Biomedical subjects

R Hamelin

Publications and source records attributed to R Hamelin.

At least 55 records · Page 3Linked to original sources

PubMed1996

Inverse correlation between RER+ status and p53 mutation in colorectal cancer cell lines.

The high point mutation rate of replication error-prone (RER+) cells could theoretically lead to inactivation of the p53 gene by polyclonal mutations, which might explain the conflicting results that have been published on the p53 status of RER+ colon cancers. To address this issue, we tested the p53 status of 21 human colorectal cancer cell lines, including four showing microsatellite instability (RER+ phenotype). Denaturing gradient gel electrophoresis (DGGE) followed by sequencing showed that all four RER+ cell lines were wild type for p53 while 15 of the 17 RER- cell lines contained p53 mutations (P=0.001). Eight cell lines (four RER+ and four RER-) were analysed using three complementary methods to test more rigorously the polyclonal mutation hypothesis. (i) Of 87 single-cell clones (seven to 14 per cell line) examined by DGGE, only those derived from known p53 mutant cell lines showed altered profiles. (ii) Antibody DO-7 stained more than 80% of nuclei from the p53 mutant cell lines, but only 15% of nuclei from the RER+ cell lines. (iii) A yeast functional assay which can simultaneously detect polyclonal mutations at over 500 different sites in the p53 cDNA scored all four RER+ cell lines as containing only transcriptionally active p53. These data thus do not support the polyclonal mutation hypothesis and instead suggest that mismatch repair deficiency provides a p53-independent pathway for development of colorectal cancers.

Colorectal Neoplasms↗

PubMed1996

Tumoral progression of human breast epithelial cells secreting FGF2 and FGF4.

The human breast epithelial cell line HBL100 synthesizes and secretes FGF2, is able to grow in soft agar but is not tumorigenic in nude mice. Transfection of this cell line with the FGF4 gene led to its tumorigenic conversion in a dose-dependent manner as assessed by growth under serum-free conditions, growth in soft agar and growth as xenografts in nude mice. Clones of FGF4-transfected cells producing different amounts of FGF4 secreted similar quantities of FGF2. Exogenously added recombinant FGF4 stimulated growth of clones that do not express this growth factor, but did not affect growth of FGF4-producers. Neutralizing IgGs directed against FGF2 strongly inhibited growth of clones that do not produce FGF4, but did not affect growth of FGF4-producers, indicating that the latter are FGF2-independent. Cross-linking experiments done with 125I-FGF2 showed a down-regulation of FGF receptors from the cell surface of FGF4-expressing clones. Taken together, these results indicate that the FGF signalling pathway may be involved during tumoral progression of human breast epithelial cells and that there is a dose-dependent relationship between the quantity of FGF4 produced and tumor development.

Animals↗

PubMed1996

[Profile of p53 mutations and abnormal expression of P53 protein in 2 forms of esophageal cancer].

AIMS AND METHODS: p53 tumor suppressor gene is involved in the development of esophageal cancer. However, its role is not precisely defined in the two types of cancer, squamous cell carcinoma and adenocarcinoma developed in Barrett's esophagus. The aim of this work was to compare the frequency and type of mutation of the p53 gene and the expression of the p53 protein in a series of 21 squamous cell carcinomas and 27 adenocarcinomas of the esophagus in a single institution. p53 protein accumulation was assessed by immunohistochemistry with the monoclonal antibody PAb 1801. The mutations of exons 5 to 8 of p53 gene were detected by denaturating gradient gel electrophoresis, and sequenced. RESULTS: A mutation of the p53 gene and/or an accumulation of the p53 protein were found in 85% of squamous cell carcinomas and 92% of adenocarcinomas, respectively. The profile of mutations differed with the type of tumor; large predominance of transitions on CpG dinucleotides in adenocarcinomas suggesting an endogenous mechanism, high percentage of transversions in squamous cell carcinomas suggesting a direct effect of carcinogens present in tobacco and alcohol. CONCLUSION: Mutation of the p53 gene is a very frequent event in the two types of esophageal cancer. The mechanism responsible for these mutations is different in squamous cell carcinomas and adenocarcinomas developed in Barrett's esophagus.

Adenocarcinoma↗

PubMed1995

Incidence of germ-line p53 mutations in patients with gliomas.

