The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73␣, ␤, ␥, and ␦. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

32D cells expressing v-Ha-Ras fail to show a transformed phenotype. Since Ras requires an active IGF-1R for transformation of ®broblasts, 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 ®nd 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 dierentiation 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-HaRas exhibit only a block in dierentiation capacity. Over-expression of Shc proteins, by itself, promotes dierentiation 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 dierentiation (v-HaRas) is coupled with another dierentiation 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.

Summary Recent studies support the potential application of the wt-p53 gene in cancer therapy. Expression of exogenous wt-p53 suppresses a variety of leukaemia phenotypes by acting on cell survival, proliferation and/or differentiation. As for tumour gene therapy, the final fate of the neoplastic cells is one of the most relevant points. We examined the effects of exogenous wt-p53 gene expression in several leukaemia cell lines to identify p53-responsive leukaemia. The temperature-sensitive p53vallw mutant or the human wt-p53 cDNA was transduced in leukaemia cell lines representative of different acute leukaemia FAB subtypes, including Ml (Ozturk et al, 1992;Gotz and Montenarh 1995). Therefore, the identification of leukaemia cells susceptible to wt-p53-induced apoptosis should be useful for therapeutic purposes. In spite of the evidence that cellular context determines the final outcome of the cells after wt-p53 forced expression (Canman et al, 1995;Soddu et al, 1996), it is still unclear how to classify the cellular environments as a function of the response to wt-p53 action. In this respect, we asked whether the stage of differentiation might be a good parameter to classify the final outcome of wt-p53-transduced leukaemia cells. To address this question, seven leukaemia cell lines were transfected with a temperature-sensitive p53 (tsp53)-encoding vector or infected with a wt-p53 recombinant retrovirus. Viral infection was used in addition to plasmid transfection to evaluate the feasibility of a gene therapy approach based on wt-p53 expression in leukaemia cells. Leukaemia heat-inactivated fetal bovine serum (FBS). The Psi-2 crip-ampho (Markowitz et al, 1988) amphotropic packaging cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS. The plasmids pN53cG , carrying the ts-pS3Va-13S mutant gene (Soddu et al, 1994), and pRSVneo, carrying the selectable marker for G418 resistance, were used. The pLp53SN vector was obtained by inserting the human wt-p53 cDNA (Baker et al, 1990) into the unique BamHI site of pLXSN vectors (Miller and Rosman, 1989). Transfections and infectionsAfter 10 min incubation on ice, HEL 92.1.7, U937 and K562 cells [5 x 106 in 0.3 ml of phosphate-buffered saline (PBS)] were electroporated (HEL 92.1.7: 0.25 kV, 960 [F; U937 and K562: 0.2 kV, 960 tF) with 10 gg of plasmid, incubated for an additional 10 min on ice and plated in complete medium. G418 was added after 48 h (HEL 92.1.7: 1 mg ml-'; U937 and K562: 750 gg ml-'). HL-60 cells transfected with pN53cG(Val-135) (HL-2) and with