Chemically Modified Variants of Fenofibrate with Antiglioblastoma Potential.

Anticancer effects of a common lipid-lowering drug, fenofibrate, have been described in the literature for a quite some time; however, fenofibrate has not been used as a direct anticancer therapy. We have previously reported that fenofibrate in its unprocessed form (ester) accumulates in the mitochondria, inhibits mitochondrial respiration, and triggers a severe energy deficit and extensive glioblastoma cell death. However, fenofibrate does not cross the blood brain barrier and is quickly processed by blood and tissue esterases to form the PPARα agonist fenofibric acid, which is practically ineffective effective in triggering cancer cell death. To address these issues, we have made several chemical modifications in fenofibrate structure to increase its stability, water solubility, tissue penetration, and ultimately anticancer potential. Our data show that, in comparison to fenofibrate, four new compounds designated here as PP1, PP2, PP3, and PP4 have improved anticancer activity in vitro. Like fenofibrate, the compounds block mitochondrial respiration and trigger massive glioblastoma cell death in vitro. In addition, one of the lead compounds, PP1, has improved water solubility and is significantly more stable when exposed to human blood in comparison to fenofibrate. Importantly, mice bearing large intracranial glioblastoma tumors demonstrated extensive areas of tumor cell death within the tumor mass following oral administration of PP1, and the treated mice did not show any major signs of distress, and accumulated PP1 at therapeutically relevant concentrations in several tissues, including brain and intracranial tumors.

Aging phenotype(s) in kidneys of diabetic mice are p66ShcA dependent.

The p66ShcA protein controls cellular responses to oxidative stress, senescence, and apoptosis. Here, we test the hypothesis that aging phenotype(s) commonly associated with the broad category of chronic kidney disease are accelerated in diabetic kidneys and linked to the p66ShcA locus. At the organ level, tissue stem cells antagonize senescent phenotypes by replacing old dysfunctional cells. Using established methods, we isolated a highly purified population of stem cell antigen-1-positive mesenchymal stem cells (Sca-1+ MSCs) from kidneys of wild-type (WT) and p66 knockout (p66 KO) mice. Cells were plated in culture medium containing normal glucose (NG) or high glucose (HG). Reactive oxygen species (ROS) metabolism was substantially increased in WT MSCs in HG medium in association with increased cell death by apoptosis and acquisition of the senescent phenotype. DNA microarray analysis detected striking differences in the expression profiles of WT and p66 KO-MSCs in HG medium. Unexpectedly, the analysis for p66 KO-MSCs revealed upregulation of Wnt genes implicated in self-renewal and differentiation. To test the in vivo consequences of constitutive p66 expression in diabetic kidneys, we crossed the Akita diabetic mouse with the p66KO mouse. Homozygous mutation at the p66 locus delays or prevents aging phenotype(s) in the kidney that may be precursors to diabetic nephropathy.

Saliva induces expression of antimicrobial peptides and promotes intracellular killing of bacteria in keratinocytes by epidermal growth factor receptor transactivation.

BACKGROUND: Wounds in the oral cavity, constantly exposed to both saliva and bacteria, heal quickly without infection. Furthermore, during licking of skin wounds, saliva promotes wound healing and plays a role in keeping the wound free of infection. OBJECTIVES: To investigate whether saliva induces expression of antimicrobial peptides (AMPs) in human epidermal keratinocytes and whether saliva promotes clearance of intracellular bacteria in these cells. METHODS: Expression of AMPs was investigated in the oral mucosa and ex vivo injured skin by immunohistochemistry. Human beta-defensin-3 expression was investigated in epidermal keratinocytes after saliva stimulation, using real-time polymerase chain reaction and immunofluorescence. RESULTS: We found higher expression of AMPs in the oral mucosa than in the epidermis. Saliva accelerated the injury-induced expression of AMPs in human skin ex vivo and was a potent inducer of the expression of AMPs in epidermal keratinocytes. The expression of AMPs was induced by metalloproteinase-dependent epidermal growth factor receptor (EGFR) transactivation mediated by a salivary lipid. Saliva increased the intracellular clearance of Staphylococcus aureus in keratinocytes through EGFR activation. CONCLUSIONS: These findings suggest a previously unreported role of saliva in innate immunity and demonstrate for the first time that saliva induces gene expression in epidermal keratinocytes.

Fenofibrate subcellular distribution as a rationale for the intracranial delivery through biodegradable carrier.

