Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed human cells

The adenovirus early region 1B (Ad E1B) genes have no transforming capabili ty of their own but markedly increase the transformation frequency of Ad EI A following co-transfection into mammalian cells, The larger E1B proteins o f both Ad2/5 and Ad12 bind to p53 and inhibit its ability to transcriptiona lly activate other genes. We have previously demonstrated that synthetic pe ptides identical to the binding sites for p53 on both the Ad2 and Ad12 E1B proteins will disrupt the interaction in vivo and in vitro . in the work pr esented here we have examined the effects of complex dissociation on Ad El- transformed human cells, If has been shown, using confocal microscopy, that when the peptide identical to the p53 binding site was added to Ad5 E1-tra nsformed cells it initally located in the cytoplasmic dense bodies where it caused disruption of the p53/E1B complex. Peptide and p53 then translocate d to the nucleus, In Ad12 El-transformed cells the peptide localized in the nucleus directly and there caused a reorganization of p53 staining from a highly organized, 'flecke' distribution to one in vc which nuclear staining was homogeneous and diffuse. Peptides added to either Ad5 El or Ad12 El tr ansformed cells resulted in the release of transcriptionally active p53, in terestingly, the level of p53 then fell presumably as a result of proteasom al action - this was probably a reflection of the short half-life of 'free' (i.e. dissociated) p53 compared to that of the bound protein, Free p53 did not cause apoptosis in target cells probably due to the presence of the sm aller (19K) E1B proteins. However, addition of peptide leads to a significa nt reduction in cell growth rate. We have further demonstrated that a signi ficant proportion of those cells which had taken up peptide had ceased DNA synthesis, probably due to a p53-induced cell cycle arrest. The role of the larger E1B protein during transformation is considered in view of these da ta.