[The tumor suppressor p53: "guardian of our genomes"].
Explore the source record for details and available documents.
Genes, p53↗
Biomedical subjects
W Deppert
Publications and source records attributed to W Deppert.
p22/PACAP response gene 1 (PRG1): a putative target gene for the tumor suppressor p53.
In this study we describe a novel putative p53-responsive gene, designated p22/PACAP response gene 1 (PRG1), recently identified as a proliferation-associated early-response gene in rats. By means of electrophoretic mobility shift assay and CAT-reporter gene assay, we could demonstrate that the p53 binding site residing in the promoter of p22/PRG1 is functional in vitro. Furthermore, in clone 6 cells expression of p22/PRG1 is induced in parallel to p21/Waf1 under conditions permitting mutant p53 to adopt wild-type configuration. An increase of p22/PRG1 transcription was also observed in gamma-irradiated rat splenocytes, which undergo p53-dependent apoptosis. Our findings demonstrate that p22/PRG1 fulfills all essential criteria as a p53 target gene and might be implicated in p53-dependent apoptosis.
Animals↗
Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing.
Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT "DNA-unwinding motif," which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA.
Animals↗
Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein (vimentin) scaffold.
The temperature-sensitive mutant tsp53val135 accumulates in the cytoplasm of cells kept at the non-permissive temperature (39 degrees C), but is rapidly transported into the cell nucleus at the permissive temperature (30 degrees C). tsp53 thus may serve as a model for analysing cellular parameters influencing the subcellular location of p53. Here we provide evidence that retention of tsp53 in the cytoplasm at the non-permissive temperature is due to cytoskeletal anchorage of the p53 protein. Two sublines of C6 rat glioma cells differing in their expression of the intermediate filament protein vimentin (vimentin expressing or vimentin negative cells) were stably transfected with a vector encoding tsp53. Whereas cells of vimentin expressing C6 subclones retained tsp53 in the cytoplasm at the non-permissive temperature, cells of vimentin negative subclones exclusively harbored the tsp53 within their nuclei. Intermediate filament deficient cells that had been reconstituted with a full length vimentin protein again showed a cytoplasmic localization of tsp53, whereas in cells expressing a C-terminally truncated (tail-less) vimentin tsp53 localized to the nucleus. We conclude that cytoplasmic sequestration of tsp53 requires an intact intermediate filament system.
Actins↗
High affinity MAR-DNA binding is a common property of murine and human mutant p53.
We recently reported that murine MethA mutant but not wild-type p53 specifically binds to MAR-DNA elements (MARs) with high affinity. Here we show that this DNA binding activity is exerted not only by MethA mutant p53 but also by other murine mutant p53 proteins isolated from the transformed murine BALB/c cell lines 3T3tx and T3T3 and differing in their conformational status. High affinity MAR-DNA binding was not restricted to the Xbal-IgE-MAR-DNA fragment from the murine immunoglobulin heavy chain gene enhancer locus [Cockerill et al. (1987): J Biol Chem 262:5394-5397] used in previous studies, as MethA p53 also specifically interacted with other A/T-rich bona fide MARs. Not only murine but also human mutant p53 proteins carrying the mutational hot spot amino acid exchanges 175Arg-->His, 273Arg-->Pro, or 273Arg-->His bound to the Xbal-IgE-MAR-DNA fragment. We therefore conclude that high affinity MAR-DNA binding is a property common to a variety of mutant p53 proteins.
3T3 Cells↗
The proliferation-associated early response gene p22/PRG1 is a novel p53 target gene.
The novel early response gene p22/PRG1 is linked to cell cycle entry and the induction of proliferation in various cell types although its exact function is still unknown. The p22/PRG1 promoter region contains a 20 bp sequence matching the consensus binding motif for the tumor suppressor protein p53. Gel shift assays demonstrated that p53 specifically binds to an oligonucleotide derived from the p53 binding site of the p22/PRG1 promoter. Chloramphenicol acetyltransferase (CAT) reporter gene assays confirmed that this site confers p53-dependent transcriptional activity to the p22/PRG1 promoter. In Hela cells, p22/PRG1 promoter constructs induced CAT expression only when cotransfected with an expression plasmid for wild-type, but not for mutant p53. Similarly, CAT expression was inducible at the permissive (31 degrees C) but not at the non-permissive temperature (39 degrees C) in the rat embryo fibroblast-derived cell line clone-6 that expresses a temperature-sensitive mutant p53. Conversion of this mutant p53 to a functional p53 at the permissive temperature was accompanied by a significant increase of endogenous p22/PRG1 mRNA level in this cell line. Gamma-irradiation of rat splenocytes or doxorubicin-treatment of Hela cells increased p53 levels followed by transcriptional activation of p22/PRG1 and p21/Waf1 in parallel. Our data demonstrate that p22/PRG1 transcription is induced by p53 during p53-dependent cell cycle arrest and apoptosis. Therefore, p22/PRG1 represents a novel target for transcriptional activation by p53.
