Keeping an old friend under control: regulation of p53 stability

The tumor suppressor protein p53 plays a pivotal role in protection against the development of cancer and is inactivated in many human malignancies. p53 is thought to prevent accumulation of genomic alterations by hindering cell proliferation in response to genotoxic stress, and two of the principal functions of p53 are the induction of cell-cycle arrest and the activation of apoptotic cell death. Because p53 is an extremely efficient inhibitor of cell growth, keeping p53 function under control in normal cells is critical. One of the principal mechanisms by which cells achieve this is by regulating the p53 protein level, although the ability of the protein to adopt active and latent forms and its cellular localization also contribute to the regulation of its function. Here, we summarize recently identified mechanisms that regulate the stability of the p53 protein and discuss the potentially immense clinical relevance of these observations in developing therapeutical approaches that aim to restore p53 function in human tumors.     

The polyproline region of p53 is required to activate apoptosis but not growth arrest

p53 is a pivotal regulator of apoptosis but its mechanism of action is obscure. We report that the polyproline (PP) region located between p53's transactivation and DNA binding domains is necessary to induce apoptosis but not cell growth arrest. The PP region was dispensable for DNA binding, inhibition of SAOS-2 tumor cell growth, suppression of E1A + RAS cell transformation, and cell cycle inhibition. A temperature-sensitive dominant inhibitory p53 mutant lacking PP (p53ts deltaPP) retained its ability to cooperate with adenovirus E1A in transformation of primary BRK cells. However, while activation of wt p53 induced apoptosis in E1A + p53ts-transformed cells, activation of p53 deltaPP induced cell cycle arrest but not apoptosis in E1A + p53ts deltaPP-transformed cells. Similarly, PP deletion abolished apoptosis in LoVo colon carcinoma cells, which are killed by wt p53 overexpression. Transactivation was largely unaffected by PP deletion. Significantly, BAX induction was intact, indicating that additional events are required for p53 to induce apoptosis. As a recently described site for familial mutation in at least one breast cancer family, the PP region represents a domain that may be altered in human tumors. We concluded that p53's ability to induce apoptosis is dispensable for inhibiting cell growth and transformation and that the PP region plays a crucial role in apoptotic signaling.     

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.     

A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice

The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%-50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of the INK4a-ARF locus, which encodes two gene products (p16(INK4a) and p19(ARF)) involved in cell-cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis, using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice expressing tv-a, a gene encoding the retrovirus receptor. TVA, under control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infecting tv-a transgenic mice bearing a disrupted INK4a-ARF locus with the EGFR-carrying vector alone. Moreover, EGFR-induced gliomagenesis does not occur in conjunction with p53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.     

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Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.     

MDM2 protein overexpression promotes proliferation and survival of multiple myeloma cells

The murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 (wtp53) function. In this study, we examined MDM2 expression and function in multiple myeloma (MM) cells. MDM2 is strongly and constitutively expressed in MM cell lines (ARH-77, RPMI 8226, and OCI-My5) and in the cells of plasma cell leukemia (PCL) patients, but is not expressed in normal bone marrow mononuclear cells (BM MNCs). Treatment of MM cells with MDM2 antisense, but not sense, nonsense, or scrambled, oligodeoxyribonucleotides (ODNs) decreased DNA synthesis and cell viability; it also induced G1 growth arrest, as evidenced by propidium iodide (PI) staining and induction of retinoblastoma protein (pRB) to E2F-1 binding. Moreover, inhibition of MDM2 using antisense ODNs also triggered MM cell apoptosis as evidenced by acridine orange-ethidium bromide staining. We next studied the association of MDM2 with wtp53 and/or mutant p53 (mtp53), E2F-1, CDK4, and p21. MDM2 constitutively binds to E2F-1 in all MM cells, to both wtp53 and mtp53, and to p21 in tumor cells lacking p53. These data suggest that MDM2 may enhance cell-cycle progression in MM cells both by activating E2F-1 and by downregulating cell-cycle inhibitory proteins (wtp53 and p21). Overexpression of MDM2 may therefore contribute to both growth and survival of MM cells, suggesting the potential utility of treatment strategies targeting MDM2 in MM.