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The binding of RB to MDM2 is shown to be essential for RB to overcome both the antiapoptotic function of MDM2 and the MDM2-dependent degradation of p53. The RB-MDM2 interaction does not prevent MDM2 from inhibiting p53-dependent transcription, but the RB-MDM2 complex still binds to p53. Since RB specifically rescues the apoptotic function but not the transcriptional activity of p53 from negative regulation by MDM2, transactivation by wild-type p53 is not required for the apoptotic function of p53. However, an RB-MDM2-p53 trimeric complex is active in p53-mediated transrepression. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of RB in regulating the apoptotic function of p53.

Despite the wealth of information on the regulation of wild-type p53 function by phosphorylation, nothing is known about the biological effect of phosphorylation on mutant p53. Here we show that p53H175 is phosphorylated like wild-type p53 in cells of the same background. Ser392 nonphosphorylatable p53 mutants p53H175A392 and p53W248A392 more potently transformed rat embryo fibroblasts in cooperation with the ras oncogene than p53H175S392 and p53W248S392. p53H175A392 also had an enhanced ability to confer cellular resistance to the cytotoxic effect of cisplatin and UV radiation. This correlated with p53H175A392 being a more potent dominant negative mutant than p53H175 in inhibiting the apoptotic functions of wild-type p53. Moreover, p53H175E392, which mimics the phosphorylated form of p53H175, was less able to confer cellular resistance to DNA-damaging agents. p53H175 and p53W248 are phosphorylated like wild-type p53 in cells of the same background. Ser392 nonphosphorylated p53 was present in human breast tumors expressing mutant p53 including p53H175. Together, these results demonstrated a novel function of Ser392 phosphorylation in regulating the oncogenic function of mutant p53.

The inactivation of the p53 and Rb pathways would account for the majority of human tumours. There are many levels of cross talk between p53 and Rb that have been identified. However, the identification of the mdm2-Rb interaction established a closer link between the two most well studied tumour suppressors, p53 and Rb. Recent studies of the novel trimeric complex Rb-mdm2-p53 provided us with a functional insight of how the two tumour suppressors can act together in regulating p53 induced apoptosis. Beginning with the properties of the Rb-mdm2-p53 trimeric complex, we shall review the propounding evidence suggesting that the apoptotic function of p53 is linked to its transrepression function. The uncoupling of the apoptotic function and transactivation function of p53 will also be discussed.

The p53 tumour suppressor is stabilised following exposure to genotoxic agents, such as γ-radiation. Cell responses to p53 stabilisation include induction of apoptosis and/or cell cycle arrest. Several studies have suggested that γ-radiation stabilises p53 by blocking ubiquitin mediated proteolysis. Here we have compared the biological activities of p53 stabilized following exposure to γ-radiation or treatment with the proteosome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) in MCF7 cells with wild type p53. Stabilisation of p53 by ALLN was reversible and was not blocked by caffeine. Although ALLN was a more effective p53 stabilising agent than γ-radiation, ALLN was not as effective at inducing cell cycle arrest/apoptosis as γ-radiation. Although p53 stabilised by ALLN and γ-radiation were both able to bind DNA and activate transcription, ALLN did not increase expression of BAX, which is involved in p53-induced apoptosis. Therefore, p53 stabilised by different agents is not always biologically active to the same extent and additional alterations triggered by γ-radiation may enable p53 to activate a subset of critical target genes, such as BAX, which are required for p53 responses.

We show here that the cell cycle-dependent DNA-binding and transcriptional activity of p53 correlates with E2F expression in human primary fibroblasts. E2F1 binds and stimulates DNA-binding, transactivation and apoptotic functions of p53 but not p63 and p73. E2F1 binds residues 347-370 of p53 and enhances nuclear retention of Ser315 phosphorylated p53. This regulation of p53 by E2F1 is cell cycle dependent, as the cellular distribution of Ser315 phosphorylated p53 is associated with the periodic expression of E2F and cyclin A throughout the cell cycle. This is the first demonstration that the activities of p53 are regulated during the cell cycle by E2F/p53 interactions and that phosphorylation of p53 at Ser315 is required for this regulation.

