Prenatal arsenic exposure alters keratinocyte stem cell fate through persistent activation of IGF2R-MAPK cascade leading to aggravated skin carcinogenesis in mice offspring.

Chronic exposure to arsenic (As) promotes skin carcinogenesis in humans and potentially disturbs resident stem cell dynamics, particularly during maternal and early life exposure. In the present study, we demonstrate how only prenatal arsenic exposure disturbs keratinocyte stem cell (KSC) conditioning using a BALB/c mice model. Prenatal As exposure alters the normal stemness (CD34, KRT5), differentiation (Involucrin), and proliferation (PCNA) program in skin of offspring with progression of age as observed at 2, 10, and 18 weeks. Primary KSCs isolated from exposed animal at Day-2 showed increased survival (Bax:Bcl-xL, TUNEL assay), proliferation (BrdU), and differentiation (KRT5, Involucrin) potential through the activation of pro-carcinogenic IGF2R-MAPK cascade (IGF2R-G(α)q-MEK1-ERK1/2). This was associated with reduced enrichment of histone H3K27me3 and its methylase, EZH2 along with increased binding of demethylase, KDM6A at Igf2r promoter. Altered KSCs conditioning through disturbed Igf2r imprint contributed to impaired proliferation and differentiation and an aggravated tumor response in offspring.

Bisphenol A exposure modifies methylation of imprinted genes in mouse oocytes via the estrogen receptor signaling pathway.

Bisphenol A (BPA), a synthetic additive used to harden polycarbonate plastics and epoxy resin, is ubiquitous in our everyday environment. Many studies have indicated detrimental effects of BPA on the mammalian reproductive abilities. This study is aimed to test the potential effects of BPA on methylation of imprinted genes during oocyte growth and meiotic maturation in CD-1 mice. Our results demonstrated that BPA exposure resulted in hypomethylation of imprinted gene Igf2r and Peg3 during oocyte growth, and enhanced estrogen receptor (ER) expression at the levels of mRNA and protein. The relationship between ER expression and imprinted gene hypomethylation was substantiated using an ER inhibitor, ICI182780. In addition, BPA promoted the primordial to primary follicle transition, thereby speeding up the depletion of the primordial follicle pool, and suppressed the meiotic maturation of oocytes because of abnormal spindle assembling in meiosis I. In conclusion, neonatal exposure to BPA inhibits methylation of imprinted genes during oogenesis via the ER signaling pathway in CD-1 mice.

Reperfusion using lactate Ringer's mixture partially eliminates IGF II receptor involved cardiac damage caused by hemorrhagic shock in diabetic rats.

Diabetes is a metabolic disorder that damages many organs. We investigated the effects of reperfusion using lactate Ringer's solution (LR) in a diabetic animal model. Eight-week-old rats were divided into groups: control, hemorrhagic shock induced (HS), diabetes mellitus (DM), DM plus HS (DM + HS) and DM rats that received LR after HS (DM + HS + LR). HS was induced by withdrawing blood from the femoral artery and arterial pressure was maintained at 40 mm Hg for 1 h. Animals were perfused with either withdrawn blood or LR. Rats were sacrificed and hearts were collected from all groups. Histopathological studies were performed using left ventricles and western blotting analysis was performed using protein extracted from the left ventricle. Using the TUNEL assay, we found more apoptotic cells in the DM + HS group compared to the control group, whereas in animals resuscitated with LR, the number of apoptotic cells was reduced. Western blotting showed a significant reduction in apoptotic markers, cyt c, cas 9 and cas 3, and increased survival markers, pPI3K and pAKT, in the DM + HS + LR group. Reperfusion with LR may have therapeutic effects on trauma induced HS by blocking the IGF II R facilitated apoptosis pathway in diabetic rats.

Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes.

