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Overexpression and activation of receptor tyrosine kinases (RTKs), such as the insulin-like growth factor 1 receptor (IGF1R) and the epidermal growth factor receptor (EGFR), are frequent phenomena in colorectal cancer (CRC). Here, we evaluated the effect and the cellular mechanisms of the simultaneous inhibition of these two RTKs both in vitro and in vivo in addition to a 5-fluoruracil (5-FU)-based radiochemotherapy (RCT), which is a standard treatment scheme for CRC. Using the small molecule inhibitors AEW541 and erlotinib, specific against IGF1R and EGFR, respectively, different CRC cell lines exhibited a reduced survival fraction after RCT, with the highest effect after the simultaneous inhibition of IGF1R/EGFR. In vivo, xenograft mice simultaneously treated with low dose AEW541/erlotinib plus RCT revealed a significant reduction in tumour volume and weight compared with the tumours of mice treated with either AEW541 or erlotinib alone. In vitro, the combined inhibition of IGF1R/EGFR resulted in a stronger reduction of downstream signalling, an increase in DNA double strand breaks (DSBs), apoptosis and mitotic catastrophe after RCT depending on the cell line. Moreover, the existence of IGF1R/EGFR heterodimers in CRC cells and human rectal cancer samples was proven. The heterodimerisation of these RTKs was dependent on the presence of both ligands, IGF-1 and EGF, and functional receptors. In conclusion, these results demonstrate that the strategy of targeting both IGF1R and EGFR, in addition to basic RCT, could be of intriguing importance in CRC therapy.

Overexpression and activation of tyrosine kinase receptors are common features of colorectal cancer. Using the human colorectal cancer cell lines DLD-1 and Caco-2, we evaluated the role of the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in cellular functions of these cells. We used the small interfering RNA (siRNA) technology to specifically down-regulate IGF-IR and EGFR expression. Knockdown of IGF-IR and EGFR resulted in inhibition of cell proliferation of DLD-1 and Caco-2 cells. An increased rate of apoptosis was associated with siRNA-mediated silencing of IGF-IR and EGFR as assessed by activation of caspase-3/caspase-7. The combined knockdown of both EGFR and IGF-IR decreased cell proliferation and induced cell apoptosis more effectively than did silencing of either receptor alone. Comparable effects on cell proliferation and apoptosis were observed after single and combinational treatment of cells by the IGF-IR tyrosine kinase inhibitor NVP-AEW541 and/or the EGFR tyrosine kinase inhibitor erlotinib. Combined IGF-IR and EGFR silencing by either siRNAs or tyrosine kinase inhibitors diminished the phosphorylation of downstream signaling pathways AKT and extracellular signal–regulated kinase (ERK)-1/2 more effectively than did the single receptor knockdown. Single IGF-IR knockdown inhibited IGF-I–dependent phosphorylation of AKT but had no effect on IGF-I– or EGF-dependent phosphorylation of ERK1/2, indicating a role of EGFR in ligand-dependent ERK1/2 phosphorylation. The present data show that inhibition of the IGF-IR transduction cascade augments the antipoliferative and proapoptotic effects of EGFR inhibition in colorectal cancer cells. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy.[Mol Cancer Ther 2009;8(4):821–33]

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.

Isoflavones have been shown to exert antiproliferative effects on cancer cells by steroid receptor signaling. In this study, we demonstrate the potential of plant constituents extracted from Belamcanda chinensis as anticancer drugs, which regulate the aberrant expression of genes relevant in proliferation, invasion, immortalization and apoptosis. LNCaP cells were treated with B.chinensis extract, tectorigenin or other isoflavones and mRNA expression was quantified by using real time RT--PCR. In addition, ELISA, TRAP assays and western blots were used to measure protein expression or activity. Male nude mice (n ¼ 18) were injected subcutaneously with LNCaP cells and were fed with extracts from B.chinensis, and tumor development was monitored versus a control animal group (n ¼ 18). Tectorigenin and several other phytochemicals downregulated PDEF, PSA and IGF-1 receptor mRNA expression in vitro. Furthermore, PSA secretion and IGF-1 receptor protein expression were diminished, and hTERT mRNA expression and telomerase activity decreased after tectorigenin treatments. However, TIMP3 mRNA was upregulated on tectorigenin treatment. Growth of subcutaneous tumors in nude mice was delayed and diminished in animals fed with extracts from B.chinensis. The downregulation of PDEF, PSA, hTERT and IGF-1 receptor gene expression by tectorigenin demonstrates the antiproliferative potential of these agents. The upregulation of TIMP-3 gene expression indicates a pro-apoptotic function of the drug and a reduction of the invasiveness of tumors. The animal experiments demonstrate that B.chinensis markedly inhibited the development of tumors in vivo. Thus, these compounds may be useful for the prevention or treatment of human prostate cancer.

Hepatitis C virus (HCV) frequently causes chronic liver disease. The cause of viral persistence might be an inappropriate type I interferon (IFN) induction. To analyze the host's IFN response in chronic hepatitis C, we measured the transcription level of type I IFN genes as well as type I IFN-regulated genes in liver tissue and corresponding blood samples from patients with chronic hepatitis C, nonviral liver diseases, and a suspected but later excluded liver disease. Competitive and real-time RT-PCR assays were used to quantify the messenger RNA (mRNA) levels of all known IFN-alpha, IFN-beta, and IFN-lambda genes and those of some IFN-regulated genes. We failed to detect any hepatic type I IFN mRNA induction, although liver tissue of chronic hepatitis C patients contained high numbers of some type I IFN-inducible effector mRNA molecules. Analysis of peripheral blood samples, however, showed a clear type I IFN induction. Parallel experiments employing HCV replicon cell lines revealed that replication of HCV RNA is not sufficient to induce any type I IFN nor to induce directly type I IFN-regulated genes such as MxA. In conclusion, our data provide evidence for the absence of an induction of type I IFN genes by HCV in the human liver and argue for a further development of type I IFN-based therapies.

Proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3), the major carrier of IGFs in the circulation, is an essential mechanism to regulate IGF bioavailability. To analyze naturally occurring IGFBP-3 fragments a peptide library established from human hemofiltrate was screened. Three IGFBP-3 fragments were detected with apparent molecular masses of 34, 16, and 11 kDa. Mass spectrometric and sequence analysis identified the 16 and 11 kDa peptides as glycosylated and non-glycosylated N-terminal fragments spanning residues Gly1–Ala98 of IGFBP-3. Both the circulating forms and those secreted from IGFBP-31–98 overexpressing cells bound IGF. Additionally, two smaller fragments (IGFBP-3139–157 and IGFBP-3139–159) were identified in the hemofiltrate. The data indicate that proteolysis of circulating IGFBP-3 occurs in the variable domain at residues alanine 98, phenylalanine 138, glutamine 157, and tyrosine 159.

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) is the major mechanism of releasing IGFs from their IGFBP complexes. Analysis of fibroblasts deficient for the lysosomal cysteine protease cathepsin L (CTSL) revealed an accumulation of IGFBP-3 in the medium which was due neither to alterations in IGFBP-3 mRNA expression nor to extracellular IGFBP-3 protease activity. Incubation of CTSL-deficient fibroblasts with radiolabeled IGFBP-3 followed by subcellular fractionation indicates that both intact and fragmented IGFBP-3 accumulate transiently in endosomal and lysosomal fractions of CTSL-deficient cells. This suggests the involvement of CTSL in the intracellular degradation of IGFBP-3 representing a new mechanism to regulate the extracellular concentration of IGFBP-3.

Human oncoprotein MDM2 reveals a MHC class I binding motif HMDM441 characterizing MDM2 as a potential tumor antigen. To analyze the distribution of MDM2 proteins containing this motif in liver cancer cells we produced rabbit anti-HMDM441 serum. The novel antibodies bound to an MDM2 fragment of approximately 55 kDa which lacked the N-terminal region and was present in lysate and supernatant of a human hepatoma cell line overexpressing normal 90-kDa MDM2. The 55-kDa fragment was detected in the cytoplasm and nucleoli and at the nuclear envelope of hepatoma cells, whereas normal hepatocytes were negative. Double-fluorescence labeling indicated that the MDM2 fragments and MHC class I molecules were coexpressed on the surface of the hepatoma cells. Further studies must clarify whether MDM2 fragments containing motif HMDM441 are novel targets of immunotherapy and immunochemical tumor diagnosis.

Preneoplastic hepatic foci have been demonstrated in liver acini, which drain the blood from intraportally transplanted pancreatic islets in streptozotocin-induced diabetic rats with mild persisting diabetes. In long-term studies of this animal model, hepatocellular adenomas and carcinomas (HCC) developed after a sequence of characteristic preneoplastic hepatic foci. In this experimental model, the local hyperinsulinism is thought to have a causative role. Because insulin and the insulin-like growth factor (IGF) axis are closely linked, an altered gene expression of the IGF axis components is likely. Therefore, preneoplastic hepatic foci and HCC were studied for the expression of IGF axis components. Glycogen-storing "early" preneoplastic hepatic foci were detectable several days after pancreatic islet transplantation. Northern blot analysis, in-situ hybridization, and immunohistochemical studies of these "early" lesions demonstrated increased expressions of IGF-I and IGF binding protein-4 (IGFBP-4) in altered parenchymal cells, and a decreased expression of IGFBP-1. IGF-II was not detected in these preneoplastic foci. HCC arising in this model had decreased expressions of IGF-I and IGFBP-4 but IGFBP-1 expression was not significantly altered. Some HCC showed a more than 100-fold overexpression of IGF-II, whereas other tumors were completely negative for IGF-II expression. Low IGF-I receptor expression was detected in preneoplastic foci and adjacent nonaltered liver tissue. However, HCC tissue consistently showed an increased IGF-I receptor expression, rendering these tissues susceptible to the mitogenic effects of IGF. The altered gene expression in glycogen-storing preneoplastic hepatic foci, especially the up-regulation of IGF-I and IGFBP-4 with the down-regulation of IGFBP-1, resemble the insulin-dependent regulation of these components in normal rat hepatocytes. These data agree with previous studies demonstrating a correspondence of the focal character, morphology, and enzyme pattern of preneoplastic hepatic foci with insulin effects on hepatocytes. The development from preneoplastic foci to HCC may be driven by insulin itself and/or an altered IGF axis component or yet unidentified factors.

