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PubMed1993

p53 nuclear protein accumulation correlates with mutations in the p53 gene, tumor grade, and stage in bladder cancer.

Seventy-three transitional cell carcinomas of the bladder were analyzed by immunohistochemistry for p53 nuclear accumulation, and the results were compared to mutations detected in the p53 gene by single strand conformational polymorphism analysis (SSCP) and DNA sequence analysis. Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections. A highly significant association between the presence of p53 mutations and p53 nuclear reactivity as detected by immunohistochemistry was found (P = 0.0001). Of 32 tumors that demonstrated p53 mutations by SSCP, 27 (84%) showed p53 nuclear reactivity. Of the five cases that did not demonstrate p53 nuclear reactivity, four had mutations in exon 5. However, of 41 tumors with no evidence of p53 mutation by molecular analysis, 12 (29%) showed p53 immunoreactivity. This indicates that immunohistochemical methods may be more sensitive than SSCP in detecting p53 mutations or that discordant cases represent tumors with accumulation of wild type p53 protein, without mutations at the p53 locus. Of the 15 tumors that were found to have mutations at exon 8, 13 demonstrated high-intensity homogeneous p53 nuclear reactivity by immunohistochemistry, and all mutations located at codon 280 demonstrated high-intensity homogeneous immunoreactivity. However, three of three tumors with exon 6 mutations demonstrated low-level p53 immunoreactivity, and four of six tumors with mutations in exon 5 showed no detectable p53 nuclear reactivity. This indicates that the heterogeneity of immunoreactivity observed when analyzing p53 nuclear accumulation may be related to the site of the p53 gene mutation. Information on tumor grade, stage, lymph node status, disease-free interval, and overall survival were available in 54 patients who had undergone cystectomy. A significant association was observed between p53 alterations (detected by immunohistochemistry and SSCP) and histological tumor grade (P = 0.003) and stage (P = 0.01). We conclude that the immunohistochemical detection of p53 nuclear accumulation in formalin-fixed, paraffin-embedded tissue is highly associated with mutations in the p53 gene; this association has now been demonstrated in a large number of tumors. The heterogeneity of p53 nuclear reactivity seems to be related to the site of mutation in the p53 gene. A small proportion of tumors with a p53 gene mutation do not demonstrate immunohistochemically detectable p53 nuclear accumulation. Furthermore, a small but substantial proportion of tumors demonstrate p53 nuclear reactivity but do not show detectable mutations in the p53 gene by SSCP. Finally, both grade and stage of bladder cancer are related to p53 alterations, detected by immunohistochemistry or molecular methods.(ABSTRACT TRUNCATED AT 400 WORDS)

Carcinoma, Transitional Cell↗

PubMed1994

Transactivational and DNA binding abilities of endogenous p53 in p53 mutant cell lines.

Cells with divergent mutant alleles of the p53 gene have different biological and biochemical properties in vitro. Increasing evidence indicates that p53 is a transcriptional activator, and recently, high affinity DNA binding sites for p53 have been identified. The purpose of this study was to determine in vivo, the effect that various mutant p53 proteins have on their ability to mediate transactivation and to bind specifically to DNA. Either a p53 responsive or control reporter gene was transfected into 18 human carcinoma cell lines, having various p53 mutations, either with or without a wild-type p53 expression vector. The CAT activity and DNA gel retardation were studied to measure transactivation and DNA binding by these endogenous p53s. As expected, the endogenously produced wild-type p53 binds to DNA binding sequences and can transactivate a reporter construct containing a p53 high affinity DNA binding site. Four of five cell lines with homozygous p53 mutations at codon 273 (273His), contained p53 which had the ability to bind to p53 DNA binding sequences and transactivate. In contrast, all the homozygous, non-codon 273 mutant p53s (156Pro, 175His, 223Leu, 248Gln, 248Trp, 280Lys) present in the other cell lines had no transactivating ability. These findings suggest that the biology of cancers with mutations at codon 273 may be different than those with p53 mutations at other sites. The p53 from WRO, a thyroid carcinoma cell line with p53 mutation at codon 223 (223Leu), was able to bind p53 DNA recognition sequences, but was unable to transactivate. Interestingly, in a vulvar carcinoma cell line (A431) with a p53 mutation at codon 273 (273His), the p53 was unable to transactivate and gave an aberrant band on gel retardation. Both CEM and SK-UT-1, which have compound heterozygous mutations at codons 175/248 (175His/248His), produced p53 which can complex with DNA, as well as transactivate. In contrast, the p53 in cell lines with either homozygous 175His or 248His p53 mutations, were unable either to transactivate or bind to the p53 response element. A cell line (NPA) heterozygous for 266Glu p53 mutation, was able to efficiently transactivate a reporter containing a p53 DNA binding site, therefore showing no evidence of a dominant negative effect of the endogenous p53 mutant allele. In summary, this in vivo study further supports the idea that different p53 mutant alleles have various properties which may affect their function.