At present little is known of the biochemical machinery controlling transport of newly synthesized lysosomal hydrolases from the trans-Golgi network (TGN) to endosomes. The demonstration that Vps34p (a protein required for targeting soluble hydrolases to the vacuole in Saccharomyces cerevisiae) is a phosphatidylinositol 3-kinase (PI3-K) suggested the possibility that a homologous enzyme might be involved in the equivalent step in mammalian cells. Using the PI3-K inhibitors wortmannin and LY294002, I provide evidence to support this hypothesis. Treatment of K-562 cells with wortmannin induced secretion of procathepsin D, with half-maximal inhibition of accurate targeting to lysosomes at 10-20 nM. Kinetic analysis indicated that a late Golgi (TGN) step was affected, and that other constitutive vesicular transport events were not. The M6P recognition signal was still generated in the presence of wortmannin suggesting that the drug was directly inhibiting export of the receptor-ligand complex from the TGN, while removal of the drug led to a rapid restoration of accurate sorting. At the concentrations used, wortmannin and LY294002 are presently accepted to be specific inhibitors of PI3-K. I conclude that these data implicate such an enzyme in the trafficking of M6P-receptor-ligand complexes from the TGN towards lysosomes.

A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

Effect of kainic acid treatment on insulin-like growth factor-2 receptors in the IGF2-deficient adult mouse brain.

Insulin-like growth factor-2 (IGF2) is a member of the insulin gene family with known neurotrophic properties. The actions of IGF2 are mediated via the IGF type 1 and type 2 receptors as well as through the insulin receptors, all of which are widely expressed throughout the brain. Since IGF2 is up-regulated in the brain after injury, we wanted to determine whether the absence of IGF2 can lead to any alteration on brain morphology and/or in the response of its receptor binding sites following a neurotoxic insult. No morphological differences were observed between the brains of IGF2 knockout (IGF2(-/-)) and wild-type control (IGF2(+/+)) mice. However, our in vitro receptor autoradiography results indicate that IGF2(-/-) mice had lower endogenous levels of [(125)I]IGF1 and [(125)I]insulin receptor binding sites in the hippocampus and cerebellum as compared to IGF2(+/+) mice, while endogenous [(125)I]IGF2 receptor binding showed a decrease only in the cerebellum. Seven days after kainic acid administration, the [(125)I]insulin receptor binding sites were significantly decreased in all brain regions of the IGF2(+/+) mice, while the levels of [(125)I]IGF1 and [(125)I]IGF2 binding sites were decreased only in select brain areas. The IGF2(-/-) mice, on the other hand, showed increased [(125)I]IGF1 and [(125)I]IGF2 and [(125)I]insulin receptor binding sites in selected regions such as the hippocampus and cerebellum. These results, taken together, suggest that deletion of IGF2 gene does not affect gross morphology of the brain but does selectively alter endogenous [(125)I]IGF1, [(125)I]IGF2 and [(125)I]insulin receptor binding sites and their response to neurotoxicity.

Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network.

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.

Scarring impedes regeneration at sites of peripheral nerve repair.

We have investigated the effect of scarring at a site of peripheral nerve repair by comparing regeneration of the sciatic nerve in normal mice and two transgenic strains with an increased or decreased propensity for scarring. The outcome was assessed by quantifying collagen at the repair site, recording compound action potentials and counting myelinated nerve fibres on each side of the repair. We found that higher levels of collagen scar formation were associated with smaller compound action potentials, slower conduction velocities and a reduction in fibre numbers across the repair site. We conclude that scarring impedes regeneration at sites of nerve repair and suggest that this could be amenable to therapeutic manipulation.

Cryptic receptors for insulin-like growth factor II in the plasma membrane of rat adipocytes--a possible link to cellular insulin resistance.

