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1.
Mutagenesis ; 17(6): 529-38, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12435850

ABSTRACT

Fanconi anaemia (FA) is a rare autosomal recessive disease characterized by increased spontaneous and DNA crosslinker-induced chromosome instability, progressive pancytopenia and cancer susceptibility. An increasing number of genes are involved in FA, including the breast cancer susceptibility gene BRCA2. Five of the FA proteins (FANCA, FANCC, FANCE, FANCF and FANCG) assemble in a complex that is required for FANCD2 activation in response to DNA crosslinks. Active FANCD2 then interacts with BRCA1 and forms discrete nuclear foci. FANCD2 is independently phosphorylated by ATM (the protein whose gene is mutated in ataxia telangiectasia) in response to ionizing radiation. In addition, the FA proteins are interconnected with other nuclear and cytoplasmic factors all related to cellular responses to carcinogenic stress and to caretaker and gatekeeper functions. In this review, the most recently published data on the molecular biology of the FA pathway and its molecular crosstalk with ATM, BRCA1 and BRCA2, proteins involved in xenobiotic and reactive oxygen species metabolism, apoptosis, cell cycle control and telomere stability, are summarized. The currently available data indicate that FA is a central node in a complex nuclear and cytoplasmic network of tumour suppressor and genome stability pathways fully committed to prevent cancer.


Subject(s)
Fanconi Anemia/genetics , Genes, Tumor Suppressor , Apoptosis , Ataxia Telangiectasia Mutated Proteins , Cell Cycle , Cell Cycle Proteins , Cell Nucleus/metabolism , Chromatin/genetics , DNA Repair , DNA-Binding Proteins , Fanconi Anemia/metabolism , Fanconi Anemia/pathology , Fanconi Anemia Complementation Group D2 Protein , Female , Genes, BRCA1 , Genes, BRCA2 , Humans , Male , Models, Biological , Mutation , Nuclear Proteins/genetics , Oxidative Stress , Protein Serine-Threonine Kinases/genetics , Telomere/genetics , Transcription, Genetic , Tumor Suppressor Proteins
2.
Carcinogenesis ; 22(3): 387-93, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11238177

ABSTRACT

The oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG), the product of deamination of cytosine uracil (U), and the sites of base loss [abasic (AP) sites] are among the most frequent mutagenic lesions formed in the human genome under physiological conditions. In human cells, the enzymatic activities initiating DNA base excision repair (BER) of 8-oxoG, U and AP sites are the 8-oxoG DNA glycosylase (hOGG1), the U-DNA glycosylase (UNG) and the major hydrolytic AP endonuclease (APE/HAP1), respectively. In recent work, we observed that BER of the three lesions occurs in human cell extracts with different efficacy. In particular, 8-oxoG is repaired on average 4-fold less efficiently than U, which, in turn, is repaired 7-fold slower than the natural AP site. To discriminate whether the different rates of repair may be linked to different expression of the initiating enzymes, we have determined the amount of hOGG1, UNG and APE/HAP1 in normal human cell extracts by immunodetection techniques. Our results show that a single human fibroblast contains 123 000 +/- 22 000 hOGG1 molecules, 178 000 +/- 20 000 UNG molecules and 297 000 +/- 50 000 APE/HAP1 molecules. These limited differences in enzyme expression levels cannot readily explain the different rates at which the three lesions are repaired in vitro. Addition to reaction mixtures of titrated amounts of purified hOGG1, UNG and APE/HAP1 variably stimulated the in vitro repair replication of 8-oxoG, U and the AP site respectively and the increase was not always proportional to the amount of added enzyme. We conclude that the rates of BER depend only in part on cellular levels of initiating enzymes.


Subject(s)
Base Pair Mismatch , Carbon-Oxygen Lyases/metabolism , DNA Repair , N-Glycosyl Hydrolases/metabolism , DNA Glycosylases , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Humans , Recombinant Proteins/metabolism
3.
Oncogene ; 19(50): 5781-7, 2000 Nov 23.
Article in English | MEDLINE | ID: mdl-11126365

ABSTRACT

The breast cancer predisposing genes BRCA1 and BRCA2 appear to be involved in DNA repair. In particular, the sensitivity of BRCA2-deficient mouse embryonic fibroblasts to ionizing radiation and the demonstrated interaction of the BRCA2 protein with Rad51, a major factor in recombinational repair, indicate that BRCA2 is important for double strand break repair. The human BRCA2-deficient human cell line Capan-1, whilst being sensitive to ionizing radiation, is also sensitive to the alkylating agent methymethanesulfonate. The major lesions induced by this agent are methylated bases which are removed primarily by the base excision repair (BER) pathway. We have investigated the efficiency of BER in Capan-1 cells by an in vitro assay in which plasmid substrates containing a single lesion are repaired by mammalian cell extracts. In comparison to the control cell lines BxPC-3, T24 and MCF7, Capan-1 cells exhibited a reduced rate of DNA ligation during both the single-nucleotide insertion and PCNA-dependent pathways of BER. The reduced rate of DNA ligation exhibited by Capan-1 cell extracts was complemented by addition of bacteriophage T4 DNA ligase or human DNA ligase III. BRCA2-mutant Capan-1 cells may possess reduced DNA ligase activity during BER.


