Search

The p53 tumor suppressor gene is activated in response to DNA damage resulting in either growth arrest or apoptosis. We previously demonstrated the specific involvement of homeodomain interacting protein-kinase 2 (HIPK2), a nuclear serine/threonine kinase, in inducing p53-dependent apoptosis through selective p53 phosphorylation at serine 46 after severe genotoxic damage. Here we show that HIPK2 contributes to p53 regulation, independently from serine 46 phosphorylation upon nonapoptotic DNA damage such as that induced by cytostatic doses of cisplatin. We show that HIPK2 depletion by RNA interference inhibits p53 binding to the p21Waf1 promoter affecting its p53-induced transactivation thereby allowing cell proliferation. We found that nonapoptotic DNA damage induces p53 acetylation mediated by the HAT protein PCAF and this p53 post-translational modification is abolished by HIPK2 depletion. In this regard, we found that HIPK2 cooperates with PCAF to induce selectively p53 transcriptional activity toward the p21Waf1 promoter while depletion of either HIPK2 or PCAF abolished this function. These data show that HIPK2 regulates the p53 growth arrest function through its PCAF-mediated acetylation.Oncogene (2005) 24, 5431–5442. doi:10.1038/sj.onc.1208717; published online 9 May 2005 [ABSTRACT FROM AUTHOR]

32D cells expressing v-Ha-Ras fail to show a transformed phenotype. Since Ras requires an active IGF-1R for transformation of fibroblasts, we asked whether expression of IRS-1 or Shc (two of the major substrates of the IGF-1R) could co-operate with oncogenic Ras in transforming 32D cells. We find that IRS-1, but not Shc, in combination with v-Ha-Ras generates a fully transformed phenotype in 32D cells. 32D cells expressing both IRS-1 and v-Ha-Ras (32D/IRS1/Ras) survive and proliferate in the absence of IL-3, do not undergo granulocytic differentiation in the presence of G-CSF and form tumors in nu/nu and syngeneic mice. In contrast, 32D cells expressing singly IRS-1 or v-Ha-Ras exhibit only a block in differentiation capacity. Over-expression of Shc proteins, by itself, promotes differentiation of 32D cells. Concomitant expression of IRS-1 and v-Ha-Ras synergistically phosphorylates ERK-1 and ERK-2 whereas a MEK inhibitor rapidly induces death of 32D/IRS1/Ras transformed cells. Furthermore, transformed 32D/IRS1/Ras cells display high levels of PI3-K activation and undergo rapid apoptosis when exposed to PI3-K inhibitors. The data indicate that: (1) a fully transformed phenotype in 32D cells is generated when a block in differentiation (v-Ha-Ras) is coupled with another differentiation block (IRS-1); (2) PI3-K and MAPK activity are required for the survival of transformed cells; (3) the signals generated by IRS-1 and oncogenic Ras converge on ERK and PI3-K resulting in high levels of activation. Oncogene (2000) 19, 3245–3255 [ABSTRACT FROM AUTHOR]

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32°C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32°C, a significant reduction in the proliferation rate (approx. percnt;50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32°C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli. [ABSTRACT FROM AUTHOR]

Serum and glucocorticoid regulated kinase 1 (Sgk1) is a serine–threonine kinase that is activated by serum, steroids, insulin, vasopressin, and interleukin 2 at the transcriptional and post-translational levels. Sgk1 is also important in transduction of growth factors and steroid-dependent survival signals and may have a role in the development of resistance to cancer chemotherapy. In the present paper, we demonstrate that Sgk1 activates MDM2-dependent p53 ubiquitylation. The results were obtained in RKO cells and other cell lines by Sgk1-specific RNA silencing and were corroborated in an original mouse model as well as in transiently and in stably transfected HeLa cells expressing wild-type or dominant negative Sgk1 mutant. Sgk1 contributes to cell survival, cell-cycle progression, and epithelial de-differentiation. We also show that the effects of Sgk1 on the clonogenic potential of different cancer cells depend on the expression of wild-type p53. Since transcription of Sgk1 is activated by p53, we propose a finely tuned feedback model where Sgk1 down-regulates the expression of p53 by enhancing its mono- and polyubiquitylation. [ABSTRACT FROM AUTHOR]

The p53 oncosuppressor protein is subject to negative regulation by MDM2, which efficiently inhibits its activity through an autoregulatory loop. In response to stress, however, p53 undergoes post-translational modifications that allow the protein to escape MDM2 control, accumulate, and become active. Recent studies have shown that, following DNA damage, the HIPK2 serine/threonine kinase binds and phosphorylates p53, inducing p53 transcriptional activity and apoptotic function. Here, we investigated the role of HIPK2 in the activation of p53 in the presence of MDM2. We found that HIPK2 rescues p53 transcriptional activity overcoming MDM2 inhibition, and that restoration of this p53 function induces apoptosis. Recovery of p53-dependent apoptosis is achieved by preventing p53 nuclear export and ubiquitination mediated by MDM2 in vitro and in vivo following genotoxic stress. These results shed new light on the mechanisms by which the HIPK2/p53 pathway promotes apoptosis and suppression of tumorigenesis.Oncogene (2004) 23, 5185-5192. doi:10.1038/sj.onc.1207656 Published online 3 May 2004 [ABSTRACT FROM AUTHOR]

