p53 immunolabeling in archival paraffin-embedded tissues: optimal protocol based on microwave heating for eight antibodies on lung carcinomas

The prognostic value of p53 gene mutations is dealt with by several recent reports. However, retrospective assessment of p53 tumor status on archived samples has been prevented by p53 epitope alteration during routine fixation and embedding procedures. This study aimed at establishing a reproducible low-cost protocol to retrieve not only N-terminal, but also midregion and C-terminal, epitopes, with special attention to possible artifacts induced by epitope retrieval procedures. Using microwave heating, we compared the epitope retrieval efficiency of five solutions with eight commercial antibodies on 21 lung carcinomas for which frozen tissue and samples fixed with formalin and Bouin's liquid were available. All eight epitopes were retrieved, citrate buffer proving efficient for seven. PAb 240 epitope was restored by target unmasking fluid only. No false positivity was observed. Fixation-induced loss of p53 immunoreactivity was minimal for formalin (two of 10 tumors for one antibody each), more significant for Bouin (six of 10 tumors for one to five antibodies). On the other hand, staining intensity was maintained or even improved, and nonspecific staining reduced, through fixation. We conclude that p53 stabilization can be detected on routinely processed archival tumor samples with a reliability similar to that of frozen tissue by means of a microwave-based procedure and a panel of at least three antibodies, with epitopes on the N-terminal, C-terminal, and midpart of the molecule.     

Ultrasonic debridement of mitral calcification

PURPOSE: Kaposi's sarcoma (KS) is a proliferative process of suspected viral aetiology associated with immune deficiency. In transplanted patients, lesions regress on discontinuation of immunosuppressive therapy. The purpose of this work was to analyse the expression of the p53 oncosuppressor gene product, a proliferation regulator overexpressed in both malignant and non-malignant conditions, with the aim of better qualifying KS proliferation characteristics. METHODS: We analysed p53 expression in a group of transplanted, cyclosporin A-treated, KS patients by immunohistochemistry, utilizing the DO-7 (with and without the antigen retrieval pretreatment), and the PAb 240 monoclonal anti-p53 antibodies, the latter of which is able to detect a mutated epitope, and evaluating staining intensity and localization, whether cytoplasmic or nuclear. RESULTS: Seventy five percent of KS lesions from transplanted patients presented both nuclear and cytoplasmic positive p53 immunostaining with DO-7 antibody, thus demonstrating a presumably functional inactivation; one case also presented immunoreactivity with the PAb 240 antibody. CONCLUSIONS: On the basis of the results obtained and in the presence of lesion regression upon immunosuppression withdrawal, it may be concluded that KS in transplanted patients can be considered a non-malignant proliferative process, and that the cytoplasmic expression of p53 may stand for a functional inactivation pattern.     

Modification of two distinct COOH-terminal domains is required for murine p53 activation by bacterial Hsp70

Activation of the latent DNA binding function of human p53 protein by the bacterial Hsp70, DnaK, represents a unique reaction in which a heat shock protein can interact with a native protein to affect its function. We have localized a likely DnaK interaction site on native human p53 tetramers to a motif flanking the COOH-terminal casein kinase II and protein kinase C phosphorylation sites. Murine p53 is less efficiently activated by DnaK, which has permitted a search for factors that might cooperate in p53 activation by DnaK. We show that optimal activation by DnaK may be dependent upon the phosphorylation state of murine p53, in particular, modification of p53 at the cdc2 phosphorylation site by point mutation decreases the extent of activation by DnaK. Additionally, the monoclonal antibody PAb241, binding in the vicinity of the cdc2 phosphorylation site, is able to activate the specific DNA binding function of p53. This has led us to propose a second regulatory motif flanking the tetramerization domain of p53 that cooperates with factors binding at the negative regulatory domain in the extreme COOH terminus.     

The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation

p53 accumulates after DNA damage and arrests cellular growth. These findings suggest a possible role for p53 in the cellular response to DNA damage. We have previously shown that the C terminus of p53 binds DNA nonspecifically and assembles stable tetramers. In this study, we have utilized purified segments of human and murine p53s to determine which p53 domains may participate in a DNA damage response pathway. We find that the C-terminal 75 amino acids of human or murine p53 are necessary and sufficient for the DNA annealing and strand-transfer activities of p53. In addition, both full-length wild-type p53 and the C-terminal 75 amino acids display an increased binding affinity for DNA damaged by restriction digestion, DNase I treatment, or ionizing radiation. In contrast, the central site-specific DNA-binding domain together with the tetramerization domain does not have these activities. We propose that interactions of the C terminus of p53 with damaged DNA may play a role in the activation of p53 in response to DNA damage.     

p53 protein expression in peripheral lymphocytes from atrazine chronically intoxicated rats

The p53 expression in peripheral lymphocytes of rats chronically exposed to atrazine was investigated. The experiment was performed in female Wistar rats. Atrazine was administrated in different doses (2.7 and 5.4 mg/kg body weight), each dose once a day, 5 days per week, for 6 and 12 months. The percentage of rats peripheral lymphocytes expressing p53 protein was evaluated by immunocytochemical technique, using a monoclonal antibody (clone PAb 122) against a common epitope, both for the wild type and the mutant p53 protein. The results indicate that in the atrazine long-term administration, the serum level of atrazine is associated with: (i) Significantly increased percentage of lymphocytes expressing p53 protein for all treated animals; (ii) different p53 intracellular compartmentalization (nucleus and cytoplasm), depending on dose and time of atrazine administration. The present study suggests that atrazine modifies the p53 expression, which could confirm the clastogenicity of this herbicide, and that the detection of the p53 protein may serve as a biomarker for the long-term exposure to atrazine.     

