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The tumour suppressor p53 plays a complex role in the regulation of apoptosis. High levels of wild type p53 potentiate the apoptotic response, while physiological range, low levels of the protein have an anti-apoptotic activity in serum starved immortalized fibroblasts. Here we report that primary fibroblast-like cells that show normal growth control are also efficiently protected from apoptosis by the endogenous p53 activity. The capacity to inhibit apoptosis is not restricted to the wild type protein: the R-->H175 p53 mutant fully retains the anti-apoptotic activity of the wild type p53, providing a possible explanation for its high oncogenicity. Using a series of point and deletion mutants of p53 under the control of tetracycline-regulated promoter we show that certain mutants, like the wild type, protect cells at low levels but lead to apoptosis when overexpressed. This latter effect is lost upon deletion of a proline-rich domain in the NH2 part of the protein. The anti-apoptotic activity can be mapped to the extreme carboxy-terminal part of the protein and is therefore independent of other well characterized p53 activities. Our results add a new level of complexity to the network of interactions mediated by p53 in normal physiology and pathology.

Objective: To detect evidence of abnormalities of the p53 protein in autoimmune diseases. Mutation of the p53 protein may inhibit apoptosis and thereby lead to cancer and possibly play a role in the pathogenesis of autoimmune diseases., Methods: Serum antibodies to p53 are detected in 30 to 50% of patients with cancer who have p53 mutations. Using an ELISA, we determined the prevalence of anti-p53 antibodies in the serum of 106 patients with rheumatoid arthritis (RA), 72 patients with primary Sjögren's syndrome (SS), and 14 patients with lymphoma complicating SS. The presence of anti-p53 antibodies was also measured in the synovial fluid of 16 patients with RA. Positive sera by ELISA were confirmed by immunoprecipitation., Results: Serum anti-p53 antibodies were detected in 2 of 106 patients with RA. The synovial fluid of one of these 2 patients was also studied and was positive. Anti-p53 antibodies were not detected in the other synovial fluids. Serum anti-p53 antibodies were not detected in 72 patients with primary SS alone, but were present in 2 of 14 patients with lymphoma complicating SS., Conclusion: Our results suggest that if p53 mutations have any role in the pathogenesis of some autoimmune diseases, they are rarely associated with the presence of anti-p53 antibodies in patients with RA. In patients with SS, the presence of serum anti-p53 antibodies might be an indirect sign of the development of a lymphoma.

We have evaluated the functional properties of the unique p53 mutant Y236delta (deletion of codon 236) that gave rise to apparent cell-type specific tumor development. Four family members carrying this mutation in the germline developed early onset brain tumors, as previously reported. Deletion of residue Y236, which is tightly packed in an evolutionary conserved hydrophobic pocket, results in a protein with a mutant conformation according to immunoprecipitation with the conformation-sensitive antibodies PAb240 and PAb1620. The Y236delta mutant lacks specific DNA binding to the p53-responsive element in the WAF1-promoter, and functional analysis in Saos-2 cells revealed inability to transactivate the p53-responsive elements in the WAF1-promoter and the RGC sequence. The mutant has retained a functional oligomerization domain, a key element mediating the dominant negative effect, and inhibits DNA binding of wild-type p53. In addition, transactivation of endogenous wild-type p53 in LoVo cells was inhibited upon transfection of the mutant in a dose-dependent manner. Thus, in vitro and in vivo data suggest the loss of important tumor-suppressing functions and demonstrate a dominant negative effect of this unique p53 mutant that is associated with an unusual clustering of familial brain tumors.

We report here a systematic analysis of the effects of different p53 mutations on both spontaneous and radiation-stimulated homologous recombination in mouse L cells. In order to monitor different recombination pathways, we used both direct and inverted repeat recombination substrates. In each line bearing one of these substrates, we expressed p53 proteins mutated at positions: 175, 248 or 273. p53 mutations leading to an increased spontaneous recombination rate also stimulate radiation-induced recombination. The effect on recombination may be partially related to the conformation of the p53 protein. Moreover, p53 mutations act on recombination between direct repeats as well as between inverted repeats indicating that strand invasion mechanisms are stimulated. Although all of the p53 mutations affect the p53 transactivation activity measured on the WAF1 and MDM2 gene promoters, no correlation between the transactivation activity and the extent of homologous recombination can be drawn. Finally, some p53 mutations do not affect the G1 arrest after radiation but stimulate radiation-induced recombination. These results show that the role of p53 on transactivation and G1 cell cycle checkpoint is separable from its involvement in homologous recombination. A direct participation of p53 in the recombination mechanism itself is discussed.

