p53 and mdm2 are expressed independently during cellular proliferation

We analysed p53 expression during proliferation of serum stimulated Swiss mouse 3T3 cells and of concanavalin A stimulated mouse spleen lymphocytes and correlated it to rate of DNA synthesis and to expression of PCNA. We also analysed mdm2 gene expression, as rising p53 levels during proliferation might require MDM2 protein expression to functionally antagonize p53 mediated growth inhibition. p53 protein synthesis closely paralleled DNA synthesis and PCNA expression, suggesting a direct involvement of p53 in cellular DNA synthesis. mdm2 expression in 3T3 cells could not be correlated with p53 expression and DNA synthesis and was not detected at all in stimulated lymphocytes. We conclude that p53 and mdm2 expression during proliferation are not functionally related and that mdm2 expression is not required for proliferation.     

Tumor suppressor protein p53 binds preferentially to supercoiled DNA

Wild type human tumor suppressor protein p53 (expressed in insect cells) binds strongly to negatively supercoiled (sc) plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of scDNA in agarose gels and imaging by scanning force microscopy (SFM). The binding occurs both in the presence and absence of the p53 consensus sequence. At relatively low p53/DNA ratios, binding of p53 to scDNA results in the appearance of several retarded DNA bands on the gels, similar to a conventional topoisomer ladder generated enzymatically. However, after removal of p53 by deproteination, the original mobility of the scDNA is recovered, indicating that the reduction of torsional stress accompanying p53 binding does not reflect changes in linking number. In DNA samples partially relaxed by topoisomerase I p53 binds preferentially to the scDNA molecules with the largest negative superhelix density. SFM imaging of the p53/scDNA complex reveals a partial or total relaxation of the compact scDNA, the degree of which increases with the number of bound p53 molecules. Competition assays with linear DNA reveal a preference of p53 for scDNA. In addition, scDNA induces dissociation of p53 from a preformed complex with a DNA fragment (474 bp) containing the consensus sequence. We conclude that the affinity of p53 for negatively supercoiled DNA is greater than that for the consensus sequence in linear fragments. However, thermally denatured linearized plasmid DNA is efficient in competing for the binding of p53 to scDNA, although the first retarded band (presumed to contain one bound p53 molecule) is retained in the case of the plasmid containing the consensus sequence. Thus, it appears that interactions involving both the core domain and the C-terminal domain regulate the binding of p53 to scDNA. The above results are not restricted to human p53; the wild type rat p53 protein also results in the retardation of scDNA on agarose gels. The biological implications of the novel DNA binding activities of p53 are discussed.     

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.     

The proliferation of normal human fibroblasts is dependent upon negative regulation of p53 function by mdm2

Loss of function of the tumour suppressor gene p53 is a key event in most human cancers. Although usually occurring through mutation, in some tumour types this appears to be achieved via an indirect mechanism involving inappropriate expression of a functional inhibitor, mdm2, which binds to the transactivation domain of p53. This interaction offers an ideal potential target for novel cancer therapies. However, therapeutic specificity may depend on the extent to which this p53-inhibitory action of mdm2 is also required by normal cells. Transgenic data have already established that mdm2 is needed to prevent embryonic lethality, but the situation in adult cells is still unclear. Here we show that micro-injection of normal human fibroblasts with an antibody directed against the p53-binding domain of mdm2 induces expression of p53-responsive genes, and furthermore results in p53-dependent growth arrest. We conclude that normal cell proliferation can be dependent on negative regulation of p53 by mdm2, a finding which raises an important note of caution for mdm2-directed cancer therapies.     

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-small-cell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.     

Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers

We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.     

p53 as a sensor of carcinogenic exposures: mechanisms of p53 protein induction and lessons from p53 gene mutations

The p53 tumor suppressor gene encodes a nuclear phosphoprotein with growth inhibiting properties, which is activated in cell exposed to various forms of DNA damaging stress. The development of human cancer often involves inactivation of this suppressor through various mechanisms, including gene deletions and point mutations. Most mutations impair the specific DNA-binding capacity of p53, therefore allowing cells to proliferate in conditions where cells with intact p53 function are suppressed or eliminated. Thus, mutation of p53 may provide a selective advantage for the clonal expansion of preneoplastic or neoplastic cells. The diversity of p53 mutations provides a valuable tool to identify important sources of cancer-causing mutation in the human setting. Mutagens and carcinogens damage the genome in characteristics ways, leaving "mutagen fingerprints" in DNA. Well-characterised examples of such "fingerprints" include G: C to T: A transversions in lung cancers in association with cigarette smoke, G: C to T: A transversions at codon 249 in liver cancers in association with dietary exposure to Aflatoxin B1 (AFB1) and CC: GG to TT: AA tandem dipyrimidine transitions in skin cancers in association with UVB exposure. In addition, mutations at different codons are not functionally equivalent. The availability of crystal structures of p53 protein represents an essential development in the understanding of the functional properties of p53 mutants. In the future, it is expected that analysis of p53 mutations may provide useful information for the diagnosis, prognosis and therapy of cancer.     