Subject(s)
DNA Ligases/metabolism , DNA Repair/genetics , Mutation , Neoplasm Proteins/genetics , Transcription Factors/genetics , Adenocarcinoma/enzymology , Adenocarcinoma/genetics , BRCA2 Protein , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Humans , Neoplasm Proteins/deficiency , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/genetics , Transcription Factors/deficiency , Tumor Cells, Cultured , Urinary Bladder Neoplasms/enzymology , Urinary Bladder Neoplasms/genetics
4.
Mutagenesis ; 14(1): 77-82, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10474826

ABSTRACT

The presence of the 4977 bp deletion ('common deletion') in the mitochondrial DNA (mtDNA) is associated with defects in the metabolic machinery acquired during ageing as a hallmark of a degenerative phenotype. We analysed 27 samples (18 from surgical patients and nine from autopsy cases) of DNA extracted from smooth muscle cells of abdominal aorta fragments affected by atherosclerotic lesions. The deletion was detected by PCR amplification gel electrophoresis and characterized by sequencing of the PCR product. The mtDNA 'common deletion' was detected in all analysed samples. However, its levels were not particularly high, which may be ascribed to the fact that smooth muscle cells in atherosclerotic lesions have a lower energy requirement and an appreciable proliferation rate, as compared for instance with cardiac myocytes. When the subjects were divided into two numerically equivalent age classes (60-72 years plus a 45-year-old subject versus 73-95 years), the deletion had significantly higher levels in the older subjects. Conversely, its presence did not correlate with source (surgical or autoptic), sex, cigarettes consumption, other clinical and anamnestic parameters or with the levels of adducts and 8-hydroxy-2'-deoxyguanosine measured in the nuclear DNA of the same samples. A previously unreported deletion of 5111 bp was additionally found in the mtDNA from a 45-year-old woman. The origin of this lesion seems to be compatible with the slipped mispairing model proposed for the 'common deletion'.


Subject(s)
Arteriosclerosis/genetics , DNA, Mitochondrial/genetics , Sequence Deletion/genetics , Age Factors , Aged , Aged, 80 and over , Aorta, Abdominal/chemistry , Base Composition , DNA, Mitochondrial/isolation & purification , Female , Humans , Male , Middle Aged , Risk Factors , Sex Factors
5.
Anal Cell Pathol ; 17(1): 1-12, 1998.
Article in English | MEDLINE | ID: mdl-9807634

ABSTRACT

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases. Five tumours (25%) were DNA diploid, while 15 tumours (75%) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60% of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p < 0.04, Fisher's exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86%, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation. The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.


Subject(s)
Adenocarcinoma/genetics , Aneuploidy , Colorectal Neoplasms/genetics , Genes, p53/genetics , Mutation/genetics , Adult , Aged , Aged, 80 and over , Base Sequence , Female , Flow Cytometry , Humans , Lymphatic Metastasis/genetics , Male , Metaplasia/genetics , Middle Aged , Molecular Sequence Data , Ploidies
6.
Int J Cancer ; 73(6): 816-21, 1997 Dec 10.
Article in English | MEDLINE | ID: mdl-9399658

ABSTRACT

This study investigates the timing of p53 mutations detected in the malignant cells of a Burkitt's lymphoma cell line (BRG-P) with respect to other maturation or transforming events. The BRG-P cell line, derived from an AIDS patient, was of special value since it displayed subclones that had undergone an isotype switch from IgM to IgA1 (BRG-M and BRG-A cells). BRG-M and BRG-A cells were characterized by the same monoclonal c-myc and VDJ rearrangements and by the expression of Ig receptors with specificity for a 45 kDa protein of human breast cells. Analysis of p53 mutations in the different BRG subclones showed that 1) BRG-M cells displayed 2 different p53 mutations in trans; since the original BL cells also showed the same mutations, this finding indicated that both occurred in vivo; 2) one of the p53 alleles of BRG-A cells was lost, while the other showed a mutation different from those seen in BRG-M cells; and 3) all 3 mutations observed in BRG-M or BRG-A cells resulted in the functional inactivation of the transcriptional activation function of p53. Together, our data demonstrate that p53 mutations were relatively late events during lymphomagenesis. Moreover, in view of the role of p53 in cell apoptosis, it is conceivable that BRG cells were subjected to a strong selective pressure that favored p53 inactivation. Such inactivation was possibly required to counterbalance other potentially apoptotic events, including the presence of a deregulated c-myc oncogene and signals delivered by the host environment in situ.