The insulin-like growth factors I and II (IGF-I, -II) are circulating peptides known to participate in the regulation of metabolism, growth, and cellular differentiation. In the present study, "early cultured" (days 2-3 of culture) and "culture-activated" (days 6-7 of culture) rat hepatic stellate cells (HSCs) were analyzed for expression of individual components of the IGF axis. Northern blot analysis of IGF-I messenger RNA (mRNA) revealed transcripts of 7.5, 4, 2, and 1.0 to 1.5 kb in culture-activated HSCs, while early cultured HSCs did not express IGF-I mRNA. In culture-activated HSCs, an IGF-I secretion of 8.3+/-2.5 ng/10(6) cells per 24 hours was determined radioimmunologically. In media from early cultured HSCs, IGF-I was not detectable. The IGF-I receptor (IGF-I-R) mRNA expression was three-fold higher in early cultured HSCs than in culture-activated HSCs. By immunohistochemistry, a decrease of IGF-I-R expression of HSCs in vivo following CCl4-induced liver damage was noted as well. IGF binding proteins (IGFBPs) were detected in conditioned media from HSCs by 125I-IGF-I ligand blotting at apparent molecular masses of 24 and 41 to 45 kd that were immunologically identified as IGFBP-4 and -3, respectively. Synthesis of these IGFBPs increased with time of culture. At neutral pH, no IGFBP proteolysis was observed in conditioned media of early cultured and culture-activated HSCs, whereas at acidic pH, protease activities against IGFBP-3 and -4 were detectable. IGFBP protease activities were completely abolished by inhibitors of aspartyl and cysteine proteases. Addition of 100 nmol/L IGF-I stimulated cell proliferation of early cultured HSCs 5.6+/-1.1- and 4.6+/-0.2-fold as measured by [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation, respectively. In culture-activated HSCs, proliferation was increased 1.2+/-0.1-fold in the presence of 100 nmol/L IGF-I in both proliferation assays. It can be concluded that due to a higher expression of the IGF-I-R and lower levels of IGFBPs, early cultured HSCs are more susceptible to the mitogenic actions of IGFs than the culture-activated HSCs. The present data suggest a role for the IGF axis components in the initiation rather than the perpetuation of HSC proliferation during hepatic fibrogenesis.

Hepatic lipase (HL) gene transcription is almost exclusively limited to hepatocytes. Here we have studied sequences and transcription factors regulating basal and hepatocyte-restricted HL promoter activity. Sequencing of a cloned 3.4-kb HL promoter fragment revealed three Alu repeat sequences and a consensus hepatocyte-enriched nuclear transcription factor 1 (HNF1) binding site located upstream of one major and one minor transcription initiation site. By transfection of cell lines of hepatic and non-hepatic origin and of primary hepatocyte cultures, sequences controlling basic HL promoter activity and negative elements located downstream and upstream thereof which extinguish or enhance this activity were defined. Some HL-promoter fragments with internal deletions were active only in primary hepatocyte cultures. Human HNF1 protein was shown to bind to the HL-specific HNF1 response element and the activity of a heterologous promoter was enhanced by HL-HNF1 in rat primary hepatocyte cultures but not in the context of the authentic 3.4-kb HL promoter sequences. In cell lines the presence of HNF4 but not of HNF1 and vHNF1 mRNA was found to correlate with HL gene expression although no perfect consensus HNF4 binding motif was detected in the promoter region tested. Taken together, these data indicate that hepatocyte-specific HL gene transcription is controlled by positive and negative transcription regulatory proteins which bind to sequence motifs within and outside of the proximal 3.4-kb promoter fragment studied. For the elucidation of the control of HL promoter activity in vivo the use of primary hepatocyte cultures is essential.

Background/Aims: Serum concentrations of insulin-like growth factor-I are decreased in liver cirrhosis. However, this growth factor is bound for the most part to specific binding proteins that are known to modulate biological actions. Plasma insulin-like growth factor binding proteins are predominantly synthesized in the liver. Methods: The effect of liver disease on basal and on growth hormone-stimulated serum concentrations of total and "free" insulin-like growth factor-I and on insulin-like growth factor binding protein patterns is reported. Sera were obtained from 20 patients with non-cirrhotic chronic liver diseases and from 20 patients with cirrhosis before and 24 h after a single subcutaneous dose of growth hormone. Samples were analyzed using radioimmunoassays, gel chromatography, ligand blotting and immunoblotting. Results: In cirrhosis, serum concentrations of total and "free" insulin-like growth factor-I were decreased, the binding protein pattern was changed profoundly showing a reduction in the 150 kD complex and an increase in the 30–40 kD complexes. Concentrations of binding protein-1 and -2 were increased, while that of binding protein-3 was decreased in cirrhosis. The response to growth hormone was blunted. These changes were related to the degree of liver dysfunction as assessed by the Child-Pugh classification. Conclusions: A pathogenetic link of altered bioavailability of insulin-like growth factor-I to clinical characteristics of advanced liver disease, e.g. insulin resistance or skeletal muscle wasting, may be suggested by the present data.

In the circulation, most of the IGFs are bound to a high molecular weight binding protein complex of 150 kDa that consists of IGF-I (or IGF-II), IGFBP-3 and the acid-labile subunit (ALS). Within rat liver, individual components of the 150 kDa complex are synthesized in different cellular compartments: ALS expression in localized in hepatocytes, but not in non-parenchymal cells. IGFBP-3 mRNA, however, is exclusively expressed in non-parenchymal and among them in endothelial and Kupffer cells. Co-cultures of hepatocytes and Kupffer cells were used as a model to study the hormonal regulation of biosynthesis of the components of the 150 kDa complex. Although expressed in different liver cell populations IGFBP-3 and ALS were regulated synergistically. Insulin stimulated both the expression of ALS and IGFBP-3 in co-cultures in a dose-dependent manner, while expression of IGFBP-1 was decreased. Regulation of IGFBP-3 synthesis of Kupffer cells required a mediator that is secreted by hepatocytes, since IGFBP-3 expression in cultures of pure Kupffer cells did not respond to the stimulating effect of insulin.

The external envelope glycoprotein gp130 of a simian immunodeficiency virus isolated from an African green monkey (SIVagmTYO-7) was purified as micellar complexes. The molecular weight of the gp130 micelles was about 700 K. On electron microscopy, the micelles appeared as spherical particles with a diameter of 15 to 20 nm. Such aggregates consisted of about 4 to 5 gp130 monomers. Hyperimmune sera raised in rabbits and rhesus monkeys against these gp130 micelles exhibited titers between 10 5 and 10 6 . Such sera inhibit the CD4 binding of gp130 and neutralize SIVagmTYO-7 and SIVmac251 but not HIV-2ben.

