Biomedical subjects
Publications and source records attributed to T Soussi.
A computerized database containing DNA sequence information regarding human p53 mutants has been created. The database itself is in the dBASE format and contains information on nearly 3000 mutants. In addition, an IBM PC compatible software package to analyze the information in the database has been developed. Both the database and software are freely available via the Internet.
A data base is described in which over 2,500 mutations in the p53 gene of human tumors and tumor cell lines are compiled from a systematic search of reports published before 1 January 1994. Data from 1994 are being added intermittently, with a systematic search and update scheduled for December, 1994. The compilation has been deposited with the EMBL Data Library and is available in electronic form free of charge. This report contains a rationale for the compilation, a brief summary of the major findings and a description of the data base.
The eponym Li-Fraumeni syndrome is given to a particular form of cancer-prone family in which the main encountered tumors are sarcomas occurring in childhood and breast cancers affecting young adult females. There is also an increased frequency of cerebral tumors, leukemias and adrenal carcinomas among these families. The transmission of the cancer-proneness is autosomal dominant and related to the loss of function of the p53 tumor suppressor gene located on the short arm of the chromosome 17. The related p53 protein is identified but its precise mechanism of action and its regulation are still unclear. It seems to activate the genes which negatively regulate the multiplication of the cell and to act as a factor of transcription. A germline mutation of the p53 gene must be looked for in every cancer-prone family but also in case of multifocal tumors, particularly osteosarcomas and glioblastomas, and in case of second malignant neoplasm. This is of major scientific as well clinical interest. Indeed, the study of new families will help to better understand the molecular mechanisms underlying the syndrome. For the family itself this allow to identify the cancer prone members and to offer them preventive measures and early detection of cancers, particularly early breast cancer detection.
Using a set of overlapping peptides of the human p53 protein, we analysed the epitopes recognized by 18 monoclonal antibodies specific for human p53. We showed that most of these epitopes correspond to linear antigenic determinants which lie predominantly in the amino- or carboxy-terminus of the p53 protein. Using either truncated p53 or the set of human p53 peptides, we directly analysed the sera of animals immunized with human p53. These sera contained antibodies which also recognized the regions corresponding to the extremity of the p53 protein. These p53 regions were similar to those recognized by p53-specific antibodies present in sera of patients with cancer. Preferential recognition of these regions by antibodies specific for non conformational epitopes suggested that these regions are localized at the surface of the p53 protein as unfolded structures.
p53 antibodies have been found in sera of patients with breast and lung carcinomas and in children with B-lymphomas. We report here the presence of p53 antibodies in sera of patients with 11 different types of cancer. The frequency of seropositives for p53 varied among the different types of cancer, but a correlation with the frequency of p53 gene alteration was established. Using a powerful peptide enzyme-linked immunosorbent assay, we demonstrated that the immune response of patients with p53 antibodies was restricted to a small subset of peptides localized in the amino and carboxy termini of p53, whatever the type of cancer. Given the similarities of the patterns of immune responses in patients with p53 antibodies and animals hyperimmunized with human p53, we propose that the p53 humoral response is the result of a self-immunization process which is itself the consequence of p53 protein accumulation in tumor cells.
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We previously isolated a p53 cDNA from a Xenopus oocyte library. To determine if p53 has a function in the developmental period, we have studied its expression at the RNA and protein levels during the early development of Xenopus laevis. Two p53 transcripts (3 and 2.2 kb) are expressed from the beginning of Xenopus oogenesis, and the major one (2.2 kb) reaches a level of 7 x 10(5) to 7 x 10(6) transcripts per mature oocyte. After fertilization only the 2.2-kb RNA is detected, but its level decreases and at the neurula stage p53 RNA becomes undetectable. The p53 protein is highly expressed during Xenopus development, in contrast to an undetectable level in Xenopus cells in culture. Most of the p53 protein is synthesized during late oogenesis and a stage VI oocyte contains 7 x 10(11) molecules of p53 protein. This maternal p53 store is maintained at a constant level during Xenopus development, at least until the tadpole stage. This high level of expression is mainly due to stabilization of the p53 protein. Unusually for p53, the protein is strictly located in the cytoplasm of oocytes and this localization might indicate that it is stored in an inactive form at this stage. These data are discussed relative to previous observations made in transgenic mice.