Subject(s)
Burkitt Lymphoma/genetics , Gene Rearrangement , Genes, Immunoglobulin/genetics , Genes, myc/genetics , Genes, p53/genetics , Lymphoma, AIDS-Related/genetics , B-Lymphocytes/metabolism , Chromosome Deletion , Female , Heterozygote , Humans , In Situ Hybridization, Fluorescence , Tumor Cells, Cultured
7.
Chem Biol Interact ; 102(1): 55-62, 1996 Sep 27.
Article in English | MEDLINE | ID: mdl-8827062

ABSTRACT

In the framework of a project investigating the possible involvement of cancer biomarkers in human atherogenesis, we evaluated the occurrence of K-ras mutations in the DNA extracted from smooth muscle cells of abdominal aorta atherosclerotic lesions. The molecular analysis of the DNA from 32 surgical specimens, using PCR-based denaturing gradient gel electrophoresis (DGGE), did not reveal any variant in K-ras codons 12 and 13, which are the most frequently involved codons among the ras genes mutated in various types of human tumors. Analysis of the DNA extracted from four cell lines carrying known K-ras mutational alleles showed typically positive DGGE patterns. Thus, on the whole, the conclusions of this study and of previous studies using the same biological material are consistent with the occurrence of DNA adducts in human atherosclerotic lesions but in the absence of p53 involvement or of K-ras mutations in codons 12 and 13. The search for candidate genes which may possibly be involved in the atherogenetic process warrants further studies.


Subject(s)
Arteriosclerosis/genetics , Codon/genetics , Genes, ras/genetics , Aged , Aged, 80 and over , Animals , Aorta, Abdominal/chemistry , Base Sequence , Cell Line , DNA/analysis , DNA/isolation & purification , Female , Humans , Male , Mice , Middle Aged , Molecular Sequence Data , Mutation , Nucleic Acid Heteroduplexes/analysis , Polymerase Chain Reaction
8.
Cancer Epidemiol Biomarkers Prev ; 5(7): 559-65, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8827361

ABSTRACT

Barrett's Esophagus (BE) is a complication of gastroesophageal reflux in which the normal squamous epithelium of the lower esophagus is replaced by metaplastic tissue. The clinical significance of this condition is the associated predisposition to adenocarcinomas (ADCs). Three types of BE have been characterized: the gastric fundic (F) type, the gastric cardial (C) type, and the intestinal (I) type. The latter is the most closely associated with the development of ADCs; the causes of this bias remain unknown. To determine whether p53 and/or K-ras gene alterations (a) are present in preneoplastic lesions and (b) are associated with a specific histotype, we performed PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of exon 1 (codons 12-13) of K-ras gene and of exons 5-8 of the p53 gene in biopsies obtained from 30 patients with BE of the I type (9 patients), combined I type (I + C +/- F; 10 patients) and non-I type (C, F, or C + F; 11 patients). None of the cases under study revealed K-ras mutations, whereas biopsies from 12 patients showed at least one p53 DGGE variant. Four patients showed the exact same variants in leukocytes also (polymorphisms), whereas eight cases revealed specific DGGE variants only in biopsies. The molecular characterization of these variants revealed that four of them showed a single base pair substitution, and four showed multiple mutations. Of 17 somatic mutations, all but 1 were base pair substitutions located mainly in exons 7 and 8. The majority of these mutations were GC targeted (13 of 16; 81%), 54% (7 of 13) of which were transitions occurring at CpG sites. All somatic mutations were found in BE with at least one I component. The association with the histotype was statistically significant (P < 0.03; pure I type versus non-I type; P < 0.04, combined I type versus non-I type; Fisher's exact test). Loss of heterozygosity in the vicinity of the p53 locus was evaluated by PCR using a highly polymorphic variable number of tandem repeats marker on 25 out of 30 cases. Ninety-two % of the cases analyzed were informative, and none of them showed LOH. In conclusion, we showed that p53 mutations are frequently observed in specimens from BE patients of the I-type, whereas no involvement of K-ras (exon 1) mutational activation was observed. In light of the key roles that the p53 protein plays in controlling cell cycle and cell diploidy, this result may suggest why this type of metaplasia is the most closely associated to the development of ADCs.


Subject(s)
Barrett Esophagus , Esophageal Neoplasms/etiology , Genes, p53 , Genes, ras , Precancerous Conditions/pathology , Adult , Aged , Aged, 80 and over , Barrett Esophagus/genetics , Barrett Esophagus/pathology , Biopsy , Cell Transformation, Neoplastic/genetics , Culture Techniques , Esophageal Neoplasms/pathology , Esophagoscopy , Female , Genes, p53/physiology , Genes, ras/physiology , Humans , Intestines/pathology , Male , Middle Aged , Mutation , Polymerase Chain Reaction , Sensitivity and Specificity
9.
Cancer Genet Cytogenet ; 88(2): 95-102, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8640734

ABSTRACT

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.


Subject(s)
Aneuploidy , Astrocytoma/genetics , Brain Neoplasms/genetics , Chromosome Deletion , Genes, p53/genetics , Glioblastoma/genetics , Point Mutation/genetics , Adult , Aged , Chromosomes, Human, Pair 10/genetics , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 15/genetics , DNA Mutational Analysis , Electrophoresis, Polyacrylamide Gel , Exons/genetics , Female , Humans , In Situ Hybridization, Fluorescence , Male , Middle Aged , Regression Analysis
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