Biomedical subjects
Publications and source records attributed to A Sarasin.
The relationships between mutations, in a general sense, and cancer are reviewed with a special emphasis on the description of hereditary tumors. These rare diseases provide good biological models to better understand cancer initiation at the molecular level. Tumors transmitted with a recessive mode are usually linked with DNA repair deficiencies. Tumors transmitted with a dominant mode are usually linked with deletions in anti-oncogene sequences.
Oncogenes capable of transforming 3T3-Vill cells were not detected in 'normal' Xeroderma pigmentosum (XP) fibroblasts but were detected in two out of six XP epitheliomas. Preliminary results concerning the transfection of 'normal' XP fibroblasts with activated ras genes seem to indicate that these cells are as resistant as the healthy controls to the transforming action of the group II oncogenes. However, after transfection with v-myb oncogene in XP fibroblasts several cellular clones have been isolated showing some new phenotypic characteristics.
The chemical carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), which induces well characterized DNA lesions, strongly inhibits Simian virus 40 (SV40) DNA replication. By using SV40 mutants which were temperature-sensitive for replication initiation, we were able to synchronize SV40 DNA replication and therefore to introduce AAAF-induced lesions only on unreplicating SV40 molecules. One to two acetylaminofluorene (AAF)-adducts per SV40 genome inhibit more than 90% of normal semi-conservative DNA synthesis. SV40 replicative intermediates (RIs) from AAAF-treated infected cells, purified through neutral sucrose gradients and BND-cellulose column, possess a structure different from the usual Cairns molecules found in the untreated cultures. Both by neutral and alkaline sucrose gradients and by electron microscopy, the RIs isolated from treated cells appear as complex molecules with single-stranded portions and sometimes with a tailed structure. Moreover, the newly synthesized strands found in these molecules are equal in size to the average distance between AAF-adducts on the template strand, indicating that AAF-adducts represent a block for the SV40 DNA replication. By using specific anti Guo-AAF antibodies and electron microscopy, we show the presence of an AAF adduct at halted replication forks, i.e. showing a DNA replication block in a mammalian replicon for the first time. We therefore assume that AAF-adducts severely block the progression of the replication forks by inhibiting, at least, the in vivo polymerization of the leading strand.
The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. (i) In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.
Ultraviolet light at 254 nm induces various kinds of DNA damage. We have located and quantified the pyrimidine (6-4) pyrimidone photoproducts along three hundred and forty two nucleotides of SV40 DNA. The level of photoproduct induction varies greatly according to the position on the DNA, but unlike what happens with pyrimidine dimers, the very adjacent nucleotides do not play a major role in the frequency of formation. A new alkali-sensitive site has been found on the ACA sequence after UV irradiation. This complex lesion is insensitive to the T4 endonuclease V and the E. coli photolyase, and may be involved with mutagenesis.
DNA ligase was partially purified from normal and regenerating rat liver. Its structure was studied using the activity gel procedure that identifies the functional polypeptides. Two slightly different purification procedures were followed leading to the isolation of one or two peaks (fractions A and B) of DNA ligase by hydroxyapatite chromatography. When analyzed on activity gels, all these enzyme fractions corresponded to a single active 130-kDa polypeptide both in normal and regenerating liver. A limited trypsin digestion of ligase fractions A and B gave rise to an identical pattern of smaller polypeptides of 110 kDa, 100 kDa and 75 kDa. Also storage at 4 degrees C of fractions A and B produced smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa, which were identical for the two fractions. Our results indicate that the same ligase polypeptide of 130 kDa can be isolated from stationary or regenerating rat liver cells. However, physiological or artifactual proteolysis during various purification procedures can lead to the isolation of two enzyme fractions with different chromatographic behaviour but with the same molecular mass.
We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.
We describe SV40-based Escherichia coli shuttle vectors which can be packaged as pseudovirions without excision of plasmid sequences and which can be rescued in bacteria. These vectors replicate and are transmitted as virus in monkey COS cells without requiring a helper virus. Extrachromosomal vector DNA isolated from infected cells can be rescued in E. coli, so that DNA alterations can be easily screened. Indeed, some of the constructions give rise to very stable plasmids with no detectable rearrangements after multiple lytic cycles in COS cells. The spontaneous mutation frequency measured in bacteria, on the lacO target, is smaller than those usually found with shuttle vectors. We also constructed an expression vector derived from one of our infectious viruses by inserting the chloramphenicol acetyl transferase gene, expressed from the SV40 early promoter, which is efficiently transduced to cells by infection. In this system, the shuttle virus combines the convenience of plasmid rescue and analysis in bacteria, with the advantages of infectious virus.
