The role of the insulin receptor substrate-1 in the differentiation of rat hippocampal neuronal cells.

H19-7/IGF-IR cells are rat hippocampal cells expressing a human IGF-I receptor, which differentiate to a neuronal phenotype when stimulated by IGF-I at 39 degrees C. H19-7/IGF-IR cells have low levels of expression of insulin receptor substrate-l (IRS-1), a major substrate of the IGF-IR. IGF-I induces serine-phosphorylation and down-regulation of the endogenous IRS-1 upon differentiation of H19-7/IGF-IR cells. The profound influence of IRS-1 on differentiation of H19-7/IGF-IR cells was confirmed by transfecting these cells with a plasmid expressing mouse IRS-1. Over-expression of wild type IRS-1 in H19-7/IGF-IR cells abolishes IGF-I-induced differentiation at 39 degrees C. A mutant of IRS-1 lacking the PTB domain loses the ability to inhibit the differentiation program. H19-7/IGF-IR/IRS-1 cells at 39 degrees C show a stronger and prolonged activation of Akt, when compared to H19-7/IGF-IR cells. The role of Akt in the inhibition of the differentiation program was confirmed by using the inhibitor of Class I PI3 kinases LY29400, which restores IGF-I-induced differentiation of H19-7/IGF-IR/IRS-1 cells. H19-7/IGF-IR/IRS-1 cells show a strong reduction in MAP kinases signaling, which is related to the superactivation of Akt. This was confirmed by expressing in H19-7/IGF-IR cells a constitutively active Akt, which inhibited MAP kinases activation in these cells. These experiments confirm the importance of MAPK in the mechanism of IGF-I-mediated differentiation of H19-7/IGF-IR cells

Cooperative transformation of 32D cells by the combined expression of IRS-1 and V-Ha-Ras.

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.

Insulin-like growth factor I receptor signaling in differentiation of neuronal H19-7 cells.

The type I insulin-like growth factor receptor (IGF-IR) is known to send two seemingly contradictory signals inducing either cell proliferation or cell differentiation, depending on cell type and/or conditions. H19-7 cells are rat hippocampal neuronal cells immortalized by a temperature-sensitive SV40 large T antigen that grow at 34 degrees C in epidermal growth factor or serum but differentiate at 39 degrees C when induced by basic fibroblast growth factor. At 39 degrees C, expression of the human IGF-IR in H19-7 cells induces an insulin-like growth factor (IGF) I-dependent differentiation. We have investigated the domains of the IGF-IR required for differentiation of H19-7 cells. The tyrosine 950 residue and serines 1280-1283 in the COOH terminus of the receptor are required for IGF-I-induced differentiation at 39 degrees C, although they are dispensable for IGF-I-mediated growth at 34 degrees C. Both domains have to be mutated to inactivate the differentiating function. The inability of these mutant receptors to induce differentiation correlates with mitogen-activated protein kinase activation. In contrast, inhibitors of phosphatidylinositol 3'-kinase have no effect on IGF-I-mediated differentiation of H19-7 cells, although they do inhibit the mitogenic response.

Domains of the insulin-like growth factor I receptor required for the activation of extracellular signal-regulated kinases.

The type 1 insulin-like growth factor receptor (IGF-IR) activates the extracellular signal-regulated kinases (ERK1 and -2). The two major substrates of the IGF-IR, insulin receptor substrate-1 (IRS-1) and the Shc proteins, are known to contribute to this activation. We investigated the domains of the IGF-IR required for the activation of the ERK proteins. To facilitate this study, we used a cell line (32D cells) that lacks IRS-1. In the absence of IRS-1, ERK activation is inhibited if the IGF-IR is mutated at two domains: tyrosine Y950 and a serine quartet at 1280-1283. Expression of IRS-1 in 32D cells expressing the double mutant IGF-IR restores ERK activation. The importance of the C-terminus of the IGF-IR in ERK activation (in the absence of IRS-1) is confirmed by the failure of the insulin receptor to give a sustained activation of ERK. In this model system, there is a good, but not exact, correlation between ERK activation and cell survival after withdrawal of growth factors.

Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis.

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.

Co-operation of simian virus 40 T antigen and insulin receptor substrate-1 in protection from apoptosis induced by interleukin-3 withdrawal.