The binding and binding inhibition of the SIVagmTYO-7 external glycoprotein gp130 in micellar form to the CD4 molecule on human Molt-4 clone 8 cells was investigated. The best binding of gp130 to Molt-4 clone 8 cells occurred at pH 5.5 to 6.5 at 37 degrees C after 4 h or at room temperature after 10 h. The dissociation constant of this reaction was 0.2-0.4 nM, with both soluble CD4 or CD4 on Molt-4 clone 8 cells. This value is close to 0.15 nM determined for the antihuman CD4 monoclonal antibody 30F16H5. After partial deglycosylation of gp130, a 90 kD product arose which still bound to CD4. Fully deglycosylated gp130 of 60 kD was still immunoprecipitable, but had lost the CD4 binding activity. Lens culinaris agglutinin was able to inhibit the gp130-CD4 interaction very efficiently, while the agglutinin of Phaseolus vulgaris was half as efficient and Canavalia ensiformis was inefficient. CD4 binding of gp130 micelles was also inhibited with several anti CD4 monoclonal antibodies directed against the OKT4a epitope as well as with soluble recombinant CD4.

Insulin-like growth factors (IGFs), IGF receptors and IGF binding proteins (IGFBPs) participate in the regulation of proliferation and differentiation of epithelial cells. Expression of the growth-inhibitory murine IGFBP-6 in epithelial Madin–Darby canine kidney (MDCK) cells followed by 2D analysis revealed the presence of multiple isoforms. Metabolic labelling experiments showed that several IGFBP-6 isoforms are modified by phosphate and sulfate groups. Expression analysis of mutant IGFBP-6 further demonstrated that serine residue 143 is O-glycosylated. Substitution of serine 143 by alanine did slightly reduce the preferential sorting of mIGFBP-6 to the apical site in MDCK cells grown on semipermeable filters. Both the presence of multiple and heterogeneously modified isoforms of murine IGFBP-6 in MDCK cells, and the preferential secretion of non-glycosylated IGFBP-6 mutants to the apical side suggest that the major apical sorting signal is the protein moiety.

The function of the androgen-regulated homeobox protein NKX3.1 in prostate cancer is controversial. NKX3.1 is necessary for correct prostate development and undergoes frequent allelic loss in prostate cancer. However, no mutations occur in the coding region and some particularly aggressive cancers over-express the protein. Nevertheless NKX3.1 is often referred to as candidate tumor suppressor gene. Recent findings suggest a function in protection against oxidative damage involved in prostate carcinogenesis. Thus NKX3.1 may act differently at various stages of prostate cancer. Unlike a classical tumor suppressor NKX3.1 is up-regulated by androgens and down-regulated by phytoestrogens. In this study we performed RNAi based functional analysis by knocking down NKX3.1 expression in LNCaP prostate cancer cells and analyzing the impact of NKX3.1 on gene expression and cell proliferation. Knock-down of NKX3.1 evoked a massive down-regulation of NKX3.1 expression, followed by reduction in mRNA expression of the androdrogen receptor (AR) and the insulin-like growth factor receptor (IGF-1R). Western blot analysis showed strong decreases of NKX3.1, AR, and IGF-1R protein expression. Concomitantly, cell proliferation decreased and expression of prostate-specific antigen (PSA) mRNA and its secretion were diminished, whereas expression of IGF binding protein 3 (IGFBP-3) and MMP tissue inhibitor 3 (TIMP-3) was up-regulated. In tumor cells not deprived of NKX3.1 expression this gene still has a function which might differ from its role in prostate development and carcinogenesis. NKX3.1 knock-down altered the expression of genes highly relevant in prostate cancer cell proliferation and apoptosis. In LNCaP NKX3.1 most probably plays the role of an androgen-regulated transcription factor whose down-regulation is paralleled by anti-proliferative and pro-apoptotic effects. Since NKX3.1 can regulate AR expression it may become a target for interference in hormone refractory prostate carcinoma.

Objective The acute-phase response (APR), a cytokine-induced defense reaction of the body that enhances the innate immunity mechanisms directed to eliminate the noxious agent and restrict the area of damage, is accompanied by numerous alterations of the IGF axis. The liver is a central organ of both the IGF system and the APR because it releases most of IGF-I and IGFBP-1 in the circulation and is the main target organ for acute-phase-cytokines such as IL-6. Methods In the current work the expression of IGF-I and IGFBP-1 was studied in the liver and extrahepatic tissues in a rat model of localized inflammation induced by intramuscular injection of turpentine oil (TO). The mRNA expression of IGF-I and IGFBP-1 was determined by Northern blot analysis and quantitative RT-PCR. Circulating levels of IGF-I and IGFBP-1 were evaluated by radioimmunoassay and [ 125 I]-IGF-I ligand blotting, respectively. Results Administration of TO to the rats led to a significant reduction of IGF-I gene expression in the liver and spleen. These changes were accompanied by a reduction of serum IGF-I concentrations to approximately 50% of levels observed in control rats. In contrast to IGF-I, IGFBP-1 mRNA expression was rapidly elevated in the livers of TO-treated rats. IGFBP-1 transcripts were already detectable at 30min after TO injection and reached their maximal levels by 6h. IGFBP-1 gene expression was also increased in the kidneys. This elevation, however, was delayed and less prominent than in the liver. Conclusions Our data demonstrate that localized inflammation induced by intramuscular TO injection is accompanied not only by decreased IGF-I but also by increased IGFBP-1 gene expression explaining at least in part the catabolic changes of metabolism observed during the acute-phase response.