Epidemiological studies on intracranial tumors have suggested that the observed familial aggregation of a proportion of gliomas may be due to inherited predisposition to their development. In the Li-Fraumeni syndrome (LFS) associated with germ-line mutations of the p53 gene, nervous-system tumors are observed with increased frequency. However, the contribution of germ-line p53 mutation to the incidence of brain tumors has not been investigated. In order to address this point, we have performed 2 independent investigations. First, we have examined an unselected series of brain tumors. Whenever the presence of a p53 mutation in the tumor was observed, the possible germ-line origin of the mutation was investigated. Germ-line p53 mutations were also analyzed in constitutional DNA of patients with gliomas that had been selected for an unusual personal or familial history of cancer. Germ-line p53 mutations were detected in 1 out of 80 unselected cases and in 3 out of 15 selected cases (20%). We conclude that germ-line p53 mutation may contribute to a small fraction of gliomas that develop in the general population. The presence of a personal or familial history of cancer in a patient with glioma should prompt the search for a germ-line p53 mutation. However, the low frequency of p53 germ-line mutation suggests that alterations of this gene may not account for most familial cases of gliomas.

Amino Acid Sequence↗

PubMed1995

Frequent loss of heterozygosity on chromosome 9, and low incidence of mutations of cyclin-dependent kinase inhibitors p15 (MTS2) and p16 (MTS1) genes in gliomas.

Four cyclin-dependent kinase inhibitors called p15, p16, p21 and p27 have been identified in mammals. Because these proteins participate in the control of cell cycle, they are potential targets for somatic mutations during carcinogenesis. In order to document the prevalence of p15 and p16 alterations in gliomas, we looked for loss of heterozygosity of chromosome 9p where these genes are localized. Allelic losses were observed in 31 of 44 investigated cases. In all cases they involved the p15/p16 locus. We then looked for mutations in the p16 and p15 genes in 46 gliomas. A total of three DNA variants were observed which were all present in the matched constitutional DNA. They may be unrelated to tumor development. A single somatic mutation was detected. It involved a C to G substitution in codon 93 of p16 and is predicted to change a threonine into an arginine. Taken together, these data indicate that inactivation by point mutation of these two cyclin-dependent kinase inhibitors is uncommon in glial tumor carcinogenesis, but that there may be a tumor suppressor gene on 9p in the vicinity of p16 and p15 genes.

Base Sequence↗

PubMed1995

Polymorphisms and probable lack of mutation in the WAF1-CIP1 gene in colorectal cancer.

WAF1/CIP1, a gene up-regulated by p53 encodes an inhibitor of cyclin-dependent kinases. Induction of WAF1/CIP1 in cells with intact p53 is believed to be instrumental in cell cycle arrest and apoptosis caused by DNA damage. In a model system, WAF1/CIP1 has been shown to have tumor suppressive activity. It is not known however whether WAF1/CIP1 is mutated in human primary tumors. Cells from colorectal cancer have been shown to acquire a series of genetic alterations, including frequent p53 mutations. Thus colorectal tumors, particularly those without identified p53 mutations, are good candidate to search for putative WAF1/CIP1 mutations. DNA extracted from 45 tumors, (including 28 tumors for which p53 mutations had previously been searched for and not found) were PCR amplified for exon 2 of WAF1/CIP1. A search for point mutations was performed in each amplified product using a denaturing gradient gel electrophoresis (DGGE) technique which enables the efficient screening of codons 9 to 139 (i.e. 80% of the WAF1/CIP1 coding sequence). Two different DNA variants were identified and shown to be present in constitutional DNAs of the corresponding patients. The first variant, a C to A transversion at codon 31, changes a serine for an arginine and was detected in eight tumors (18% of the cases). The second variant, detected in a single case (2%) is a silent A to T transversion at the third base of codon 91. DNA extracted from 70 unrelated members from the Centre d'Etude du Polymorphisme Humain (CEPH) was screened for these polymorphisms. The ser/arg polymorphism of codon 31 was detected in seven cases (10%) thus suggesting that it is not associated with a marked colorectal cancer predisposition. The polymorphism on codon 91 was not detected. Two additional variants (arginine to histidine at position 67 and threonine to methionine at position 80) were observed once each in the CEPH family members. Somatic mutation of the WAF1/CIP1 gene was not observed, indicating that, unless there are hot spots for mutations outside the screened region, this gene is not a frequent site of point mutation in colorectal cancer.

Base Sequence↗

PubMed1995

[Molecular biology in cerebral gliomas: recent advances].

Meanwhile first gene therapy assays in cerebral gliomas are in progress, we present in this article a review of the major genetic aberrations reported to date in these tumors. The analysis of these alterations enabled, these last few years, significative advances in the understanding of the mechanisms involved in glioma tumorigenesis. It is now well recognized that tumors result from a multistep process requiring in particular proto-oncogenes activation and tumor suppressor genes inactivation. The different genes implicated in the development of gliomas, their place in the tumoral progression and their potential therapeutic relevance are detailed in this review. The continuation of the identification of glioma altered genes is essential to provide new diagnostic and pronostic markers, and specific targets for more efficient therapy.