32D cells are interleukin-3 (IL-3) dependent murine hemopoietic cells, that undergo apoptosis after IL-3 withdrawal. An overexpressed insulin-like growth factor I receptor (IGF-IR) protects these cells from apoptosis induced by IL-3 withdrawal. When 32D cells are stably transfected with plasmids expressing either IRS-1 (a major substrate of the IGF-IR) or the Simian virus 40 large T antigen, singly, they still undergo apoptosis after IL-3 withdrawal, although IRS-1 offers partial protection. The cells, however, are fully protected when they are stably transfected with both IRS-1 and SV40 T antigen. Protection from apoptosis in these cells is characterized by the stabilization of the Stat1 and Stat5 protein levels, whose synthesis is inhibited when IL-3 is withdrawn.

IGF-I receptor protection from apoptosis in cells lacking the IRS proteins.

The type I insulin-like growth factor receptor (IGF-IR) plays a crucial role in cell growth, transformation and protection from apoptosis. Although the mitogenic function of the IGF-IR may require the activation of insulin receptor substrate-1 (IRS-1) or IRS-2, an overexpressed IGF-IR is able to protect 32D cells, which lack IRS-1 and IRS-2, from apoptosis caused by Interleukin-3 (IL-3) withdrawal. Here, using mutational analysis, the authors identify domains of the IGF-IR necessary to protect from apoptosis without downstream signaling from IRS-1 and IRS-2. A receptor mutant of the tyrosine kinase (TK) domain only partially inhibited antiapoptotic signaling, whereas a mutant displaying constitutive autophosphorylation of the receptor did not show enhanced survival activity. Surprisingly, survival signaling was dependent upon tyrosine 950, the binding site for IRS-1, IRS-2, and Shc proteins. Yet, overexpressed Shc and/or IRS-1 could not replace the IGF-IR survival signal, suggesting the existence of other critical substrates. Finally, the C-terminus may encode a proapoptotic signal, as receptors truncated at C-terminal residues 1229 or 1245 were found to inhibit apoptosis better than the wild type (WT) IGF-IR.

Induction of chromosomal aberrations, cytotoxicity, and morphological transformation in mammalian cells by the antiparasitic drug flubendazole and the antineoplastic drug harringtonine.

The antiparasitic drug flubendazole and the antineoplastic compound harringtonine were studied for ability to induce chromosomal damage in Chinese hamster lung (CHL) cells and cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts. Flubendazole caused a dose- and time-dependent induction of polyploidy in CHL cells. In cells treated with 0.78 micrograms/ml flubendazole, the yield of polyploid cells was 95%. Harringtonine caused a dose- and time-dependent induction of chromosome breaks, and 0.195 micrograms/ml harringtonine induced chromosome breaks in 47% of CHL cells. Both flubendazole and harringtonine caused dose-dependent cytotoxicity to 10T1/2 cells at concentration ranges of 0.04-1.60 and 0.05-0.8 micrograms/ml, respectively. Flubendazole and harringtonine at concentrations of 0.08-0.4 and 0.4-0.8 micrograms/ml, respectively, induced morphological transformation (predominantly type II foci) in 10T1/2 cells. Three of four harringtonine-transformed cell lines and two of four flubendazole-transformed cell lines formed foci in reconstruction experiments with non-transformed 10T1/2 cells. All four harringtonine-transformed and all four flubendazole-transformed cell lines formed colonies in soft agar. Similar concentrations of flubendazole and harringtonine induced chromosome damage in CHL cells and cytotoxicity and morphological transformation in 10T1/2 cells. The ability of flubendazole to induce polyploidy may be part of the mechanism by which this compound induces morphological transformation. Similarly, the ability of harringtonine to induce chromosomal aberrations may be part of the mechanism by which this compound induces morphological transformation. Therefore, flubendazole and harringtonine induce cytotoxicity and morphological and anchorage-independent transformation, harringtonine induces chromosome aberrations (breakage, translocation, and rings), and flubendazole induces polyploidy in cultured mammalian cells. The clastogenic and cell transformation-inducing properties of these compounds suggest that these drugs may have carcinogenic potential. This should be investigated rigorously in animal carcinogenesis bioassays. The genotoxicity of these drugs should be considered during their development as antiparasitic and antineoplastic agents.