Recombinant human p53 isolated either from E. coli or from insect cells is poorly active for binding to DNA but it can be dramatically stimulated by phosphorylation, antibody binding to the carboxy-terminal negative regulatory domain, short peptides derived from this negative regulatory domain or short single strands of DNA. We report here that Xenopus p53 has a very similar behavior. Using a new set of monoclonal antibodies directed either to the amino- or the carboxy-terminus of Xenopus p53, we demonstrate that the frog protein can be activated by specific carboxy-terminus monoclonal antibodies in order to bind to human p53 DNA response element. In addition, we report that such activation of both humans and frogs protein can also be achieved by small peptides derived from the carboxy-terminus of both p53. Although, the sequence of this region is not conserved in the various p53 species, the presence of conserved basic residues indicates that such activation is charge-dependent. This is confirmed by the finding that small poly-lysine peptides can activate both human and Xenopus p53. In vivo expression of Xenopus p53 indicates that this protein is able to transactivate a wide variety of human p53 response elements as long as the experiments are performed at 32 degrees C since activity at 37 degrees C, a temperature well above the natural temperature of Xenopus, is lost. Finally, we demonstrate that human mdm2 is able to down regulate the transcriptional activity of Xenopus p53.

In several types of solid tumours, circulating antibodies to p53 are seen in about a third of cases with a p53 mutation, but are absent in cases without p53 mutation. Therefore detection of those antibodies has relatively low sensitivity but high specificity in the detection of p53 mutations. We looked for circulating p53 antibodies by ELISA in 56 adult non-Hodgkin's lymphoma (NHL) and 80 multiple myeloma cases. A certain or highly probable p53 mutation was found by SSCP analysis, immunocyto- or immunohistochemistry in 8/35 (23%) NHL cases and 2/19 (10%) MM cases analysed by these techniques. None of the 80 MM cases and only one of the 56 cases of NHL had circulating p53 antibodies. The positive case had Burkitt's lymphoma and a p53 missense mutation at codon 273. Thus, very few MM and NHL patients with a p53 mutation develop p53 antibodies and this test does not appear to be useful in haematological malignancies.

The role of p53 in modulating apoptosis has suggested that it may affect efficacy of anti cancer agents. For this reason, we have evaluated p53 alterations in 282 consecutive patients with infiltrating node-negative breast cancer who underwent primary surgery and were randomized either to CMF (Cyclophosphamide 400 mg/m2, Fluorouracil 400 mg/m2, and Methotrexate 40 mg/m2) or control arm (no adjuvant therapy) from 1980 to 1989. p53 alterations were analyzed by immunohistochemistry using DO7 MoAb, revealed by immunoperoxidase technique, and quantitated in term of percentage of positive cells. We observed a positive staining in 24% of the tumors. Among them, 10% had a positive staining in more than 75% of the cells. There was a highly significant association between the proportion of positive cells and histologic grade of the infiltrating ductal carcinomas (p<0.004). However, there was no association with age, tumor size, hormone receptor content, or vascular embolism. There was a trend but no significant relationship between positive staining and overall survival either in each arm of the trial or in the overall population. Interestingly, we observed a higher relative risk of local relapse after conservative therapy in the boosted area in the group of mutated p53 (RR=4.41; p<0.0005). We conclude that, in this node-negative breast tumor population, alteration of p53 cannot predict the response to the chemotherapy. However, it may represent a useful marker of risk of local relapse and of radio resistance.

