Tolerability of long-term temperature controlled whole-body thermal treatment in advanced cancer-bearing dogs.

Aim: In oncology, thermal therapy is the application of external heat to fight cancer cells. The goal of whole-body thermal treatment (WBTT) is to raise the patient's core temperature to 39-42 °C, and represents the only thermal treatment modality that can act on both the primary tumor and distant metastases. However, WBTT carries potential risks for toxicity when applied without accurate thermometry and monitoring.Methods: ElmediX has developed a medical device, HyperTherm, to deliver long-term controlled and accurate WBTT (41.5 °C, up to 8 h). The safety of the device and thermal treatment protocol was initially evaluated in minipigs, and we present the confirmation of tolerability of WBTT in dogs with advanced cancer, in combination with a reduced dose of radiotherapy or chemotherapy.Results: Thermometry in liver, rectum, and tumor confirmed a homogeneous heating of these body parts. Monitoring of clinical parameters showed acceptable and reversible changes in liver, cardiac, muscle and coagulation parameters, as was expected. Combination of WBTT with both radiotherapy and chemotherapy only caused some low-grade adverse events.Conclusion: We conclude that our findings support the safe use of HyperTherm-mediated WBTT for canine patients with advanced malignancies. They also tend to support a genuine therapeutic potential for long-term WBTT which needs to be confirmed on a larger dog patient population. Combined with previously reported safety results in minipigs, these contribute to support the ongoing clinical evaluation of WBTT in advanced human cancer patients.

An antisense oligonucleotide targeting TGF-ß2 inhibits lung metastasis and induces CD86 expression in tumor-associated macrophages.

BACKGROUND: The transforming growth factor (TGF)-ß pathway is a well-described inducer of immunosuppression and can act as an oncogenic factor in advanced tumors. Several preclinical and clinical studies show that the TGF-ß pathway can be considered a promising molecular target for cancer therapy. The human genome has three TGF-ß isoforms and not much is known about the oncogenic response to each of the isoforms. Here, we studied the antitumor response to ISTH0047, a recently developed locked nucleic acid-modified antisense oligonucleotide targeting TGF-ß2. MATERIALS AND METHODS: We have studied the anticancer response to ISTH0047 using gymnotic delivery in tumor cell cultures and in in vivo preclinical orthotopic mouse models for primary tumors (breast and kidney tumors) and lung metastasis. RESULTS: We observed that ISTH0047 is able to significantly reduce TGF-ß2 mRNA and protein levels without altering the levels of TGF-ß1 and TGF-ß3. ISTH0047 prevented lung metastasis in syngeneic orthotopic renal cell carcinoma (RENCA) and breast cancer (4T1) tumor models. In addition, using an orthotopic xenograft model of a lung cancer cell line (CRL5807) that mainly expresses TGF-ß2, we observed that ISTH0047 had an important effect on the lung microenvironment inhibiting the growth of lung lesions. ISTH0047 treatment re-educated macrophages in the lung parenchyma to express the tumor-suppressive factor, CD86. CONCLUSION: Overall, our data point to TGF-ß2 as a therapeutic target and ISTH0047 as a novel anticancer drug to prevent lung metastasis by impacting on the tumor niche, in part, through the induction of CD86 in tumor-associated macrophages.

Delivery of a DNAzyme targeting c-myc to HT29 colon carcinoma cells using a gold nanoparticulate approach.