Insulin-like growth factor I (IGF-I) and platelet-derived growth factor (PDGF) have been identified as significant mitogens for liver myofibroblasts (LMFs), one of the cell populations playing a role in liver fibrogenesis. In the present work, we aimed to elucidate a possible interaction between PDGF receptor (PDGFR) and IGF-I receptor (IGF-IR) signaling in LMFs. Among different rat liver cells, PDGFR alpha- and beta-subunits were mainly expressed in hepatic stellate cells and LMFs, and were upregulated during their in vitro cultivation. In LMFs, PDGF-BB (10 ng/ml) stimulated DNA synthesis approximately two-fold and this effect was similar to that of IGF-I. IGF-I and PDGF-BB differentially affected IGF-IR and PDGFR signaling. High concentrations of IGF-I decreased levels of IGF-IR and IRS-1 and inhibited the expression and activation of PDGFRalpha. PDGF-BB prevented IGF-I-induced downregulation of the IGF-IR, but did not affect expression of its cognate receptor subunits. Transphosphorylation of PDGFR and IGF-IR was not observed. PDGF effectively activated terminal MAP kinases, PI3 kinase and Akt kinase, whereas IGF-I demonstrated weaker effects. PLCgamma(1) was phosphorylated only in response to PDGF, but not to IGF-I. In rat LMFs, blockade of the IGF-IR via inhibition of the IGF-IR kinase completely abrogated IGF- and PDGF-induced mitogenesis and the ability of PDGF to phosphorylate PLCgamma(1). In conclusion, the presented data demonstrate that the PDGFR signaling requires a functional IGF-IR and that PDGF-BB stabilizes the IGF-IR function through preventing the IGF-I-induced downregulation of the IGF-IR. These interactions might be relevant in vivo for the fibroproliferative response during liver injury.

Oxygen has important functions as substrate for biochemical reactions and as modulator of gene expression. In the liver, the physiologically occurring oxygen gradient is a major effector of metabolic zonation. In addition, cross-talks between the O2 signaling and nutrient signaling chains initiate a dynamic zonation pattern. Under pathological situations, hypoxia appears to be a major determinant for liver diseases and cancer. Thereby transcription factors of the HIF family are activated whereas USF proteins have the potential to counteract HIFs. In addition, feedback mechanisms between hypoxia, HIF and the IGF axes appear to exist. Thus, the knowledge of these mechanisms may help to initiate new therapies in diseases with disturbed O2 availability.

Higher levels of IGF-binding protein 1 (IGFBP-1) mRNA are expressed in the less aerobic perivenous zone of the liver. Because gradients in oxygen tension (pO(2)) may contribute to zonated gene expression, the influence of arterial and venous pO(2) on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA and protein levels were observed under venous pO(2), whereas less than 30% of maximal levels were observed under arterial pO(2). In contrast, the expression of IGFBP-4 was greatest under arterial pO(2), indicating that this effect of hypoxia on IGFBP-1 gene expression is specific. The response to hypoxia appears to involve reactive oxygen species, because treatment with H(2)O(2) results in a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO(2), whereas IGFBP-1 mRNA expression under arterial pO(2) was not affected. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, and inhibition of IGFBP-1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO(2) suggested the involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF-3alpha and HIF-2alpha, and to a lesser extent HIF-1alpha, contribute to the induction of IGFBP-1 mRNA expression in isolated hepatocytes, whereas experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD-2 in destabilization of HIFs, attenuating the induction of IGFBP-1 under venous pO(2). Reporter gene studies indicate that hypoxia stimulates IGFBP-1 expression through a putative HIF response element located approximately 250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, and the HIF-2 and -3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP-1 gene expression in the liver.

Objective The liver plays a central role in insulin-like growth factor (IGF) homeostasis providing the majority of circulating IGF-I and some of its binding proteins (IGFBPs). In liver cirrhosis the IGF axis is severely disturbed, and these alterations are associated with reduced IGF-I, IGFBP-3 but elevated IGFBP-1 serum levels. Methods By Northern blotting and in situ hybridization (ISH), hepatic expression of IGF-I and of IGFBP was studied in a rat model of liver cirrhosis induced by thioacetamide. Results ISH revealed a homogeneous distribution of IGFBP-1, IGFBP-4 and IGF-I mRNA over hepatic parenchyma in normal and cirrhotic liver. Fibrous septa of cirrhotic liver were IGFBP-1 mRNA negative, whereas IGFBP-4 and IGF-I transcripts were detected in single cells. In normal liver, IGFBP-3 mRNA was distributed within nonparenchymal cells of the hepatic lobule and in the wall of the portal vein. In cirrhotic liver, IGFBP-3 transcripts were abundant in mesenchymal cells of fibrous tissue. IGFBP-3 mRNA expression was also prominent in cells at the septal–nodular interface most likely representing monocyte infiltration. IGFBP-3 mRNA expression was reduced in nonparenchymal liver cells located more distantly from the septal–nodular interface in the cirrhotic nodule that correlated with reduced IGFBP-3 mRNA expression observed in Kupffer cells (KC) and sinusoidal endothelial cells (SEC) isolated from macronodular cirrhotic livers. Conclusion Cirrhosis is accompanied by an altered spatial expression of IGFBP-3 in liver tissue, which is characterized by decreased levels of IGFBP-3 mRNA in KC and SEC, but elevated IGFBP-3 expression in myofibroblast-like cells and inflammatory infiltrate.