p53 is a nuclear protein that acts like a tumor suppressor and is involved in regulation of cellular growth. In Xenopus, the p53 protein is highly expressed during oogenesis and is strictly cytoplasmic in the oocyte. We have analysed its participation in DNA replication and transcription during early development, using the egg and oocyte as model-systems. The injection of sperm nuclei into Xenopus eggs is followed by DNA replication and mitotic events. We show that the endogenous p53 enters the nuclei and moves through a series of discrete sub-nuclear loci whose distribution is S-phase specific. A specific peripheral nuclear localization of p53 is observed before entry into S-phase, followed by an internal localization which is strictly dependent on ongoing DNA synthesis. At no stage in the cell cycle, however, did we observe any co-localization with RPA or PCNA, which were used as initiation or elongation markers for DNA replication. We also show that injection into the nucleus of the oocyte of small amounts of either Xenopus or human p53 - less than 10% of the cytoplasmic storage - is sufficient to block RNA polymerase II-dependent transcription from a coinjected TATA-box-containing reporter plasmid. Transcription is rescued by microinjection of the TATA-box binding protein (TBP), suggesting that nuclear exclusion of p53 during oogenesis may be necessary for transcription of maternal genes. These characteristics are discussed in relation to the regulation of nuclear activities during early embryogenesis.

The occurrence of non-Hodgkin's lymphoma (NHL) is the most serious complication of Sjogren's syndrome (SS). We performed a study of 16 NHLs occurring in patients with an underlying SS. These lymphomas arose not only in salivary glands (7 cases) but also in other mucosal extranodal sites (the stomach [4 cases], the lung [3 cases], the skin [3 cases], the buccal mucosa [1 case], the thymus [1 case]) and in nodal sites (8 cases). Low-grade marginal zone lymphomas (MZL) were diagnosed in 12 of the 16 patients, 9 of mucosa-associated lymphoid tissues (MALT) type in mucosal sites and 3 exclusively nodal. The 4 other patients presented with a high-grade B-cell lymphoma that was probably a histological transformation of an underlying low-grade MZL at least in 3 of the cases involving skin, stomach, and parotid, respectively. A t(14;18) translocation was detected in 1 of 8 lymphomas tested. We detected serum anti-p53 antibodies in 2 of the 14 studied patients. p53 protein was detected in 1 of 11 lymphomas tested. LMP protein and Eber RNAs of Epstein-Barr virus (EBV) were not detected in the 16 NHL biopsies. Using polymerase chain reaction, EBV was never detected except in 1 of 4 parotid lymphomas. No human T-lymphotropic virus 1 or human herpes virus 8 DNAs were detected in NHL biopsies. None of the patients had hepatitis C virus infection found using serological methods. Chemotherapy was usually efficient. In conclusion, lymphomas occurring in patients with an underlying SS are in most cases MZL. These lymphomas are not associated with viruses known to be present in other types of lymphomas. Some of the translocations or mutations of oncogenes or antioncogenes described in other lymphomas are detected in SS-associated lymphomas.

Homologous recombination plays an essential role in processes involved in genome stability/instability, such as molecular evolution, gene diversification, meiotic chromosome segregation, DNA repair and chromosomal rearrangements. p53 devoid cells exhibit predisposition to neoplasia, defects in G1 checkpoint and high genetic instability but a normal rate of point mutations. We investigated the effect of a p53 mutation, on spontaneous homologous recombination between intrachromosomal direct repeat sequences, in mouse L cells. In these cells, wild type for the p53 gene, we have overexpressed the mutant p53(175(Arg>His)) protein leading to a p53 mutant phenotype, as verified by the absence of a G1 arrest after gamma-irradiation. We show that the rate of spontaneous recombination is increased from five- to 20-fold in the mutant p53 lines. Moreover, this increase is observed in gene conversion as well as in deletion events. Our results provide new insights into the molecular mechanisms of genetic instability due to a defect of p53.

In recent years, there has been an exponential increase in the number of p53 mutations identified in human cancers. The p53 mutation database consists of a list of point mutations in thep53 gene of human tumors and cell lines, compiled from the published literature and made available through electronic media. The database is now maintained at the International Agency for Research on Cancer (IARC) and is updated twice a year. The current version contains records on 5091 published mutations and is expected to surpass the 6000 mark in the January 1997 release. The database is available in various formats through the European Bioinformatics Institute (EBI) ftp server at: ftp://ftp.ebi.ac.uk/pub/databases/p53/ or by request from IARC (p53database@iarc.fr) and will be searchable through the SRS system in the near future. This report provides a description of the criteria for inclusion of data and of the current formats, a summary of the relevance ofp53 mutation analysis to clinical and biological questions, and a brief discussion of the prospects for future developments.

