Thierry Soussi

APC Gene: Database of Germline and Somatic Mutations in Human Tumors and Cell Lines

A database (http://perso.curie.fr/tsoussi ) is described, in which over 1000 mutations in the human APC gene of tumors (colon cancer predominantly) are compiled from the literature. It includes both molecular information about the mutations and clinical data about the patients. Software has been designed to analyse all this information in the database.

The humoral response to the tumor-suppressor gene-product p53 in human cancer: implications for diagnosis and therapy

Abstract Mutation of the tumor-suppressor gene p53 appears to be an important trigger for the development of a humoral response in cancer patients. As discussed here, diagnosis of an alteration in p53 can be used as a marker of unfavorable prognosis and indicate a potential target for immunotherapy.

Can we predict solar ultraviolet radiation as the causal event in human tumours by analysing the mutation spectra of the p53 gene

The tumour suppressor gene, p53, has proved to be one of the genes most often modified in human cancers. These alterations consist mainly of point mutations located in the evolutionarily conserved sequences which render the protein inactive for its normal biological functions. In fact the p53 gene presents nearly 300 potential mutation sites whose analysis should enable the correlation of specific mutation spectra with different causal agents in cancer development. In this study we have analysed the mutation spectrum of the p53 gene in skin tumours from normal individuals and repair-deficient xeroderma pigmentosum (XP) patients in comparison with mutations found in internal cancers. Point mutations are mainly GC-->AT transitions in skin tumours (74% in non-XP, 87% in XP), and also to a lesser extent in internal tumours (47%) where, however, they are mainly located at CpG (63%) sequences probably due to the deamination of the unstable 5-MeC. Moreover, mutations are targeted at py-py sequences in over 90% of skin tumours whereas the distribution of mutations in internal malignancies is proportional to the frequency of py-py sites (61%) and other sequences (39%) at mutable sites. Indeed, in XP skin tumours 100% of the mutations are targeted at py-py sequences and 55% of these are tandem CC-->TT transitions considered as a signature of UV-induced lesions. In skin tumours from normal individuals, 14% of the p53 mutations are double mutations and as in XP skin tumours all these are CC-->TT transitions. In contrast, internal tumours rarely contain tandem mutations (0.8%), and of these only 2/14 were CC-->TT transitions. Finally, nearly all (95%) of the mutations in XP are located on the non-transcribed strand while internal or non-XP skin tumours do not show this strand bias. Hence, the mutation spectrum analysed in XP skin tumours also demonstrates for the first time the existence of preferential repair in humans. In conclusion, the specificity of UV-induced p53 mutation spectra in skin tumours shows that this gene is a particularly appropriate candidate for the correlation of mutation spectra with specific damaging agents.

REGULATION OF MUTANT P53 TEMPERATURE-SENSITIVE DNA BINDING

We have examined in detail the DNA binding properties of several immunopurified tumor-derived mutant p53 proteins (Val-143 → Ala, Arg-175 → His, Arg-248 → Trp, Arg-249 → Ser, and Arg-273 → His). While all mutants were defective for binding to DNA at 37°C, each bound specifically to several cognate p53 binding sites at sub-physiological temperatures (25-33°C), and several mutants activated transcription from a p53-responsive promoter at 26°C in transfected H1299 cells. Heating mutant p53 proteins at 37°C irreversibly destroyed their ability to subsequently bind at 25°C. However, several different monoclonal antibodies that each share the ability to recognize an epitope encompassing amino acids 46-55 markedly stabilized binding by mutant p53 proteins at 37°C. Both intact antibody and FAb fragments allowed mutant p53 to bind to DNA. By contrast, antibodies that recognize epitopes located elsewhere within p53 stabilized mutant p53 binding significantly less effectively. Our data show that the major hot-spot p53 mutants have the intrinsic ability to bind to DNA and that a unique region within the N terminus of p53 may be critical for rescuing them from loss of binding at physiological temperatures. This suggests the possibility of developing small molecules that can stabilize mutant p53 proteins under physiological conditions.