Animals↗
Relationship among immunodominance of single CD8+ T cell epitopes, virus load, and kinetics of primary antiviral CTL response.
The primary CTL response of BALB/c mice infected with the lymphocytic choriomeningitis (LCM) virus strain WE is directed exclusively against one major epitope, n118, whereas a viral variant, ESC, that does not express n118 induces CTL against minor epitopes. We identified one minor epitope, g283, that induces primary lytic activity in ESC-infected mice. Infections of mice with WE and ESC were used to study the hierarchical control of a T cell response. Presentation of minor epitopes is not reduced in WE-infected cells. Generation of CTL against n118 does not suppress the generation of minor epitope-specific CTL systemically, as mice coinfected with WE and ESC developed CTL against n118 and g283. However, elimination of ESC and development of minor epitope-specific CTL in ESC infection were slower than elimination of WE and development of CTL against n118. CD8+ T cells against the minor epitope were activated in ESC and WE infection, but did not expand in the latter to show lytic activity in a primary response. We explain the absence of minor epitope-specific lytic activity in WE infection by the fast reduction of virus load due to the early developing n118-specific CTL. Immunodominance of CTL epitopes in primary virus infections thus can be explained as a kinetic phenomenon composed of 1) expansion of CD8+ T cells specific for individual epitopes, 2) stimulatory effect of virus load, and 3) negative feedback control on virus load by the fastest CTL population.
Animals↗
Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role in fidelity control of homologous recombination.
We demonstrate that wild-type p53 inhibits homologous recombination. To analyze DNA substrate specificities in this process, we designed recombination experiments such that coinfection of simian virus 40 mutant pairs generated heteroduplexes with distinctly unpaired regions. DNA exchanges producing single C-T and A-G mismatches were inhibited four- to sixfold more effectively than DNA exchanges producing G-T and A-C single-base mispairings or unpaired regions of three base pairs comprising G-T/A-C mismatches. p53 bound specifically to three-stranded DNA substrates, mimicking early recombination intermediates. The KD values for the interactions of p53 with three-stranded substrates displaying differently paired and unpaired regions reflected the mismatch base specificities observed in recombination assays in a qualitative and quantitative manner. On the basis of these results, we would like to advance the hypothesis that p53, like classical mismatch repair factors, checks the fidelity of homologous recombination processes by specific mismatch recognition.
Animals↗
DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53.
Sequence-specific transactivation of target genes is one of the most important molecular properties of the tumor suppressor p53. Binding of p53 to its target DNAs is tightly regulated, with modifications in the carboxy-terminal regulatory domain of the p53 protein playing an important role. In this study we examined the possible influence of DNA structure on sequence-specific DNA binding by p53, by analysing its binding to p53 consensus elements adopting different conformations. We found that p53 has the ability to bind to consensus elements which are present in a double-helical form, as well as to consensus elements which are located within alternative non-B-DNA structures. The ability of a consensus element to adopt either one of these conformations is dependent on its sequence symmetry, and is strongly influenced by its sequence environment. Our data suggest a model according to which the conformational status of the target DNA is an important determinant for sequence-specific DNA binding by p53. Modifications in the carboxy-terminal regulatory region of p53 possibly determine the preference of p53 for a given DNA conformation.
Base Sequence↗
Overexpression of p53 protein during pancreatitis.