Hepatic stellate cells (HSCs) and liver myofibroblasts (LMFs) represent major cell populations involved in liver fibrogenesis. Several lines of evidence demonstrate that in contrast to LMFs, HSCs undergo spontaneous apoptosis both in vitro and in vivo, in parallel with their activation. Therefore, LMFs appear to be an essential cell type with the fibrogenic potential in the liver. The IGF system including the insulin-like growth factors I and II (IGF-I, -II), their receptors (IGF-I receptor, IGF-IR; IGF-II/mannose 6-phosphate receptor, IGF-II/M6-PR) and six high affinity IGF binding proteins (IGFBPs) participate in the regulation of growth and differentiation of cells of the fibroblast lineage, possibly contributing to the fibrogenic process. Therefore, the purpose of the current work was to study the expression and regulation of the IGF axis components in rat LMFs. Since IGF-I is known as a progression factor for the growth-promoting effects of platelet-derived growth factor (PDGF) in many cell types, the aim of this work was also to study the role of PDGF in proliferation of LMFs and to investigate a possible cross-talk between PDGFR and IGF-IR signalling systems in rat LMFs.LMFs from passages 1 to 7 constitutively expressed transcripts encoding IGF-I, IGF-IR and IGF-II/M6-PR. A soluble form of the IGF-II/M6-PR was abundantly produced by LMFs, and its release was stimulated by IGF-II and TGF-s. In LMFs, biosynthesis of IGFBP-3 and -2 was observed that was stimulated by IGF-I, insulin and TGF-s and inhibited by PDGF-BB. During cultivation of LMFs IGFBP-3 gene expression was down-regulated, whereas that of IGFBP-2 was up-regulated.IGF-I stimulated de novo synthesis of type I collagen and had mitogenic activity, whereas long-R3-IGF-I, an IGF-I analogue which binds to the IGF receptors but not to IGFBPs, had no effect on DNA synthesis in LMFs. Simultaneous addition of recombinant human IGFBP-2 or -3 with IGF-I diminished the mitogenic effects of IGF-I on LMFs, whereas preincubation of LMFs with IGFBP-2 or -3 potentiated DNA synthesis induced by IGF-I. Exogenous IGFBP-3 revealed also mitoinhibitory activity in LMFs that was independent from IGF-I. Moreover, a relatively high amount of endogenous IGFBP-3 in LMFs was accumulated in the nucleus that might be linked with the intrinsic antiproliferative activity of IGFBP-3.Recombinant PDGF-BB stimulated DNA synthesis in LMFs and this effect was similar to that of IGF-I. Blockade of the IGF-IR with a selective inhibitor completely abrogated IGF-I- and PDGF-induced mitogenesis in cultures of rat LMFs. In rat liver, alpha and beta subunits of the PDGF receptor (PDGFR) were exclusively expressed in HSCs and LMFs, and were substantially up-regulated during their in vitro cultivation. IGF-I and PDGF-BB differentially affected the IGF-IR and PDGFR signalling systems. High concentrations of IGF-I induced down-regulation of the IGF-IR and decreased amount of IRS-1, a principal adaptor protein of the IGF-IR. Expression and activation of the PDGFR-alpha was also inhibited by IGF-I. In contrast PDGF-BB increased the IGF-IR expression and effectively prevented its IGF-I-induced down-regulation. However, PDGF-BB inhibited the IGF-I-induced tyrosine phosphorylation of IRS-1 and substantially decreased the abundance of several IRS proteins in the cell, in particular IRS-1, IRS-2 and Gab-1. PDGF-BB did not affect expression of the PDGFR. Transphosphorylation of the PDGFR and the IGF-IR was not observed in LMFs. PDGF-BB effectively induced phosphorylation of all terminal MAP kinases (ERK1/2, JNK, p38 kinase) in LMFs in contrast to IGF-I, which had only a weak effect. Inhibition of MEK, p38 kinase and JNK effectively blocked IGF-I-induced DNA synthesis in LMFs. Inactivation of JNK and p38 kinase also resulted in abrogation of mitogenic effects induced by PDGF-BB. However, the rate of PDGF-induced DNA synthesis was unaffected when phosphorylation of ERK1/2 was blocked. Inhibition of phospholipase C (PLC) in LMFs was associated with a substantial reduction of both PDGF- and IGF-I-induced DNA synthesis, although in LMFs PLC-gamma-1 was activated only in response to PDGF-BB, but not to IGF-I. Blockade of the IGF-IR kinase considerably impaired the ability of PDGF-BB to stimulate PLC-gamma-1 activity in LMFs.In conclusion, the present study demonstrates that the IGF axis via complex interactions with the PDGFR signalling system may play an important role in the proliferation of LMFs in vitro that might be relevant in vivo for fibroproliferative response during acute and chronic liver injury.

Hepatic stellate cells (HSC) play a pivotal role in hepatic tissue repair and fibrogenesis. IGF-I has been considered a mitogenic signal for activation and proliferation of HSC in vitro. In the present study IGF-I and IGF-binding protein (IGFBP) gene expression was studied in a model of acute liver injury induced by a single intragastric dose of carbon tetrachloride (CCl4) in adult rats. Northern blot analysis revealed a marked increase in IGFBP-1 mRNA levels, with a maximum between 3 and 9 h after CCl4 application, whereas steady state mRNA levels of IGF-I were only moderately altered. In situ hybridization experiments demonstrated that this increase in IGFBP-1 mRNA was due to a strong expression of IGFBP-1 in the perivenous region 6–12 h after CCl4 application, extending to the midzonal region of the acinus within 24–48 h. Consequently, a prominent immunostaining for IGFBP-1 was observed in perivenous areas, with a maximum 24–48 h after intoxication. Preincubation of early cultured HSC with a nonphosphorylated IGFBP-1 from human amniotic fluid resulted in a 3.4-fold increase in IGF-I-induced DNA synthesis. The mitogenic effect of IGF-I was also potentiated when HSC were cocultivated with IGFBP-1-overexpressing BHK-21 cells compared with nontransfected cells. These data suggest that IGFBP-1 released during the early steps of liver tissue damage and repair may interact with HSC and potentiate the sensitivity of IGF-I to mitogenic signals.

Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) may be an important mechanism to regulate IGF availability and IGF-independent functions of IGFBPs. We analyzed the secretion of IGFBP proteases in Madin-Darby canine kidney (MDCK) cells. The results showed that several specific proteases were secreted, cleaving IGFBP-2 to -6 at neutral pH. The proteolytic activity against IGFBP-6 differed at least from IGFBP-5 protease activity in its sensitivity both to IGF-II and to the hydroxamic acid-based disintegrin metalloprotease inhibitor, as well as serine protease inhibitors. During partial purification steps, the serine protease inhibitor-sensitive fraction with IGFBP-6 protease activity was separated from fractions characterized by the presence of a 30-kDa disintegrin immunoreactive band. Whereas the IGFBP-4 and -6 proteases are predominantly secreted across the basolateral membrane, the majority of IGFBPs are sorted to the apical medium from filter-grown cells. These studies indicate that the side-specific secretion of several distinct IGFBP proteases with partially overlapping IGFBP specificities may be another level in the regulation of IGF-dependent epithelial functions.

Background/Aims : Catabolism is associated with decreased serum concentrations of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 associated with elevated IGFBP-3 protease activity and increased concentrations of IGFBP-1 and -4. The effects of the acute phase mediators interleukin (IL)-6, IL-1 β and tumor necrosis factor α (TNF α ) on the biosynthesis of IGF-I and IGFBPs were studied in primary rat liver cells. Methods : mRNA levels of IGF-I and of IGFBPs were analyzed by Northern blotting, secretion of IGFBPs by [ 125 I]IGF-I ligand blotting. Proteolytic activity was measured using iodinated recombinant IGFBP-3 as the substrate. Results : In hepatocytes, Kupffer cells (KC) and cocultures of hepatocytes with KC, IL-6 reduced IGF-I biosynthesis dose-dependently. IL-6 stimulated mRNA expression and protein secretion of IGFBP-1 and -4 in hepatocytes and that of IGFBP-3 in KC, respectively. In cocultures, biosynthesis of IGFBP-1, -3 and -4 was increased dose-dependently by IL-6, while the effects of IL-1 β or TNF α were less prominent. At neutral pH, proteolytic activity against IGFBP-3 was not detected in media of cocultures treated with IL-6. Conclusions : The alterations of IGF-I, IGFBP-1 and -4 observed in catabolism correlate with the effects of IL-6 on the biosynthesis of these components in primary rat liver cells, while a neutral IGFBP-3 protease was not detectable.

Deregulation of the insulin-like growth factor (IGF) axis, including the autocrine production of IGFs, IGF binding proteins (IGFBPs), IGFBP proteases, and the expression of the IGF receptors, has been identified in the development of hepatocellular carcinoma (HCC). Characteristic alterations detected in HCC and hepatoma cell lines comprise the increased expression of IGF-II and the IGF-I receptor (IGF-IR), which have emerged as crucial events in malignant transformation and the growth of tumours. Alterations of IGFBP production and the proteolytic degradation of IGFBPs resulting in an excess of bioactive IGFs, as well as the defective function of the IGF degrading IGF-II/mannose 6-phosphate receptor (IGF-II/M6PR), may further potentiate the mitogenic effects of IGFs in the development of HCC.

In the circulation, most of IGFs are bound to a high molecular mass complex of 150 kDa that consists of IGF-I (or IGF-II), IGFBP-3 and the acid-labile subunit (ALS). Within rat liver, biosynthesis of these components has been localized to different cell populations with hepatocytes as source of ALS and nonparenchymal cells (endothelial and Kupffer cells (KC)) as source of IGFBP-3. In the present study, the regulatory effects of the cAMP analogs dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP (8-br-cAMP) on IGF-I, ALS, and IGFBP expression were evaluated in primary cultures of rat hepatocytes, KC as well as in cocultures of hepatocytes and KC. In cocultures, biosynthesis of IGFBP-3 and ALS was inhibited dose-dependently by db-cAMP and 8-br-cAMP while that of IGF-I, IGFBP-1, and -4 was stimulated as demonstrated by ligand and Northern blotting. IGFBP-3 expression in primary cultures of pure KC did not respond to cAMP treatment indicating the importance of a cellular interaction between KC and hepatocytes for the decreased IGFBP-3 synthesis. The inhibition of IGFBP-3 in db-cAMP-treated cocultures was due to a decrease of IGFBP-3 mRNA level accompanied by a reduced cellular degradation of IGFBP-3. We conclude that cAMP stimulate the biosynthesis of IGF-I, IGFBP-1, and -4 in cocultures of hepatocytes and KC thereby enabling the formation of binary IGF/IGFBP complexes while the formation of the 150 kDa complex is impaired through downregulation of IGFBP-3 and ALS. This complex regulation may be a prerequisite for the effects of cAMP-dependent hormones on the transfer of IGFs from circulation to peripheral tissues.

