The expression and role of insulin-like growth factor II in malignant hemangiopericytomas

Hemangiopericytoma is a rare soft tissue tumor originating from contractile pericapillary pericytes. To address the issue of molecular genetic events that participate in genesis and progression of hemangiopericytoma we analyzed insulin-like growth factor (IGF) II and IGF I receptor in 29 tumors collected from a human tumor bank network. Seven of these tumors were associated with severe hypoglycemia; six were retroperitoneal and one was located in the leg. Of 22 tumors tested 12 (54.5%) exhibited IGF II mRNA, while almost 90% (17 of 19) of hemangiopericytomas exhibited IGF I receptor mRNA. Sera from some patients whose tumors expressed IGF II mRNA contained elevated levels of IGF II. Removal of the tumor eliminated most of the IGF II immunoreactivity from the sera. The potential role of IGF II as a growth-promoting factor was examined on three malignant primary hemangiopericytoma cell cultures. Extracellular addition of IGF II significantly enhanced cell proliferation in a dose-dependent manner. Antisense oligodeoxynucleotides that specifically inhibit IGF II mRNA, at a concentration of 40 or 80 micrograms/ml, inhibited the growth of hemangiopericytoma cells significantly, by 40%. Simultaneous administration of antisense deoxyoligonucleotides to both IGF II and IGF I receptor inhibited tumor cell proliferation by even 80%. Our data suggest that tumor cells produce IGF II, and that this in turn stimulates their proliferation by autocrine mechanisms.     

Parental allele specific methylation of the human insulin-like growth factor II gene and Beckwith-Wiedemann syndrome.

In an attempt to elucidate the role of methylation in parental imprinting at the IGF-II gene locus, for which imprinting has already been described in the mouse, we undertook an allele specific methylation study of the human IGF-II gene (mapped to 11p15.5) in a control population and in patients with Beckwith-Wiedemann syndrome. In control leucocyte DNA (16 unrelated adults and eight families), the maternal allele of the IGF-II gene was specifically hypomethylated, whereas no such allele specific methylation was found for either the insulin or the calcitonin genes which are located in 11p15.5 and 11p15.1, respectively. Furthermore, the IGF-II gene specific hypomethylation was localised on the 5' portion of exon 9. In the patients with Beckwith-Wiedemann syndrome in which the IGF-II gene is thought to be involved and where paternal isodisomy has been described, hypomethylation of the maternal allele was conserved in leucocyte DNA, but abnormal methylation was detected in malformed tissues where the paternal allele was also demethylated. Some specific mechanism linked to methylation therefore seems to be involved in the pathogenesis of Beckwith-Wiedemann syndrome.     

Genetic changes and expression of the mannose 6-phosphate/insulin-like growth factor II receptor gene in human hepatitis B virus-associated hepatocellular carcinoma

It was reported that 60-70% of hepatitis B virus (HBV)-negative hepatocellular carcinoma (HCC) had loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus and this gene was mutated in 55% of these patients with LOH. In this study, genomic DNA from 29 pairs of HBV-positive HCC and corresponding non-tumor tissues was used to analyze LOH at the M6P/IGF2R locus and single deoxyguanosine deletion in this gene by PCR. Total RNA from 19 of the 29 patients was utilized to determine a 192 bp insert in the M6P/IGF2R mRNA and expression of this gene by RT-PCR. Twenty-eight of 29 (97%) HBV-positive HCC were found to be informative at the M6P/IGF2R locus but LOH at this region was only detected in 4/28 (14%) informative patients. Neither single deoxyguanosine deletion in this gene nor 192 bp insert in its mRNA occurred in these patients. Compared with corresponding non-tumor tissues, expression of the M6P/IGF2R mRNA was decreased in 13/19 (68%) HBV-positive HCC tissues, suggesting that M6P/IGF2R may be involved in HBV-associated hepatocarcinogenesis by the regulation of its expression level. In the development of HBV-associated HCC, M6P/IGF2R mutation may not be a major agent.     

Cloning and expression of human insulin-like growth factor binding protein 3.

Insulin-like growth factors (IGFs) found in plasma and other body fluids circulate in association with specific binding proteins. We report here the cloning and the nucleotide sequence of cDNAs for the growth-hormone-dependent acid-stable IGF binding protein, hIGF-BP3. The derived protein begins with a putative 27-amino acid signal peptide followed by 264 residues of the mature polypeptide. The predicted sequence contains three potential N-linked glycosylation sites and shares two region of homology with the low-molecular-weight non-growth-hormone-dependent binding proteins BP-1 and BP-2. The protein contains 18 cysteine residues clustered in the amino and carboxy termini. Chinese Hamster ovary cells transfected with this cDNA secrete a 43-45 kD protein doublet, which bound IGF. The expressed IGF-binding protein is indistinguishable from the native BP-3 found in human plasma.     