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.
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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.
Treatment of monkey kidney cells with mitomycin C (MMC) 24 h prior to infection with UV-irradiated simian virus 40 (SV40) enhanced both virus survival and virus mutagenesis. The use of SV40 as a biological probe has been taken as an easy method to analyse SOS response of mammalian cells to the stress caused by DNA damage or inhibition of DNA replication. The mutation assay we used was based on the reversion from a temperature-sensitive phenotype (tsA58 mutant) to a wild-type phenotype. The optimal conditions for producing enhanced survival and mutagenesis in the virus progeny were determined with regard to the multiplicity of infection (MOI). Results showed that the level of enhanced mutagenesis observed for UV-irradiated virus grown in MMC-treated cells was an inverse function of the MOI, while enhanced survival was observed at nearly the same level regardless of the MOI. For the unirradiated virus, almost no increase in the mutation of virus progeny issued from MMC-treated cells was observed, while a small amount of enhanced virus survival was obtained. These results show that enhanced virus mutagenesis and enhanced virus survival can be dissociated under some experimental conditions. Enhanced virus mutagenesis, analogous to the error-prone replication of phages in SOS-induced bacteria, was observed, at least for SV40, only when DNA of both virus and host cells was damaged and when infection occurred with a small number of viral particles. We therefore hypothesize that an error-prone replication mode of UV-damaged templates is observed in induced monkey kidney cells.
It has been previously shown that skin biopsies isolated from various xeroderma pigmentosum (XP) patients present a permanent decline in catalase activity from the onset of the disease to the tumor formation. We report here that cultured XP cell strains are also markedly deficient in the catalase activity with about only 25% of the activity measured in normal human cells. No direct correlation between catalatic activity and excision repair ability has been found, since a XP variant line is as deficient as an XP-C strain. The exact cause of the catalase deficiency is still unknown but could be due to the synthesis of a modified enzyme or to an abnormal regulation leading to a limited enzyme synthesis. Furthermore, simian virus 40 transformation of normal and radiosensitive cells (XP, ataxia telangiectasia) provokes a decrease in catalase activity of about 80% compared to the control derivatives. Mathematical analysis performed on our data shows a clearcut distinction between XP and normal cells while some of the XP heterozygote cells exhibit an intermediate behavior. Although most of the XP syndrome could be explained by the impairment in the excision repair ability, the decrease in catalase activity leading to a probable increase in intracellular H2O2 concentration and/or to a higher sensitivity to any oxygen-activated species could represent an additive effect in inducing the carcinogenic process.
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Using a method that detects catalytically active DNA ligase in NaDodSO4-polyacrylamide gels (activity gels) we have characterized ligase produced in CV1-P monkey kidney cells infected with SV40 or treated with mitomycin C. Purification on hydroxylapatite columns of DNA ligase from control cells results in two peaks of activity called ligases I and II, respectively. Analysis of ligase I on activity gels revealed major catalytic peptides with Mr of 120, 110, 70 and 58 kDa, while analysis of ligase II revealed two major peptides of 65 and 58 kDa. Infecting CV1-P cells with SV40 produced a significant increase in the 120, 110, 70 and 58 kDa peptides while treating them with mitomycin C produced a significant increase in the 70 and 58 kDa peptides and a decrease in the 120 and 110 kDa ones. Autoproteolysis of partially purified ligase under several conditions resulted in an increase in the 58 kDa peptide and in the disappearance of other peptides. These results suggest that at least one active polypeptide is common to ligases I and II.
Mammalian cells are constantly facing various types of mutagens. However, due to the high complexity of the cell genome, the molecular analysis of mutagenesis has not yet been possible. Therefore, we have used simian virus 40 (SV40) as a biological and molecular probe to characterize mutagenesis at the nucleotide level. By using a reversion assay from a temperature-sensitive phenotype towards a wild-type phenotype, we have analysed mutagenesis induced by u.v.-light and by apurinic sites (Ap sites). We report here experiments allowing us to quantify and to compare the mutagenic efficiency of various DNA lesions measured on the SV40 genome. The Ap sites are very mutagenic in this type of assay. The molecular analysis of u.v.-induced mutagenesis reveals that mutations correspond to single base-pair substitutions always located opposite Py-Py lesions. The mutations are almost equally distributed between transition and transversion types, and between the 5' and the 3' side of the Py-Py targets. These results demonstrate for the first time in animal cells the existence of targeted mutations induced by u.v.-light. We propose therefore, the use of SV40 as an efficient biological and molecular probe for assaying mutagenic pathways in mammalian cells.
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A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed. After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP. Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography. Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity. Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells. The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000. Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity. Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000.