Fenofibrate, a well-known normolipidemic drug, has been shown to exert strong anticancer effects against tumors of neuroectodermal origin including glioblastoma. Although some pharmacokinetic studies were performed in the past, data are still needed about the detailed subcellular and tissue distribution of fenofibrate (FF) and its active metabolite, fenofibric acid (FA), especially in respect to the treatment of intracranial tumors. We used high performance liquid chromatography (HPLC) to elucidate the intracellular, tissue and body fluid distribution of FF and FA after oral administration of the drug to mice bearing intracranial glioblastoma. Following the treatment, FF was quickly cleaved to FA by blood esterases and FA was detected in the blood, urine, liver, kidney, spleen and lungs. We have also detected small amounts of FA in the brains of two out of six mice, but not in the brain tumor tissue. The lack of FF and FA in the intracranial tumors prompted us to develop a new method for intracranial delivery of FF. We have prepared and tested in vitro biodegradable poly-lactic-co-glycolic acid (PLGA) polymer wafers containing FF, which could ultimately be inserted into the brain cavity following resection of the brain tumor. HPLC-based analysis demonstrated a slow and constant diffusion of FF from the wafer, and the released FF abolished clonogenic growth of glioblastoma cells. On the intracellular level, FF and FA were both present in the cytosolic fraction. Surprisingly, we also detected FF, but not FA in the cell membrane fraction. Electron paramagnetic resonance spectroscopy applied to spin-labeled phospholipid model-membranes revealed broadening of lipid phase transitions and decrease of membrane polarity induced by fenofibrate. Our results indicate that the membrane-bound FF could contribute to its exceptional anticancer potential in comparison to other lipid-lowering drugs, and advocate for intracranial delivery of FF in the combined pharmacotherapy against glioblastoma.

Anti-leukemic mechanisms of pegylated arginase I in acute lymphoblastic T-cell leukemia.

New treatments for adults with acute lymphoblastic T-cell leukemia (T-ALL) are urgently needed, as the current rate of overall remission in these patients is only about 40 percent. We recently showed the potential therapeutic benefit of the pegylated-human-arginase I (peg-Arg I) in T-ALL. However, the mechanisms by which peg-Arg I induces an anti-T-ALL effect remained unknown. Our results show the induction of T-ALL cell apoptosis by peg-Arg I, which associated with a global arrest in protein synthesis and with the phosphorylation of the eukaryotic-translation-initiation factor 2 alpha (eIF2α). Inhibition of eIF2α phosphorylation in T-ALL cells prevented the apoptosis induced by peg-Arg I, whereas the expression of a phosphomimetic eIF2α form increased the sensibility of T-ALL cells to peg-Arg I. Phosphorylation of eIF2α by peg-Arg I was mediated through kinases PERK and GCN2 and down-regulation of phosphatase GADD34. GCN2 and decreased GADD34 promoted T-ALL cell apoptosis after treatment with peg-Arg I, whereas PERK had an unexpected anti-apoptotic role. Additional results showed that phospho-eIF2α signaling further increased the anti-leukemic effects induced by peg-Arg I in T-ALL-bearing mice. These results suggest the central role of phospho-eIF2α in the anti-T-ALL effects induced by peg-Arg I and support its study as a therapeutic target.

Gliomas and seizures.

Glial neoplasms account for nearly 50% of all adult primary brain tumors. They originate from glial cells in the brain and/or spinal cord and include low-grade diffuse astrocytomas, anaplastic-astrocytomas, and glioblastomas. Of all brain tumors, glioblastoma multiforme (GBM) is the most aggressive and is characterized by rapid glial cell growth, resistance to radio- and chemo- therapies, and relentless infiltration and spreading throughout the central nervous system (CNS). In glioblastomas, primary tumor growth and CNS invasion are associated with the activation of complex structural molecular and metabolic changes within the tumor tissue, which profoundly affect the surrounding neuronal networks and may in part explain induction of epilepsy. In fact, epileptic seizures are very common among patients with glial tumors, reaching nearly 50% in glioblastoma patients and almost 90% in low-grade astrocytomas. The overall hypothesis presented here discusses the possibility that the aberrant tumor cell metabolism may act directly on neuronal network, and this leads to seizure susceptibility. Further invasion and growth of the malignant glial cells exacerbate this initial pathologic state which promotes recurrent seizures (epileptogenesis).

IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.