The insulin-like growth factors (IGF-I and -II) are structurally related peptides participating in the regulation of metabolism, growth and cellular differentiation. In the present study, the human hepatoma cell line PLC was studied for the expression of individual components of the IGF axis. Northern blot analysis using IGF-I and -II coding cDNAs failed to detect IGF-I- or -II-specific transcripts in total RNA from PLC cells. Biosynthesis of type I and II IGF receptors was demonstrated by northern blotting and binding studies as well as cross-linking of the respective radiolabeled ligand. Both IGF-I and -II stimulated [3H]-thymidine incorporation dose-dependently. The mitogenic activity of exogenously added IGFs was reduced by the presence of IGF-binding proteins of 24, 30, 34, 41 and 45 kDa in supernatants of PLC cells identified as IGFBP-4, -1, -2 and -3, respectively, by [125I]IGF-I ligand-, immuno- and northern blotting. Biosynthesis of IGFBP-3 was stimulated dose-dependently by IGF-I and -II, while IGFBP-1, -2 and -4 were not affected. The increase of IGFBP-3 in response to IGF-I and -II was due to a stimulation of IGFBP-3 specific mRNA as well as to an inhibition of IGFBP-3 endocytosis. Proteolytic activity for rhIGFBP-3 was detected in media from PLC cells at acidic pH that was inhibited by the aspartyl protease inhibitor pepstatin A as well as after immunodepletion of cathepsin D from media of PLC cells. Thus, a role of cathepsin D for the regulation of IGFBP-3 bioavailability via endocytosis in acidic prelysosomal compartments was suggested. The susceptibility of PLC for IGF-I and -II was restricted by their ability to increase the abundance of inhibitory IGFBPs and to decrease the level of IGF-I receptor expression. The present data point to the IGF axis as a complex regulatory system that self limits the mitogenic activity of exogenous IGFs.

The mitogenic and metabolic activities of insulin-like growth factors (IGF) are modulated by a family of six high affinity IGF binding proteins (IGFBPs). This study describes the expression of the mouse IGFBP-6 which is unique among IGFBPs in its preferential binding of IGF II, in insect cells using the baculovirus system. The purified, O-glycosylated IGFBP-6 was functional as shown by IGF binding and by inhibition of IGF II-stimulated DNA synthesis in human fibroblasts. Specific antibodies generated in chicken against the recombinant IGFBP-6 were used for Western blotting analysis and immunohistochemistry. Strong immunoreactivity was found in ossifying bones of the cranial base, in cell clusters of the pancreas anlage, in the trigeminal ganglion, on myoblasts, on motoneurons of the spinal cord of embryonic mice. In tissues of adult mouse, strong IGFBP-6 immunostaining was present in epidermal and peridermal layers of the skin, in meningeal layers, in long-striated skeletal muscle, and in the Langerhans' islets of the pancreas. No immunopositive staining was observed in lung and liver indicating that sites of synthesis and IGFBP action are different.

Background/Aims: The liver is the main source of circulating insulin-like growth factor binding proteins. In man, the cellular origin of insulin-like growth factor binding proteins has remained obscure. Methods: Human hepatocytes isolated from surgical specimens were purified and cultured using a collagen gel immobilization technique. Gene expression of individual insulin-like growth factor binding proteins and of the acid-labile subunit of the insulin-like growth factor ternary binding protein complex was studied by Northern blotting, secretion of insulin-like growth factor binding proteins by Western ligand blotting and immunoblot analysis. Neutral size chromatography of medium samples was used to detect insulin-like growth factors binding protein complexes. Results: In cultured hepatocytes transcripts for insulin-like growth factor binding protein-1, -2, -3, -4 and for acid-labile subunit could be demonstrated. Ligand blotting revealed the secretion of insulin-like growth factor binding proteins of molecular weights of 24 kD, 30 kD, 34 kD, 43 kD and 46 kD, respectively. Using polyclonal antisera, these proteins were identified as insulin-like growth factor binding protein-1, -2 and the insulin-like growth factor binding protein-3 doublet. Neutral size chromatography of culture supernatants showed the presence of an insulin-like growth factor binding protein complex of approximately 40 kD, but absence of the high molecular weight ternary complex of 150 kD. Conclusions: It is concluded that in man parenchymal liver cells have to be regarded as a source of acid-labile subunit and of circulating insulin-like growth factor binding proteins including insulin-like growth factor binding protein-3.

Expression, ribosomal frameshifting, and proteolytic processing of HIV-1 GAG and POL proteins were investigated in heterologous mammalian cells in order to elucidate the influence of the cellular background on these events. DNA fragments encoded by the gag and pol region were expressed in two rodent cell lines, LTK- and BHK. Both stably transfected cell lines continuously produce recombinant proteins which react with HIV-specific antisera. The GAG precursor and a 39-kDa proteolytic fragment thereof were the major recombinant proteins detected. Expression of the gag-pol region leads to the production of the GAG-POL precursor. Ribosomal frameshifting at the HIV-1 shifty sequence to a typical extent could be positively demonstrated by an enzyme assay. Despite the presence of the viral protease within the GAG-POL precursors, proteolytic processing of the HIV-derived polyproteins was extremely inefficient. The efficiency could not be enhanced by overexpression of the HIV-1 protease encoding region.

Adenocarcinoma of the small intestine arising from heterotopic gastric mucosa is extremely rare. In this report, we present the case of a 68-year-old woman who complained of abdominal pain, weight loss and subileus. Gross examination of resected small bowel revealed multiple flat polypous lesions with cysts in the ileal submucosa, one of which containing an ulcerated, stenosing tumour. On microscopic examination, an adenocarcinoma of the ileum arising from multifocal gastric heterotopia with secondary gastritis cystica profunda was diagnosed. Comparative genomic hybridization of the adenocarcinoma revealed chromosomal gains at 1q, 3q, 5p, 8q, 11p, 12p, 13q and losses at Xp, 4q, 8p, 10p, 14q, 17p, 20p, compatible with a high degree of genomic instability. peerReviewed