Base Sequence↗

PubMed2001

Knock-in mice with a chimeric human/murine p53 gene develop normally and show wild-type p53 responses to DNA damaging agents: a new biomedical research tool.

The high prevalence and great diversity of p53 tumor suppressor gene mutations in human tumors call for development of therapeutic molecules that rescue function of aberrant p53 protein. P53 mutations also offer new approaches to the study of the origins of mutations in human cancer. An experimental mouse model with a genetically modified but normal functioning p53 gene harboring the human rather than the murine core domain, would be of considerable benefit to research on both cancer therapeutics and etiology; however, it is uncertain whether such mice would permit biological functions of p53 to be retained. Using a Cre/lox P gene-targeting approach, we have constructed a human p53 knock-in (hupki) mouse strain in which exons 4-9 of the endogenous mouse p53 allele were replaced with the homologous, normal human p53 gene sequence. The chimeric p53 allele (p53(KI)) is properly spliced, transcribed in various tissues at levels equivalent to wild-type mice, and yields cDNA with the anticipated sequence, that is, with a core domain matching that of humans. The hupki p53 protein binds to p53 consensus sequences in gel mobility shift assays and accumulates in the nucleus of hupki fibroblasts in response to UV irradiation, as is characteristic of wild-type p53. Induction of various p53-regulated genes in spleen of gamma-irradiated homozygous hupki mice (p53(KI/KI)), and the kinetics of p53-dependent apoptosis in thymocytes are similar to results with wild-type (p53(+/+)) mice, further indicating normal p53 pathway function in the hupki strain. The mice are phenotypically normal and do not develop spontaneous tumors at an early age, in contrast to knock-out (p53(-/-)) strains with a defective p53 gene. The chimeric (p53(KI)) allele thus appears to provide a biological equivalent to the endogenous murine (p53(+)) gene. This strain is a unique tool for examining in vivo spontaneous and induced mutations in human p53 gene sequences for comparison with published human tumor p53 mutation spectra. In addition, the hupki strain paves the way for mouse models in pre-clinical testing of pharmaceuticals designed to modulate DNA-binding activity of human p53.

Animals↗

PubMed2001

Role of p53-dependent activation of caspases in chronic obstructive uropathy: evidence from p53 null mutant mice.

