Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with DeltaNp73.

TP63, a member of the TP53 gene family, encodes two groups of three isoforms (alpha, beta and gamma). The TAp63 isoforms act as transcription factors. The DeltaNp63 isoforms lack the main transcription activation domain and act as dominant-negative inhibitors of transactivation (TA) isoforms. To clarify the role of these isoforms and to better understand their functional overlap with p53, we ectopically expressed each p63 isoform in the p53-null hepatocellular carcinoma cell line Hep3B. All TA isoforms, as well as DeltaNp63alpha, had a half-life of <1 h when transiently expressed and were degraded by the proteasome pathway. The most stable form was DeltaNp63gamma, with a half-life of >8 h. As expected, TA isoforms differed in their transcriptional activities toward genes regulated by p53, TAp63gamma being the most active form. In contrast, DeltaNp63 isoforms were transcriptionally inactive on genes studied and inhibited TA isoforms in a dose-dependent manner. When stably expressed in polyclonal cell populations, TAp63beta and gamma isoforms were undetectable. However, when treated with doxorubicin (DOX), p63 proteins rapidly accumulated in the cells. This stabilization was associated with an increase in phosphorylation. Strikingly, in DOX-treated polyclonal populations, increase in TAp63 levels was accompanied by overexpression of DeltaNp73. This observation suggests complex regulatory cross talks between the different isoforms of the p53 family. In conclusion, p63 exhibits several transcriptional and stress-response properties similar to those of p53, suggesting that p63 activities should be taken into consideration in approaches to improve cancer therapies based on genotoxic agents.

Analysis of p53 antibodies in patients with various cancers define B-cell epitopes of human p53: distribution on primary structure and exposure on protein surface.

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.

Cloning and characterization of a cDNA from Xenopus laevis coding for a protein homologous to human and murine p53.

A Xenopus laevis oocyte cDNA library was screened with a murine p53 cDNA probe for the presence of p53-related clones. Several such clones were isolated and analysed. The nucleotide sequence of the largest cDNA clone (2.2 kb) showed a high degree of homology with the human (68%) and murine (70%) p53 coding sequences. This clone contains a single large open-reading frame, coding for a protein of 363 amino acids, which is 51% homologous to human p53 and 57% homologous to murine p53. Furthermore, five highly conserved internal regions were found in all three proteins. The three proteins have a highly similar amino acid composition (including, notably, the presence of a high proportion of proline residues), and they display a comparable distribution of charged amino acids and hydropathic index profile. The in vitro transcription-translation products of the X. laevis clone were successfully immunoprecipitated by human anti-p53 sera, demonstrating that there is at least one epitope in common between the X. laevis protein and human, and possibly murine, p53.

The consequence of p53 overexpression for liver tumor development and the response of transformed murine hepatocytes to genotoxic agents.

To analyse the effect of p53 on liver tumor development, we generated transgenic mice overexpressing wild-type p53 in the liver and crossed them with transgenic mice in which the expression of the SV40 large T antigen (TAg) induces hepatic tumors. Remarkably, whereas preneoplastic TAg liver exhibited anisocaryosis and anisocytosis, TAg/p53 liver never presented any dysplastic cells. Moreover, whereas expression of p53 did not affect hepatic development, its constitutive expression in tumorigenic livers resulted in a significantly enhanced apoptosis once nodules had appeared. In contrast, p53 overexpression did not modify the elevated proliferation of TAg-transformed hepatocytes and had no effect on hepatocarcinoma progression. In vitro analysis of primary hepatocytes exposed to various genotoxic agents showed that p53 failed to sensitize normal or TAg-transformed hepatocytes to apoptosis, except when high doses of doxorubicin, UV-B and UV-C radiation were used. Our results confirmed that the hepatocyte cell type is very resistant to genotoxic agents and showed that constitutive expression of p53 failed to improve their responsiveness. In addition, our results showed that suppression of dysplastic cells, probably by restoring normal cytokinesis and karyokinesis, and enhancement of apoptosis by means of p53 overexpression were insufficient to counteract or delay the TAg-induced liver tumoral progression. Oncogene (2000) 19, 3498 - 3507

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments.

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

A tumor specific single chain antibody dependent gene expression system.

The design of conditional gene expression systems restricted to given tissues or cellular types is an important issue of gene therapy. Systems based on the targeting of molecules characteristic of the pathological state of tissues would be of interest. We have developed a synthetic transcription factor by fusing a single chain antibody (scFv) directed against p53 with the bacterial tetracycline repressor as a DNA binding domain. This hybrid protein binds to p53 and can interact with a synthetic promoter containing tetracycline-operator sequences. Gene expression can now be specifically achieved in tumor cells harboring an endogenous mutant p53 but not in a wild-type p53 containing tumor cell line or in a non-transformed cell line. Thus, a functional transactivator centered on single chain antibodies can be expressed intracellularly and induce gene expression in a scFv-mediated specific manner. This novel class of transcriptional transactivators could be referred as 'trabodies' for transcription-activating-antibodies. The trabodies technology could be useful to any cell type in which a disease related protein could be the target of specific antibodies.

