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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.

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.

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.