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ing apoptosis but are as active as wild-type p53 in inducing G1 arrest (Friedlander et al., 1996;Ludwig et al., 1996;Smith et al., 1999). They are also less able to

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.

Intact p73 function is shown to be an important determinant of cellular sensitivity to anticancer agents. Inhibition of p73 function by dominant-negative proteins or by mutant p53 abrogates apoptosis and cytotoxicity induced by these agents. A polymorphism encoding either arginine (72R) or proline (72P) at codon 72 of p53 influences inhibition of p73 by a range of p53 mutants identified in squamous cancers. Clinical response following cisplatin-based chemo-radiotherapy for advanced head and neck cancer is influenced by this polymorphism, cancers expressing 72R mutants having lower response rates than those expressing 72P mutants. Polymorphism in p53 may influence individual responsiveness to cancer therapy.

A single-nucleotide polymorphism (SNP) in exon 4 results in expression of either arginine (72R) or proline (72P) at codon 72 of p53. We demonstrate that the in vitro response of cells exposed to anticancer agents is strongly influenced by this SNP in wild-type p53. In inducible systems and in cells expressing the endogenous protein, expression of 72P wild-type p53 results in a predominant G1 arrest, with only a minor apoptosis, at drug concentrations causing extensive apoptosis in cells expressing the 72R wild-type variant. The superior apoptosis-inducing activity of the 72R form correlates with more efficient induction of specific apoptosis-associated genes, and is maximal in the presence of serine 46 (S46). In vivo, the outcome of chemo-radiotherapy of squamous carcinomas is more favourable in cancers retaining a wild-type 72R allele, such cases having higher response rates and longer survival than those with wild-type 72P. Together, these results reveal that this SNP is an important determinant of response to anticancer agents in cells expressing wild-type p53. Analysis of complete p53 genotype (mutation and SNP) merits detailed investigation as a simple means for prediction of treatment response and survival in clinical oncology.

iASPP is one of the most evolutionarily conserved inhibitors of p53, whereas ASPP1 and ASPP2 are activators of p53. We show here that, in addition to the DNA-binding domain, the ASPP family members also bind to the proline-rich region of p53, which contains the most common p53 polymorphism at codon 72. Furthermore, the ASPP family members, particularly iASPP, bind to and regulate the activity of p53Pro72 more efficiently than that of p53Arg72. Hence, escape from negative regulation by iASPP is a newly identified mechanism by which p53Arg72 activates apoptosis more efficiently than p53Pro72.

We recently showed that ASPP1 and ASPP2 stimulate the apoptotic function of p53. We show here that ASPP1 and ASPP2 also induce apoptosis independently of p53. By binding to p63 and p73 in vitro and in vivo, ASPP1 and ASPP2 stimulate the transactivation function of p63 and p73 on the promoters of Bax, PIG3, and PUMA but not mdm2 or p21 WAF-1/CIP1 . The expression of ASPP1 and ASPP2 also enhances the apoptotic function of p63 and p73 by selectively inducing the expression of endogenous p53 target genes, such as PIG3 and PUMA, but not mdm2 or p21 WAF-1/CIP1 . Removal of endogenous p63 or p73 with RNA interference demonstrated that (16) the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated mainly by p63 and p73. Hence, ASPP1 and ASPP2 are the first two identified common activators of all p53 family members. All these results suggest that ASPP1 and ASPP2 could suppress tumor growth even in tumors expressing mutant p53.The p53 gene is mutated in around 35 to 40% of human tumors. Pathways that activate p53 are also disrupted in many other tumors. The p53 protein modulates cellular functions, such as gene transcription, DNA synthesis, DNA repair, cell cycle arrest, senescence, and apoptosis. Mutations of the gene may result in inhibited protein function, and it is this dysfunction that is linked to tumor progression and genetic instability. In response to a variety of cellular stresses, p53 is posttranslationally modified, and protein levels increase dramatically. Activation of the protein results in either arrest of the cell at G 1 or commitment to death through apoptosis. Research has demonstrated the role of p53 transcriptional transactivation in cell cycle arrest through the up-regulation of the p21 WAF-1/CIP1 cyclin-dependent kinase inhibitor (cdki). However, many reports have shown that p53 can induce apoptosis by both transcription-dependent and -independent mechanisms (19,20).p53 is a member of a family of three proteins: p53, p63, and p73. p63 and p73 have more than 60% amino acid identity within the DNA binding region of p53 (12,13,22). DNA binding specificity among p53 family members is very similar but not identical. As a result, a large number of p53 target genes are also transactivated by p63 and p73. Hence, p63 and p73 share some p53 functions, such as cell cycle arrest and apoptosis. However, there are many other structural and functional differences between p53, p63, and p73. For example, mutations in p63 and p73 are rare in human cancer. Studies of p53-, p63-, and p73-deficient mice established that the expression of p63 and p73 is more important for mouse development than the expression of p53 and that the loss of p73 or p63 does not predispose mice to cancer (21). Cellular regulators of p53, such as mdm2, do not have the same effects on p63 and p73.While the binding of mdm2 to p53 inhibits the transactivation function of p53 and targets it for degradation (11,14), it fails to target p63 and p73 for degradation (4,8). In contrast, the binding of mdm2 to p63 stimulates the transactivati...