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 degrees C in transfected H1299 cells. Heating mutant p53 proteins at 37 degrees C irreversibly destroyed their ability to subsequently bind at 25 degrees 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 degrees 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.

Background: Mutation of the p53 tumor suppressor gene (also known as TP53) often leads to the synthesis of p53 protein that has a longer than normal half-life. Mutant p53 protein that accumulates in tumor cell nuclei can be detected by means of immunohistochemical staining techniques. Serum antibodies directed against p53 protein (p53-Abs) have been detected in some cancer patients., Purpose: We assayed serum samples from 80 patients with head and neck squamous cell carcinoma (HNSCC) for the presence of p53-Abs, and we evaluated potential associations between the presence of these antibodies and other histopathologic and clinical features., Methods: Serum was collected from each patient at the time of diagnosis. In addition, tumor biopsy specimens were obtained before the initiation of treatment. An enzyme-linked immunosorbent assay was used to detect p53-Abs. The accumulation of p53 protein in tumor cell nuclei was assessed immunohistochemically by use of the anti-p53 monoclonal antibody DO7. Patient treatment consisted of radiotherapy alone, primary chemotherapy followed by radiotherapy, or surgery and postoperative radiotherapy. Relapse-free and overall survival from the beginning of treatment were estimated by use of the Kaplan-Meier method; survival comparisons were made by use of the logrank statistic. Univariate and multivariate analyses were conducted to identify factors associated with survival. Reported P values are two-sided., Results: Fifteen (18.8%) of the 80 patients had p53-Abs. Tumor cell nuclei in 43 (58.9%) of 73 assessable biopsy specimens exhibited strong p53 immunostaining. Patient treatment method and the accumulation of p53 protein in tumor cell nuclei were not associated with increased risks of relapse or death. In univariate analyses, advanced tumor stage (> T1 [TNM classification]) and the presence of p53-Abs were significantly associated with an increased risk of death (P for trend = .007 and P = .002, respectively), whereas advanced tumor stage, substantial regional lymph node involvement (> N1), and the presence of p53-Abs were associated with an increased risk of relapse (P for trend = .002, P = .02, and P < .0001, respectively). In multivariate analyses, advanced tumor stage and the presence of p53-Abs were significantly associated with increased risks of relapse (p for trend = .04 and P = .003, respectively) and death (P for trend = .04 and P = .03, respectively). At 2 years of follow-up, the overall survival proportion was 63% (95% confidence interval [CI] = 47%-80%) when no p53-Abs were detected compared with 29% (95% CI = 4%-54%) when p53-Abs were detected. Relapse-free survival at 2 years was 62% (95% CI = 49%-76%) if no p53-Abs were detected compared with 13% (95% CI = 0%-31%) if p53-Abs were detected., Conclusions and Implications: The proportion of patients with HNSCC who have serum p53-Abs is smaller than that of patients exhibiting tumor cell accumulation of p53 protein. The presence of p53-Abs is significantly associated with increased risks of relapse and death.

Several years ago, a comparison of the amino acid sequences of p53 proteins from a variety of species enabled us to reveal structural features of this protein, giving clues to its function. Since then, numerous studies on the biochemical, immunological and biological functions of p53 as well as on its structure (including crystallography data) have provided considerable insight into the multifunctional aspects of p53. The purpose of this review is to present the most recent data concerning the various structural features of the p53 protein with special emphasis on its flexibility, which plays a key role in regulation of its biological activity.

p53 alterations are the most common alteration found in human cancer. Protein p53 was studied in 102 patients with early lesions or advanced squamous cell carcinoma of the head and neck (SCCHN). Biopsies and sera samples were collected before the initiation of treatment. Protein p53 expression was evaluated by immunohistochemistry using Pab 1801, Pab 240, DO7 and CM1 antibodies on paraffin-embedded sections. Antibodies specific for p53 protein were analysed in the sera of these patients by an ELISA procedure. We demonstrated that p53 protein overexpression (> or = 20% positive cells) was an early event in the carcinogenesis of SSCHN and correlated to the progression of carcinogenesis. Overexpression of protein p53 was frequent (56.5%) in advanced tumors. No correlation was found with clinical stage or the differentiation status of the tumor, but we demonstrated differences in protein p53 expression according to the initial localisation of the tumor, with high expression in hypopharynx (67%) and oropharynx (65%) versus larynx (12%). The prevalence of p53 antibodies was high (44%) and was correlated with the rate of p53 overexpression (> or = 30% positive cells) in tumors (p < 0.0001 chi square test). These results suggest that the humoral response is an indicator of the presence of squamous cell carcinoma with immunogenic mutant p53 protein. Therefore the detection of anti-p53 antibodies could be used as a precocuous marker of p53 alteration and a prognostic marker, in order to screen for patients with a better prognosis.