The objective of the current study was to develop cellular delivery approaches for catalytic DNA enzymes (DNAzymes) which cleave targeted messenger RNA, using vectors based on colloidal gold. The model DNAzyme was a 32mer oligonucleotide designed to specifically interact with and cleave c-myc mRNA. Colloidal gold particles were prepared by reduction of tetrachlororauric [III] acid with sodium citrate. Particles could be produced in the 1-90 nm range. A cationic substrate linked to transferrin was electrostatically/hydrophobically bound to the gold particle. These vectors were then treated with the DNAzyme to yield the condensed DNA-cationic polymer-particulate product. The pH (4-11.5), the quantity of the DNAzymes (0.079-0.567 microg/probe), the cationic polymer (polylysine (PL) or polyethylenimine (PEI)) as well as the surfactant (PVP) concentration (0-0.5%) were varied to give stable constructs which decomplexed under the desired conditions (i.e., in lysosomes and at lower pH values). Cellular uptake of the FITC-labelled c-myc DNAzyme incorporated in this vector was measured using FACS analysis in human HT29 colon carcinoma cells. Data suggested that PEI gave better delivery efficiencies than PL. The use of PVP to stabilize the formed dispersions was detrimental to DNAzyme delivery when PL was used but had little effect in the PEI systems. In the best cases, delivery to 77% of the cells was possible using PEI with the PVP stabilizer and completing the DNA condensation at pH 5.5 with 0.118 microg of DNAzyme/probe. In contrast, the best conditions for PL gave only transfection to 43% of the cells (no PVP, condensed at pH 5.7 and with a loading of 0.079 microg DNAzyme/probe). The PL probe tended to be more toxic than the PEI-based systems (65% cell death in PL transfected cells compared to 22% for PEI). These results suggest that cellular targeting using colloidal gold appears feasible for DNAzyme delivery.

R306465 is a novel potent inhibitor of class I histone deacetylases with broad-spectrum antitumoral activity against solid and haematological malignancies.

R306465 is a novel hydroxamate-based histone deacetylase (HDAC) inhibitor with broad-spectrum antitumour activity against solid and haematological malignancies in preclinical models. R306465 was found to be a potent inhibitor of HDAC1 and -8 (class I) in vitro. It rapidly induced histone 3 (H3) acetylation and strongly upregulated expression of p21waf1,cip1, a downstream component of HDAC1 signalling, in A2780 ovarian carcinoma cells. R306465 showed class I HDAC isotype selectivity as evidenced by poor inhibition of HDAC6 (class IIb) confirmed by the absence of downregulation of Hsp90 chaperone c-raf protein expression and tubulin acetylation. This distinguished it from other HDAC inhibitors currently in clinical development that were either more potent towards HDAC6 (e.g. vorinostat) or had a broader HDAC inhibition spectrum (e.g. panobinostat). R306465 potently inhibited cell proliferation of all main solid tumour indications, including ovarian, lung, colon, breast and prostate cancer cell lines, with IC50 values ranging from 30 to 300 nM. Haematological cell lines, including acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphoblastic leukaemia, chronic myeloid leukaemia, lymphoma and myeloma, were potently inhibited at a similar concentration range. R306465 induced apoptosis and inhibited angiogenesis in cell-based assays and had potent oral in vivo antitumoral activity in xenograft models. Once-daily oral administration of R306465 at well-tolerated doses inhibited the growth of A2780 ovarian, H460 lung and HCT116 colon carcinomas in immunodeficient mice. The high activity of R306465 in cell-based assays and in vivo after oral administration makes R306465 a promising novel antitumoral agent with potential applicability in a broad spectrum of human malignancies.

Effect of an hdm-2 antagonist peptide inhibitor on cell cycle progression in p53-deficient H1299 human lung carcinoma cells.

The hdm-2 oncogene is overexpressed in several types of malignancies including osteosarcomas, soft tissue sarcomas and gliomas and hdm-2 has been associated with accelerated tumor formation in both hereditary and sporadic cancers. Among the other key binding partners, hdm-2 forms a complex with the tumor suppressor p53, resulting in a rapid proteasome-mediated degradation of the p53 protein. This positions the hdm-2-p53 complex as an attractive target for the development of anticancer therapy and recently the first small molecule hdm-2 antagonist has been reported. Development of hdm-2 antagonists is currently focused on malignancies containing a wild-type p53 genotype, which is the case in approximately half of human cancer indications. However, hdm-2 has also been implicated in oncogenesis in the absence of p53. We therefore studied the effect of hdm-2 antagonists in p53-deficient human H1299 lung carcinoma cells. The hdm-2 antagonistic peptide caused G1 cell cycle arrest, inhibited colony growth and induced expression of G1 checkpoint regulatory proteins, such as p21(waf1,cip1). These data demonstrate that hdm-2 regulates the G1 cell cycle checkpoint in a p53-independent manner, suggesting that hdm-2 antagonists represent a novel class of anticancer therapeutics with broad applicability towards tumors with different p53 genetic backgrounds.

Assay development for high throughput screening of p21(Waf1/Cip1) protein expression in intact cells using fluorometric microvolume assay technology.

