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Biomedical subjects

E Dogliotti

Publications and source records attributed to E Dogliotti.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mutation spectra resulting from carcinogenic exposure: from model systems to cancer-related genes.

The events leading to cancer are complex and interactive. Alteration of cancer genes, such as the tumor suppressor gene p53, plays a central role in this process. Analysis of the frequency, type and site of mutations in important cancer-related genes may provide clues to the identification of etiological factors and sources of exposure. In this chapter we have selected a few examples of environmental human carcinogens and have attempted to use the knowledge of their mechanisms of mutagenesis, as derived from in vitro cell systems, as a key to understanding the complexity of p53 mutation spectra in tumors arising at the putative target organ. The analysis will focus on environmental exposure to UV radiation. The examples of tobacco smoke, dietary aflatoxin and vinyl chloride will be also briefly discussed.

Aflatoxin B1↗
PubMed1997

DNA repair of UV photoproducts and mutagenesis in human mitochondrial DNA.

The induction and repair of DNA photolesions and mutations in the mitochondrial (mt) DNA of human cells in culture were analysed after cell exposure to UV-C light. The level of induction of cyclobutane pyrimidine dimers (CPD) in mitochondrial and nuclear DNA was comparable, while a higher frequency of pyrimidine (6-4) pyrimidone photoproducts (6-4 PP) was detected in mitochondrial than in nuclear DNA. Besides the known defect in CPD removal, mitochondria were shown to be deficient also in the excision of 6-4 PP. The effects of repair-defective conditions for the two major UV photolesions on mutagenesis was assessed by analysing the frequency and spectrum of spontaneous and UV-induced mutations by restriction site mutation (RSM) method in a restriction endonuclease site, NciI (5'CCCGG3') located within the coding sequence of the mitochondrial gene for tRNALeu. The spontaneous mutation frequency and spectrum at the NciI site of mitochondrial DNA was very similar to the RSM background mutation frequency (approximately 10(-5)) and type (predominantly GC>AT transitions at G1 of the NciI site). Conversely, an approximately tenfold increase over background mutation frequency was recorded after cell exposure to 20 J/m2. In this case, the majority of mutations were C>T transitions preferentially located on the non-transcribed DNA strand at C1 and C2 of the NciI site. This mutation spectrum is expected by UV mutagenesis. This is the first evidence of induction of mutations in mitochondrial DNA by treatment of human cells with a carcinogen.

Base Sequence↗
PubMed1997

p53 mutations and chromosome instability in basal cell carcinomas developed at an early or late age.

Tumor DNA from 45 primary basal cell carcinoma (BCC) biopsies was screened for p53 gene mutations, chromosome 9 allele loss, and microsatellite instability. p53 mutation frequency increased significantly as a function of the age at BCC onset ranging from 6% (1/16) in early BCC (before age 40 years) to 35% (10/29) in late BCC. All p53 mutations found implicated sunlight as the mutagen. Chromosome 9 instability (allele loss or microsatellite instability) was detected at high frequency (38%) independently of age at tumor onset. Allelic loss was confined to chromosome 9q, whereas microsatellite instability was observed prevalently on chromosome 9p often in association with a replication error (RER+) phenotype. Most of our late BCC patients reported occupational sun exposure, while early BCC patients recalled childhood (0-20 years) recreational sun exposure. These data suggest that chronic exposure to sunlight is responsible for accumulation of p53 mutations and thus for late BCC appearance, whereas acute UV exposure in childhood and adolescence leads to early skin cancer development in genetically susceptible individuals via a p53-independent pathway.

Adult↗
PubMed1996

Misincorporation rate and type on the leading and lagging strands of UV-damaged DNA.

