The p53 protein is related by sequence homology and function to the products of two other genes, p63 and p73, that each encode several isoforms. We and others have discovered previously that certain tumor-derived mutants of p53 can associate and inhibit transcriptional activation by the ␣ and ␤ isoforms of p73. In this study we have extended these observations to show that in transfected cells a number of mutant p53 proteins could bind and down-regulate several isoforms not only of p73 (p73␣, -␤, -␥, and -␦) but also of p63 (p63␣ and -␥; ⌬Np63␣ and -␥). Moreover, a correlation existed between the efficiency of p53 binding and the inhibition of p63 or p73 function. We also found that wild-type p63 and p73 interact efficiently with each other when coexpressed in mammalian cells. The interaction between p53 mutants and p63 or p73 was confirmed in a physiological setting by examining tumor cell lines that endogenously express these proteins. We also demonstrated that purified p53 and p73 proteins interact directly and that the p53 core domain, but not the tetramerization domain, mediates this interaction. Using a monoclonal antibody (PAb240) that recognizes an epitope within the core domain of a subset of p53 mutants, we found a correlation between the ability of p53 proteins to be immunoprecipitated by this antibody and their ability to interact with p73 or p63 in vitro and in transfected cells. Based on these results and those of others, we propose that interactions between the members of the p53 family are likely to be widespread and may account in some cases for the ability of tumor-derived p53 mutants to promote tumorigenesis.

The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator. Recent studies have identified a p53 relative, termed p73. We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73␣ and -␤ isoforms to transactivate various p53-responsive promoters. We show that both p73␣ and p73␤ activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299. However, a number of significant differences were observed between p53 and p73 and even between p73␣ and p73␤. Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37°C. Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay. When we examined complex formation in transfected mammalian cells, we found that p73␣ coprecipitates with mutant but not wild-type p53. Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73. By cotransfecting p73␣ along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73␣. This decrease in transcriptional activity is correlated with the reduced ability of p73␣ to promote apoptosis in the presence of tumorderived p53 mutants. Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.A new gene family whose encoded products show significant sequence similarity to the tumor suppressor protein p53 have been identified (32,33,50,59,60,72). KET, the first to be identified, was cloned from a rat circumvallate taste papilla cDNA library (59). p73, the second identified from a COS cell cDNA library, encodes for at least two splicing variants, p73␣ and p73␤ (32, 33). Finally, the human homolog of KET, referred to as either p51 or p63, encodes at least six isoforms (p63␣/p51B/p73L, p63␤, p63␥/p51A, ⌬Np63␣, ⌬Np63␤, and ⌬Np63␥) that are expressed in a tissue-specific manner and harbor different transactivation potentials (50,60,72). It has been proposed that this family of proteins is ancestral to human p53, in that all show significant amino acid similarity in their C-terminal p53-unrelated extensions to the squid p53 protein (33,59,72).The p53 protein is modular and can be divided into at least four distinct domains: (i) the amino-terminal transcriptional transactivation domain (residues ϳ1 to 70) (6,7,15,55,68), (ii) the PXXP domain (residues ϳ61 to 94) (70), (iii) the sequence-specific DNA binding domain (residues ϳ102 to 292) (1, 27, 51, 71), and (iv) the carboxy-terminal regulatory and tetramerization domains (residues ϳ320 to 393 and ϳ320 to 360, respectively) (3,4,57,71). The various isoforms of p73 and p51/p63 display a modular structure similar to that o...

No abstract

Localized inflammation of a rat's hindpaw elicits an accumulation of ␤ -endorphin-(END) containing immune cells. We investigated the production, release, and antinociceptive effects of lymphocyte-derived END in relation to cell trafficking. In normal animals, END and proopiomelanocortin mRNA were less abundant in circulating lymphocytes than in those residing in lymph nodes (LN), suggesting that a finite cell population produces END and homes to LN. Inflammation increased proopiomelanocortin mRNA in cells from noninflamed and inflamed LN. However, END content was increased only in inflamed paw tissue and noninflamed LN-immune cells. Accordingly, corticotropin-releasing factor and IL-1 ␤ released significantly more END from noninflamed than from inflamed LN-immune cells. This secretion was receptor specific, calcium dependent, and mimicked by potassium, consistent with vesicular release. Finally, both agents, injected into the inflamed paw, induced analgesia which was blocked by the co-administration of antiserum against END. Together, these findings suggest that END-producing lymphocytes home to inflamed tissue where they secrete END to reduce pain. Afterwards they migrate to the regional LN, depleted of the peptide. Consistent with this notion, immunofluorescence studies of cell suspensions revealed that END is contained predominantly within memory-type T cells. Thus, the immune system is important for the control of inflammatory pain. This has implications for the understanding of pain in immunosuppressed conditions like cancer or AIDS. ( J. Clin. Invest.