IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools.

Since 1989, about 570 different p53 mutations have been identified in more than 8000 human cancers. A database of these mutations was initiated by M. Hollstein and C. C. Harris in 1990. This database originally consisted of a list of somatic point mutations in the p 53 gene of human tumors and cell lines, compiled from the published literature and made available in a standard electronic form. The database is maintained at the International Agency for Research on Cancer (IARC) and updated versions are released twice a year (January and July). The current version (July 1997) contains records on 6800 published mutations and will surpass the 8000 mark in the January 1998 release. The database now contains information on somatic and germline mutations in a new format to facilitate data retrieval. In addition, new tools are constructed to improve data analysis, such as a Mutation Viewer Java applet developed at the European Bioinformatics Institute (EBI) to visualise the location and impact of mutations on p53 protein structure. The database is available in different electronic formats at IARC (http://www.iarc. fr/p53/homepage.htm ) or from the EBI server (http://www.ebi.ac.uk ). The IARC p53 website also provides reports on database analysis and links with other p53 sites as well as with related databases. In this report, we describe the criteria for inclusion of data, the revised format and the new visualisation tools. We also briefly discuss the relevance of p 53 mutations to clinical and biological questions.     

APC gene: database of germline and somatic mutations in human tumors and cell lines.

A database is described in which over 700 mutations in the human APC gene of tumors (colon cancer predominantly) are compiled from the literature. It includes both molecular informations about the mutations and also clinical data about the patients. A software have been designed in order to analyse all these informations in the database.     

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.     

Analysis of somatic mutations at human minisatellite loci in tumors and cell lines

Hypervariable human minisatellite loci show a substantial level of germline instability, and spontaneous mutation rates to new length alleles have been measured directly by pedigree analysis. We now show that mutation events altering the number of minisatellite repeat units are not restricted to the germline, but also arise in other tissues. Mutant alleles can be detected at a very low frequency in lymphoblastoid cell lines and at much higher frequencies in clonal tumor cell populations, most particularly in gastrointestinal adenocarcinomas. Mutant alleles in these tumors are usually present at a dosage equal to or greater than that of the progenitor allele, indicating that most or all of the tumor cells carry the same clonally derived mutant allele. As with germline mutation, the incidence of somatic mutations in tumors varies from locus to locus, with the same locus showing the highest level of germline and somatic instability. Most length changes, as those in the germline, are of only a few repeat units; however, very large changes are also observed, implying that such mutations can occur in the absence of meiosis.     

Database and software for the analysis of mutations at the human p53 gene.

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.     

p53 gene expression and activation of p53-dependent transcription in melanoma cell lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope of this protein does not derive from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, which suggested a differential effect of camptothecin on tumor-derived and primary cells.     

p53 mutations in tumors derived from irradiated human thyroid epithelial cells

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.     

Multiple mutations of the p53 gene in human mammary carcinoma

Alteration of the p53 tumor suppressor gene is the most common genetic abnormality in human cancer. In breast cancer, depending on the stage of disease and method of detection, mutation rates of 25-60% have been observed. Multiple mutations of p53 gene in the same tumor however, are rarely reported. In this study we explored the frequency of multiple mutations of p53 gene in mammary carcinoma in a cohort of south Florida patients. Three hundred eighty-four cases of primary breast cancer diagnosed between 1984 and 1986 at the University of Miami, Jackson Medical Center were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on cloned PCR-amplified DNA of formalin-fixed, paraffin-embedded tumors. Two hundred thirty-four of 384 breast cancers (61%) had p53 mutation. Of those, 36 tumors showed more than one mutation; 31 tumors had two mutations, three showed three, one tumor had five mutations, and one case carried six mutations. The majority of mutations were missense (43) followed by silent (35); and most occurred within a single exon. Our study suggests that multiple mutations of p53 suppressor gene in breast cancer are more common than currently believed.     

p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines

The p53 tumour suppressor gene is activated following cellular exposure to DNA-damaging agents. The functions of wild-type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity.
In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96-well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants α and β in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4–10 by the polymerase chain reaction (PCR), screening for mutations using single-strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild-type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4±0.1 to 2.6±0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild-type p53 allele were significantly ( P <0.01) more radioresistant (mean inactivation dose 2.23±0.15 Gy) than cell lines which lacked a wild-type gene (1.82±0.24 Gy).
Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild-type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

p53-independent downregulation of histone gene expression in human cell lines by high- and low-let radiation

Using microarrays to analyze differential gene expression as a function of p53 status and radiation quality, we observed downregulation of a large set of histone genes in p53 wild-type TK6 cells 24 h after exposure to equitoxic doses of high-LET (1.67 Gy 1 GeV/amu (56)Fe ions) or low-LET (2.5 Gy γ rays) radiation. Quantitative real-time PCR of specific subtypes of core (H2A, H2B, H3 and H4) and linker (H1) histones confirmed this result. DNA synthesis and histone gene expression are tightly coordinated during the S phase of the cell cycle, and both processes are regulated by cell cycle checkpoints in response to DNA damage caused by ionizing radiation. However, we observed similar repression of histone gene expression in both TK6 cells and their p53-null derivative NH32 after radiation exposure, although the histone gene expression was not decreased to the same extent in NH32 cells as it was in TK6 cells. We also found decreased histone gene expression that was dose- and time-dependent in the colon cancer cell line HCT116 and its p53-null derivative. These results show that both high- and low-LET radiation exposure negatively regulate histone gene expression in human lymphoblastoid and colon cancer cell lines independent of p53 status.     

Splicing mutations in TP53 in human squamous cell carcinoma lines influence immunohistochemical detection.

The mutational status of the tumor suppressor gene TP53 is often examined by immunohistochemistry. We compared the incidence of TP53 mutations in 12 permanent squamous cell carcinoma lines of the head and neck with the immunohistochemical staining obtained with two different antibodies. The mutational status of the TP53 gene was assessed by sequencing the complete coding frame of the TP53 mRNA. All 12 tumor cell lines had TP53 mutations. Six of them showed missense mutations and five had premature stop codons caused either by splicing mutations or nonsense mutations or by exon skipping. One tumor cell line was heterozygous, with a truncating splicing mutation and an additional missense mutation located on different alleles. In one case, an in-frame insertion of 23 extra codons was found. All missense mutations were positive in immunhistochemistry and Western blotting. The truncated p53 was not immunohistochemically detected in three cases with the DO-7 antibody and in five cases with the G59-12 antibody, giving false-negative results in 25% or 40%, respectively, of all tumor cell lines examined. We conclude that splicing mutations are common in squamous cell carcinoma lines and that the incidence of p53 inactiviation by erroneous splicing is higher than yet reported. Sequencing of only the exons of TP53 may miss intronic mutations leading to missplicing and may therefore systematically underestimate the TP53 mutation frequency.     

Detection of human somatic cell structural gene mutations by two-dimensional electrophoresis

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.