The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. Mutations in p53 occur in at least 50% of human tumors. We hypothesized that reactivation of mutant p53 in such tumors should trigger massive apoptosis and eliminate the tumor cells. To test this, we screened a library of low-molecular-weight compounds in order to identify compounds that can restore wild-type function to mutant p53. We found one compound capable of inducing apoptosis in human tumor cells through restoration of the transcriptional transactivation function to mutant p53. This molecule, named PRIMA-1, restored sequence-specific DNA binding and the active conformation to mutant p53 proteins in vitro and in living cells. PRIMA-1 rescued both DNA contact and structural p53 mutants. In vivo studies in mice revealed an antitumor effect with no apparent toxicity. This molecule may serve as a lead compound for the development of anticancer drugs targeting mutant p53.

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.

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The tumor-suppressor p53 can induce various biological responses. Yet, it is not clear whether it is p53 in vivo promoter selectivity that triggers different transcription programs leading to different outcomes. Our analysis of genome-wide chromatin occupancy by p53 using chromatin immunoprecipitation (ChIP)-seq revealed ‘p53 default program', that is, the pattern of major p53-bound sites that is similar upon p53 activation by nutlin3a, reactivation of p53 and induction of tumor cell apoptosis (RITA) or 5-fluorouracil in breast cancer cells, despite different biological outcomes. Parallel analysis of gene expression allowed identification of 280 novel p53 target genes, including p53-repressed AURKA. We identified Sp1 as one of the p53 modulators, which confer specificity to p53-mediated transcriptional response upon RITA. Further, we found that STAT3 antagonizes p53-mediated repression of a subset of genes, including AURKA.

We demonstrate here that synthetic 22-mer peptide 46, corresponding to the carboxy-terminal amino acid residues 361-382 of p53, can activate specific DNA binding of wild-type p53 in vitro and can restore the transcriptional transactivating function of at least some mutant p53 proteins in living cells. Introduction of peptide 46 in Saos-2 cells carrying a Tet-regulatable His-273 mutant p53 construct caused growth inhibition and apoptosis in the presence of mutant p53 but not in its absence, confirming that the effect of the peptide is mediated by reactivation of mutant p53. Moreover, peptide 46 caused apoptosis in mutant as well as wild-type p53-carrying human tumor cell lines of different origin, whereas p53 null tumor cells were not affected. These findings raise possibilities for developing drugs that restore the tumor suppressor function of mutant p53 proteins, thus selectively eliminating tumor cells.

Mutant p53-carrying tumors are often more resistant to chemotherapeutical drugs. We demonstrate here that the mutant p53-reactivating compound PRIMA-1 MET acts synergistically with several chemotherapeutic drugs to inhibit tumor cell growth. Combined treatment with cisplatin and PRIMA-1 MET resulted in a synergistic induction of tumor cell apoptosis and inhibition of human tumor xenograft growth in vivo in SCID mice. The induction of mutant p53 levels by chemotherapeutic drugs is likely to increase the sensitivity of tumor cells to PRIMA-1 MET . Thus, the combination of PRIMA-1 MET with currently used chemotherapeutic drugs may represent a novel and more efficient therapeutic strategy for treatment of mutant p53-carrying tumors.

The p53 tumor suppressor protein inhibits the formation of tumors through induction of cell cycle arrest and/or apoptosis. In the present study we demonstrated that p53 is also a powerful inhibitor of human telomerase reverse transcriptase (hTERT), a key component for telomerase. Activation of either exogenous temperature-sensitive (ts) p53 in BL41 Burkitt lymphoma cells or endogenous wild type (wt) p53 at a physiological level in MCF-7 breast carcinoma cells triggered a rapid downregulation of hTERT mRNA expression, independently of the induction of the p53 target gene p21. Co-transfection of an hTERT promoter construct with wt p53 but not mutant p53 in HeLa cells inhibited the hTERT promoter activity. Furthermore, the activation of the hTERT promoter in Drosophila Schneider SL2 cells was completely dependent on the ectopic expression of Sp1 and was abrogated by wt p53. Finally, wt p53 inhibited Sp1 binding to the hTERT proximal promoter by forming a p53-Sp1 complex. Since activation of telomerase, widely observed in human tumor cell lines and primary tumors, is a critical step in tumorigenesis, wt p53-triggered inhibition of hTERT/telomerase expression may re¯ect yet another mechanism of p53-mediated tumor suppression. Our ®ndings provide new insights into both the biological function of p53 and the regulation of hTERT/telomerase expression. Oncogene (2000) 19, 5123 ± 5133.

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long peptide, encoded by the W repeat of the EBV genome, is sufficient for binding. Point mutations of RB and p53 that inhibit their complexing with other DNA viral oncoproteins do not affect their binding to EBNA-5. p53 competes with RB for EBNA-5 binding. Our data suggest that the mechanisms involved in EBV transformation may include impairment ofRB and p53 function.Simian virus 40 (SV40), adenoviruses, and human papilloma viruses (HPVs) encode transforming proteins that can form complexes with the two major tumor suppressor proteins, the retinoblastoma (RB) and p53 proteins (1)(2)(3)(4)(5)(6)(7)(8) Herpes viruses are the largest among the DNA tumor viruses. They encode a higher number of transformationassociated proteins than the medium-sized adenoviruses or the small papova viruses, and their transforming strategy must be fundamentally different. It therefore appeared important to investigate whether any of their products interact with RB and p53 as well. We have chosen Epstein-Barr virus (EBV) as the most highly transforming member of the herpes virus family to approach this question.EBV expresses six nuclear proteins in immortalized lymphoblastoid cell lines (LCLs), designated EBV nuclear antigens 1-6 (EBNA-1-6). In a previous study, we have examined the intranuclear distribution of EBNA-1, -2, -3, and -5 and their ability to colocalize with the RB protein, stained with the aRBlCl monoclonal antibody (9). We have found a striking colocalization between EBNA-5, alternatively called EBNA-LP, and RB in the LCL IB4 (10).EBNA-5 is required for B-cell transformation (11 The monoclonal anti-RB antibody aRBlCl was found to stain similar nuclear foci in the LCL IB4. Using computerassisted overlap analysis, we found complete overlapping in the RB and EBNA-5-positive foci (10). They differed from the equally distinct dots of the small nuclear ribonucleoproteinrich splicing islands. Neither EBNA-1, -2, nor -3 showed any colocalization with RB (10, 15).In the present study, we have investigated the ability of EBNA-5 to form complexes with the RB and p53 proteins, using bacterially produced glutathione S-transferase (GST) fusion proteins. We demonstrate that EBNA-5 can bind RB and p53.MATERIALS AND METHODS Cell Lines and Cell Culture. The LCL IARC139, the Burkitt lymphoma (BL) lines BL28, E95A-BL28, Namalwa, and Ramos, the promyelocytic leukemia cell line HL60, and the colon carcinoma cell line SW480 were used in this study. E95A-BL28, a subline of the originally EBV-negative BL28, carries a fragment of EBV DNA encoding EBNA-2 and EBNA-5 but no other members of the EBNA family (16). Cel...