Molecular mechanisms of mutagenesis in mammalian cells: present and future.
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Animals↗
Biomedical subjects
A Sarasin
Publications and source records attributed to A Sarasin.
SV40-based shuttle viruses.
We summarize in this paper the advantages of the shuttle virus system. These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells. Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid DNA can be obtained. The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E. coli, using various gene mutation targets. Moreover, we describe a new series of vectors which can be produced as single-stranded DNA in bacteria. They allow the transfection of a plasmid genome into mammalian cells, as either single-stranded or double-stranded DNA.
Animals↗
Comparative study of Epstein-Barr virus and SV40-based shuttle-expression vectors in human repair-deficient cells.
Shuttle vectors and expression vectors have been used in human cells to examine various aspects of DNA repair including effects of DNA damage on mutagenesis, transcription, replication and recombination. A combined shuttle-expression system should provide further advantages for the stable expression of and perhaps selection/rescue strategies for DNA repair genes. We describe 2 such systems. The first is a simian virus 40 (SV40) shuttle system which allows a quasi-stable episomal vector/host relationship in which the shuttle vector may be recovered in extrachromosomal DNA preparations many months after transfection and selection but in which a high proportion of the plasmids rescued in bacteria are heavily mutated and rearranged. Secondly, we describe Epstein-Barr virus-based shuttle-expression vectors which exist as stable, multicopy episomes in human cells. Using a reporter gene and a metal-inducible promoter we have obtained low basal and very high induced expression from episomal vectors in a variety of human cells including xeroderma pigmentosum and ataxia telangiectasia cell lines. This should facilitate many molecular genetic experiments in human cells and may have particular application to molecular cloning, expression and analysis of DNA repair genes.
Animals↗
Enhanced reactivation and mutagenesis after transfection of carcinogen-treated monkey kidney cells with UV-irradiated simian virus 40 (SV40) DNA.
Monkey kidney cells, either untreated or pretreated with UV-light at 254 nm or mitomycin C, were transfected 24 hours later with the intact or UV-irradiated DNA from the thermosensitive tsB201 simian virus 40 mutant unable to grow at 41 degrees C. The survival of the viral progeny obtained from the UV-irradiated DNA is increased in pretreated cells compared to the survival of the viral progeny obtained in untreated cells. Irradiation of the viral DNA enhances the reversion frequency of the viral progeny towards a wild type phenotype able to grow at 41 degrees C. Pretreatment of the cells with UV or mitomycin C does not increase the reversion frequency.
Animals↗
DNA ligase activity in carcinogen-treated human fibroblasts.
In an enzymological approach to study DNA repair mechanisms induced by carcinogen-treatment of mammalian cells, we have investigated how DNA ligase activity is affected by the treatment with several compounds producing different DNA lesions. Stationary cultures of human fibroblasts were exposed to various doses of carcinogens (UV-light at 254 nm, N-acetoxy-acetyl-aminofluorene, ethyl-methane sulfonate, N-methylnitro-nitrosoguanidine, mitomycin C and 4-nitroquinoline-N-oxide) at different time-intervals before preparing crude cellular extracts and assaying for ligase activity. Results have shown that: 1. UV-irradiation, AAAF, 4NQO or MMC treatment of cells induces a two-fold increase in the ligase activity compared to control cells within 48 hours following the treatment. 2. A partial purification of the enzyme from these cellular crude extracts by sedimentation through sucrose gradients has shown: a. DNA ligase activity from control cells presents a profile composed of two distinct peaks sedimenting respectively at about 4S and 7S; b. the carcinogen treatment of either repair-proficient human fibroblasts or repair-deficient xeroderma pigmentosum cells (complementation group A) seems to induce a specific increase of the 4S-form of DNA ligase.
Carcinogens↗
Error-prone replication of ultraviolet-irradiated simian virus 40 in carcinogen-treated monkey kidney cells.
To analyze the molecular mechanism of mutagenesis in carcinogen-treated mammalian cells, we developed a model system composed of various simian virus 40 (SV40) mutants as a biological probe to detect inducible DNA repair and mutagenesis in carcinogen-treated monkey kidney cells (CV1-P). Results have shown that treatment of cells with UV-light, acetoxy-acetyl-aminofluorene, or mitomycin C, increases the mutagenesis of UV-irradiated SV40 ts mutant measured as a reversion frequency from a thermosensitive phenotype toward a thermoresistant phenotype. This increased mutagenesis is not observed in the case of undamaged virus indicating that we are looking at targeted mutagenesis. The molecular analysis of several revertant genomes indicates that some DNA rearrangements may occur in the revertant genomes but in some cases the reversion site is a single basepair substitution located at positions different from the original thermosensitive mutation, which is still present. The general interpretation of our results leads to the conclusion that carcinogen treatment of monkey cells activates some kind of error-prone replication mode able to better replicate UV-damaged templates but leading to a higher level of mutagenesis. This activity may represent a SOS-like function in mammalian cells.