大肠癌中胰岛素样生长因子2基因的印迹状态和表达

目的：研究胰岛素样生长因子2（insulin-like growth factor 2,IGF2)基因的印迹状态和表达与大肠癌的关系，为研究大肠癌的发生机理提供线索。方法：用逆转录－聚合酶链反应半定量检测IGF2的表达量，比较其在大肠癌及癌旁组织中有无差异，用限制性片段长度多态检测IGF2的印迹状态，分析印迹状态、表达量与大肠癌的关系。结果：82．4％（28／34）大肠癌有IGF2的表达增加，IGF2的表达量在肿瘤组织与癌旁组织中差异有显著性（P＜0．01，t=3.01)。IGF2在87．5％（14／16）大肠癌组织中发生了印迹丢失，但其相对应的癌旁组织也有71．4％（10／14）存在IGF2的印迹丢失。结论：IGF2的表达增加是大肠癌发生的相关因素，IGF2的印迹丢失可能是大肠癌发生的前期表现。     

Microdialysis of insulin-like growth factor-I in human muscle

The determination of the insulin like growth factor I (IGF-I) concentration in the interstitial milieu is of outstanding importance to explore its autocrine/paracrine function. We previously reported a method to calibrate microdialysis probes for lactate and glucose (slope method). In the present study, we investigated the ability of our method to determine the concentration of larger molecules, such as IGF-I. We observed in vitro a close linear relationship (r=0.86, P<0.0005) between the recoveries of lactate (RecLac) and of IGF-I (RecIGF-I), giving access to the measurement of IGF-I with the same accuracy as the one previously found for lactate and glucose. In seven human volunteers, we calibrated each probe with the slope method: we first determined in vivo for every probe the specific RecLac/loss ethanol relationship and thereafter, using that relationship, we deduced RecLac from the loss ethanol value measured in every dialysate. This allowed calculation of RecIGF-I from the calculated RecLac value and the in vitro RecLac/RecIGF-I relationship, and finally free IGF-I concentration in muscle interstitial fluid. The mean free IGF-I interstitial concentration was 6.8±3.2 ng/ml while the mean plasma concentration was 0.4±0.2 ng/ml. This large gradient from interstitium to plasma for free IGF-I could be related to the local action of this growth factor.     

Cellular pattern of type-I insulin-like growth factor receptor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II.

Insulin-like growth factors have a number of potent trophic effects on cultured neural tissue and most if not all of these effects appear to be mediated by the type-I insulin-like growth factor receptor. In order to establish the identity of cell types expressing this receptor in the rat central nervous system during development and maturity, we have used in situ hybridization to map sites of type-I insulin-like growth factor receptor mRNA synthesis in the developing and adult rat brain. In order to identify possible local sources of peptide ligands for this receptor, we have also mapped the sites of insulin-like growth factors I and II mRNA synthesis in parallel brain sections. From early development onward, there is a uniform and stable pattern of type-I insulin-like growth factor receptor gene expression in all neuroepithelial cell lineages, in which regional variations reflect primarily differences in cell density. In addition to this generalized pattern, during late postnatal development, high levels of type-I insulin-like growth factor receptor gene expression are found in specific sets of sensory and cerebellar projection neurons in conjunction with abundant insulin-like growth factor-I gene expression in these same neurons. While insulin-like growth factor-I expression is confined to the principal neurons in each system, receptor mRNA is also found in local interneurons. In the cerebral cortex and hippocampal formation, type-I insulin-like growth factor receptor mRNA and insulin-like growth factor-I are concentrated in different cell populations: receptor mRNA is abundant in pyramidal cells in Ammon's horn, in granule cells in the dentate gyrus, and in pyramidal cells in lamina VI of the cerebral cortex. Insulin-like growth factor-I mRNA is found in isolated medium- to large-sized cells which are rather irregularly distributed throughout the hippocampus and isocortex. In the hypothalamus, receptor mRNA is concentrated in the suprachiasmatic nucleus but is in low abundance elsewhere, including the median eminence, while insulin-like growth factor-I mRNA is not detected in this region at all. Type-I insulin-like growth factor receptor and insulin-like growth factor-II mRNAs are both abundant in choroid plexus, meninges and vascular sheaths from early development to maturity, but insulin-like growth factor-II mRNA is not detected in cells of neuroepithelial origin at any stage of development. This study provides evidence for two fundamentally different patterns of gene expression for the brain type-I insulin-like growth factor receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

