We have recently characterized a 95 kDa protein, p95, which exhibits enhanced binding to temperature‐sensitive p53 (ts‐p53) when cells are shifted down to 32.5 degrees C, a temperature at which ts‐p53 possesses wild‐type (wt)‐like activities. In the present study we show that p95 is a product of the mdm2 putative proto‐oncogene. The enhanced complex formation of mdm2 with ts‐p53 in cells maintained at 32.5 degrees C is due to an elevation in total mdm2 protein levels following the temperature shift. We further demonstrate that the induction of mdm2 expression by t p53 activity is at the mRNA level. The induction occurs with very rapid kinetics and does not require de novo protein synthesis, suggesting a direct involvement of p53 in the process. Based on these data and on recent findings implicating p53 as a transcription factor, we suggest that the mdm2 gene is a target for activation by wt p53. In view of the ability of mdm2 to act as a specific antagonist of p53 activity, this induction process may serve to tightly autoregulate p53 activity in living cells.

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Direct interactions between the genes that regulate development and those which regulate the cell cycle would provide a mechanism by which numerous biological events could be better understood. We have identified a direct role for PAX5 in the control of p53 transcription. In primary human diffuse astrocytomas, PAX5 expression inversely correlated with p53 expression. The human p53 gene harbours a PAX binding site within its untranslated first exon that is conserved throughout evolution. PAX5 and its paralogues PAX2 and PAX8 are capable of inhibiting both the p53 promoter and transactivation of a p53‐responsive reporter in cell culture. Mutation of the identified binding site eliminates PAX protein binding in vitro and renders the promoter inactive in cells. These data suggest that PAX proteins might regulate p53 expression during development and propose a novel alternative mechanism for tumour initiation or progression, by which loss of p53 function occurs at the transcriptional level.

The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G 0 and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors.Members of the E2F family of transcription factors are thought to regulate cell cycle progression by activating the transcription of a set of genes necessary for the induction of S phase (30, 53). E2F DNA binding activities are dependent on growth factors (52), and their function as transcription factors is temporally regulated throughout the cell cycle by complex formation with the tumor suppressor protein pRb and the pRb-related proteins p107 and p130 (9,10,13,43,69). E2F is activated by adenovirus E1A binding to pRb and its related proteins, and release of E2F from pRb is critical for transformation induced by both E1A (18, 61) and pRb inactivation (29,31,57,58).The DNA binding activity originally termed free E2F (3) is now recognized to be a heterodimer containing the product of an E2F gene family member (E2F1 to E2F5) and a DP family member (5, 21-23, 27, 34, 38, 44, 72, 76). E2F can bind DNA in vitro, whereas DP proteins bind DNA only weakly (23). Dimerization of DP proteins with E2F proteins increases the transcriptional activity of E2F and is required for association of E2F with pRb or pRb-related proteins (4,5,21,28,41). Furthermore, enforced DP-1 expression augments E2F-mediated transformation of primary rat embryo fibroblast cells in cooperation with an activated ras oncogene (5,21,35).Microinjection of serum-starved fibroblasts with an E2F-1 expression plasmid (37) or glutathione S-transferase-E2F-1 fusion protein (17), or activation of E2F-1 expression in transfected cell lines (59,67), is sufficient to drive quiescent cells into S phase. Inappropriate entry of these cells into S phase, in the absence of survival factors, is associated with the activation

Overexpression of wild‐type p53 in p53‐deficient leukemic cells induces apoptosis, which can be inhibited by hematopoietic survival factors. This suggests that p53 may contribute to survival factor dependence. To assess the role of wild‐type p53 in mediating apoptosis following survival factor withdrawal, we interfered with endogenous p53 activity in interleukin‐3 (IL‐3)‐dependent cells. Extended survival without IL‐3 was conferred by recombinant retroviruses encoding either a full‐length p53 mutant or a C‐terminal p53 miniprotein, both of which can act as negative‐dominant inhibitors of wild‐type p53. On the other hand, excess wild‐type p53 activity failed to elicit apoptosis as long as IL‐3 was present. We propose that p53 is a positive, though not exclusive, mediator of survival factor dependence in hematopoietic cells.