Rainbow trout p53 : cDNA cloning and biochemical characterization

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.

The p53 tumour suppressor gene: a model for molecular epidemiology of human cancer

The gene encoding the tumour suppressor protein p53 is one of the most commonly mutated genes in human cancers. Analysis of the mutational events that target the p53 gene has revealed evidence for both exogenous and endogenous mutational mechanisms. For example, the p53 mutational spectrum reveals evidence for a direct causal effect of ultraviolet radiation in skin cancer, of aflatoxin B1 in liver cancer and of tobacco smoke in lung cancer. This novel field, molecular epidemiology of human cancer risk, has added a new dimension to classical associative epidemiology by providing a direct link between human cancer and carcinogen exposure.

Expression of p53 in oral squamous cell carcinoma is associated with the presence of IgG and IgA p53 autoantibodies in sera and saliva of the patients.

Around 50% of head and neck cancers are known to have aberrations of the p53 gene. Overexpression of the mutant p53 protein can induce a specific humoral response in cancer patients. Matched saliva, serum, and tissue samples from 26 patients with histologically confirmed oral squamous and verrucous carcinoma were investigated. p53 protein expression was evaluated by immunohistochemistry and antibodies specific for 53 protein were analysed in sera and whole mouth saliva by ELISA, immunoprecipitation, and competition assays; 16/25 (64%) samples demonstrated the stabilized p53 protein in tissues and 7/26 (27%) had a high level of p53 antibody in serum. In samples where matching saliva was available, p53 antibody was also present in saliva. In some tumours, only IgA‐type p53 antibody was detected. p53 antibodies were found only in the serum and saliva of patients who showed p53 overexpression in their tumour tissues. These results demonstrate that detection of p53 antibodies can offer a specific and non‐invasive method for the detection of a subset of tumours with p53 aberrations. Copyright

ATF/CREB site mediated transcriptional activation and p53 dependent repression of the cyclin A promoter

Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated through tight control of its promoter. We have previously shown that the ATF/CREB site, present in the cyclin A promoter, mediates transcriptional regulation by CAMP responsive element binding proteins. The main goal of the present study was to investigate whether this site is involved in transcriptional regulation of the gene. We have constructed stable NIH‐3T3 cell lines that express the luciferase reporter gene under the control of normal or mutated versions of the cyclin A promoter. We show that the ATF/CREB is required to achieve maximal levels of transcription from the cyclin A promoter starting in late G1. We also show that down‐regulation of the cyclin A promoter by p53 does not implicate a direct binding of p53 to its cognate consensus sequence but occurs probably by interference with trans‐activating factors. This result suggests that p53 can interfere with transcription of the cyclin A gene, in the absence of a TATA sequence in the promoter.

Absence of p53 germ-line mutations in bilateral breast cancer patients

SummaryThe 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.

Stimulation of rat liver α‐ and β‐type DNA polymerases by an homologous DNA‐unwinding protein

Stimulation of DNA polymerases by homologous DNA-unwinding proteins is one of the strongest arguments to assign a role to these proteins in the in vivo DNA synthesis [I]. In the field of prokaryotic replication, Molineux et al. [2] have shown in E. coli that only DNA polymerase II was stimulated in vitro by the homologous DNA-unwinding protein. Moreover, a significant inhibition of both DNA polymerase I and DNA polymerase III was observed. Nevertheless, Weiner et al. [3] have reported that the DNAunwinding protein was also absolutely required in the conversion of the single stranded DNA of phage G4 into replicative form, catalyzed by DNA polymerase III holoenzyme. In both cases, optimum of stimulation occured with an amount of unwinding protein sufficient to cover all the single stranded regions of the template DNA. In the field of eukaryotic replication, DNAunwinding proteins had been described by Herrick and Alberts in calf thymus [4,5] and by Otto et al. in mouse cells [7]. These proteins are able to stimulate specifically the homologous DNA polymerase CK [6,7]. A DNA-unwinding protein was purified to homogeneity from rat liver and its main properties had been studied. In the native state, the protein is a tetramer