To further elucidate the mechanisms for short-term regulation of the receptor for insulin-like growth factor II (IGF-II), we investigated effects of insulin, cAMP and phosphatase inhibitors on cell surface 125I-IGF-II binding in rat adipocytes. Preincubation with the serine/threonine phosphatase inhibitor okadaic acid (OA, 1 microM) or the non-hydrolysable cAMP analogue N6-mbcAMP (4 mM) markedly impaired insulin-stimulated 125I-IGF-II binding. Furthermore, addition of OA enhanced the inhibitory effect exerted by N6-mbcAMP. N6-mbcAMP also induced an insensitivity to insulin which was normalized by concomitant addition of the tyrosine phosphatase inhibitor vanadate (0.5 mM). In contrast, vanadate did not affect the impairment in maximal insulin-stimulated 125I-IGF-II binding produced by either OA or N6-mbcAMP. Phospholipase C (PLC), which cleaves phospholipids at the cell surface, markedly enhanced cell surface 125I-IGF-II binding in a concentration-dependent manner. Scatchard analysis demonstrated that the effect of PLC was due to an increased number of binding sites suggesting that "cryptic' IGF-II receptors are associated with the plasma membrane (PM). PLC (5 U/ml) also reversed the N6-mbcAMP-induced decrease of 125I-IGF-II binding at a low insulin concentration (10 microU/ml). Taken together, these data indicate that cAMP, similar to its effects on the glucose transporter GLUT 4 and the insulin receptor, may increase the proportion of functionally cryptic IGF-II receptors in the PM through mechanisms involving serine phosphorylation, possibly of a docking or coupling protein. Tyrosine phosphorylation appears to exert an opposite effect promoting the full cell surface expression of receptors.

IGF-II receptor number is increased in TE-85 osteosarcoma cells by combined magnetic fields.

Human osteosarcoma-derived osteoblast-like cells, TE-85, were used to assess the effect of a low frequency alternating magnetic field in combination with a controlled static magnetic field (combined magnetic fields, CMF) on insulin-like growth factor receptor regulation. In our culture system, application of a 15.3 Hz CMF induces a calculated maximum electrical potential in the culture media of 10(-5) V/m. Initial characterization of TE-85 cells demonstrated that (a) TE-85 cells contain both type I insulin-like growth factor (IGF-I) and IGF-II receptors and (b) dose dependence for IGF-stimulated cell proliferation were comparable to the affinities of the IGF's binding to membrane binding sites (i.e., receptors had dissociation constants in the low nanomolar concentration range). The studies with CMF exposure revealed that CMF treatment for 30 minutes increased the number of IGF-II receptors in a frequency-dependent manner without affecting the number of IGF-I receptors. The CMF-dependent increase in IGF-II receptor number was associated with a significant increase in the IGF-II dissociation constant. These results indicate that a membrane receptor levels can be altered by short-term exposure to low-energy, low-frequency electromagnetic fields and suggest a potential biochemical mechanism for electromagnetic effects on bone formation and remodeling.

Cortisol represses insulin-like growth factor II receptor transcription in skeletal cell cultures.

Glucocorticoids have a number of effects on bone cell function, some of which might be mediated by changes in the synthesis or activity of insulin-like growth factors (IGFs). Glucocorticoids inhibit IGF-I, but not IGF-II, synthesis in osteoblasts and decrease the expression of selected IGF-binding proteins. The effects of glucocorticoids on IGF-I and -II receptor messenger RNA (mRNA) expression in osteoblasts are not known, and changes in IGF-I or -II receptor levels could result in changes in IGF activity. We examined the effects of glucocorticoids on IGF-I and -II receptor mRNA expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Cortisol at 1 microM for 2-48 h did not alter IGF-I receptor transcripts, as determined by Northern blot analysis and ribonuclease protection assay. In contrast, cortisol caused a time- and dose-dependent inhibition of IGF-II receptor mRNA levels. The effect was maximal at 0.1-1 microM for 24-48 h and was accompanied by a decrease in IGF-II receptor levels, as determined by affinity labeling, cross-linking and polyacrylamide gel electrophoresis, Western immunoblot, and Scatchard analysis. The effect of cortisol on IGF-II receptor transcripts was not dependent on de novo protein synthesis. Cortisol did not modify the IGF-II receptor mRNA half-life in transcriptionally arrested Ob cells and decreased the rate of IGF-II receptor RNA transcription in nuclear run-on assays. In conclusion, cortisol decreases transcription of the IGF-II receptor in Ob cell cultures, an effect that could mediate selected actions of glucocorticoids in bone.

Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK.

We have earlier shown that migration and invasiveness of first trimester human extravillous trophoblast cells are stimulated by IGF-II, independently of IGF type 1 receptor and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by IGF-II. In the present study we examined the functional role of IGF type II receptor in IGF-II stimulation of extravillous trophoblast cell migration and the underlying signal transduction pathways including the participation of inhibitory G protein(s) and MAPK. The migratory ability of a well characterized in vitro propagated human first trimester extravillous trophoblast cell line expressing the phenotype of extravillous trophoblast cells in situ was quantitated with a Transwell migration assay under different experimental conditions. We found that the extravillous trophoblast cells expressed an abundance of IGF type 2 receptor as detected by immunostaining and Western blots, and recombinant human IGF-II promoted their migration in a dose- and time-dependent manner. Both polyclonal and monoclonal IGF type 2 receptor-blocking antibodies blocked migration-stimulating effects of IGF-II. Two synthetic IGF-II analogs ([Leu27]IGF-II, which can bind to IGF type 2 receptor and IGF-binding proteins, but not IGF type 1 receptor, and [QAYL-Leu27]IGF-II, which can bind to IGFR-II, but neither IGFR-I nor IGF-binding proteins) both stimulated extravillous trophoblast cell migration to levels higher than those induced by wild-type IGF-II. These results reveal that IGF-II action was mediated by IGF type 2 receptor, independently of IGF type 1 receptor and IGF-binding proteins. Treatment of extravillous trophoblast cell membrane preparations with IGF-II decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitory G proteins in IGF-II action. This was substantiated further with the findings that increasing intracellular cAMP using forskolin or (Bu)2cAMP inhibited basal extravillous trophoblast cell migration and blocked IGF-II stimulation of migration. IGF-II treatment rapidly stimulated phosphorylation of MAPK (ERK-1 and -2), which was blocked by pretreatment of extravillous trophoblast cells with the MAPK kinase (MEK) inhibitor PD98059. Treatment with this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of IGF-II. These results, taken together, reveal that IGF-II stimulates extravillous trophoblast cell migration by signaling through IGF type 2 receptor, involving inhibitory G proteins and activating the MAPK pathway.

Effect of endocrine therapy on growth of T61 human breast cancer xenografts is directly correlated to a specific down-regulation of insulin-like growth factor II (IGF-II).

Insulin-like growth factors I and II (IGF-I and IGF-II) are potent mitogens for some human breast cancer cell lines, and expression of IGF-II mRNA in the oestrogen receptor-positive (ER+) and oestradiol (E2) stimulated human breast cancer cell line T47D is increased by E2, suggesting a role for IGF-II in the mitogenic response to E2. Very little information is available from the literature on the relation between growth inhibition by endocrine therapy and cellular production of IGF-II. Here we report on the effect of E2 and tamoxifen (TAM) on IGF-II mRNA and protein expression in the ER+T61 human breast cancer xenograft. Growth of the T61 tumour is inhibited by treatment with E2 and TAM. Ribonuclease (RNAse) protection assays with human- and mouse-specific IGF-II antisense probes were used to study the regulation of IGF-II mRNA by E2 and TAM in the tumour. IGF-II protein expression was studied by radioimmunoassay. Untreated T61 tumours have a high baseline expression of IGF-II mRNA. TAM treatment of T61 tumours, which results in inhibition of tumour growth without tumour regression, reduced IGF-II mRNA expression approximately 10-fold after 48 h of treatment. E2 treatment of T61 tumours, which results in tumour regression, was accompanied by a more pronounced decrease in IGF-II mRNA expression in the tumour cells; 96 h after initiation of E2 treatment, there was almost no detectable IGF-II mRNA. Analyses of IGF-II protein showed that both treatments significantly reduced the concentration of IGF-II protein in the tumours. This down-regulation was found to be specific for IGF-II, since analyses of the effect of E2 on the expression of IGF-I mRNA, 36B4 mRNA, transforming growth factor alpha(TGF-alpha) mRNA, and epidermal growth factor (EGF) receptor mRNA in T61 tumours did not reveal any down-regulation. To further study the relation between inhibition of tumour growth and down-regulation of IGF-II, we exposed T61 tumours to a monoclonal antibody, alpha-IR3, which abolishes the physiological effect of IGF-I and IGF-II by blocking the binding of both growth factors to the type I IGF receptor. Treatment with alpha-IR3 resulted in inhibition of tumour growth during treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of beta2-glycoprotein 1 in late endosomes of human endothelial cells.

Antiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards beta2-glycoprotein 1 (beta2GP1)phospholipid complexes, we investigated the localisation of beta2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti-beta2GP1 antibodies, we detected beta2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti-beta2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti-beta2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti-beta2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

GH regulation of the Type 2 IGF receptor in regenerating skeletal muscle of rats.

GH enhances skeletal muscle growth, and IGF-II peptide is highly expressed during regeneration. We have therefore investigated the effect of GH administration on IGF-II binding and expression in regenerating rat skeletal muscle using the techniques of receptor autoradiography and in situ hybridisation. Notexin, a myotoxin, was injected into the right M. biceps femoris (day 0), causing affected fibres to undergo necrosis followed by rapid regeneration. Animals were administered either GH (200 micrograms/100 g body weight) or saline vehicle daily. Contralateral muscles were used as regeneration controls. GH administration during regeneration resulted in significant increases in body weight, and damaged and undamaged muscle weights (P < 0.001). IGF-II expression, which was examined in regenerating fibres, survivor fibres and undamaged fibres, varied according to tissue type (P < 0.001). Specifically, IGF-II expression in regenerating fibres was elevated relative to control and survivor fibres after day 3 (P < 0.05), with a peak on day 9 (P < 0.001). GH did not affect IGF-II message levels. 125I-IGF-II binding in regenerating muscle was examined in the same fibre types as well as in connective tissue. 125I-IGF-II binding in regenerating fibres was higher (P < 0.001) than in other tissue types on day 5. GH administration increased 125I-IGF-II binding in all damaged muscle tissues on day 5 (P < 0.001, regenerating fibres; P < 0.01, others). We believe that this shows for the first time an effect of GH on the Type 2 IGF receptor in regenerating skeletal muscle.

Mono- and bivalent ligands bearing mannose 6-phosphate (M6P) surrogates: targeting the M6P/insulin-like growth factor II receptor.

[reaction: see text] Mannose 6-phosphate mimics locked into the alpha-configuration and bearing hydrolase-resistant phosphate surrogates were synthesized and evaluated for binding affinity to the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R). Affinity increases as the phosphate surrogate is varied in the order malonyl ether < malonate < phosphonate. An alkene cross-metathesis approach to sought-after bivalent M6P-bearing ligands is also described. These compounds were designed to map onto biantennary sectors of high-mannose-type oligosaccharides carried by glycoprotein M6P/IGF2R ligands.

IGF receptor function and regulation in autocrine human neuroblastoma cell growth.

Insulin-like growth factor-II (IGF-II) and its receptors (type I and II IGF receptors) are expressed in the nervous system in a tissue and developmentally specific manner. We have previously shown that SH-SY5Y human neuroblastoma cells synthesize and secrete high levels of IGF-II, and respond to it with increased neuritic outgrowth, DNA synthesis, and cell proliferation. SH-SY5Y cells also produce type I IGF and IGF-II/M6P receptors; however, it is not known whether these receptors mediate the observed growth promoting effects of IGF-II. In this study, we assayed the role of type I IGF receptor and IGF-II/M6P receptor expression in mediating autocrine IGF-II induced growth. Using anti-receptor antibodies, we found that IGF-II stimulates cell proliferation via the type I IGF receptor but not via the IGF-II/M6P receptor. By Northern analysis, we detected increased mRNA expression of both receptors, with more dramatic changes in type I IGF receptor expression. Collectively, our results indicate a role for the type I IGF receptor in mediating IGF-II induced autocrine neuroblastoma cell growth.