Chronic obstructive uropathy (COU) created by unilateral ureteric ligation is associated with increased renal cell apoptosis and p53 expression. Genetically engineered mice were used to examine the role of p53 in renal cell apoptosis in COU and the involved molecular pathways. Obstructed kidneys in p53+/+, p53+/-, and p53-/- mice were examined at days 4, 7, 15, 20, and 30 for apoptosis, and mRNA were examined for p53, members of the bcl-2 family, the death receptor family, and the common effectors of apoptosis. Obstructed kidneys in p53+/- and p53-/- mice exhibited equal attenuation of tubular and interstitial cell apoptosis (70 and 50%, respectively), compared with p53+/+ mice. However, p53 gene deficiency did not confer complete protection from apoptosis. Obstructed kidneys from p53-/- mice did not express p53 mRNA, whereas those from p53+/- and p53+/+ mice displayed mild and marked increase in their expression, respectively. Obstructed kidneys in p53+/+, p53+/-, and p53-/- mice displayed upregulation of mRNA for members of the bcl-2 family and most of the death receptor family, except for a lower level of tumor necrosis factor receptor-1, TRAIL, and FAP in p53+/+ mice. Obstructed kidneys in p53-/- and p53+/- mice showed virtual absence of caspase 11 and marked attenuation of caspases 1 and 12, contrasted with their strong expression in p53+/+ kidneys. These data suggest that apoptosis in obstructed kidneys involves p53-dependent as well as p53-independent pathways. The p53-dependent pathway may involve activation of caspases 1, 11, and 12, whereas the p53-independent pathway may involve activation of members of the bcl-2 and death receptor families.

Animals↗

PubMed2004

C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells.

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.

Amino Acid Sequence↗

PubMed1992

Growth suppression mediated by transfection of p53 in Hut292DM human lung cancer cells expressing endogenous wild-type p53 protein.

This study was undertaken to analyze the effect of wild-type p53 transfection on the growth potential of a human lung cancer cell line Hut292DM expressing endogenous wild-type p53. Transfection efficiencies obtained with either the wild-type or a mutant p53 complementary DNA revealed a significant decrease in the number of colonies obtained with the wild-type p53 as compared to the mutant p53 complementary DNA (27%) or control vector DNA only (20%), suggesting that wild-type p53 inhibited the growth of Hut292DM cells. A series of wild-type and mutant p53 transfection clones were then analyzed for the presence and expression of the exogenous p53 gene. Polymerase chain reaction amplification revealed that 98% of mutant p53 transfection clones analyzed contained the exogenous p53 gene as opposed to 47% for wild-type p53 clones. The majority of mutant p53 clones expressed high levels of exogenous p53 mRNA and protein as analyzed by Northern and Western blots, respectively. In contrast, all wild-type p53 clones analyzed failed to express exogenous p53 mRNA transcript or protein of a normal size. Aberrant-size p53 mRNA was detected in two wild-type p53 clones (X833.W2 and W18), and Western blot analysis revealed that these clones expressed truncated p53 proteins (M(r) 45,000 and 33,000 respectively). No difference in proliferation rates in vitro or in tumorigenic potential in nude mice were observed between mutant p53 clones or control cell lines. In contrast, a wild-type p53 clone (X833.W2) exhibited a significantly reduced tumorigenic potential in nude mice, whereas its in vitro proliferation rate was comparable to parental Hut292DM cells. The data indicate that exogenous expression of wild-type p53 is incompatible with Hut292DM lung cancer cell proliferation in vitro and suggest that p53-mediated growth control in vitro and in vivo may be dissociated and exerted by separate domains of the p53 protein.

Animals↗

PubMed1994

Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene-induced mammary tumors in BALB/c and p53 hemizygous mice.

We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12-dimethylbenz[a]anthracene (DMBA)-treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53-overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild-type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild-type p53 was related to in vitro culture conditions. The effect of DMBA on mammary-tumor development was also tested in mice rendered hemizygous for p53. These mice and wild-type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridization detected no gross alterations in the wild-type p53 allele in mammary tumors from the p53-deficient mice. Point mutation of the wild-type p53 allele was also infrequent in the DMBA-induced mammary tumors from hemizygous p53 mice; it occurred in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA-induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha-ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA-induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild-type p53 was retained in both DMBA-induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild-type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2001

Enhanced apoptotic activity of a p53 variant in tumors resistant to wild-type p53 treatment.