Overexpression of p53 correlates with neoplasia in many cytological specimens. To test the specificity of overexpressed p53 as a tumour marker for the detection of pancreatic cancer, we analysed cytological specimens of pancreatic juice samples from patients with pancreatitis or pancreatic carcinoma (n = 42) for p53 protein overexpression. p53 protein overexpression was found in 59% of patients with pancreatitis and 67% of patients with pancreatic carcinoma. Thus, the assessment of p53 protein overexpression is not useful in the diagnosis of pancreatic cancer. Overexpressed p53 during pancreatitis appears to be wild-type p53. Overexpression of p53 may result from DNA damage occurring during chronic inflammation. It is well established that p53 can induce apoptosis upon DNA damage. Consequently, we found apoptotic cell death in five out of five tested cytological preparations from patients with pancreatitis as well as in one out of one pancreatic carcinoma specimen.
Adenocarcinoma↗
MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection.
Phosphopeptide analyses of the simian virus 40 (SV40) large tumor antigen (LT) in SV40-transformed rat cells, as well as in SV40 lytically infected monkey cells, showed that gel-purified LT that was not complexed to p53 (free LT) and p53-complexed LT differed substantially in their phosphorylation patterns. Most significantly, p53-complexed LT contained phosphopeptides not found in free LT. We show that these additional phosphopeptides were derived from MDM2, a cellular antagonist of p53, which coprecipitated with the p53-LT complexes, probably in a trimeric LT-p53-MDM2 complex. MDM2 also quantitatively bound the free p53 in SV40-transformed cells. Free LT, in contrast, was not found in complex with MDM2, indicating a specific targeting of the MDM2 protein by SV40. This specificity is underscored by significantly different phosphorylation patterns of the MDM2 proteins in normal and SV40-transformed cells. Furthermore, the MDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of MDM2 by SV40 is aimed at preventing MDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells.
3T3 Cells↗
p53 Protein exhibits 3'-to-5' exonuclease activity.
Highly purified p53 protein from different sources was able to degrade DNA with a 3'-to-5' polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV-cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a "guardian of the genome."
Antibodies, Monoclonal↗
Protective immunity in BALB/c mice against the simian virus 40-induced mKSA tumor resulting from injection of recombinant large T antigen. Requirement of CD8+ T lymphocytes.
BALB/c mice are often considered "low responders" or even "nonresponders" with regard to cytolytic CD8+ T lymphocytes and SV40 large T Ag (TAg). Large TAg and fragments thereof were produced by recombinant technology and injected into BALB/c mice that were subsequently challenged by i.p. injection of syngeneic TAg-expressing mKSA tumor cells. Two portions of the TAg were found to induce protective immunity, one stretching from amino acid residues 1-272 and the other from amino acid residues 683-708. In mice thus protected, the spleens were virtually free of cytotoxic T cells but CD8+ T lymphocytes obtained from the peritoneal cavity during rejection of the mKSA cells were directly lytic for TAg-expressing target cells. Depleting immune mice of CD4+ or CD8+ T lymphocytes by treatment with mAb abolished their ability to resist tumor development. We conclude that immunity against SV40 TAg-expressing tumor cells in BALB/c mice is dependent on both CD4+ and CD8+ T lymphocytes.
Animals↗
Specific binding of MAR/SAR DNA-elements by mutant p53.
Inactivation of the tumor suppressor p53 by single missense point mutations characterizes a large number of human tumors. At least some mutant p53 proteins not only have lost the tumor suppressor function, but at the same time reveal a variety of dominant oncogenic properties. The molecular basis of this 'gain of function' is still unknown. In this report we describe a new biochemical activity of mutant p53, the specific high-affinity interaction with MAR/SAR DNA-elements (nuclear matrix/scaffold attachment regions). This DNA-binding activity can be distinguished from the previously reported DNA-binding activities of p53 by its specificity for mutant p53, the high binding affinity, and the domains of the mutant p53 molecule involved in MAR/SAR DNA-binding. The MAR/SAR-binding region of mutant p53 maps to a bipartite domain consisting of the mutated core region and the C-terminal 60 amino acids, carrying the unspecific DNA-binding domain and the oligomerization motif. MAR/SAR elements are considered as important regulatory elements in a variety of nuclear processes. We propose a model according to which the specific interaction of mutant p53 with MAR/SAR elements might interfere with these processes, thereby exerting pleiotropic oncogenic effects.
Animals↗
Abrogation of wild-type p53 mediated growth-inhibition by nuclear exclusion.