Brain Neoplasms↗

PubMed1994

p53 mutations in human tumors with chimeric EWS/FLI-1 genes.

The Ewing family of tumors is recurrently characterized at the molecular level by the presence of a fusion transcript between the EWS gene on chromosome 22 and either the FLI-I or ERG genes, 2 closely related members of the Ets family of transcription factors. We have investigated 12 primary human tumors, 11 xenografts and 11 cell lines, which have been shown to express chimeric EWS transcripts in search of p53 mutations. Fragments of exons 5 to 8 and the corresponding consensus splice sequences were amplified by PCR and analyzed by denaturing gradient gel electrophoresis (DGGE). In 12 of 34 samples p53 mutations were detected (including 4 samples with multiple p53 mutations). The distribution of the mutations in the various samples was as follows: primary tumors 2/12; cell lines 5/11; xenografts 5/11. No correlation between the presence or absence of p53 mutations and the presence of a specific EWS chimeric transcript was observed. In addition, we observed that p53 mutations were almost always associated with a second hit (either deletion or second mutation) on the other p53 allele.

Animals↗

PubMed1994

Single base pair germ-line deletion in the p53 gene in a cancer predisposed family.

A family with an aggregation of adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, and early onset breast cancer was referred to our laboratory. Because this aggregation was reminiscent of Li-Fraumeni syndrome, germ-line mutation of the p53 tumor suppressor gene was sought in the DNA of two affected members. The highly conserved regions spanning exons 5 to 8 of the p53 gene were screened by a previously validated denaturing gradient gel electrophoresis method. A single base pair deletion at codon 215 was detected in constitutional DNA of the two patients, and in the DNA extracted from an adrenocortical carcinoma tumor specimen of the propositus. This deletion is predicted to lead to the formation of a truncated p53 protein, a relatively rare event in Li-Fraumeni families. The spectrum of tumors observed in this family does not differ markedly from the spectrum observed in families with missense p53 mutations.

Adult↗

PubMed1994

TP53 gene mutations and p53 protein immunoreactivity in malignant and premalignant Barrett's esophagus.

BACKGROUND/AIMS: Limited data are available regarding TP53 gene alterations in Barrett's esophagus. This study was undertaken to characterize TP53 mutations and p53 protein immunoreactivity in cancers and preinvasive lesions of Barrett's esophageal mucosa. METHODS: Seventeen Barrett's adenocarcinomas were examined by polymerase chain reaction amplification, denaturant gradient gel electrophoresis, and sequencing for the presence of TP53 mutations in exons 5-8. In 9 cases, Barrett's epithelium adjacent to the cancer was investigated. p53 protein immunoreactivity was studied with PAb 1801. RESULTS: Sixteen mutations were found in 15 adenocarcinomas, including 10 missense, 3 nonsense, 1 frameshift, and 2 mutations located within consensus splice donor and acceptor sequences. All nucleotide substitutions were transitions. Eight of the 12 transitions involving a GC base pair occurred within the context of a CpG dinucleotide. p53 immunostaining was present in all 10 cases with missense mutations and in 1 case without a detectable mutation. The surrounding Barrett's mucosa showed TP53 mutations identical to that observed in the carcinoma in only 3 of 5 specimens showing high-grade dysplasia. CONCLUSIONS: TP53 gene mutations and p53 protein immunostaining are present in a majority of Barrett's adenocarcinomas. Our results suggest that these mutations are involved at an early stage during malignant transformation of Barrett's esophagus.

Adenocarcinoma↗

PubMed1994

Association of p53 mutations with short survival in colorectal cancer.

BACKGROUND/AIMS: Mutations in p53, a tumor suppressor gene located on chromosome 17p, are the most frequent genetic alterations found in human cancers. Increased intracellular concentration of p53, which is frequently but not systematically related to p53 mutation, has been proposed to be associated with poor prognosis in some tumor types. In colorectal cancer, this significance is still a matter of debate. To directly investigate the relationship between prognosis and p53 mutation, this study screened a series of 85 colorectal carcinomas for mutations in exons 5-8 of this gene. METHODS: Polymerase chain reaction-amplified products from tumor DNA were analyzed by denaturing gradient gel electrophoresis and direct DNA sequencing. RESULTS: Forty-four tumors were found to be mutated (52%). A strong correlation between the presence of a mutation and short survival was observed (P = 0.003). When tumors were classified according to their histological stage, a multivariate Cox model analysis showed that p53 mutation, rather than 17p allelic loss (previously proposed to convey prognostic information), was retained as the only independent prognostic factor (relative risk, 2.25; 95% confidence interval, 1.06-4.80; P < 0.029). CONCLUSIONS: Combined with staging, direct monitoring of p53 mutation improves prognostic accuracy for colorectal cancer.

