Two different forms of p53 localized differently within cells of urogenital tumours.

We analyzed the subcellular localization of p53 in prostate and bladder carcinoma cells. Using laser scanning microscopy and PAb1620, a monoclonal antibody recognizing the wildtype conformation of p53, and another monoclonal antibody directed against the mutant conformation of the protein (PAb240), we found two different subsets of p53 within the same cell. The wildtype subgroup was found in the nucleolus, whereas the mutant protein was confined to the nucleus. The results obtained by immunofluorescence were verified by Western blot analysis and immunoprecipitation. Thus, our findings demonstrate an unusual subcellular localization pattern of p53 in prostate and bladder cancer cells which may indicate another mechanism of inactivation of p53.

Humoral immune response to p21WAF1/CIP1 in tumor patients, non-tumorous patients and healthy blood donors.

We performed a serological analysis for anti-p21WAF1/CIP1 antibodies in sera of patients with different gynecological diseases such as breast cancer, ovarian carcinoma, cervix carcinoma and benign gynecological tissue alterations and from healthy blood donors using the immunoblotting technique with recombinant p21WAF1/CIP1 as antigen as well as a newly designed ELISA. We detected antibodies specific for p21WAF1/CIP1 in sera derived from cancer patients, as well as from patients with non-malignant diseases and from healthy blood donors. Thus, the presence of antibodies against p21WAF1/CIP1 is not a marker for malignancies. Some of the sera with antibodies against p21WAF1/CIP1 also contained antibodies against the oncoprotein mdm2, and/or the growth suppressor gene product p53. The presence of antibodies against p53 correlates with a malignant disease.

Detection of p53 in inflammatory tissue and lymphocytes using immunohistology and flow cytometry: a critical comment.

AIMS: To analyse the expression of p53 in lymphatic cells found in inflammatory tissues and the peripheral blood by immunological methods. METHODS: Immunohistological analysis of synovial tissues from patients with rheumatoid arthritis and flow cytometric analysis of peripheral blood lymphocytes were performed with anti-p53 antibodies from different sources. RESULTS: The anti-p53 antibodies PAb240, PAb421, and PAb1801 from one supplier bound to the cytoplasm of lymphocytes, fibroblasts, and endothelial cells in rheumatoid synovial tissue, while the same anti-p53 antibodies from other sources and the p53 specific antibodies PAb1620 and DO1 were negative. Using flow cytometry, the antibodies that labelled cells in inflammatory tissues were shown to bind also to peripheral lymphocytes, while the antibodies that were negative in immunohistology did not react with peripheral blood lymphocytes. p53 expression could be confirmed by western blot in rheumatoid synovial tissue, but not in peripheral blood lymphocytes using PAb421 and PAb240 antibodies from our own laboratory, which had been negative in immunohistology. CONCLUSIONS: Demonstration of p53 by western blot is more sensitive and reliable than immunohistology and flow cytometry. Western blot is the gold standard for the demonstration of p53 expression and should be used, whenever possible, to confirm p53 expression in normal tissue shown by immunohistology or flow cytometry. All other reports on p53 expression, especially those obtained using antibodies with an unusual staining pattern must be interpreted with caution.

Antibodies against murine double minute-2 (mdm2) in sera of patients with various gynaecological diseases.

We performed a serologic analysis for anti-murine double minute-2 (anti-mdm2) antibodies in sera of patients with different gynaecological diseases using immunoblotting technique with recombinant mdm2 as antigen. In addition, for large scale screening we established an anti-mdm2 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients with breast cancer, ovarian carcinoma, cervix carcinoma and benign gynaecological tissue alterations were tested. We detected antibodies specific for mdm2 in sera derived from cancer patients, as well as from patients with non-malignant diseases. Some of the sera with antibodies against mdm2 also contained antibodies against the growth suppressor gene product p53.

Different subnuclear localization of wild-type and mutant p53 in human prostate-cancer cells.