We have examined the fidelity of replication of the leading and lagging strands of UV-irradiated DNA by using an EBV-derived shuttle vector system which contains as marker gene for mutation analysis the bacterial gpt gene in both orientations relative to the EBV oriP. Human cells stably transformed with this vector were UV irradiated and gpt mutation rate and type were analysed. An increased mutagenicity associated with UV irradiation was observed, but the average error frequency was unaffected by the direction of replication of the target gene. Some variability by position and sequence context of leading and lagging strand errors was detected, suggesting that the different architecture of the replication complex for the two strands might, to some extent, affect mutation spectra. The comparable fidelity of translesion replication on the leading and lagging strands is in agreement with the current model for eukaryotic replication that postulates the simultaneous synthesis of both strands by a DNA polymerase with a proof-reading exonuclease.

Bacterial Proteins↗
PubMed1996

Two pathways for base excision repair in mammalian cells.

Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

Animals↗
PubMed1996

Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay.

The single cell gel electrophoresis (SCGE) or comet assay, which measures DNA strand breaks in individual cells, was used to analyse DNA damage and repair induced by the SN1-type alkylating carcinogens N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea in CHO cells. The comet assay was comparable in sensitivity to the alkaline elution assay. The alkyl-adducts detected as DNA single-strand breaks (ssb) by this technique were completely repaired within 24 h after treatment. These data indicate that long-lived lesions, such as alkylphosphotriesters, are not converted into ssb under the standard SCGE alkaline conditions (pH 13.5). The lesions revealed by the comet assay are mainly apurinic/apyrimidinic (AP) sites and breaks formed as intermediates in the base excision repair process of N-alkylpurines. When SCGE was performed at pH 12.5 instead of pH 13.5 a lower level of ssb was detected and these breaks were completely resealed within 2 h after treatment. These data suggest that different subsets of lesions are detected under different pH conditions. The SCGE combined with inclusion within the cells of endonuclease III revealed that a high portion of AP sites induced by alkylation damage were not converted into ssb by alkali. The level of endonuclease III-sensitive sites decreased as a function of the repair time and by 24 h after treatment no sites were left on the DNA. The use of this modified SCGE assay allows the estimation of the total amount of unrepaired AP sites present on DNA. Alkylation-induced ssb as detected by the comet assay should be regarded as an indicator of repair rate and balance more than a measure of actual DNA damage.

Alkylation↗
PubMed1996

Genetic alterations in skin cancer.

Cancer is a multi-stage process in which the accumulation of genetic changes allows clonal expansion of abnormal cells that will eventually form a tumor. Skin cancer is the most common malignancy affecting human beings. Mutations of the tumor suppressor gene p53 are often found in non-melanoma skin cancer and pre-invasive lesions, like actinic keratosis. The type of mutations detected in the p53 gene strongly indicate UV light as the initiating and promoting agent in skin cancer development. Chromosome instability is also an early event in skin tumor formation. However, despite the huge amount of information available in the literature on molecular markers of skin cancers, much remains to be uncovered about the progression of genetic events that separate normal sun-exposed epidermis from skin cancer. In this paper the following issue will be addressed: how far are we from being able to define a human model for multistage skin carcinogenesis in humans?

Animals↗
PubMed1995

Mutation analysis in two newly identified rat p53 pseudogenes.

We have analysed the genomic organization of the rat p53 gene in normal intestinal rat cells. Exons 5-8 of the p53 gene were amplified by PCR from rat genomic DNA using primers complementary to stretches of nucleotides identical in rat and human cDNAs. Two amplification products of 727 and 339 bp were obtained from the rat DNA. The PCR products were subcloned into M13mp18 and sequenced. By comparison with the rat cDNA sequence the 727 bp band was identified as the functional p53 gene containing exons 5, 6 7 and 8 and introns 5 and 7. The sequences corresponding to human intron 6 are absent from the rat p53. The second amplification product was a mixture of two different 'processed' pseudogenes, in which 42 mutations accumulated in a sequence corresponding to the cDNA between exons 5 and 8. Analysis of these mutations shows that in both pseudogenes the vast majority is constituted by base substitutions, with transitions being more frequent than transversions. The most prominent mutational class is formed by G-->A transitions which are predominantly located at CpG sites. Since a high level of homology is present between the rat and the human cDNA, the type and the positions where mutations occur in the two rat pseudogenes is discussed in relation to the possible origin of these mutations in human tumors.