The p53 oncosuppressor is strictly maintained in an inactive form under normal conditions, while it is post-translationally activated by a variety of stresses, enacting different protective biological functions. Since one critical issue in cancer gene therapy is tumor specificity, we asked whether the tight p53 regulation applies also to exogenously transferred p53. In principle, this type of regulation could allow p53 gene transfer in both normal and tumor cells to produce detrimental effects only in the latter ones. Here, we report that primary bone marrow cells infected with a p53 recombinant retrovirus and transplanted into irradiated mice reconstitute the hematopoietic system, with no detectable alterations in any of its compartments. Furthermore, simultaneous infection of leukemia and bone marrow cells depleted the neoplastic contamination, allowing lifelong, disease-free survival of 65% of the transplanted animals. These results show that exogenous p53 is controlled as tightly as the endogenous one, and opens the way to p53 gene therapy, without requiring tumor targeting.Oncogene (2004) 23, 418-425. doi:10.1038/sj.onc.1207042 [ABSTRACT FROM AUTHOR]

The onco-suppressor gene TP53 has potential use in the gene therapy of many human cancers including leukemias. The latter indication derived from numerous experimental reports of p53-mediated suppressing effects on human and murine leukemia cells in vitro. However, few in vivo experiments have been performed, and those that have used a subcutaneous injection of p53-transduced leukemia cells. Thus, we developed an orthotopic leukemia model in adult, syngenic mice to evaluate the feasibility of TP53-mediated therapeutic approaches. We found that among other cells, v-src-transformed 32D myeloid progenitors induce leukemia when injected intravenously in syngenic mice. The resulting malignancy resembles the clinical manifestations of human acute myeloid leukemia because it is characterized by a massive invasion of bone marrow compartments, splenomegaly, generalized lymphadenopathy, and a macroscopic or microscopic infiltration of the kidneys, liver, and lungs. When these 32Dv-src cells were infected with a TP53-recombinant retrovirus before intravenous injection, we found a decreased mortality and, in those animals that develop leukemia, a drastic reduction of the generalized organ infiltration, suggesting that exogenous TP53 expression might be used for ex vivo bone marrow purging from leukemia cells. [ABSTRACT FROM AUTHOR]

Experiments previously performed on 32D and C2C12 cell lines indicated that wild type p53 (wtp53) protein has a role in granulocyte and myotube differentiation. Since these are immortal cells, we asked whether the inhibition of differentiation induced by the expression of dominant-negative p53 (dnp53) proteins was dependent on the immortalization-determined microenvironment. Thus, we evaluated the effects produced by interfering with the endogenous p53 gene in murine primary hemopoietic and muscle cells. Expression of dnp53 protein reduced the differentiation of bone marrow cells into granulocytes and macrophages. Moreover, p53 activation was measurable during the differentiation process of primary myoblasts, while interference with this activation led to a consistent slow down of terminal differentiation. Since the impairment of the differentiation was not accompanied by alterations in the cell cycle withdrawal and in the rate of apoptosis which are coupled with these types of differentiation, the data here reported support a specific role for p53 in the differentiation process. However, the difference in the intensity of inhibition between immortal and primary cells, i.e., complete versus slow down, respectively, suggests that the immortalization process might render the cells more sensitive to the loss of wtp53 activity for the differentiation process. [ABSTRACT FROM AUTHOR]

Expression of exogenous wt-p53 in different tumor cell lines can induce growth arrest, apoptosis, or differentiation. Several experimental works have highlighted the relevance of cellular context in the determination of p53-mediated final outcomes. We recently observed that these diverse wt-p53 effects can also be induced by overexpressing wt-p53 in a single cell type – the 32D myeloid progenitors – transformed with different activated oncogenes. Here we show that 32D cells transformed with two different oncogenes, v-src or c-fms [S301,F969], both belonging to the CSF-1 transduction pathway, respond to exogenous wt-p53 expression with the same final outcome – monocytic differentiation. This result is particularly significant since 32D cells do not spontaneously express the CSF-1 receptor, whereas they undergo granulocytic differentiation upon G-CSF stimulation. These data strongly support the idea that wt-p53 suppressing effects result from interactions between p53 activity and the signaling pathways activated in different transformed cells. [ABSTRACT FROM AUTHOR]