Animals↗
Distribution of ultraviolet-induced lesions in simian virus 40 DNA.
In order to analyze the molecular mechanisms of mutagenesis in mammalian cells, we devised an analytical assay using Simian Virus 40 as biological probe. To study the possible correlations between the distribution of the lesions on the treated DNA and the distribution of mutations, we have located and quantified the lesions induced by ultraviolet light (254 nm) on a SV40 DNA fragment. At a fluence of 2,000 J/m2, our results show that the formation frequency of thymine-thymine dimers (TT) is three to four times higher than the formation frequency of the other types of dimers (TC, CT, CC). On the other hand, the formation frequency of a dimer is influenced by the adjacent sequence. In particular, a pyrimidine in the 5' position of a thymine-thymine dimer enhances its formation frequency. At the dose used the formation frequency of the pyrimidine (6-4) pyrimidone photoproducts is twenty times less than the formation frequency of pyrimidine dimers. This paper shows the distribution of the major lesions induced by UV-light on a defined fragment of SV40 genome after UV irradiation. This work is necessary to get an insight into the molecular mechanisms of UV-mutagenesis.
Base Sequence↗
Survival and mutagenesis of ultraviolet irradiated simian virus 40 in foetal human fibroblasts.
Survival and mutagenesis of UV-irradiated, temperature-sensitive simian virus 40 mutants (SV40) have been studied after infection of human fibroblasts. Survival of the viral progeny obtained after 6,8 or 10 days at permissive temperature decrease as a function of the UV-dose delivered to the virus. In cels which have been pretreated with 10 Jm-2 of UV 24 hours before infection, progeny survival was increased as compared to survival in control cells. The reactivation factor varies from one to ten, depending on the number of lytic cycles carried out at permissive temperature. The level of mutation frequency, as measured by the reversion from a temperature sensitive growth phenotype towards a wild type phenotype, increases with the dose of UV-irradiation given to the virus. Moreover, the mutation frequency is increased in the viral progeny produced in UV-irradiated human cells. Similar experiments carried out with SV40-transformed human fibroblasts, which constitutively express SV40 T antigen, gave comparable results. These experiments show that, as in monkey cells, a new error-prone recovery pathway can be induced by pretreating human cells with UV-light before infection.
Cell Line↗
Repair and mutagenesis survey of 8-hydroxyguanine in bacteria and human cells.
8-Hydroxyguanine is one of the major products formed by the reactive oxygen species which are generated in living cells as a consequence of either the normal metabolic pathways or an exogeneous chemical or physical stress. The production of the oxidative damage is described and the different repair pathways of the oxidative lesions are analyzed from bacteria to human cells. Analysis of repair in human cells harboring different deficiencies in the nucleotide excision repair mechanism such as xeroderma pigmentosum cells from different complementation groups and cells from Cockayne's syndrome patients allows us to emphasize the possibility of the intervention of this repair mechanism on the elimination of oxidative damages. Finally, a repair model of oxidative lesions is proposed.
DNA Damage↗
Mechanisms of mutagenesis in mammalian cells. Application to human thyroid tumours.
Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements. These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway. The molecular process leading to mutagenesis depends largely on the type of DNA lesions. Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions. The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors. Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone. These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell. In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process. The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells. IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals. The breakage of the double-helix is a difficult lesion to repair. Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements. Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor. The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis. In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes. These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5. The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84%. This frequency was only 15% in 'spontaneous' PTC. The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different. Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones. The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%). The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test). In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present. A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation. The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one. It will be interesting to use this time lag in accidental or therapeutic-irradiated p
DNA Damage↗
Targeted mutagenesis of SV40 DNA induced by UV light.