TP53 is the most commonly altered tumor-suppressor gene in cancer and is currently being tested in Phase II/III gene replacement trials. Many tumors contain wild-type TP53 sequence with elevated MDM2 protein levels, targeting p53 for degradation. These tumors are more refractory to treatment with exogenous wild-type p53. Here we generate a recombinant adenovirus expressing a p53 variant, rAd-p53 (d 13-19), that is deleted for the amino acid sequence necessary for MDM2 binding (amino acids 13-19). We compared the apoptotic activity of rAd-p53 (d 13-19) with that of a recombinant adenovirus expressing wild-type p53 (rAd-p53) in cell lines that differ in endogenous p53 status. rAd-p53 (d 13-19) caused higher levels of apoptosis in p53 wild-type tumor lines compared with wild-type p53 treatment, as measured by annexin V-FITC staining. In p53-altered tumor lines, rAd-p53 (d 13-19) showed apoptotic activity similar to that seen with wild-type p53 treatment. In normal cells, no increase in cytopathicity was detected with rAd-p53 (d 13-19) compared with wild-type p53 treatment. This variant protein displayed synergy with chemotherapeutic agents to inhibit proliferation of ovarian and breast cell lines. The p53 variant showed greater antitumor activity in an established p53 wild-type tumor compared with treatment with wild-type p53. The p53 variant represents a means of expanding TP53 gene therapy to tumors that are resistant to p53 treatment due to the cellular responses to wild-type p53.

Adenoviridae↗

PubMed1997

p53 expression in CMV-infected cells: association with the alternative expression of the p53 transactivated genes p21/WAF1 and MDM2.

The p53 tumour suppressor gene is a cell cycle regulator, able to induce cell cycle arrest to allow DNA repair or apoptosis. The molecular mechanisms underlying p53 action imply transactivation of p53 dependent genes such as WAF1 (for wild type p53 associated fragment 1) and the murine double minute (MDM2) gene. In some cases, inactivation of the p53 gene results from p53 gene mutations leading to p53 protein accumulation, but in others it may results from mechanisms other than mutation, such as interaction with viral or cellular proteins. The expression of p53 protein and p53 transactivated gene proteins p21/WAF1 and MDM2, combined with in situ detection of apoptosis, was studied in specimens of CMV-infected patients as an in vivo model of p53 alteration not due to point mutation. p53 positivity was found in CMV + cells in different tissues, in cells with typical inclusion bodies, and in in situ hybridization and immunohistochemistry CMV + cells without inclusions (hidden infection). Although this p53 reactivity was accompanied by the expression of MDM2 and p21/WAF1 proteins, the patterns of MDM2 and p21/WAF1 protein expression were mutually exclusive, and were associated with the presence or absence of inclusion bodies. Nuclei bearing inclusion bodies were usually MDM2+, p21/ WAF1-, while hidden infected cells were usually MDM2-, p21/WAF1+. Apoptosis was not detected in any tissue section from CMV-infected patients. Two alternative patterns were found in CMV-infected tissues: p53+, p21/WAF1+, MDM2-, or p53+, p21/WAF1-. MDM2+ protein expression. These may represent examples of p53 dependent alternative effects in the course of CMV infection. Early stages are represented by CMV + cells without inclusion bodies, which display p53 and p21/ WAF1 expression, suggesting that p53 could be acting as a growth suppressor protein. Late CMV infection is represented by cells harbouring inclusion bodies. These cells showed a p53+, p21/WAF1-, MDM2+ profile, consistent with MDM2 mediated p53 inactivation. The absence of p21/WAF1 expression and lack of apoptosis suggest that the p53 protein expressed by MDM2+ cells could be functionally inactivated in CMV-infected cells with inclusion bodies. Previous studies have suggested that p53 inactivation by MDM2 over-expression occurs in sarcomas and lymphomas. Our observations seem to indicate that this mechanism of MDM2 mediated p53 inactivation may play a role in the late phase of CMV infection.

Adrenal Glands↗

PubMed2001

Is p53 haploinsufficient for tumor suppression? Implications for the p53+/- mouse model in carcinogenicity testing.