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.

In 1994 we described a list of approximately 2500 point mutations in the p53 gene of human tumors and cell lines which we had compiled from the published literature and made available electronically through the file server at the EMBL Data Library. This database, updated twice a year, now contains records on 4496 published mutations (July 1995 release) and can be obtained from the EMBL Outstation-the European Bioinformatics Institute (EBI) through the network or on CD-ROM. This report describes the criteria for inclusion of data in this database, a description of the current format and a brief discussion of the current relevance of p53 mutation analysis to clinical and biological questions.

Germline and somatic mutations in the p53 and APC genes contribute to neoplasia. The patterns of these and other acquired mutations in cancers reflect environmental mutagens and endogenous factors that contribute to carcinogenesis. Herein, we describe a database of almost 2,300 mutations in the p53 and APC genes published until September 1, 1993. In addition to cataloging the mutations, multiple fields of information have been added to facilitate future molecular epidemiological analyses of human cancer. The accuracy of the database has been checked by the present authors and, by soliciting feedback from the original corresponding authors. The strengths and limitations of the primary literature are discussed.

Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were described at first in the heritable connective tissue disorder, Marfan syndrome (MFS). More recently, FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS and many mutations will have to be accumulated before genotype/phenotype relationships emerge. To facilitate mutational analysis of the FBN1 gene, a software package along with a computerized database (currently listing 63 entries) have been created.

We have created databases and software applications for the analysis of DNA mutations in the human p53 gene, the human hprt gene and the rodent transgenic lacZ locus. The databases themselves are stand-alone dBase files and the software for analysis of the databases runs on IBM- compatible computers. The software created for these databases permits filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web (WWW). Open home page http://sunsite.unc.edu/dnam/mainpage.ht ml with a WWW browser. Alternatively, the databases and programs are available via public ftp from anonymous@sunsite.unc.edu. There is no password required to enter the system. The databases and software are found in subdirectory pub/academic/biology/dna-mutations. Two other programs are available at the WWW site, a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

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.

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.

A large number of different mutations in the tumor suppressor gene p53 gene have been identified in all types of cancer. As of September 1995, this database contains over 4200 mutations. This substantial increase since our previous report can enable epidemiological analyses which were not previously possible. In order to capture all these new data, the software permitting analysis has been improved. This report describes the various improvements since first release of the database.

Prostate cancer is one of the most common malignancies in men. Few authors have attempted to identify consistent genetic alterations at the molecular level in adenocarcinoma of the prostate, but those most frequently reported are loss of heterozygosity (LOH) involving chromosome arms 8p, 10q, 16q, and 18q and inactivation of the TP53 tumor suppressor gene. In order to determine if alterations frequently found in other adenocarcinomas (breast, ovarian, colorectal), including losses of genetic material from chromosome arms 1p, 3p, 7q, 8p, 11p, 17p, 17q, and 18q, are also involved in prostate cancer, we examined 20 localized early-stage prostate tumors. We detected no mutations of the TP53 gene. Allelic losses were found from 7q (33%), 8p (50%), 10q (20%), and 18q (33%). Furthermore, as the first step toward isolating tumor suppressor genes on 18q, we used six polymorphic markers and identified a small common deleted region between the chromosome 18 centromere and the D18S19 locus.

The p53 alteration is the most common alteration found in human cancer. It usually involves missense mutations that stabilize the p53 protein, which in turn accumulates, reaching levels detectable by immunohistochemistry. We and others have demonstrated that this overexpression of mutant p53 protein can induce a specific humoral response in cancer patients. This result was assessed by the presence of p53 antibodies in sera of patients with various types of cancers, whereas normal populations do not exhibit such antibodies. In lung cancer, the prevalence of p53 antibodies is high (30%) and is correlated with a very high rate of p53 mutations in this cancer (60-70%). We show that these antibodies are always present at the time of diagnosis, but never appear during tumour development, an observation strengthened by the fact that these antibodies are mostly IgG, corresponding to a secondary immune response. These results suggest that the humoral response is an early event and that p53 antibodies can be used as a precocious marker of p53 alteration before clinical manifestation of the disease.