The insulin-like growth factor-1 receptor (IGF-IR) and the human polyomavirus JCV protein, T-antigen cooperate in the transformation of neuronal precursors in the cerebellum, which may be a contributing factor in the development of brain tumors. Because it is not clear why T-antigen requires IGF-IR for transformation, we investigated this process in neural progenitors from IGF-IR knockout embryos (ko-IGF-IR) and from their wild-type nontransgenic littermates (wt-IGF-IR). In contrast to wt-IGF-IR, the brain and dorsal root ganglia of ko-IGF-IR embryos showed low levels of the antiapoptotic protein Survivin, accompanied by elevated numbers of apoptotic neurons and an earlier differentiation phenotype. In wt-IGF-IR neural progenitors in vitro, induction of T-antigen expression tripled the expression of Survivin and accelerated cell proliferation. In ko-IGF-IR progenitors induction of T-antigen failed to increase Survivin, resulting in massive apoptosis. Importantly, ectopic expression of Survivin protected ko-IGF-IR progenitor cells from apoptosis and siRNA inhibition of Survivin activated apoptosis in wt-IGF-IR progenitors expressing T-antigen. Our results indicate that reactivation of the antiapoptotic Survivin may be a critical step in JCV T-antigen-induced transformation, which in neural progenitors requires IGF-IR.

Molecular mimicry in inducing DNA damage between HIV-1 Vpr and the anticancer agent, cisplatin.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (vpr) gene is an evolutionarily conserved gene among the primate lentiviruses. Several functions are attributed to Vpr including the ability to cause cell death, cell cycle arrest, apoptosis and DNA damage. The Vpr domain responsible for DNA damage as well as the mechanism(s) through which Vpr induces this damage is unknown. Using site-directed mutagenesis, we identified the helical domain II within Vpr (aa 37-50) as the region responsible for causing DNA damage. Interestingly, Vpr Delta(37-50) failed to cause cell cycle arrest or apoptosis, to induce Ku70 or Ku80 and to suppress tumor growth, but maintained its capability to activate the HIV-1 LTR, to localize to the nucleus and to promote nonhomologous end-joining. In addition, our cytogenetic data indicated that helical domain II induced chromosomal aberrations, which mimicked those induced by cisplatin, an anticancer agent. This novel molecular mimicry function of Vpr might lead to its potential therapeutic use as a tumor suppressor.

Tracking TrkA's trafficking: NGF receptor trafficking controls NGF receptor signaling.

Growth factors such as the neurotrophins promote neuronal survival and shape neuronal morphology. Neurotrophin receptors are located on the surface of axons and dendrites and must convey their signal retrogradely to the nucleus to influence transcription of target genes. The distance between the site of receptor activation and the nucleus is tremendous. How is the retrograde transmission of survival signals being achieved? Recent work showed that signaling endosomes containing neurotrophin receptors and associated downstream kinases undergo retrograde vesicular transport along microtubules, propelled by the molecular motor dynein. The next objective in the "neurotrophin receptor trafficking meets signal transduction field" will be to elucidate the traffic control mechanisms governing the directed movement of signaling endosomes. Much is already known on the trafficking of the receptor for epidermal growth factor, EGFR. We will summarize the known traffic control mechanisms for EGFR and hypothesize whether EGFR-relevant traffic control mechanisms might also be relevant for neurotrophin receptor traffic control. Moreover, we speculate about potential implications of neurotrophin receptor traffic jams for neurodegenerative diseases.

The estrogen receptor alpha:insulin receptor substrate 1 complex in breast cancer: structure-function relationships.

BACKGROUND: Insulin receptor substrate 1 (IRS-1) is a signaling molecule that exerts a key role in mediating cross talk between estrogen receptor alpha (ERalpha) and insulin-like growth factor 1 (IGF-1) in breast cancer cells. Previously, we demonstrated that a fraction of IRS-1 binds ERalpha, translocates to the nucleus, and modulates ERalpha-dependent transcription at estrogen response elements (ERE). Here, we studied structure-function relationships of the ERalpha:IRS-1 complex under IGF-1 and/or estradiol (E2) stimulation. MATERIALS AND METHODS: ERalpha and IRS-1 deletion mutants were used to analyze structural and functional ERalpha/IRS-1 interactions. IRS-1 binding to ERE and IRS-1 role in ERalpha-dependent ERE transcription was examined by chromatin immunoprecipitation and gene reporter analysis, respectively. The requirement for IRS-1 in ERalpha function was tested with RNAi technology. RESULTS: Nuclear translocation of IRS-1 was induced by E2, IGF-1, and a combination of both stimuli. ERalpha/IRS-1 binding was direct and involved the activation function-1 (AF-1)/DNA binding domain (DBD) region of ERalpha and two discrete regions of IRS-1 (the N-terminal pleckstrin homology domain and a region within the C-terminus). IRS-1 knock down abrogated IGF-1-dependent transcriptional activity of unliganded ERalpha, but induced the activity of liganded ERalpha. CONCLUSIONS: ERalpha/IRS-1 interactions are direct and involve the ERalpha AF-1/DBD domain and IRS-1 domains mapping within N- and C-terminus. IRS-1 may act as a repressor of liganded ERalpha and coactivator of unliganded ERalpha.