Inactivation of the p53 gene expression by a splice donor site mutation in a human T-cell leukemia cell line.

An abnormally sized 3.5 kb p53 transcript was detected in the KE-37R human leukemic T-cell line in which no p53 protein could be detected by immunoprecipitation. S1 nuclease protection experiments and sequencing analysis indicated conservation of the entire intron 4 (755 bp) in the 3.5 kb transcript and the presence of a G to A substitution in the last exonic nucleotide of the splice donor site. These data support the notion that p53 gene inactivation by point mutations in splice junctions also exists in hemopoietic neoplasia.

cDNA cloning and immunological characterization of rabbit p53.

We have cloned and sequenced the p53-encoding rabbit cDNA (Oryctolagus cuniculus). The encoded product is 86% and 80% homologous to human and mouse p53, respectively. It features many characteristics found in all p53 proteins: (i) the five domains highly conserved during evolution, (ii) an acidic N terminus, (iii) a hydrophilic C terminus and (iv) a penultimate serine residue. Immunoprecipitation of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 has confirmed the identity of the protein.

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.

Functional interplay between p63 and p53 controls RUNX1 function in the transition from proliferation to differentiation in human keratinocytes.

The interfollicular epidermis is continuously renewed, thanks to a regulated balance between proliferation and differentiation. The ΔNp63 transcription factor has a key role in the control of this process. It has been shown that ΔNp63 directly regulates Runt-related transcription factor 1 (RUNX1) transcription factor expression in mouse keratinocytes. The present study showed for the first time that RUNX1 is expressed in normal human interfollicular epidermis and that its expression is tightly regulated during the transition from proliferation to differentiation. It demonstrated that ΔNp63 directly binds two different RUNX1 regulatory DNA sequences and modulates RUNX1 expression differentially in proliferative or differentiated human keratinocytes. It also showed that the regulation of RUNX1 expression by ΔNp63 is dependent on p53 and that this coregulation relies on differential binding and activation of RUNX1 regulatory sequences by ΔNp63 and p53. We also found that RUNX1 inhibits keratinocyte proliferation and activates directly the expression of KRT1, a critical actor in early keratinocyte differentiation. Finally, we described that RUNX1 expression, similar to ΔNp63 and p53, was strongly expressed and downregulated in basal cell carcinomas and squamous cell carcinomas respectively. Taken together, these data shed light on the importance of tight control of the functional interplay between ΔNp63 and p53 in regulating RUNX1 transcription factor expression for proper regulation of interfollicular epidermal homeostasis.

TP63 P2 promoter functional analysis identifies ß-catenin as a key regulator of ΔNp63 expression.

The ΔNp63 protein, a product of the TP63 gene that lacks the N-terminal domain, has a critical role in the maintenance of self renewal and progenitor capacity in several types of epithelial tissues. ΔNp63 is frequently overexpressed in squamous cell carcinoma (SCC) and in some other epithelial tumours. This overexpression may contribute to tumour progression through dominant-negative effects on the transcriptionally active (TA) isoforms of the p53 family (TAp63, TAp73 and p53), as well as through independent mechanisms. However, the molecular basis of ΔNp63 overexpression is not fully understood. Here, we show that the expression of ΔNp63 is regulated by the Wnt/ß-catenin pathway in human hepatocellular carcinoma (HCC) and SCC cell lines. This regulation operates in particular through TCF/LEF sites present in the P2 promoter of TP63. In addition, we show that ΔNp63 and ß-catenin are frequently coexpressed and accumulated in oesophageal SCC, but not in HCC. These results suggest that activation of the ß-catenin pathway may contribute to overexpression of ΔNp63 during tumour progression, in a cell type-specific manner.

[Role of the epithelial-mesenchymal transition during tumor progression]. / Rôle de la transition épithéliomésenchymateuse dans la progression tumorale.

The epithelial-mesenchymal transition (EMT) is a morphogenetic program that converts epithelial into mesenchymal cells during the embryonic development. This mechanism is frequently reactivated during tumor progression and provides cells with motility and invasive capabilities favoring the metastatic dissemination from epithelial tumors. Various EMT-inducing transcription factors, such as the TWIST proteins, were also shown to inhibit oncogene-induced fail-safe programs (senescence and apoptosis), thereby promoting the progression from benign to malignant stages. Altogether, these observations suggest that EMT could play an important role in favoring both tumor development and metastatic dissemination.

TP53 tumor suppressor gene: a model for investigating human mutagenesis.

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.

SV40 large T-antigen and transformation related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells.