The p53 tumor suppressor gene has been shown to be critical in preventing cancer in humans and mice. We have generated and extensively characterized p53-deficient mice lacking one (p53+/-) or both (p53-/-) p53 alleles. The p53-deficient mice are much more susceptible to an array of different tumor types than their wild-type (p53+/+) littermates. The enhanced tumor susceptibility of the p53+/- mice has made them one of several transgenic mouse models that are being considered as substitutes for standard 2-year rodent carcinogenicity assays. In order to fully exploit this model, it will be important to understand some of the basic biological and molecular mechanisms that underlie its enhanced tumor susceptibility. With this in mind, we have explored the fate of the remaining wild-type p53 allele in spontaneously arising p53+/- tumors and have shown that over half of these tumors retain an intact, functional wild-type p53 allele. This suggests that p53 is haploinsufficient for tumor suppression and that mere reduction in p53 dosage is sufficient to promote cancer formation. To support the idea that p53 is indeed a haploinsufficient tumor suppressor, we show here that normal p53+/- cells exhibit reduced parameters of growth control and stress response compared to their p53+/- counterparts. We hypothesize that the reduced p53 dosage in the p53+/- cells provides an environment more conducive to the development of further oncogenic lesions and the initiation of a tumor. Finally, we have assessed p53 loss of heterozygosity (LOH) in carcinogen-induced p53+/- tumors and have found that some agents induce tumors that almost invariably exhibit p53 LOH, whereas other agents induce tumors that often retain the wild-type p53 allele. Our preliminary data suggest that LOH is dependent on both the mechanism of genotoxicity of the agent utilized and the tissue type targeted.

Animal Testing Alternatives↗

PubMed2005

Human monocyte-derived dendritic cells pulsed with wild-type p53 protein efficiently induce CTLs against p53 overexpressing human cancer cells.

PURPOSE: Dendritic cells are the most potent antigen-presenting cells for initiating cellular immune responses. Dendritic cells are attractive immunoregulatory cells for cancer immunotherapy, and their efficacy has been investigated in clinical trials. The tumor suppressor gene p53 is pivotal in the regulation of apoptosis, and p53-based immunization is an attractive approach to cancer immunotherapy because of the accumulation of p53 protein in malignant but not in normal cells. It has been shown that dendritic cells transduced with an adenoviral wild-type p53 (wt-p53) construct mediate the antitumor immune responses against p53-overexpressing tumor cells. We examined whether monocyte-derived human dendritic cells pulsed with the purified full-length wt-p53 protein were also capable of inducing the specific antitumor responses against p53-overexpressing tumors in vitro. EXPERIMENTAL DESIGN: Immature dendritic cells generated in the presence of interleukin-4 and granulocyte/macrophage colony-stimulating factor from monocytes of HLA-A2- or HLA-A24-positive healthy individuals were pulsed with the purified p53 protein. Uptake of p53 protein by human dendritic cells was assessed by Western blotting and immunohistochemical staining using anti-p53 antibody. Induction of p53-specific CTL response was also evaluated by the cytotoxic assay against p53-overexpressing human tumor cells. RESULTS: Both Western blot and immunohistochemical analysis showed the accumulation of p53 protein in human immature dendritic cells. T cells obtained from HLA-A2- or HLA-A24-positive healthy donors were stimulated twice with p53 protein-pulsed dendritic cells and then applied to the cytotoxicity assay against p53-overexpressing target cells. The CTL activity was specific for p53-overexpressing tumor cells and MHC class I restricted. Moreover, the CTL activity generated by p53 protein-pulsed dendritic cells was nearly identical with that induced by adenoviral wt-p53-transduced dendritic cells. CONCLUSIONS: Our results indicate that monocyte-derived human dendritic cells pulsed with the wt-p53 protein could induce the specific antitumor effect against p53-overexpressing tumors and that this in vitro model offers a new and more simple approach to the development of p53-based immunotherapy.