Aged↗

PubMed1994

FGF2 as an autocrine growth factor for immortal human breast epithelial cells.

HBL100 is a human breast epithelial cell line which has been immortalized by the defective genome of SV40 but which is not tumorigenic in nude mice. Growth experiments in defined serum-free and protein-free media revealed its ability to grow without exogenous supply of serum growth factors. Addition of heparin to the serum-free medium increased its growth rate, suggesting the release of heparin-binding autocrine growth factor(s) in the culture medium. HBL100-conditioned medium was mitogenic for HEMG (human embryonic mammary gland) and CCL39 fibroblasts. Western blot analysis of growth factors revealed the presence of the M(r) 18,000, 22,000 and 24,000 forms of basic fibroblast growth factor (bFGF or FGF2) both in HBL100 cell extracts partially purified on heparin-Sepharose beads and in concentrated conditioned medium. Northern blot analysis of HBL100 RNA demonstrated the synthesis of 7-, 3.7-, and 1.8-kilobase (kb) FGF2-specific mRNAs by these cells. Anti-FGF2 neutralizing IgG inhibited HBL100 growth in serum-free medium in a dose-dependent manner. Affinity cross-linking of 125I-FGF2 to the HBL100 cell surface revealed an FGF receptor of 130 kDa. Taken together, these results suggest that FGF2 may be implicated as an autocrine growth factor in the early events leading to neoplastic transformation of human breast epithelial cells.

Animals↗

PubMed1994

Relative efficiency of denaturing gradient gel electrophoresis and single strand conformation polymorphism in the detection of mutations in exons 5 to 8 of the p53 gene.

p53 is the most commonly mutated gene in a large variety of human tumors including familial cancers. Because p53 mutations have in a number of human cancer types, been related to a negative outcome of the disease and the importance of pre-symptomatic diagnosis in cancer-prone families, screening for p53 mutations is becoming more and more widely used. In order to avoid sequencing of the complete coding sequence, several pre-screening methods have been developed and applied to the p53 gene. Among them, Single Strand Conformation Polymorphism (SSCP) and Denaturing Gradient Gel Electrophoresis (DGGE) appear to be highly sensitive. In this work, we used 52 different p53 variants to compare the two methods. In our conditions, DGGE is more sensitive than SSCP since 100% of the variants were detected. SSCP detected 90% of the variants, but efficiency of the method can still be improved by additional optimization experiments.

Base Sequence↗

PubMed1993

[Genetic heterogeneity of certain sporadic colorectal cancers].

Intratumoral heterogeneity provides information concerning the timing of genetic events which occur during colorectal carcinogenesis. In a series of 14 colorectal adenocarcinomas, the following genetic alterations were characterized: loss of heterozygosity on chromosomes 17 p, 1 p, 18 q, 5 q and 22 q, point mutations on TP53 and K-RAS genes, change in DNA index. Six of the 14 initial samples were investigated for putative genetic heterogeneity. In two cases, variations in genetic alterations on chromosome 17 p and gene TP53 were demonstrated. These events seem to occur at a late stage in colorectal carcinogenesis.

Adenocarcinoma↗

PubMed1993

Efficient screening of p53 mutations by denaturing gradient gel electrophoresis in colorectal tumors.

Alterations in exons 5-8 of the p53 gene have been found in the vast majority of human tumors. Although some specific codons have been revealed as preferential mutational sites in defined tumor types, mutations are generally so scattered within this region that various screening methods for their detection have been widely used. However, the capacity of these techniques to detect exhaustively all mutations has not been evaluated. In this report, conditions for analysing exons 5-8 of the p53 gene by denaturing gradient gel electrophoresis (DGGE) have been elaborated using the Melt87 and SQHTX computer programs. The procedure requires five different amplifications. The screening of 90 colorectal tumors revealed 56 amplification products demonstrating abnormal DGGE behavior. Direct sequencing of these amplification products after asymmetric polymerase chain reaction (PCR) showed, besides polymorphism, 49 somatically mutated DNA samples (54.4%) corresponding to 38 different p53 variants. Moreover, 25 additional p53 variants identified in other tumor types were also detectable with our conditions. Thus, altogether the procedure has been successfully used in the detection of 63 different p53 variants. The experimental differences in migration between the wild-type homoduplex DNA molecule and the heteroduplex(es) containing one mismatch were systematically compared with those calculated by the SQHTX program. The computer program was found to be reliably predictive. This DGGE procedure appears thus to be effective and reliable for detecting mutations in exons 5-8 of the p53 gene.