The localization of SV40 large T-antigen (T-Ag) and the cellular protein p53 in the nuclei of mouse and human SV40-transformed cells and of a methylcholanthrene-transformed mouse cell line, was studied. Their detection by ultrastructural immunocytochemistry with specific monoclonal antibodies employed two complementary methods used in parallel. These consisted of indirect immunoperoxidase labelling carried out before embedment on Triton-permeabilized cells, or indirect immunogold labelling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that in SV40-transformed cells both proteins are chiefly localized on peri- and interchromatin RNP fibrils. This shows that they occur in structures involved in the synthesis and processing of hnRNA. The nucleoli and chromatin did not appear to be labelled. In methylcholanthrene-transformed cells the protein p53 (in the absence of large T-Ag) was also detected on peri- and interchomatin fibrils. Taken together with recent results which demonstrated that, during lytic infection, T-Ag was associated chiefly with cellular chromatin (Harper, F, Florentin, Y & Puvion, E, Exp cell res 161 (1985) 434) [33], our experiments provide evidence that the transforming function of SV40 large T-Ag is dissociable from its function in SV40 lytic infection in terms of its subnuclear distribution.

p53 genetic abnormalities and myc activation in human lung carcinoma.

p53 mutations and myc gene amplification and expression were studied in 119 lung carcinomas of all histological types. A mutant p53 immunophenotype was previously found in 47% of these tumors by immunohistochemical analysis. Seven cases exhibited p53 genomic rearrangements on Southern blots. Elevated levels of p53 transcript were found in 12 carcinomas (10%) and decreased levels in 27 carcinomas (23%) on Northern blots. In most of the cases, low levels of transcript were associated with negative immunostaining, whereas elevated levels of mRNA were related to positive immunostaining (mutant immunophenotype). p53 RT/PCR analysis in 10 tumors with absence of transcript on Northern blots revealed only weak or absent expression of normal and/or altered size transcripts. These abnormal transcripts showed deletions, insertions or splicing abnormalities. Taken together, p53 abnormalities were found in 66% of lung carcinomas [52% of neuroendocrine (NE) carcinomas and 75% of NSCLC]. c-myc was found to be activated in 24% (10/42) of these NE and in 48% (33/69) of these NSCLC carcinomas using Southern- and Northern-blot techniques. In addition, L- and N-myc genes were also activated in 26% (10/42) of NE carcinomas. No correlation was found between p53 mutations and myc activation in SCLC or in NSCLC, but their association was significantly more frequent in NSCLC than in SCLC. These results indicate that the p53-positive immunophenotype uncovers the occurrence of p53 point mutations in lung cancer and that p53 and c-myc gene alterations are important but represent independent occurrences in the development of lung tumors.

Presence of circulating antibodies against cellular protein p53 in a notable proportion of children with B-cell lymphoma.

Serum samples taken from children bearing a wide variety of tumors were screened for the presence of circulating antibodies against the cellular tumor antigen p53. There was a significant correlation (p less than 0.001, n = 119) between the presence of such antibodies and the occurrence of cancer; 12% of the sera tested were positive. These sera were derived from children with a wide range of tumor types. In particular, 21% of the sera obtained from children suffering from a B-cell lymphoma contained anti-p53 antibodies. We were not able to establish a correlation between the secretion of p53-reactive antibodies and any other parameters, such as the age or sex of the child, presence of metastasis, stage or prognosis of disease, or treatment regimen. These results therefore show that the development of p53 immunogenicity is associated with a wide range of neoplastic diseases in children, and in particular with the presence of a B-cell lymphoma.

Immunohistochemical study of p53 in human lung carcinomas.

Immunohistochemical analysis of p53 protein was carried out on 95 lung carcinomas from all histological types, including 60 primary tumors, 35 lymph node metastases, and 36 corresponding nude mice xenografts, using four antibodies: PAb240 specific for some mutant conformations; PAb421, PAb1801, and CM1 reactive with most of the forms of p53. Nuclear staining with at least two of those four antibodies revealed the presence of an accumulated protein, considered as indicative of a missense mutation in the p53 gene, in 50% of primary tumors of all histological types, except carcinoids. Some defect of messenger RNA expression was detected by Northern blot analysis in an additional 26% of tumors. p53 immunophenotype of the original tumor was fairly maintained on nude mice. p53 accumulation was not correlated with survival, but with disease extension (P = 0.01). Finally, immunohistochemical analysis allowed the recognition of p53 mutant immunophenotype in 41% of tumors where p53 DNA and messenger RNA were apparently normal, using standard molecular biology. Thus, this method provides a rapid and efficient approach for studying p53 mutations leading to an accumulated protein in lung tumors cells.

[Lack of expression of the protein p53 gene in a human T-cell leukemia line]. / Absence d'expression du gène de la protéine p53 dans une lignée humaine issue d'une leucémie de type T.

Expression of the gene encoding the nuclear phosphoprotein p53 (a proto-oncogene classified in the same functional family as c-myc and E1a adenovirus gene) was examined in a human T-cell leukemia (KE-37R cell line). No p53 (or a modified product) could be detected by immunoprecipitation with monoclonal antibodies P Ab 421 and P Ab 122 in KE-37R cell extracts, and no p53-specific RNA was characterized by Northern blot analysis. Southern blot using a murine p53 cDNA clone as a probe, did not reveal any gross rearrangement in the structure of the gene. However, this molecular probe was not suited for investigating the 5' end of the gene which contains the promoter and the non coding exon 1. It is interesting to notice that in KE-37R cells, c-myc has been activated by a t(8; 14) (q24; q11) translocation, suggesting that the c-myc product might substitute to some functions normally requiring p53.