The p21(Waf1/Cip1) protein represents a broad-acting cyclin-dependent kinase inhibitor that plays a key role in cell cycle regulation. Furthermore, p21(Waf1/Cip1) protein has been described as a direct participant in regulating genes involved in growth arrest, senescence and aging. In response to genotoxic insults (e.g., following chemotherapeutic treatment), p21(Waf1/Cip1) protein accumulates mainly through p53-mediated transcriptional activation and is also regulated at the post-transcriptional level. In tumor cells, p53 is frequently mutated leading to reduced p21(Waf1/Cip1) protein induction that may contribute to resistance to treatment by DNA-damaging agents. In order to identify compounds capable of restoring p21(Waf1/Cip1) protein level, we have developed a 96-multi-well plate-based high throughput screening assay in intact cells using the Applied Biosystems Fluorometric Microvolume Assay Technology (FMAT) macro-confocal system. Briefly, following incubation with test compounds, human MCF7 breast carcinoma cells were fixed and p21(Waf1/Cip1) protein was detected using anti-p21(Waf1/Cip1) monoclonal antibody and anti-mouse IgG conjugated to the red fluorescent dye Alexafluor 633. FMAT provides a set of raw images at a high magnification, in which fluorescence concentrated in a cell is detected as a specific signal. The mean fluorescence of a population of cells is calculated independently of the number of cells as with a classical FACS analysis. This is of particular interest for screening anticancer drugs that may affect cell number and therefore may impact on the readout. This assay was validated using reference compounds such as camptothecin and actinomycin D, known inducers of p21(Waf1/Cip1) protein.

Inhibition of all-TRANS-retinoic acid metabolism by R116010 induces antitumour activity.

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

p53-dependent G2 arrest associated with a decrease in cyclins A2 and B1 levels in a human carcinoma cell line.

In vivo transfer of wild-type (wt) p53 gene via a recombinant adenovirus has been proposed to induce apoptosis and increase radiosensitivity in several human carcinoma models. In the context of combining p53 gene transfer and irradiation, we investigated the consequences of adenoviral-mediated wtp53 gene transfer on the cell cycle and radiosensitivity of a human head and neck squamous cell carcinoma line (SCC97) with a p53 mutated phenotype. We showed that ectopic expression of wtp53 in SCC97 cells resulted in a prolonged G1 arrest, associated with an increased expression of the cyclin-dependent kinase inhibitor WAF1/p21 target gene. A transient arrest in G2 but not in G1 was observed after irradiation. This G2 arrest was permanent when exponentially growing cells were transduced by Ad5CMV-p53 (RPR/INGN201) immediately after irradiation with 5 or 10 Gy. Moreover, levels of cyclins A2 and B1, which are known to regulate the G2/M transition, dramatically decreased as cells arrived in G2, whereas maximal levels of expression were observed in the absence of wtp53. In conclusion, adenoviral mediated transfer of wtp53 in irradiated SCC97 cells, which are mutated for p53, appeared to increase WAF1/p21 expression and decrease levels of the mitotic cyclins A2 and B1. These observations suggest that the G2 arrest resulted from a p53-dependent premature inactivation of the mitosis promoting factor.

Radiosensitization of human tumor cell lines induced by the adenovirus-mediated expression of an anti-Ras single-chain antibody fragment.

The expression of activated ras genes has been implicated as a contributing factor to the radioresistance of tumor cells. As a strategy for compromising Ras protein activity and potentially enhancing the radiosensitivity of tumor cells, we have investigated the application of the AV1Y28 adenovirus, which expresses a single-chain antibody fragment directed against p21 Ras proteins. The ability of AV1Y28 transduction to modulate radioresponse was investigated using four human tumor cell lines--U251 glioblastoma, MIA PaCa-2 pancreatic carcinoma, and the colon carcinomas SW620 and HT29. Cultures were exposed to sufficient levels of AV1Y28 to transduce more than 90% of the cells; 24 h later, cultures were exposed to ionizing radiation, and clonogenic cell survival was determined. Tumor cell survival was reduced by 40-50% when the tumor cell lines were exposed to AV1Y28 only. In addition, for each tumor cell line, AV1Y28 exposure enhanced the level of radiation-induced cell killing. Dose enhancement factors at a surviving fraction of 0.1 ranged from 1.3 to 1.5. Furthermore, for each of the cell lines, the surviving fraction at 2 Gy was significantly reduced by AV1Y28 exposure. In contrast to the results seen in tumor cells, the radiosensitivity of a normal human fibroblast cell line was not affected by AV1Y28. These data indicate that this anti-Ras adenovirus enhances the radiosensitivity of tumor cells but does not affect the radiosensitivity of normal cells.

Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes.

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50-70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (alpha2,beta2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor beta subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.

Uptake of injected 125I-ricin by rat liver in vivo. Subcellular distribution and characterization of the internalized ligand.

Subcellular-fractionation techniques were used to characterize the endocytic pathway followed by ricin in rat liver in vivo and tentatively identify the site(s) at which the ricin interchain disulphide bridge is split. After injection of 125I-ricin, hepatic uptake of radioactivity was maximum at 30 min (40% of injected dose). At 5 min, about 80% of the radioactivity in the homogenate was recovered in the microsomal (P) fraction, but later on the recovery of the radioactivity in the mitochondrial-lysosomal (ML) fractions progressively increased (50% at 30 min) at the expense of that in the P fraction. Subfractionation of the P and ML fractions on analytical sucrose-density gradients revealed a time-dependent translocation of the radioactivity from low- to high-density endocytic structures, with median relative densities at 5 and 60 min of about 1.15 and 1.16 (P fraction) and 1.19 and 1.22 (ML fraction) respectively. The late distribution of the radioactivity in the ML fraction was similar to that of the lysosomal marker acid phosphatase. Studies with co-injected lactose and mannan showed that ricin was internalized mainly via the mannose receptor. In the presence of mannan, the late recovery of radioactivity in the ML fraction was decreased, and the distribution of the radioactivity associated with the P fraction was shifted toward lower densities (median relative density 1.13), indicating a different pathway of endocytosis. Analysis of the radioactivity associated with the ML and S fractions by SDS/PAGE revealed a time-dependent increase in the amount of intact A- and B-chains and low-molecular-mass products. When ML fractions containing partially processed ricin were incubated at 37 degrees C at pH 5 or at pH 7.2 in the presence of ATP, only low-molecular-mass products were generated. We conclude that internalized ricin associates with endocytic structures whose size and density of equilibration increase with time, and that, although detectable in these structures, reduction of the ricin interchain disulphide bridge occurs to a large extent in the cytosol.

Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

Heterologous proteins can be expressed in Xenopus laevis oocytes by cytoplasmic microinjection of mRNA. To circumvent limitations inherent in this approach we investigate direct nuclear injection of strong viral expression vectors to drive transcription and subsequent translation of cDNAs encoding cytoplasmic, secreted, and plasma membrane proteins. After several viral promoters had been tested, the pMT2 vector was found to be a superior expression vector for X. laevis oocytes capable of directing expression of high levels of functional heterologous proteins. Typically the amount of protein derived from transcription-translation of the microinjected cDNA accounts for approximately 1% of total non-yolk protein. Moreover, the inefficiency usually associated with nuclear injections was overcome by coinjection of pMT2 driving expression of a secreted alkaline phosphatase as an internal control to select positive-expressing oocytes. Using this method, we have successfully expressed high levels of chloramphenicol acetyltransferase, the adipocyte-specific cytosolic 422(aP2) protein, and the membrane-associated glucose transporter GLUT1. The system described should be applicable to a wide variety of proteins for which cDNAs are available. Hence, the cumbersome and often inefficient in vitro synthesis of mRNA for studying ion channels, receptors, and transporters as well as for expression cloning in Xenopus oocytes should no longer be necessary.

Expression of a type I insulin-like growth factor receptor with low affinity for insulin-like growth factor II.