Immunity to p53 induced by an idiotypic network of anti-p53 antibodies: generation of sequence-specific anti-DNA antibodies and protection from tumor metastasis

The general overexpression of p53 by different types of tumor cells suggests that p53 immunity might be generally useful for tumor immunotherapy. We describe here the induction of immunity to p53 and resistance to tumor metastasis using an idiotypic network. Mice were immunized with domain-specific anti-p53 monoclonal antibodies (Ab1): PAb-248 directed to the N-terminus; PAb-246 directed to the specific DNA-binding region; or PAb-240 directed to a mutant p53 that does not bind specific DNA. Immunized mice responded by making anti-idiotypic antibodies (Ab2) specific for the Ab1 inducer. Ab1 PAb-246 induced Ab2 that, like p53 itself, could bind the specific DNA oligonucleotide sequence of the p53 responsive element. Mice immunized with Ab1 PAb-240 or PAb-246 spontaneously made Ab3 anti-p53 antibodies that reflected the specificity of their Ab1 inducers: Ab1 PAb-246 induced Ab3 specific for wild-type p53; PAb-240 induced Ab3 specific for mutant p53. Ab1 PAb-248 induced only Ab2. The spontaneously arising Ab3 were of T cell-dependent IgG isotypes. Peptides from the complementarity determining regions of the Ab1 antibodies PAb-240 and PAb-246 could also induce Ab3 anti-p53. Finally, mice that produced Ab3 anti-p53 acquired resistance to tumor metastases. Therefore, an anti-idiotypic network built around certain domains of p53 seems to be programmed within the immune system, specific Ab2 antibodies can mimic the DNA binding domain of p53, and Ab3 network immunity to p53 can be associated with resistance to tumor cells.     

Morphology of brain stem lesion and bera findings after 60Co irradiation

BACKGROUND: We assessed the frequency and molecular basis of p53 mutations in clinically localized prostatic adenocarcinoma. METHODS: Prostate specimens were examined from 100 patients with clinically localized prostatic adenocarcinoma and 13 patients with benign prostatic hyperplasia (BPH). Mutations producing nuclear accumulation of p53 were detected immunohistochemically. Exon-specific mutations were analyzed by polymerase chain reaction amplification and single strand conformation polymorphism (PCR-SSCP) and sequenced. RESULTS: p53 accumulation was detected in 5 tumors using antibody DO-1, and in 4 of these using antibody PAb 1801, but not in BPH. PCR-SSCP detected mutations in all 5 tumors, with alterations in exon 5 for 1 tumor, exon 6 for 3 tumors, and exon 7 for 1 tumor. An exon 6 mutation was also found in a tumor with no anti-p53 staining. CONCLUSIONS: p53 mutations are uncommon in clinically localized prostatic adenocarcinoma and absent from BPH. 5 of the 6 mutations were derived from locally invasive, prostate carcinomas, supporting the hypothesis that mutation of p53 is a late event in prostate carcinoma progression.     

Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot-start-PCR-single-strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin-embedded sections was amplified by hot-start-PCR using oligonucleotide primers specific for exons 4-9 of p53. The mutations were analyzed by PCR-SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild-type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR-SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR-SSCP analysis; (2) DNA sequencing of samples showing frame-shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC-->TGC) and exon 5 at codon 132 [AAG (Lys)-->AGG (Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti-human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR-SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.     

Conformational dependence of Anaplasma marginale major surface protein 5 surface-exposed B-cell epitopes

The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940-2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denatured A. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.     

A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.     

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.     

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

The C-terminal domain of p53 catalyzes DNA-renaturation and strand exchange toward annealing between intact ssDNAs and toward eliminating damaged ssDNA from duplex formation through preferential recognition of damaged DNA by a duocarmycin

The C-terminal domain of p53 may bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules, suggesting a possible direct role for p53 in DNA repair (Proc. Natl. Acad. Sci. USA, 92, 9455-9459, 1995). We found that DU-86, a duocarmycin derivative which alkylates DNA, bound ssDNA and enhanced the DNA binding activity of the p53 C-terminus. DU-86 weakened p53-mediated catalysis of complementary ssDNA renaturation. p53 C-terminus catalyzed DNA strand transfer toward annealing between intact ssDNAs and toward eliminating DU-86-damaged ssDNA from duplex formation. These results suggest that p53, via the C-terminal domain, may play a direct role in DNA repair by preferential recognization and elimination of damaged DNA.     

An engineered four-stranded coiled coil substitutes for the tetramerization domain of wild-type p53 and alleviates transdominant inhibition by tumor-derived p53 mutants

The tetramerization domain of p53 is required for efficient tumor suppressor activity. This domain, however, also allows wild-type p53 to heterooligomerize with dominant negative tumor-derived p53 mutants. We explored the feasibility of substituting the native tetramerization domain of wild-type p53 with an engineered leucine zipper that assembles as a four-stranded coiled coil. The engineered zipper drove p53 tetramerization in vitro and p53 function in vivo. Furthermore, it alleviated transdominant inhibition by tumor-derived p53 mutants, implying that dominant negative mutants act by hetero-oligomerizing with wild-type p53. The ability of the engineered zipper to drive tetramerization was critical for p53 function, since p53 dimers, formed by substituting the p53 tetramerization domain with a native leucine zipper, were weak tumor suppressors.