UV light is a potent mutagen in most living cells, but molecular analysis of its mode of action in animal cells has not been possible because of the high complexity of the cell genome. Therefore, we have used simian virus 40 (SV40) as a biological probe to study the mutagenic effect of UV-irradiation at the molecular level. A thermosensitive SV40 mutant of the large T antigen (tsA58), which is defective at high temperature for initiation of viral DNA replication and for repression of early transcription, was UV-irradiated in vitro, then replicated in monkey kidney cells. Revertants of tsA58 were selected for growth at the nonpermissive temperature, then the reversion sites were mapped by using the marker rescue technique, and precisely localized by DNA sequencing. We report here that all revertants analysed showed one or two base pair substitutions localized in the C-terminal half of the T antigen gene. The original tsA58 mutation was still present in all revertant genomes. In 16 DNAs sequenced, seven reversion sites were found on the large T antigen gene, all of which localized opposite a possible UV-induced pyrimidine-pyrimidine lesion, suggesting targeted mutagenesis. UV-irradiation of the host cell before infection did not change the pattern of reversion sites at the molecular level, although it strongly increased the mutation frequency. These results demonstrate for the first time in animal cells the specificity of UV-induced mutagenesis.
Animals↗
[Rothmund-Thomson syndrome and defective DNA excision-resynthesis repair].
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Adolescent↗
Cellular genes possibly involved in the transformation process of the human melanoma cell line XP44 RO (Mel).
P44 Ro (Mel) is a human malignant melanoma cell line derived from a testicular metastasis in a DNA repair deficient, xeroderma pigmentosum patient. This line harbors a N-ras gene mutated in codon 61. To investigate other cellular genes possibly contributing to the expression of its transformed phenotype, four XP44 revertant cell lines were isolated by different selection procedures and the association of the level of expression of various oncogenes (including N-ras) and tumor suppressor genes with the selection for the revertant phenotype was determined. The revertants exhibited a significant but variable degree of phenotypic reversion, according to the selective pressure to which they were submitted, and a phenotypic stability dependent on their constant maintenance in selective medium. Back-revertant lines were isolated by culturing revertant lines in control medium for several weeks. The comparison between parental, revertant and back-revertant cells has revealed that, beyond the mutation in codon 61 of N-ras, two groups of genes appear to be also implicated in the transformation process of XP44 RO (Mel) cells: one group, comprising pim A, trk, Rb and p53, whose expression is independent of the cell selection conditions; the other group, comprising Ha-ras, N-ras, neu 1, fos and met H, whose expression is more or less dependent upon such conditions. The myc gene is apparently not involved in this phenomenon. These results, besides strengthening the concept that carcinogenesis is a multigenic process, suggest that diverse mechanisms can lead to the transformed phenotype, but that these mechanisms might have some pathway(s) in common.
3T3 Cells↗
The alkylating antitumor drug tallimustine does not induce DNA repair.
Tallimustine, an alkylating benzoyl mustard derivative of distamycin A (FCE 24517), is a novel anti-tumor agent. Both its cytotoxic activity against human LoVo cells and nicking efficiency on isolated plasmid DNA were studied in relation to hyperthermic treatment and compared to the effect of doxorubicin, a known non-alkylating anti-tumor agent. The results of this analysis indicate that the cytotoxic activity of tallimustine reflects its direct interaction with the DNA target. The ability of tallimustine to induce DNA repair in human primary normal fibroblasts was monitored by determining both the stimulation of unscheduled DNA synthesis (UDS) and the ability to reactivate a plasmid containing a reporter gene, treated in vitro with tallimustine, in comparison with the effect of UV-C irradiation. The results suggest that human cells able to repair UV-damage arc unable to overcome DNA damage induced by tallimustine. Therefore, the hypothesis that the biological activity of tallimustine is related to its alkylating properties is further supported by the temperature studies and strengthened by the observed inability of cells to repair tallimustine-induced DNA damage.
Antibiotics, Antineoplastic↗
Antisense insulin-like growth factor I transferred into a rat hepatoma cell line inhibits tumorigenesis by modulating major histocompatibility complex I cell surface expression.
We have established a hepatocarcinoma cell line (LFCI2 A) that produces voluminous tumors when injected subcutaneously into syngeneic Commentry rats. These neoplastic cells express both insulin-like growth factors (IGF) I and II. When transfected with an episomal cassette-expressing IGF-I antisense RNA, the modified LFCI2 A cell lines become poorly tumorigenic and, when injected subcutaneously, are associated with inhibition of the growth of the parental tumoral cells and/or induction of regression of established tumors. By contrast, cell lines isolated after transfection with the IGF-II antisense-expressing vector were as tumorigenic as the parental cell lines. The results are discussed in terms of protective immunity induced by the tumoral cells transfected by the IGF-I antisense vector. In the transfected hepatocarcinoma cells that do not produce IGF-I, the expression of major histocompatibility complex class I antigen was increased at least 4-fold compared with parental cells. The introduction of these cells in vivo induced a tumor-specific immunity that was associated with CD8 T cells.
Animals↗