Antigens, CD↗

PubMed2004

Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNA-binding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein. To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.

Cell Cycle↗

PubMed1991

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.

Animals↗

PubMed1993

The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription.

Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation.

Amino Acid Sequence↗

PubMed1995

Effects of p53 mutants on wild-type p53-mediated transactivation are cell type dependent.

The spectrum of p53 mutations differs among human cancer types. We have hypothesized that the p53 mutational spectrum observed in particular tumor types reflects the functional ability of different p53 mutants to modulate wild-type (WT) p53-dependent gene transcription. Missense p53 mutants representing several mutational hotspot codons were cotransfected with WT p53 and analysed for their effects on p53-dependent transactivation of a reporter construct containing a specific p53 binding sequence (PG13-CAT) in human tumor cell lines lacking endogenous p53. Our results show that the ability of p53 mutants to inhibit WT p53-mediated transactivation is cell type dependent. In cell lines derived from a lung adenocarcinoma and a mesothelioma, the transactivation function of WT p53 was strongly inhibited by all p53 mutants examined. However, in cell lines derived from a prostate carcinoma and an osteosarcoma, the mutants examined generally had only minimal dominant negative effects. In cell lines derived from a hepatocellular carcinoma and an ovarian carcinoma, two mutants (248trp and 273his) enhanced WT p53-mediated transactivation of the reporter construct. Additional mutants retained the ability to inhibit WT p53-mediated transactivation in these cell lines. In addition, in a series of four breast tumor cell lines, the p53 mutants examined had similar effects on WT p53 transactivation ability including enhanced transactivation activity in the 273his cotransfectants. The p53 mutants were incapable of transactivating the PG13-CAT reporter in the absence of WT p53 expression. Therefore, the dominant negative effects of p53 mutants on WT p53 function may vary depending on the particular cell type. In addition, mutants with stronger inhibitory capabilities may confer a selective advantage during the tumorigenic process.

Base Sequence↗

PubMed2001

Heterogeneous distribution of P53 immunoreactivity in human lung adenocarcinoma correlates with MDM2 protein expression, rather than with P53 gene mutation.

Although the tumor suppressor p53 protein (P53) immunoreactivity and its gene (p53) mutation were reported to be significant prognostic indicators for human lung adenocarcinomas, little is known regarding the relationship between the heterogeneous distribution of P53 and its genetic status in each tumor focus and the clinicopathological significance. To determine how P53 is heterogeneously stabilized in patients, we compared P53 expression to both the p53 allelic mutation in exon 2 approximately 9 by polymerase chain reaction-single strand conformation polymorphism using microdissected DNA fractions, and the immunohistochemical MDM2 expression. Of the 48 positive to P53 in 118 lung adenocarcinomas examined, 10 with heterogeneous P53 expression were closely examined. The higher P53 expression foci in 7 of 10 cases were less differentiated, histologically in respective cases, and were frequently associated with fibrous stroma. Two had genetic mutations in exon 7 of the p53 gene in both the high and low P53 expression foci of cancer tissue indicating no apparent correlation between heterogeneous P53 expression and the occurrence of gene mutation. Immunohistochemical expression of MDM2 was significantly lower in high P53 expression areas (p < 0.05, the mean labeling indices of high and low P53 expression areas being 4.2 +/- 5.4% and 13.6 +/- 12.2%, respectively). In addition, among all the 118 cases examined, MDM2 expression was significantly suppressed in cases of p53 gene mutation, simultaneously with P53 overexpression, as compared with cases without both the p53 mutation and expression (p < 0.001). These findings suggest that the heterogeneous stabilization of P53 in human lung adenocarcinomas could be partly due to suppressed MDM2 expression. The overexpression of non-mutated P53 may afford a protective mechanism in human lung adenocarcinomas.

Adenocarcinoma↗

PubMed2001

Expression of p53 protein in T- and natural killer-cell lymphomas is associated with some clinicopathologic entities but rarely related to p53 mutations.