Base Sequence↗

PubMed1993

Frequently elevated content of immunochemically defined wild-type p53 protein in colorectal adenomas.

Mutations of the p53 tumour-suppressor gene are considered to be rare in human colorectal adenomas, a premalignant state of the digestive tract. We have analysed a series of 32 exophytic tumours of the colon and rectum for the presence of p53 protein. In 26 of the 28 pure adenomas, the presence of significant levels of p53 proteins was established by a sensitive two-point enzyme-linked immunosorbent assay. The detectability of p53 protein was frequently limited to PAb 1801, recognizing an N-terminal epitope. Immunoblotting of the fractions captured by the monoclonal antibodies revealed that PAb 421 reacted exclusively with a 53-kDa species, whereas an additional 48-kDa band was detected after incubation with PAb 1801. In the adenomas, the mutant conformation-specific PAb 240 was always negative and no mutations were detected on exons 5-8 in three large and highly dysplastic lesions, selected for their high p53 protein content. The remaining four of the 32 tumours presented foci of cancer. Three of these were shown to contain 'mutant' PAb 240-reactive p53, and gene mutations were identified in two by denaturing gradient gel electrophoresis and sequencing of the amplified products. Intense p53 nuclear immunohistochemical staining was associated with the malignant areas. We conclude that a novel mechanism affecting the regulation of p53 protein could occur in colorectal adenomas.

Adenoma↗

PubMed1993

Short direct repeats flanking deletions, and duplicating insertions in p53 gene in human cancers.

Compilation of 740 independent p53 mutations from a wide variety of human cancers was performed between 1989 and April 1992. Deletions or insertions were observed in 10% of the cases. Insertions ranged from 1 to 14 nucleotides. In 14 out of 16 cases, the inserted nucleotide(s) duplicated the sequence where it was inserted. The deletions ranged from one to 37 nucleotides. Twenty six cases were single nucleotide deletions and 19 of them were localized at iterated nucleotides. In all 26 deletions of two nucleotides or more, a direct repeat of 2-8 base pairs was present on the unaltered sequence in the close vicinity of the deletion. In 15 of these cases, the deletion removed a complete repeat and the entire region in between repeats. In the remaining cases, the deletion imperfectly removed one or both repeats and/or the intercalating sequence. Both p53 insertions and deletions can be explained by a slipped-mispairing mechanism as proposed for germinal mutations of a small number of eukaryotic genes. Less frequent than the deamination of 5-methyl cytosine in CpG dinucleotides, mutations resulting in loss or gain of nucleotide base pairs may represent the second highest endogenous mutagenic event for p53 gene in human cancers.

Base Sequence↗

PubMed1992

p53 from basic research to clinical applications.

Mutations on the TP53 tumor suppressor gene and allele loss on chromosome 17p, where this gene has been located, are among the most frequent alterations yet identified in human malignancies. The p53 protein is highly conserved through evolution and expressed in most normal tissues. Wild-type p53 has been shown to control normal cell proliferation possibly through transcriptional regulation. The wild-type TP53 gene can suppress cell transformation and neoplastic cell growth. In contrast, mutant TP53 has lost the tumor suppressing ability and, in most cases, has gained a transformation promoting ability. Many different TP53 mutations have common conformational and functional consequences on the p53 protein. However, all the TP53 mutants are not necessarily equivalent in terms of biological activity: some mutations confer a strong transforming ability, while others lead to truncated products with probably no biological function. Some mutants may exhibit a dominant behavior over wild-type TP53, others may be recessive. Point mutations of TP53 tend to occur on evolutionary conserved positions. However, the TP53 mutational spectrum differs among cancer types, and this fact may reflect different exogenous mutagens and endogenous factors contributing to human carcinogenesis. In defined types of malignancies, the tumors with TP53 alteration or tumors with allele loss on 17p are associated with more aggressive phenotypes than those without these alterations. For these malignancies, the monitoring of TP53 alteration should now be included in therapeutic trials. Germ line mutations on TP53 may also occur. Individuals with constitutional TP53 mutation have a predisposition to a wide variety of neoplasms. Further characterization of this predisposition will enable the definition of the best follow-up for these at-risk patients.

Animals↗