We have previously shown that ASPP1 and ASPP2 are specific activators of p53; one mechanism by which wild-type p53 is tolerated in human breast carcinomas is through loss of ASPP activity. We have further shown that 53BP2, which corresponds to a C-terminal fragment of ASPP2, acts as a dominant negative inhibitor of p53 (ref. 1). Hence, an inhibitory form of ASPP resembling 53BP2 could allow cells to bypass the tumor-suppressor functions of p53 and the ASPP proteins. Here, we characterize such a protein, iASPP (inhibitory member of the ASPP family), encoded by PPP1R13L in humans and ape-1 in Caenorhabditis elegans. iASPP is an evolutionarily conserved inhibitor of p53; inhibition of iASPP by RNA-mediated interference or antisense RNA in C. elegans or human cells, respectively, induces p53-dependent apoptosis. Moreover, iASPP is an oncoprotein that cooperates with Ras, E1A and E7, but not mutant p53, to transform cells in vitro. Increased expression of iASPP also confers resistance to ultraviolet radiation and to cisplatin-induced apoptosis. iASPP expression is upregulated in human breast carcinomas expressing wild-type p53 and normal levels of ASPP. Inhibition of iASPP could provide an important new strategy for treating tumors expressing wild-type p53.

We demonstrate here that the E2F1 induced by DNA damage can bind to and promote the apoptotic function of p53 via the cyclin A binding site of E2F1. This function of E2F1 does not require its DP-1 binding, DNA binding, or transcriptional activity and is independent of mdm2. All the cyclin A binding E2F family members can interact and cooperate with p53 to induce apoptosis. This suggests a novel role for E2F in regulating apoptosis in response to DNA damage. Cyclin A, but not cyclin E, prevents E2F1 from interacting and cooperating with p53 to induce apoptosis. However, in response to DNA damage, cyclin A levels decrease, with a concomitant increase in E2F1-p53 complex formation. These results suggest that the binding of E2F1 to p53 can specifically stimulate the apoptotic function of p53 in response to DNA damage.

We identified a family of proteins termed ASPP. ASPP1 is a protein homologous to 53BP2, the C-terminal half of ASPP2. ASPP proteins interact with p53 and specifically enhance p53-induced apoptosis but not cell cycle arrest. Inhibition of endogenous ASPP function suppresses the apoptotic function of endogenous p53 in response to apoptotic stimuli. ASPP enhance the DNA binding and transactivation function of p53 on the promoters of proapoptotic genes in vivo. Two tumor-derived p53 mutants with reduced apoptotic function were defective in cooperating with ASPP in apoptosis induction. The expression of ASPP is frequently downregulated in human breast carcinomas expressing wild-type p53 but not mutant p53. Therefore, ASPP regulate the tumor suppression function of p53 in vivo.

The ability of Mdm2 to inhibit the activities of a C-terminal truncated p53 mutant, p53-Delta30, which can bind Mdm2 but is resistant to Mdm2-mediated protein degradation was investigated. The inhibitory function of an Mdm2 mutant, Mdm2-Delta(222-437), which can bind p53 but is defective in targeting p53 for degradation was also studied. We have demonstrated that targeting p53 for degradation is the most effective way for Mdm2 to inhibit the apoptotic function of p53. However, we have also shown that Mdm2 can inhibit the transactivation function of p53 without targeting it for degradation, although Mdm2 releases the transrepression ability of p53 mainly by targeting it for degradation. The ability of Mdm2 to inhibit the apoptotic function of p53 was linked to its ability to inhibit the transrepression but not the transactivation function of p53. Furthermore, we have demonstrated that the transrepression function of p53 was specific to p53-induced apoptosis and was not simply a result of cell death.