Accumulation of an inactive form of p53 protein in cells treated with TNF alpha.

In MCF-7 cells, TNF alpha induces a G1 arrest with an increased expression of p21/Waf1, an activation of NF-kappa B and an accumulation of p53. NF-kappa B and p53 are two transcriptional factors known to activate p21/Waf1 gene expression. Here we show that p53 inhibition has no effect on p21/Waf1 mRNA accumulation following TNF alpha treatment. In contrast, inactivation of NF-kappa B inhibits p21/Waf1 expression without affecting G1 arrest. The fact that p21/Waf1 gene expression is still stimulated when p53 is inactivated strongly suggests that TNF alpha induces accumulation of an inactive form of p53 protein. This assumption was further supported by the following observations: (i) the p53 DNA-binding activity to its consensus sequence was not stimulated following TNF alpha treatment, (ii) phosphorylation at Ser-15, -20 or -392 was not detected in response to TNF alpha, (iii) the transcription rate of Ddb2, another p53 target gene, was not stimulated by TNF alpha. Finally, the accumulation of p53 in the nuclei of TNF alpha-treated MCF-7 cells was concomitant with an increase in p53 mRNA level, suggesting a regulation at the transcription level.

Reciprocal down-regulation of p53 and SOD2 gene expression-implication in p53 mediated apoptosis.

p53 regulates the transcription of a number of genes among which are different redox-related genes. It has been proposed that these genes can induce a cellular oxidative stress leading to p53-dependent apoptosis (Polyak et al., 1997). MnSOD, the product of superoxide dismutase 2 (SOD2) gene, is one of the major cellular defences against oxidative stress. We demonstrate here that p53 is able to repress SOD2 gene expression and that this repression takes place at promoter level. We show the importance of this regulation for the p53 function, by demonstrating that an overexpression of MnSOD decreases p53-mediated induction of apoptosis. Moreover, we demonstrate that MnSOD overexpression decreases p53-gene expression at the promoter level. These findings raise the hypothesis that p53 and SOD2 genes are mutually regulated leading to the modulation of various cellular processes including apoptosis.

Identification of RB18A, a 205 kDa new p53 regulatory protein which shares antigenic and functional properties with p53.

Immunological screening with the anti-p53 moAb, PAb1801 of a cDNA expression library, prepared from human B lymphoma cells, led us to identify a new human 205 kDa protein called RB18A for 'Recognized By PAb1801 moAntibody'. Immunoblotting or immunoprecipitation of fusion protein or in vitro translated protein, respectively, demonstrated that RB18A protein was recognized by several anti-p53 moAb reacting with the N or C-terminal domains of p53. Full length sequence of RB18A cDNA and computer analysis demonstrated that despite common antigenic determinants between RB18A and p53 proteins, nucleotide and deduced protein sequences did not reveal any significant homologies. RB18A mRNA was detected in all tissues tested except in kidney. In addition, RB18A protein shared identical functions with p53 protein: binding to DNA or to p53 and self-oligomerization. Furthermore, RB18A regulated p53 specific binding on his DNA consensus binding site. These functions were associated to the C-terminal domain of RB18A protein and more specifically to the PAb421 binding site present in this domain. The activation by RB18A of p53 binding on DNA was induced through an unstable interaction between both proteins. Altogether, our data demonstrated that RB18A protein shares antigenic and functional properties with p53 and regulated p53 functions.

Pep34, a synthetic peptide whose sequence corresponds to the intracytoplasmic domain of the Epstein-Barr virus receptor (CR2, CD21), regulates human B lymphocyte proliferation triggered through CR2.

CR2 is involved in regulation of human B lymphocyte proliferation by interacting, through distinct domains, with extracellular, cell surface or intracellular components. Contribution of CR2 intracytoplasmic domain in CR2 regulatory functions remains unclear. Thus, we used pep34, a 34 amino acid synthetic peptide whose sequence corresponds to CR2 intracytoplasmic domain. Pep34 was incorporated into B lymphocytes which were then activated by EBV or C3d through CR2. Our data demonstrate that pep34 inhibits 100% B lymphocyte proliferation triggered by EBV or C3d. Irrelevant peptide had no effect. When B lymphocyte proliferation was triggered by a multipotent B cell activator as SAC, pep34 did not exert any inhibitory effect. Our data demonstrate that pep34 inhibits B lymphocyte proliferation only when lymphocytes are triggered through CR2. Thus, this strongly supports that despite its short length. CR2 intracytoplasmic domain participates to regulatory functions of this receptor.

Epstein-Barr virus/C3d receptor (CR2, CD21) activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways.

We previously demonstrated that human C3d or pep16, a 16-amino acid synthetic peptide derived from human C3d, induced in vivo and in vitro tyrosine phosphorylation of pp105, an intracellular component found only in human cells that express CR2 at their surface. To determine the contribution of CR2 molecules to this enzymatic regulation, we first analyzed whether activation of CR2 by other extracellular CR2 ligands could trigger such regulation in cell extracts. Subsequently, we used cell extracts of either CR2-positive cells depleted in CR2 molecules by absorption with anti-CR2 antibodies or CR2-negative cells transfected with CR2 cDNA. We demonstrate here that pp105 phosphorylation was induced when CR2 was activated by C3d and pep16 as well as by gp350, the Epstein-Barr virus capsid protein or OKB7, an anti-CR2 monoclonal antibody (mAb). HB5, another anti-CR2 mAb, which did not activate B lymphocytes through CR2, did not induce pp105 phosphorylation. Thus, C3d, pep16, gp350, and OKB7 presented similar properties in activating CR2 to trigger pp105 phosphorylation and in regulating B lymphocyte proliferation, while HB-5 had no effect on either assays. Furthermore, our data demonstrate that the presence of CR2 activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways: one which also requires the presence of non-activated CD19, and one which is independent of CD19. The involvement of CD19 in the first pathway was not due to the formation of putative CR2-CD19 complexes. Both pathways were TAPA-1 independent. This is the first demonstration that activated CR2 molecules can play a regulatory role in enzymatic function, such as phosphorylation, despite the absence of CD19 and TAPA-1.