p53 tumor-suppressor gene: clues to molecular carcinogenesis

Pig oocytes were examined to test their ability to undergo cortical granule exocytosis upon penetration by spermatozoa during meiotic maturation. Immature or maturing oocytes (cultured in vitro for 0 h, 26 h and 46 h) were inseminated with ejaculated boar spermatozoa in vitro. Before and after insemination, oocytes were stained with peanut agglutinin labelled with fluorescein isothiocyanate and the cortical granule distributions were examined under the fluorescent microscope and the laser confocal microscope. Before insemination, all the oocytes at the germinal vesicle stage showed a uniform distribution of cortical granules throughout the cortical cytoplasm. The granules migrated centrifugally during maturation and were distributed just beneath the oolemma in the oocytes after germinal vesicle breakdown, forming a monolayer in metaphase I or metaphase II. Cortical granules were still present in all penetrated oocytes at the germinal vesicle stage 18 h after insemination; in contrast, 26% and 84% of the oocytes inseminated at the stages of germinal vesicle breakdown or at metaphase I and II, respectively, completely released their cortical granules. Nuclear activation rates of penetrated oocytes were 0%, 38% and 96% in oocytes cultured for 0 h, 26 h and 46 h, respectively. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 88% (oocytes cultured for 46 h) released cortical granules completely. Complete cortical granule exocytosis was not observed in nuclear-inactivated oocytes. Of the nuclear-activated oocytes, 67% (oocytes cultured for 26 h) and 80% (oocytes cultured for 46 h) of monospermic oocytes and 67% (oocytes cultured for 26 h) and 91% (oocytes cultured for 46 h) of polyspermic oocytes released cortical granules, and no statistical difference was observed between oocytes cultured for 26 h or 46 h, or between monospermic and polyspermic oocytes. The proportion of oocytes with cortical granule exocytosis increased as insemination time increased and was greatest 18 h after insemination in oocytes cultured for 26 h and 46 h; no obvious changes were observed when the insemination time was prolonged to 24 h. These results indicate that pig oocytes develop the ability to release cortical granules after penetration by spermatozoa following germinal vesicle breakdown, and that this ability is not fully developed until metaphase II. Cortical granule exocytosis is accompanied by nuclear activation, suggesting that both nuclear and cytoplasmic maturation are responsible for the cortical reaction. Polyspermy may be a result of a complete failure of cortical granule exocytosis in immature oocytes and delayed CG exocytosis in matured oocytes.     

A PAb240+ conformation of wild type p53 binds DNA

It is generally accepted that wild type (growth suppressing) p53 is capable of binding to a consensus DNA sequence and is in a conformation recognizable by antibody PAb246 (for murine p53), but not by antibody PAb240. Conversely, mutant forms of p53 incapable of DNA binding often assume conformations that display the PAb240, but not the PAb246 epitope. Exposure of these two epitopes on p53 is therefore believed to be mutually exclusive. We show that wild type p53 translated in vitro in rabbit reticulocyte lysate (RRL) has a PAb240 epitope that is not always cryptic, even on p53 that is bound sequence-specifically to DNA (presumably as a tetramer). All of the DNA-bound, PAb240+ p53 concurrently displays the PAb246 epitope, and both epitopes can be occupied by antibody while p53 is bound to DNA. This novel 'dual positive' conformation also exists in the absence of DNA and suggests that p53 is not necessarily inactive when the PAb240 epitope is displayed. When the C-terminal 58 amino acids of p53 containing the dimer/tetramerization domains are replaced with a heterologous dimerization domain, the resultant dimeric p53 manifests only the PAb246+/PAb240- conformation while bound to DNA. Thus, the C-terminal 58 amino acids of p53 are required for the PAb246+/PAb240+ phenotype, possibly due to tetramerization. This novel 'dual positive' p53 conformation exists in an excess of wild type p53 that has the PAb246-/PAb240+ 'mutant' conformation, suggesting that the 'mutant' conformation is not dominant negative in and of itself.