We describe the production of 13 new monoclonal antibodies induced by the product of the human p53 tumor suppressor gene. All these monoclonal antibodies recognize human p53 irrespective of the detection technique: ELISA, immunoblot or immunoprecipitation. These antibodies can be divided into four groups according to such criteria as localization of the recognized epitopes and reactivity with p53 originating from other organisms. One monoclonal antibody was successfully used for immunohistochemistry detection of p53 accumulation in breast carcinoma. This novel panel of monoclonal antibodies represents a further tool for the study of the p53 protein.
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Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder that predisposes individuals to multiple forms of cancer including breast carcinoma, soft tissue sarcoma, osteosarcoma, leukemia and adrenocortical carcinoma. These diverse tumor types develop at unusually early ages. Analysis of the tumor suppressor gene p53 in family members with LFS have demonstrated that germline mutations in the p53 gene were present in most of the LFS family tested so far. Furthermore, germline p53 mutations were also found in cancer-prone individuals which were not indicative of the LFS.
Alteration of the p53 gene is the most frequent genetic feature of human cancer and leads to overexpression of the altered protein in the tumor cell nucleus. Two diagnostic procedures are currently available to assess p53 mutations: (a) molecular analysis of the gene sequence; and (b) immunohistochemical analysis of p53 protein accumulation. We now report a third approach, serological analysis. Fifteen % of primary breast cancer patients were found to have circulating antibodies to p53 protein by immunoprecipitation or immunoblotting. We have found a close correlation between the presence of such antibodies and bad prognosis such as high histological grade and the absence of hormone receptors. Furthermore, we found that the B-cell response to p53 protein is induced by two immunodominant regions located at the carboxy and amino termini of the protein, outside the central mutational hot spot region. These findings suggest that serological analysis, combined with molecular and histochemical methods, may be suitable for assessing the state of the p53 gene in cancer patients.
We have cloned and sequenced the p53-encoding cDNA of rainbow trout (Salmo gairdneri). The encoded product contains the characteristics found in all p53 proteins: (i) the five highly conserved domains, (ii) an acidic N terminus, (iii) a hydrophilic C terminus, and (iv) a penultimate serine residue. Furthermore, we demonstrate that the rainbow trout p53 is able to specifically interact with the SV40 large T antigen.
We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75% homologous to human and mouse p53, respectively. Immunoprecipitations of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.
More than 350 independent point mutations of the TP53 gene, found in a wide variety of human cancers, were compiled and analysed. From this study, we confirm the presence of four hot-spot regions which colocalize with some highly conserved domains of the protein. We also define a new hot-spot region which is observed predominantly in lung tumors. Analysis of the mutational events suggests the direct involvement of environmental carcinogens in lung tumors and hepatocarcinomas, and spontaneous mutagenesis generating essentially CpG transitions in most of the remaining ones. Furthermore, we demonstrate in this work that the TP53 gene is an informative model with which to study the molecular mechanisms of mutagenesis in the human genome.
The cause of Li-Fraumeni syndrome, a rare group syndrome of familial cancers, has recently been identified. Patients with this inherited condition are highly susceptible to specific neoplasms, including early-onset breast cancers. The available evidence links Li-Fraumeni syndrome to inherited mutations of the tumor suppressor gene p53. Moreover, somatically acquired p53 mutations and gene deletions are common feature in breast cancer of sporadic origin. These findings suggest that germline p53 mutations are important in familial and, possibly sporadic, breast tumors. We have therefore screened lymphocyte DNA from 19 unrelated bilateral cancer patients for germline p53 mutations in exons 5, 6, 7 and 8. We have however detected no germline mutations by means of the single-strand confirmation polymorphism technique in any of the lymphocyte DNAs examined and conclude that p53 mutations are not generally involved in bilateral breast cancer.
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