Search

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

LNCaP cells are human prostatic cancer cells that have a frame-shift mutation of the tumor suppressor gene PTEN and do not express the insulin receptor substrate1 (IRS-1), a major substrate of the type 1 insulin-like growth factor receptor (IGF-IR). Ectopic expression of IRS-1 in LNCaP cells increases cell adhesion and decreases cell motility by an IGF-I-independent mechanism. We show now that these eAects of IRS-1 are accompanied by serine phosphorylation of IRS-1 and are inhibited by inhibitors of phosphatidylinositol 3-kinase (PI3K). We have confirmed the requirement for PI3K activity and serine phosphorylation by the use of IRS-1 mutants, expressed in LNCaP cells. Serine phosphorylation inhibits IGF-I-induced tyrosyl phosphorylation of IRS-1, which is restored by the expression of wild-type PTEN or by inhibition of PI3K activity. Finally, IRS-1 in LNCaP cells co-immunoprecipitates with integrin a 5 b 1, and the association is again IGF-I-independent. We conclude that in LNCaP cells, IRS-1 is serine phosphorylated by PI3K, generating eAects that are diAerent, and even opposite, from those generated by IGF-I. Oncogene (2001) 20, 490‐500.

LNCaP prostatic cancer cells are characterized by having a PTEN mutation, low levels of type 1 insulin-like growth factor receptor (IGF-IR) and no IRS-1, one of the major substrates of the IGF-IR. The absence of IRS-1, an activator of PI3-kinase, is compensated in these cells by the mutation in PTEN, an inhibitor of PI3-kinase. However, IGF-IR signaling in the absence of IRS-1 can cause cell differentiation and growth arrest. We hypothesized that these three characteristics may not be unrelated, specifically that, together, they may favor the metastatic spread of prostatic cancer cells without decreasing their growth potential. In support of this hypothesis, we report here that: (1) IRS-1 expression increases cell adhesion and decreases cell motility; (2) over-expression of the IGF-IR, in the absence of IRS-1, causes growth arrest and (3) a combination of IGF-IR and IRS-1 restores the transformed phenotype of LNCaP cells. These findings suggest a mechanism by which prostatic cancer cells can achieve metastatic potential without interfering with their growth potential. Oncogene (2000).

Insulin-like growth factor I receptor (IGF-IR) has been implicated in the normal and malignant growth of many cell types including cells from the central nervous system. In the cerebellar cortex IGF-IR mRNA is found in granular cells and IGF-I stimulation is mitogenic and protects cells from low-potassium-induced apoptosis. Since primitive neuroectodermal tumors/medulloblastomas (PNETs/medulloblastomas) are suspected to originate from the external cerebellar granular layer, it is reasonable to postulate that IGF-IR and/or its signaling molecules may contribute to the transformation of these poorly differentiated cells. To study activation of the IGF-IR system in medulloblastomas, we have utilized an antibody (anti-pY1316) that specifically recognizes the phosphorylated (active) form of the IGF-IR. Medulloblastoma biopsy specimens were positive when examined immunohistochemically with anti-Y1316 antibody. Further analysis of the IGF-IR system was performed in three human (Daoy, TE-671, D283 Med) and four mouse (BsB8, BsB13, Bs-1b, Bs-1c) medulloblastoma cell lines. All the murine cell lines examined express IGF-IR and PI3-kinase at relatively normal levels, and grossly overexpress IRS-1, when compared with normal mouse cerebellum. Within 15 min following IGF-I stimulation both mouse and human cell lines phosphorylate the beta subunit of the IGF-IR, IRS-1, Akt, and MAP kinases. They respond with cell proliferation when stimulated solely with IGF-I and are strongly inhibited when challenged with a dominant negative mutant of the IGF-IR (486/STOP), or with antisense oligonucleotides against the IGF-IR mRNA.