Amino Acid Sequence↗
PubMed1994

Repair of abasic sites by mammalian cell extracts.

Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

Animals↗
PubMed1994

Spontaneous mutations at aprt locus in a mammalian cell line defective in mismatch recognition.

Clone B is a CHO cell line that shows a moderate mutator phenotype as a consequence of a defect in mismatch recognition. To identify the classes of mutation that accumulate spontaneously in a functional gene, we isolated and sequenced 54 clone B spontaneous mutants at the adenine phosphoribosyltransferase gene. This spectrum was compared to 42 mutants collected in the parental cells. Rates of AT-->TA transversions and frameshifts were strikingly increased in clone B (almost eight- and sixfold, respectively). Minor increases were also observed for GC-->TA transversions and GC-->AT transition rates. Frameshifts occurred in repeated sequences, and a large proportion were losses of 2 bases occurring in dinucleotide runs of a type similar to microsatellite sequences. AT-->TA transversions clustered in regions of secondary structure and their formation might be explained by slippage-mediated mechanisms. These data indicate that an important function of mismatch recognition is in repair of extrahelical bases generated by misalignment during DNA replication.

Adenine Phosphoribosyltransferase↗
PubMed1994

Molecular analysis of mutations induced by chemical carcinogens in mammalian cells. I. The use of selectable gene loci.

Several lines of evidence indicate the important role of mutation in the process of carcinogenesis, however our understanding of the factors that influence the formation of mutation is still limited. Recent developments in the analysis of gene mutations at selectable loci in cultured mammalian cells has allowed to accumulate a significant amount of information on the type and distribution of mutations induced by a variety of chemical and physical agents. The purpose of this review is to summarize the data relative to chemical carcinogens and to discuss how their mechanism of interaction with DNA and the repair processes active on the induced lesions affect mutational spectra.

Alkylating Agents↗
PubMed1994

Molecular analysis of mutations induced by chemical carcinogens in mammalian cells. II. The use of recombinant DNA vectors.

One approach to molecular and mechanistic studies of mutagenesis in mammalian cells is to introduce a mutational target gene into the cells as part of a shuttle vector which is capable of replication in both mammalian cells and bacteria. Following mutagenesis in the mammalian cell host, the shuttle vector sequences are recovered from the mammalian cells and introduced into bacteria, where large amounts of the mutant gene can be produced for sequence analysis. The variety of shuttle vector systems which have been developed for this purpose will be described. Shuttle vectors have been widely used for the molecular analysis of mutations induced by physical and chemical agents and to investigate the factors which modulate mutation fixation. The data regarding chemically induced mutational spectra will be reviewed with particular emphasis on the studies aimed to dissect the complex process which lead from DNA lesion to mutation.

Alkylating Agents↗
PubMed1993

O6-methylguanine in DNA inhibits replication in vitro by human cell extracts.

To study the effects of methylation damage on DNA replication in vitro, the plasmid pSVori containing the SV40 origin of replication was reacted with N-methyl-N-nitrosourea and used as a substrate for SV40 T antigen dependent replication by HeLa cell extracts. The plasmid was methylated with a range of N-methyl-N-nitrosourea concentrations that introduced an average of 0.3-2.5 O6-methylguanine and equal amounts of 3-methyladenine lesions per DNA molecule. When methylated plasmid was incubated with extract of Mex-HeLaMR cells under conditions favoring DNA replication, an impairment of replication was observed as the accumulation of incompletely replicated form II plasmid molecules. These extracts simultaneously performed a T antigen independent, DpnI-sensitive DNA repair synthesis that increased with increasing DNA damage. Subtraction of this repair DNA synthesis revealed that methylation inhibited overall replication. At low levels of methylation (< or = 1 O6-methylguanine and < or = 1 3-methyladenine lesion per plasmid), inhibition was transient, while more extensive damage resulted in apparently irreversible inhibition of replication. Removal of O6-methylguanine by pretreatment of the methylated plasmid with purified human O6-methylguanine-DNA methyltransferase restored replication to almost normal levels. When the methylated plasmid was replicated by extracts of Mex+ HeLaS3 cells proficient in the repair of O6-methylguanine, a lower level of inhibition and less repair DNA synthesis was observed. The inhibition of DNA synthesis and the stimulation of repair DNA synthesis are thus both largely due to the presence of O6-methylguanine in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Polyomavirus Transforming↗
PubMed1993