To determine if p53 abnormalities could be involved in the pathogenesis of T- or natural killer (NK)-cell lymphomas, we investigated 51 cases of these lymphomas for the expression of p53 and its relationship with p53 gene mutations, the expression of the p21 protein as well as the proliferative and apoptotic indices. Overexpression of p53 was found in 19 cases (37%), whereas mutations of the p53 gene were observed in only 5 of 28 tested cases. The analysis of immunohistochemical data showed some entity-related phenotypic profiles. Anaplastic large cell lymphomas showed a frequent overexpression of p53 (7/8 cases) and p21 (6/8 cases) proteins and rare p53 mutations (1/7 cases), suggesting accumulation of a functional wild type p53 protein able to induce p21 expression. Nodal peripheral T-cell lymphomas unspecified showed relatively frequent overexpression of p53 protein (5/7 cases), infrequent p21 expression (2/7 cases), and rare p53 gene mutations (1/6 cases). In angioimmunoblastic lymphomas, the common phenotype was p53-/p21- (15/17 cases), with only a few scattered p53-positive cells, which, on the basis of double staining results, were mostly Epstein-Barr virus-infected B cells. A p53 gene mutation was only found in 1 case (1/8 cases) of angioimmunoblastic lymphoma, which showed cytologic tumor progression. Mycosis fungoides showed p53 overexpression in 2 of 4 cases, including 1 case with p53 gene mutation and features of cytologic tumor progression. Nasal NK/T lymphomas showed p53 overexpression in 2 of 5 cases, 1 of which had a p53 gene mutation. Finally, all lymphoblastic T-cell lymphomas (5 cases) and gammadelta hepatosplenic T-cell lymphomas (3 cases) were negative for expression of p53 and p21 proteins. We conclude that p53 protein overexpression is a common finding in some entities of T- and T/NK-cell lymphomas, whereas a p53 gene mutation is a rare, sporadic, and rather late event associated with tumor progression in some instances. The p53/p21 expression pattern appears to be variable in T- and T/NK-cell lymphoma entities, reinforcing the concept of distinct, entity-related mechanisms of pathogenesis in these tumors.

Adolescent↗

PubMed1996

p53 transactivation through various p53-responsive elements.

p53 is a nuclear phosphoprotein whose function is classified as tumor suppression. Studies have shown that p53 functions by binding to p53 DNA recognition sequences and regulates transcription of growth-regulatory genes. Various p53 recognition sequences have recently been identified. pOST2 contained two copies of a palindromic high-affinity DNA-binding sequence for p53; the other p53 recognition sequences included p53-binding fragments found in the human ribosomal gene cluster (pRGC) region and in the murine muscle creatine kinase promoter (pMCK). The purpose of this study was to compare the abilities of various p53 recognition sequences to mediate transcription in the presence of endogenously produced wild-type (wt) or mutant p53. Three p53-responsive chloramphenicol acetyltransferase (CAT) reporter constructs (pOST2, pRGC, and pMCK) that contain one or two copies of p53 recognition sequences upstream of a herpes thymidine kinase (TK) promoter and CAT reporter cDNA were constructed. Either a p53-responsive gene or a control reporter gene was transfected into human carcinoma cell lines (having various p53 mutations) either with or without a wt or mutant p53 expression vector. CAT activity was assayed to measure transactivation through the various p53-responsive elements. We showed that pOST2 had a greater ability to mediate transactivation by p53 than either pRGC or pMCK. p53 with a mutation at either codon 175 or 248 was unable to transactivate a reporter gene with pOST2, pRGC, or pMCK. We found it interesting that pOST2, but not pRGC or pMCK, was able to mediate transactivation in cell lines that produce codon 273-mutant p53. These findings suggest that various sensitivities of the different p53-responsive elements to specific mutant and wt p53s may be an important factor in the role of p53 as a transcriptional activator both under normal physiological conditions and during carcinogenesis.

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