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death.

The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death.

Inhibition of IGF-I receptor in anchorage-independence attenuates GSK-3beta constitutive phosphorylation and compromises growth and survival of medulloblastoma cell lines.

We have previously reported that insulin-like growth factor-I (IGF-I) supports growth and survival of mouse and human medulloblastoma cell lines, and that IGF-I receptor (IGF-IR) is constitutively phosphorylated in human medulloblastoma clinical samples. Here, we demonstrate that a specific inhibitor of insulin-like growth factor-I receptor (IGF-IR), NVP-AEW541, attenuated growth and survival of mouse (BsB8) and human (D384, Daoy) medulloblastoma cell lines. Cell cycle analysis demonstrated that G1 arrest and apoptosis contributed to the action of NVP-AEW54. Interestingly, very aggressive BsB8 cells, which derive from cerebellar tumors of transgenic mice expressing viral oncoprotein (large T-antigen from human polyomavirus JC) became much more sensitive to NVP-AEW541 when exposed to anchorage-independent culture conditions. This high sensitivity to NVP-AEW54 in suspension was accompanied by the loss of GSK-3beta constitutive phosphorylation and was independent from T-antigen-mediated cellular events (Supplementary Materials). BsB8 cells were partially rescued from NVP-AEW541 by GSK3beta inhibitor, lithium chloride and were sensitized by GSK3beta activator, sodium nitroprusside (SNP). Importantly, human medulloblastoma cells, D384, which demonstrated partial resistance to NVP-AEW541 in suspension cultures, become much more sensitive following SNP-mediated GSK3beta dephosphorylation (activation). Our results indicate that hypersensitivity of medulloblastoma cells in anchorage-independence is linked to GSK-3beta activity and suggest that pharmacological intervention against IGF-IR with simultaneous activation of GSK3beta could be highly effective against medulloblastomas, which have intrinsic ability of disseminating the CNS via cerebrospinal fluid.

Role of the interaction between large T antigen and Rb family members in the oncogenicity of JC virus.

Human polyomaviruses (JC virus, BK virus and simian virus 40) are causative agents of some human diseases and, interestingly, are involved in processes of cell transformation and oncogenesis. These viruses need the cell cycle machinery of the host cell to complete their replication; so they evolved mechanisms that can interfere with the growth control of infected cells and force them into DNA replication. The retinoblastoma family of proteins (pRb), which includes pRb/p105, p107 and pRb2/p130, acts as one of the most important regulators of the G1/S transition of the cell cycle. Rb proteins represent an important target for viral oncoproteins. Early viral T antigens can bind all members of the pRb family, promoting the activation of the E2F family of transcription factors, thus inducing the expression of genes required for the entry to the S phase. The interaction between early viral antigens and cell cycle regulators represents an important mechanism through which viruses deregulate cell cycle and lead to cell transformation. In this review, we will discuss the effects of the interaction between large T antigen and Rb proteins in JC virus-mediated oncogenesis.

p73 modulates HIV-1 Tat transcriptional and apoptotic activities in human astrocytes.

HIV-1 Tat is a potent transcriptional activator of the viral promoter with the ability to modulate a number of cellular regulatory circuits including apoptosis. Tat exerts its effects through interaction with viral as well as cellular proteins. Here, we studied the influence of p73, a protein that is implicated in apoptosis and cell cycle control, on Tat apoptotic function in the central nervous system. We recently demonstrated the ability of Tat to associate with p73, and that this association modulates Tat transcriptional activity (Amini et al., Mol Cell Biol 2005; 18: 8126-8138). We demonstrated that p73 interferes with Tat-mediated apoptosis by preventing the up-regulation of Bax and down-regulation of Bcl-2 proteins in astrocytes. Thus, the interplay between Tat and p73 may affect Tat contribution to apoptotic events in the brain, limiting its involvement in the neuropathology often observed in the brains of HIV-1 patients.

Polyomaviruses and cancer--interplay between viral proteins and signal transduction pathways.