O6-methylguanine residues elicit DNA repair synthesis by human cell extracts.

We have investigated the processing of O6-methylguanine (O6-MeGua) in plasmid DNA by extracts of human cells defective in O6-MeGua-DNA methyltransferase. Cell extracts of HeLaMR cells performed viral T antigen-independent DNA synthesis on plasmids that had been treated with low concentrations of methylating agents. The in vitro DNA synthesis was non-semiconservative and depended on the presence of O6-MeGua in the substrate. The involvement of DNA polymerase delta or epsilon and proliferating cell nuclear antigen but not single-strand binding protein was indicated by partial fractionation, inhibitor, and antibody studies. Processing of O6-MeGua is not via the UV nucleotide excision repair pathway since additional component(s) are apparently required to perform repair synthesis on the methylated substrate. This is the first direct demonstration of DNA repair synthesis provoked by O6-MeGua in DNA. Since O6-MeGua is not excised from DNA by Mex- cells, it represents a novel type of processing of the methylated base that may be involved in its cytotoxicity.

Antibodies↗
PubMed1993

Mutagenic processing of ethylation damage in mammalian cells: the use of methoxyamine to study apurinic/apyrimidinic site-induced mutagenesis.

The aldehyde reagent methoxyamine is able to interact with apurinic/apyrimidinic sites formed in vivo within cells and displays both an anti-cytotoxic and an antimutagenic activity on N-ethyl-N'-nitro-N-nitrosoguanidine-induced DNA damage in Chinese hamster ovary cells. To clarify the underlying mechanism we have examined the mutational spectra induced by N-ethyl-N'-nitro-N-nitrosoguanidine alone and in the presence of methoxyamine in the hypoxanthine-guanine phosphoribosyltransferase gene of Chinese hamster ovary cells. In both cases all mutations were base pair substitutions, and their distribution among various classes did not differ significantly. Almost 60% were transitions, predominantly GC to AT, and the remaining 40% were transversions, mainly at AT base pairs. The analysis of the proportion of the different types of mutations showed that in the presence of methoxyamine, GC to AT transitions decreased by a factor of 1.8, and AT to CG transversions were reduced by a factor of 13. These data indicate that in mammalian cells the fixation of ethylation damage into mutations occurs by both (a) direct mutagenesis likely driven by O6-ethylguanine adducts and to a minor extent by O4-ethylthymine and (b) apurinic/apyrimidinic site-mediated mutagenesis. These apurinic/apyrimidinic sites are formed during the processing of ethylation at critical sites and are likely to involve O6-ethylguanine and O2-ethylthymine adducts.

Alkylation↗
PubMed1993

Quantitative relationship between ethylated DNA bases and gene mutation at two loci in CHO cells.

In a previous study we showed that the formation of O6-ethylguanine (O6-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethylsulfate (DES) at the hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was extended to another ethylating agent, ethyl methanesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N7 and O6 positions of guanine and at the N3 site of adenine were measured and the possible correlations with 6-thioguanine resistance (6-TGr) and ouabain resistance (ouar) mutations were investigated. A good correlation between the levels of ethylation at O6 guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the ouar locus, the frequency of O6-EtGua adducts correlated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specificity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multicomponent phenomena like mutation formation.

Adenine↗