This study examined the sensitivity and specificity of serum auto-antibodies to p53 protein as a non-invasive marker of p53 genetic alterations or protein accumulation in lung cancer cases. A sensitive ELISA to detect serum p53 antibodies was developed and used to examine sera from 186 patients undergoing pulmonary surgery for a suspected lung cancer. Target antigens in ELISA were wild-type p53 protein and 5 peptides covering the N- and C-terminal parts of the protein. Sixteen sera were positive for serum p53 antibodies in both ELISAs and all were among the 136 patients with confirmed primary lung carcinoma. Of 50 patients with other pulmonary diseases, none had p53 antibodies. In 92 cancer patients exons 5 to 8 of the p53 gene were examined for mutations by denaturing gradient gel electrophoresis and direct sequencing of PCR products. Forty-seven tumours had a p53 mutation and 7 (15.2%) of these were positive for p53 antibodies. Two patients had serum antibodies but no detectable mutation in exons 5 to 8. Frequencies of p53 mutations and serum antibodies were higher in squamous cell carcinoma patients than in adenocarcinoma. Accumulation of p53 protein in tumour tissue was observed in 32 patients, but only 5 were positive for p53 antibodies. In conclusion, serum p53 antibodies were detected only in a proportion of lung cancer cases, but the majority were specifically associated with a detectable p53 mutation in the tumour.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. There are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half-life than wild-type p53 and accumulate in the nucleus of tumor cells. Anti-p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild-type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti-p53 vaccine development in tumor immunology.

P53 mutations can lead to the production of P53 antibodies in serum of cancer patients. Here we studied the prognostic value of P53 antibodies in 353 primary breast cancer patients. P53 antibodies were detected in 42 cases (12%) and were negatively related to oestradiol and progesterone receptors. The median duration of follow-up for live patients was 5.3 years. In actuarial analyses, overall survival was worse in patients with P53 antibody (p < 0.0005); in Cox's multivariate analysis, P53 antibody was an independent prognostic variable. Thus plasma assay of P53 antibody would be useful to select rapidly and easily a population of patients with poor prognosis.

Specific markers for pancreatic or biliary cancer have been developed in the past few years. Ca 19-9 has a good sensitivity but it is also increased in benign cholestasis. Mutations in the p53 gene are commonly reported in pancreatic cancer and can be detected by a serological analysis. The aim of this work was to find out the sensitivity and specificity of this new assay in diagnosing cancer of the pancreas or of the bile ducts. The presence of antibodies against p53 was determined by an enzyme linked immunosorbent assay (ELISA) in 29 patients with pancreatic cancer, 33 with biliary tract cancer, and 33 with benign biliary or pancreatic diseases as controls. p53 Antibodies were detected in eight of 29 patients with pancreatic cancer (28%), in five of 33 patients with biliary tract (15%), and in one patient (3%) with stones of the common bile duct. The sensitivity and the specificity for the diagnosis of malignant biliary or pancreatic diseases were 21% and 96% respectively. It is concluded that the presence of p53 antibodies in the serum of patients with pancreatic and biliary diseases is specific for malignancy and independent from the presence of cholestatic disease.

Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

The well conserved human and murine p53 proteins are tetramers that can activate transcription from templates bearing p53 binding sites. Since the normal function of mammalian p53 is necessary for preserving the stability of genome, we examined the properties of purified Xenopus p53 (Xp53) to determine whether it shares similar biochemical activities. Xp53 was shown to bind specifically to sites containing the p53 consensus sequence derived for human p53. Moreover, Xp53 transactivates reporter genes containing a human p53 response element in vivo. Finally, Xp53 can be cross-linked into tetramers in a manner similar to human p53. However, Xp53 forms hetero-oligomers with human or murine p53 only very ineffectively, in contrast to the efficient hetero-oligomer formation that occurs between human and murine p53 polypeptides. Taken together, our data indicate that sequence specific DNA binding, transcriptional regulation and oligomerization of p53 are common properties of vertebrate p53 proteins, and thus they are likely to be required for the biological activity of the protein.