We investigated the binding properties of the type I insulin-like growth factor (IGF) receptor expressed in NIH-3T3 fibroblasts transfected with a human type I receptor cDNA. Cell surface receptors bound IGF-I with KD = 1 nM as predicted. Although recent studies have suggested that IGF-I and IGF-II bind to type I receptors with near-equal affinity, the receptors in this system bound IGF-II with much lower affinity (KD = 15-20 nM). When type I receptors from the transfected cells were solubilized and immunopurified, however, both 125I-IGF-I and 125I-IGF-II bound to the purified receptors with extremely high and relatively similar affinities (KD = 8 and 17 pM respectively). Thus the immunopurified receptors had higher affinity but lower specificity for the two ligands. The monoclonal antibody alpha IR-3 effectively inhibited IGF-I binding to cell surface receptors (75 +/- 10%), but did not inhibit IGF-II binding. In the purified receptor assay, alpha IR-3 also inhibited IGF-I binding more effectively than IGF-II binding (38 +/- 7% versus 10 +/- 4%). We conclude that the products of this cDNA can account for the binding patterns that we previously observed in receptors immunopurified from human placenta. The differential effect of alpha IR-3 on IGF-I versus IGF-II raises the possibility that these homologous growth factors bind to immunologically distinct epitopes on the type I receptor.

Role of acidic subcellular compartments in the degradation of internalized insulin and in the recycling of the internalized insulin receptor in liver cells: in vivo and in vitro studies.

Upon interaction with liver cells, insulin is internalized along with its receptor into nonlysosomal endocytic structures termed endosomes. In this work, the biochemical evidence supporting the role of endosomal acidity in the degradation of internalized insulin and in the recycling of the internalized insulin receptor is described. Treatment of rats by chloroquine and/or quinacrine, two acidotropic drugs, increases by 5-10 fold the amount of endogenous insulin associated with endosomal fractions and, in rats injected by 125I-labeled or native insulin, the endosomal uptake of these ligands at late times after injection. With 125I-insulin, these drugs inhibit the degradation of internalized hormone as judged on physical, biological and immunological criteria. Chloroquine and quinacrine treatment also increases the insulin receptor content of endosomal fractions and, in rats injected by native insulin, the ligand-induced accumulation of receptors in endosomal fractions at late times after injection. Subfractionation of endosomal fractions on Percoll gradients shows that chloroquine treatment shifts the distribution of both insulin and the insulin receptor towards higher densities, the receptor shift being slightly more pronounced in insulin-injected rats. Incubation of isolated endosomes containing internalized insulin at 30-37 degrees C results in a rapid degradation of this ligand, with a maximal at pH 5-6. Addition of ATP, by decreasing the endosomal pH, stimulates insulin degradation above pH 7, whereas addition of chloroquine and quinacrine, by elevating endosomal pH, exerts opposite effects. These data indicate that endosomal acidity is required for optimum degradation of internalized insulin within endosomes and recycling of the internalized receptor.

Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes.

Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation. Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min. Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane. In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme. Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5. Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8. It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal.

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.

Transcriptional repression of the mouse insulin-responsive glucose transporter (GLUT4) gene by cAMP.

Glucose uptake by adipose tissue is mediated by two glucose transporters: GLUT4, which is most abundant, and GLUT1. While GLUT1 is expressed in many tissues, GLUT4 is unique to tissues that exhibit insulin-stimulated glucose uptake (heart and skeletal muscle and adipose tissue). In the diabetic state and during starvation, insulin-stimulated glucose uptake and GLUT4 expression are decreased in tissue adipocytes. Using 3T3-L1 adipocytes in culture, we investigated the possibility that these effects are mediated by elevated cellular cAMP. When 3T3-L1 adipocytes were treated for 16 hr with forskolin or 8-Br-cAMP, GLUT4 mRNA and protein were decreased by approximately 70%, while expression of GLUT1 mRNA and protein was increased 3-fold. These changes were accompanied by an increased basal rate of 2-deoxyglucose uptake and a loss of acute responsiveness of hexose uptake to insulin. The magnitude of GLUT4 mRNA depletion/GLUT1 mRNA accumulation was dependent upon the concentration of 8-Br-cAMP. The decrease of GLUT4 mRNA caused by 8-Br-cAMP was the result of a decreased transcription rate, while the half-life of the message was unaffected. The increase in GLUT1 mRNA caused by 8-Br-cAMP was the result of both transient transcriptional activation and mRNA stabilization. We suggest that down-regulation of GLUT4 mRNA in adipose tissue in the diabetic state and during starvation is the result of repression of transcription of the GLUT4 gene caused by cAMP.