Insulin-like growth factor II promotes in vitro cholinergic development of mouse septal neurons: comparison with the effects of insulin-like growth factor I.

We investigated the effect of insulin-like growth factors II and I (IGFII and IGFI) on septal primary cultures from mouse embryonic day 15 brains. The addition of IGFII to septal cultures enhanced total choline acetyltransferase (ChAT) activity in a dose-dependent manner. Maximal stimulation of ChAT activity was observed at 10 ng/ml IGFII. The effect of IGFII on ChAT activity was completely blocked by anti-IGFII/M-6-P receptor antibodies, whereas the antisera alone had no effect on the enzyme activity. Double-labeled immunohistochemical studies revealed that most ChAT-positive neurons expressed IGFII/M-6-P receptor immunoreactivity. These results indicate that the trophic effect of IGFII results from the direct action of this molecule through the IGFII/M-6-P receptor in septal cholinergic neurons. IGFI also stimulated ChAT activity, but with less potency than IGFII. Antibodies against the IGFII/M-6-P receptor inhibited approximately 50% of the IGFI response, suggesting that the effect of IGFI is mediated in part by the IGFII/M-6-P receptor. Thus, it appears that IGFII and IGFI are potent trophic factors for central cholinergic neurons and could potentially play a significant role in the differentiation, maintenance and regeneration of these neurons.

The influence of ethanol exposure on insulin-like growth factor (IGF) type II receptors in fetal rat tissues.

The effect of ethanol (ETOH) exposure on the IGF type II receptor concentration was examined in 18 and 20 day fetal rat tissues. Pregnant dams were fed an ETOH (36% of calories derived from ETOH; 6.6% v/v) liquid diet. Control fetuses were offspring of dams either pair-fed a control liquid diet or ad libitum-fed a standard pelleted lab chow. Fetal brain, heart, kidney, liver, lung and skeletal muscle were removed and whole homogenates from individual animals were analyzed. Results of immunoquantification of IGF type II receptors in whole tissue homogenates show that there is a trend towards increased receptor concentration between 18 and 20 days in all tissue and this trend is significant for lung, liver and muscle. There were no significant differences in receptor concentration between treatment groups. These studies suggest that during the later stages of fetal development, there is an increase in total IGF type II receptors and this increase is undisturbed by ETOH exposure.

Modulating IGFBP-3 expression by trichostatin A: potential therapeutic role in the treatment of hepatocellular carcinoma.

The Hep3B cell line analyzed in the present study is a widely used in vitro model in studies characterizing pathogenetic, functional, and therapeutic aspects of human hepatocellular carcinoma (HCC). Here we have determined the chromosomal composition using a combination of cytogenetic techniques. In agreement with the original description for this cell line, Hep3B was found to have a hypotriploid chromosome content carrying 59-63 chromosomes and no cytogenetic differences were demonstrated between early and late passages suggesting that this cell line has remained stable after repeated subculturing. Mutations and alterations of the IGF-axis as well as of chromosome 1p34, where the genes for histone deacetylase 1 (HDAC1) and transforming growth factor beta receptor interacting protein-1 (TRIP-1) map, are frequent events in hepatocarcinogenesis. This study characterizes the Hep3B cell line in detail at the karyotypic level, using comparative genomic hybridization (CGH), spectral karyotyping (SKY), G-banding and FISH techniques. We have also examined the effects of the histone deacetylase inhibitor trichostatin A (TSA) on members of the IGF-axis, and analysed them with regard to the karyotype. The results show that expression of one member of the IGF-axis, IGFBP-3, is greatly upregulated by treatment of Hep3B cells with TSA. As IGFBP-3 has been shown to induce apoptosis, these results suggest a possible use for histone deacetylase inhibitors and/or IGFBP-3 in the treatment of HCC.