The growth suppressor protein p53 is abnormally expressed in a variety of different human tumor cells. We have analyzed the expression of p53 in cell cultures derived from tissues of radical prostatectomies and in the permanent prostate carcinoma cell line PC-3 using two different p53 specific monoclonal antibodies. With the wild-type specific monoclonal antibody PAb1620 we found p53 localized in nucleoli whereas only a few cells were positively stained in the nucleus with the mutant specific monoclonal antibody PAb240. Control experiments with p53 from SV80 cells which express wild-type p53 and HT29 cells expressing mutant p53 documented the specificity of the monoclonal antibodies. The specificity of the antibodies in recognizing indeed p53 was demonstrated further by immunoprecipitation analysis of p53 from the same cell cultures. Since p53 is usually localized to the nucleus our results may represent a specific feature of the wild-type phenotype of p53 in prostate carcinoma cells. The localization of p53 in nucleoli may be another mechanism of the inactivation of wild-type p53.

The emerging picture of p53.

1. The cellular phosphoprotein p53 is a negative regulator of the cell growth. A great majority of human malignancies expresses a mutated p53 that represents an oncogenic version of the protein. 2. However, in the meantime many tumors were identified containing a p53 protein without any mutation. Here also other events than genomic alterations of p53 might be implicated in the process of cell transformation. 3. The expression of wild-type or mutant conformation is not exclusively defined by the p53 DNA sequence but also influenced by the subcellular environment and the interaction of cellular proteins with p53. 4. In particular, the mdm-2 gene product appears to be an important partner of p53 somehow involved in these complex regulatory processes. 5. Recent findings supported a role for p53 in transcriptional regulation, perhaps by reducing the expression of genes that are needed for ongoing cell proliferation. 6. This property may be based upon the ability of p53 to bind DNA as well as different proteins from viral or cellular origin. 7. Especially transcription factors or further cellular proteins connected in any way with the regulation of cell proliferation are possible candidates. 8. Thus, it is not surprising that p53 is implicated in the regulation of the cell cycle and in the decision of a cell to replicate DNA or to go into apoptosis.

Properties of a C-terminal polypeptide fragment of simian-virus 40 T-antigen.

Certain biological activities of SV40 large T antigen can be attributed to distinct regions on the polypeptide chain of T antigen. Some of these properties are influenced by the interaction of T antigen with cellular proteins. We established a mouse cell line expressing full length T antigen and another cell line expressing a truncated T antigen (T600C) consisting of 600 C-terminal amino acids of authentic T antigen. Morphology, growth properties and actin cable organization indicated that cells expressing full length T antigen were transformed whereas the T600C T antigen expressing cells were non-transformed. Full length T antigen and the T600C fragment of T antigen were both phosphoproteins, both were located in the nucleus and both formed complexes with p53. Rate of synthesis as well as stability of the T600C T antigen and of p53 were reduced in the non-transformed cells when compared to full length T antigen and p53 in transformed cells. Stoichiometry of T antigen and p53 in T-p53 complexes seemed to be different for the T600C T antigen expressing cells when compared to cells expressing full length T antigen. Immunopurified complexes of the T600C T antigen and of full length T antigen with p53 were associated with a protein kinase phosphorylating both proteins. Interestingly uncomplexed full length T antigen was also associated with a protein kinase whereas the T600C T antigen lacked this ability. These data may indicate a correlation between cell transformation and distinct biochemical properties of T antigen and p53 including the association of T antigen with a protein kinase.

Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation.

The synthesis of a new class of antisense oligonucleotide compounds with 3'-3' and 5'-5' end inversion (INV-oligonucleotides) is described. Besides the advantage of simplicity of synthesis, physico-chemical studies show that these compounds do not disturb Watson-Crick base-pairing. INV-oligonucleotides have a half-life of 30 h in human serum. We show that they are capable of inhibiting SV40 large T-antigen expression in COS-1 cells, both in vitro and in vivo, and by modulation of the expression of cellular oncoprotein p53 in vitro.