Summaryp73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, ␣ , or a shorter ␤ mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, ␥ (splicing out exon 11) and ␦ (splicing out exons 11, 12, and 13). Both ␥ and ␦ p73 variants are expressed in human peripheral blood lymphocytes, primary keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73 ␥ nor p73 ␦ interact with p53, whereas p73 ␥ showed strong interactions with all p73 isoforms, and p73 ␦ binds efficiently p73 ␣ and p73 ␥ but only weakly p73 ␤ . At the functional level, p73 ␥ is significantly less efficient in activating transcription of the p21 Waf1/Cip1 promoter than p53 or p73 ␤ , whereas the effect of p73 ␦ is intermediate and comparable to that of p73 ␣ . The ability of the different p73 variants to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional activity on the p21 Waf1/Cip1 promoter: p73 ␤ is the most efficient in inhibiting colony formation, whereas p73 ␥ is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and growth suppression activities by affecting heterodimer formation.

In this study, we characterized the molecular events involved in the activation of the ubiquitous transcription factor NF-B by the viral transactivator pX. pX expression in HeLa cells determines a manyfold increase in NF-B-dependent transcription, which is associated with an increase in p50/p65 heterodimer DNA-binding activity. Since the IB-␣ inhibitory subunit proteolytic degradation, which follows its phosphorylation/modification, is a key event in NF-B activation by different stimuli (such as growth factors, phorbol esters, tumor necrosis factor, UV irradiation, and oxygen radicals), we investigated pX effects on IB-␣, as well as the possible involvement of known signalling pathways in pX-induced NF-B-dependent transcription. We observed that although pX had no direct effect on p50 or p65, it was able to restore the IB-␣-suppressed p50/p65 activity. More directly, the stable expression of pX in HeLa cells resulted in reduced levels of IB-␣ in the cytoplasm. Pretreatment of the cells with H7, calphostin C, tyrphostin 25, or N-acetylcysteine did not impair the effects of pX on NF-B, thus ruling out the involvement of protein kinase C, tyrosine kinases, and oxygen radicals. Finally, while most of the known NF-B-activating agents converge on Raf-1 protein kinase, when Raf-1 activity is blocked by overexpression of a dominant negative mutant, the effects of pX on NF-B are not impaired. Thus, we suggest that although pX is able to activate the Ras/Raf-1-signalling pathway, it triggers NF-B activation by an as yet unidentified Raf-1-independent pathway.

No abstract