which have been obtained with a mutational assay based on the use of Simian Virus 40 (SV40) as a biological probe. This mutational assay allowed us first to study the mutation potency of some chemical and physical DNA damaging agents such as acetoxy-acetylaminofluorene and UV-light and of apurinic sites created by heat treatment under acidic conditions, and second to study at the molecular level the modifications induced by these treatments. A correlation between the location of the DNA adducts and the location of the hot spots of mutagenesis has tentatively been researched. No direct link has been found. Our results suggest that mutation hot spots are correlated with local DNA conformations which could be modified by the DNA damaging agents.

Carcinogens↗

PubMed1987

Strategies to analyse mutagenesis in mammalian cells using simian virus 40 or shuttle vectors.

The use of exogeneous DNA probes, which replicate extrachromosomally, is proposed in order to study spontaneous and induced mutagenesis in mammalian cells. Simian virus 40 has already proved to be very useful, since it has provided much important information in this field. Recently, several shuttle vectors have been designed for this purpose; however, it seems that these molecules have high spontaneous mutation frequencies when replicating in mammalian cells. We have developed new alternative systems, such as Epstein-Barr virus-based shuttle vectors that can be episomally maintained in human cells. Furthermore, we have constructed packageable shuttle vectors, which appear to be stable in the host cell and thus suitable for analysis of mutagenesis.

Animals↗

PubMed1986

2-(N-acetoxy-N-acetylamino)fluorene mutagenesis in mammalian cells: sequence-specific hot spot.

Mutations induced by 2-(N-acetoxy-N-acetylamino)fluorene were studied using temperature-sensitive simian virus 40 (SV40) mutants as probe in monkey kidney cells. In vitro treatment of the SV40 virions with 2-(N-acetoxy-N-acetylamino)fluorene increased mutagenesis and decreased survival in the viral progeny. A lethal hit of approximately 85 acetylaminofluorene adducts per SV40 genome was calculated. UV irradiation of cells prior to infection did not modify the results. Molecular analysis of independent SV40 revertants showed that 2-(N-acetoxy-N-acetylamino)fluorene induces base substitutions that are located not opposite putative acetylaminofluorene adducts but next to them. Moreover, a hot spot of mutation restoring a true wild-type genotype was observed in 10 of the 16 revertants analyzed. This hot spot, not targeted opposite a major DNA lesion, was not observed using UV light as damaging agent in the same genetic assay. Two models involving the stabilization, by acetylaminofluorene adducts, of the secondary structure of a specific quasipalindromic SV40 sequence are proposed to explain this sequence-specific hot spot.

2-Acetylaminofluorene↗