Polyomaviruses are highly suspected to be involved in the development of cancer. A strong correlation has been established between the activity of an early viral genome and the development of a transformed phenotype. Polyomavirus transforming antigens (T-antigens) are the major suspects in the process of deregulating cellular equilibrium. Multiple interactions between T-antigens and cellular regulatory proteins have been detected at different regulatory levels including signal transduction, gene expression, cell cycle progression, and possible DNA repair. In this context, we are reviewing the most recent experimental evidence which, in combination with more than thirty years of studies of polyomaviruses, could help us understand whether and how viral infection contributes to the development of malignant transformation.

Regulation of Id2 gene expression by the type 1 IGF receptor and the insulin receptor substrate-1.

The Id family of helix-loop-helix proteins is known to be involved in the proliferation and differentiation of several types of cells. The type 1 IGF receptor (IGF-IR) induces either proliferation or differentiation in 32D cells, a murine hemopoietic cell line, depending on the availability of the appropriate substrates for the receptor. We have previously reported that the IGF-IR regulates the expression of the Id2 gene in 32D cells. We now show that the IGF-IR controls the increase in Id2 gene expression through at least three pathways. These three pathways originate from the tyrosine residue at 950, a domain in the C-terminus, and the activation of the insulin receptor substrate-1 (IRS-1) by the receptor. IRS-1 is the preponderant signal, and its effect on Id2 gene expression requires a functional phosphotyrosine binding domain. With wild-type IRS-1, Id2 gene expression is increased, even in those cells that express IGF-I receptors defective in Id2 signaling. Rapamycin, an inhibitor of p70(S6K), a downstream effector of IRS-1 signaling, partially inhibits (but does not completely abrogate) the increase in Id2 gene expression. A mutant IRS-1 with a deletion of the Pleckstrin domain is as effective as wild-type IRS-1 in up-regulating Id2 gene expression. In addition, it seems to increase the stability of p70(S6K). Our results indicate that the IGF-IR regulates Id2 gene expression through different pathways. At least in 32D cells, increased Id2 gene expression seems to correlate more with inhibition of differentiation than with proliferation.

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

Involvement of Wnt signaling pathway in murine medulloblastoma induced by human neurotropic JC virus.

By using the early genome of the human neurotropic polyomavirus, JCV, we have created transgenic animals that develop cerebellar primitive neuroectodermal tumors which model human medulloblastoma. Expression of T-antigen was found in some, but not all, tumor cells, and examination of the clonal cell lines derived from the tumor population showed enhanced tumorigenicity of cells expressing T-antigen in comparison to T-antigen negative cells. Considering the earlier notion on the potential involvement of beta-catenin with human medulloblastoma, we investigated various components of the Wnt signaling pathway including beta-catenin, its partner transcription factor, LEF-1, and their downstream target gene c-myc in these two cell populations. Immunohistochemical staining of the cells revealed enhanced nuclear appearance of beta-catenin in T-antigen positive cells. Results from Western blot showed higher levels of beta-catenin and LEF-1 in T-antigen positive cells in comparison to those in T-antigen negative cells. The enhanced level of LEF-1 expression correlated with the increase in DNA binding activity of this protein in nuclear extracts of T-antigen positive cells. Results from Northern and Western blot analyses revealed that the level of c-myc expression is augmented both at the RNA and protein levels in T-antigen positive cells. These observations corroborated results from transfection studies indicating the ability of JCV T-antigen to stimulate c-myc promoter activity. Further, co-transfection experiments revealed that the amount of c-myc and T-antigen protein in tumor cells may dictate the activity of JCV early promoter in these cells. These observations are interesting in light of recent discoveries on the association of JCV with human medulloblastoma and suggest that communication between JCV and the Wnt pathway may be an important event in the genesis of these tumors.

Activation of the IGF-IR system contributes to malignant growth of human and mouse medulloblastomas.

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.

Intracellular association of a mutant insulin-like growth factor receptor with endogenous receptors.

The type 1 insulin-like growth factor receptor (IGF-IR) is emerging as a powerful survival factor against a variety of apoptotic agents in many cell types. A mutant IGF-IR designated 486/STOP is known to induce apoptosis and inhibit the growth of human tumor cells in mice. We have investigated the mechanism of action of 486/STOP. To study it, we have developed a new retroviral vector in which we have combined a self-inactivating 5'-long terminal repeat with an inducible heat-shock promoter (heat shock protein 70) from Drosophila. Using this technique, we find that the polypeptide encoded by 486/STOP is partially retained within the cell and partially secreted. However, the secreted polypeptide is subsequently taken up by the cells. In both cases, a specific intracellular interaction of 486/STOP with the endogenous IGF-IRs can be demonstrated by coimmunoprecipitation.