We used clone 6 cells (rat embryo fibroblasts transformed by the temperature sensitive mutant p53val135 and an activated H-ras-gene (Michalovitz et al., 1990)), growth arrested at 32 degrees C, as a model to analyse whether and how transformed cells, growth-arrested by an overexpressed wild-type p53, might overcome p53-mediated growth inhibition. When clone 6 cells were kept at 32 degrees C for about 2 weeks, foci of cells appeared which grew temperature-independent. Analysis of individual clones of such cell demonstrated that the ectopically expressed tsp53-gene had not been altered by an additional mutation, but that the tsp53 in these cells at 32 degrees C had lost its ability to upregulate expression of the p53 target genes waf1 and mdm2. This loss of p53-specific transactivation correlated with nuclear exclusion of the tsp53 at 32 degrees C, which was most likely mediated by cytoplasmic retention of the tsp53 protein via short-lived anchor proteins. Cytoplasmic retention of the tsp53 at 32 degrees C was also observed in PC12 pheochromocytoma cells ectopically expressing tsp53val135, there occurring without specific selection. Also in these cells nuclear exclusion of the tsp53 correlated with loss of p53 mediated growth inhibition. Nuclear exclusion of p53 thus might serve as an epigenetic mechanism to eliminate the growth-inhibitory function of p53.
Animals↗
Binding of MAR-DNA elements by mutant p53: possible implications for its oncogenic functions.
The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrity of the genome. This function of wild-type p53 as "guardian of the genome" is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function.
Animals↗
Simian virus 40 small t antigen activates the carboxyl-terminal transforming p53-binding domain of large T antigen.
Expression of the simian virus 40 large T antigen (large T) in F111 rat fibroblasts generated only minimal transformants (e.g., F5 cells). Interestingly, F111-derived cells expressing only an amino-terminal fragment of large T spanning amino acids 1 to 147 (e.g., FR3 cells), revealed the same minimal transformed phenotype as F111 cells expressing full-length large T. This suggested that in F5 cells the transforming domain of large T contained within the C-terminal half of the large T molecule, and spanning the p53 binding domain, was not active. Progression to a more transformed phenotype by coexpression of small t antigen (small t) could be achieved in F5 cells but not in FR3 cells. Small-t-induced progression of F5 cells correlated with metabolic stabilization of p53 in complex with large T: whereas in F5 cells the half-life of p53 in complex with large T was only slightly elevated compared with that of (uncomplexed) p53 in parental F111 cells or that in FR3 cells, coexpression of small t in F5 cells led to metabolic stabilization and to high-level accumulation of p53 complexed to large T. In contrast, coexpression of small t had no effect on p53 stabilization or accumulation in FR3 cells. This finding strongly supports the assumption that the mere physical interaction of large T with p53, and thus p53 inactivation, in F5 cells expressing large T only does not reflect the main transforming activity of the C-terminal transforming domain of large T. In contrast, we assume that the transforming potential of this domain requires activation by a cellular function(s) which is mediated by small t and correlates with metabolic stabilization of p53.
Animals↗
Species- and tissue-specific expression of the C-terminal alternatively spliced form of the tumor suppressor p53.
Alternative splicing of the p53 transcript which so far has been demonstrated only in the murine system has been proposed as a general regulatory mechanism for the generation of functionally different p53 proteins. We analyzed by RT-PCR the pattern of p53 mRNAs within the region spanning exons 10 and 11 of the p53 gene in 13 different tissues from two independent mouse strains, in 10 different rat tissues and in six different human tissues. PCR products of the expected sizes, corresponding to the normally spliced and the alternatively spliced p53 mRNAs, were detected in mice. Alternatively spliced mRNA was found at approximately 25-20% the level of the normally spliced p53 mRNA in most tissues analyzed. In spleen and kidney the proportion of alternatively spliced p53 mRNA was much lower. Surprisingly, examination of p53 mRNAs isolated from 10 different rat tissues and six human tissues within the same region of the p53 gene showed only products of normal size. Although a potential homologous alternative 3' splice site within intron 10 of the human p53 gene is present in the genomic sequence of human p53, the expected corresponding alternatively spliced p53 mRNA was undetectable. These findings imply that the generation of functionally different forms of p53 by alternative splicing of p53 transcripts is a species-specific event, possibly indicating species-specific mechanisms for regulating p53 activities.
Alternative Splicing↗