Fanconi anemia belongs to a group of human genetic diseases characterized by chromosomal instability, sensitivity to genotoxic agents associated to impaired processing of DNA lesions, cell cycle anomalies and cancer predisposition. We recently added to this list of distinctive features reduced production of interleukin 6 and overproduction of tumor necrosis factor alpha. Since growth factor deprivation, TNF alpha treatment or DNA damage can trigger apoptosis, we monitored the apoptotic response of FA cell lines. We show here that, although the spontaneous rate of apoptosis is slightly more elevated in FA than in normal cell cultures, the apoptosis induced by gamma-irradiation is drastically reduced in FA. Since the induction of apoptosis by radiation is a p53-dependent mechanism, the induction of this protein in FA cells was also examined. We found that the p53 protein is not radio-induced in FA cells belonging to the two genetic complementation groups examined (C and D), in contrast to normal cells. Moreover, the same impairment in p53 induction is observed after exposure to mitomycin C, a chemical agent for which FA cells demonstrate a specific cellular and chromosomal hypersensitivity, as well as after u.v.-B irradiation, an agent known to cause oxidative stress. These observations are in line with recent reports showing that at least certain cell lines from other chromosome breakage syndromes, such as ataxia telangiectasia and Bloom syndrome, may be also defective for radiation-induced increase of p53 protein. As the p53 tumor suppressor gene encodes a transcriptional activator whose targets include genes that regulate genomic stability, cellular response to DNA damage and cell cycle progression, we suggest that altered expression of p53 may be relevant to the FA phenotype.

Human p53 displays two immunodominant regions localized in the amino and carboxy termini of the protein. Using a truncated p53 (residues 66 to 361), we selected eight new monoclonal antibodies directed to the central part of the protein. We identified the epitopes recognized by seven out of eight antibodies with a set of overlapping peptides. One of these antibodies had an epitope similar to PAb240, whereas the others recognized novel and diverse antigenic determinants. Using a series of 19 p53 mutants, we show that the behavior of several of the new monoclonal antibodies is similar to that of PAb240 despite their various epitope localizations. This suggests that different mutations in the p53 protein induce an overall conformational change that can be detected by various monoclonal antibodies directed toward the central part of the protein.

We have investigated the effect of Xenopus laevis p53 (Xp53) on transcription from a variety of promoters which are regulated by mouse p53 using a chloramphenicol acetyltransferase reporter system. Although Xp53 transactivated promoters that are up-regulated by mouse p53, it was unable to cause repression. This ability to transactivate gene expression was dependent on a temperature of 32 degrees C, and activity was lost at 37 degrees C. Temperature-sensitive transactivation was correlated with temperature-dependent binding of Xp53 to the adenovirus E1B58K protein. Despite the marked loss of transcriptional activation and binding to E1B58K at 37 degrees C, Xp53 was still capable of binding simian virus 40 large T antigen and inhibiting simian virus 40 origin-dependent DNA replication. These data show that Xp53 is temperature sensitive for N-terminal activities and suggest that the transactivation and repression "domains" of p53 are distinct.

Accumulation of mutations in oncogenes and tumor suppressor genes transforms a normal cell into a malignant cell by allowing it to escape from normal control of growth. In prostate tumorigenesis, the current model envisages specific mutations of the TP53 tumor suppressor gene and loss of loci, detected by loss of heterozygosity (LOH), on chromosome arms 8p, 10q, 16q, and 18q. In order to determine if alterations frequently found in other adenocarcinomas (breast, ovarian, gastric, colorectal), including losses of genetic material from chromosome arms 1p, 3p, 7q, 8p, 11p, 17p, 17q, and 18q, are also involved in prostate cancer, we examined 20 localized early-stage prostate tumors. We detected no mutations of the TP53 gene. Allelic losses were found from 7q (33%), 8p (50%), 10q (20%), and 18q (33%). Furthermore, as the first step toward isolating tumor suppressor genes on 18q, we used six polymorphic markers and identified a small common deleted region between the chromosome 18 centromere and the D18S19 locus.