重组人截短型胰岛素样生长因子1在大肠杆菌中的高效表达

目的：建立人截短型胰岛素生长因子１的原核高效表达系统。方法：利用基因重组技术将人人截短胰岛素样生长因子１「Ｄｅｓ（１－３）ＩＧＦ１」的ｃＤＮＡ片段克隆到融合蛋白表达载体ｐＭＴＹ４中,用离子交换层析法纯化蛋白并经ＳＤＳ聚丙烯酰胺凝胶电泳,放射免疫检测,Ｎ－末端前１６位氨基酸序列测定及生物活性检测等方法对所获蛋白进行了鉴定。结果;获得含人Ｄｅｓ（１－３）ＩＧＦ１基因的重组质粒,在大肠杆菌中高效表达出含     

Alterations in insulin-like growth factor-1 gene and protein expression and type 1 insulin-like growth factor receptors in the brains of ageing rats

Ageing in mammals is characterized by a decline in plasma levels of insulin-like growth factor-1 that appears to contribute to both structural and functional changes in a number of tissues. Although insulin-like growth factor-1 has been shown to provide trophic support for neurons and administration of insulin-like growth factor-1 to ageing animals reverses some aspects of brain ageing, age-related changes in insulin-like growth factor-1 or type 1 insulin-like growth factor receptors in brain have not been well documented. In this series of studies, insulin-like growth factor-1 messenger RNA and protein concentrations, and type 1 insulin-like growth factor receptor levels were analysed in young (three to four- and 10-12-month-old), middle-aged (19-20-month-old) and old (29-32-month-old) Fisher 344 x Brown Norway rats. Localization of insulin-like growth factor-1 messenger RNA throughout the lifespan revealed that expression was greatest in arteries, arterioles, and arteriolar anastomoses with greater than 80% of these vessels producing insulin-like growth factor-1 messenger RNA. High levels of expression were also noted in the meninges. No age-related changes were detected by either in situ hybridization or quantitative dot blot analysis of cortical tissue. However, analysis of insulin-like growth factor-1 protein levels in cortex analysed after saline perfusion indicated a 36.5% decrease between 11 and 32 months-of-age (P<0.05). Similarly, analysis of type 1 insulin-like growth factor receptor messenger RNA revealed no changes with age but levels of type 1 insulin-like growth factor receptors indicated a substantial decrease with age (31% in hippocampus and 20.8 and 27.3% in cortical layers II/III and V/VI, respectively). Our results indicate that (i) vasculature and meninges are an important source of insulin-like growth factor-1 for the brain and that expression continues throughout life, (ii) there are no changes in insulin-like growth factor-1 gene expression with age but insulin-like growth factor-1 protein levels decrease suggesting that translational deficiencies or deficits in the transport of insulin-like growth factor-1 through the blood-brain barrier contribute to the decline in brain insulin-like growth factor-1 with age, and (iii) type 1 insulin-like growth factor receptor messenger RNA is unchanged with age but type 1 insulin-like growth factor receptors decrease in several brain regions. We conclude that significant perturbations occur in the insulin-like growth factor-1 axis with age. Since other studies suggest that i.c.v. administration of insulin-like growth factor-1 reverses functional and cognitive deficiencies with age, alterations within the insulin-like growth factor-1 axis may be an important contributing factor in brain ageing.     

Analysis of insulin-like growth factors and insulin-like growth factor I receptor expression in renal cell carcinoma

The expression of insulin-like growth factors I (IGF-I) and II (IGF-II) and insulin-like growth factor-I receptor (IGF-IR) was studied in 137 clear cell, 23 chromophobe, and 20 papillary renal cell carcinomas (RCCs) using a tissue microarray technique. IGF-I immunoreactivity was detected in 110 (82.1%) of 134 clear cell, 8 (36%) of 22 chromophobe, and 3 (15%) of 20 papillary RCCs (P < .001). IGF-IR immunoreactivity was detected in 39 (29.5%) of 132 clear cell, 9 (41%) of 22 chromophobe, and 19 (95%) of 20 papillary RCCs (P < .001). In contrast, all tumors lacked IGF-II expression. Expression of IGF-I and IGF-IR was not related to tumor stage, grade, or prognosis. The IGF system is expressed differentially among different tumor types. The expression of IGF-I together with its receptor, IGF-IR, provides evidence for the existence of an autocrine-paracrine loop of tumor cell stimulation in RCC and makes this type of cancer a candidate for therapeutic strategies aimed to interfere with the IGF pathway.     

Activation-dependent expression of the insulin-like growth factor binding protein-2 in human lymphocytes.

The expression of the insulin-like growth factor binding protein-2 (IGFBP-2) was assayed in mononuclear cells originating from different organs of the immune system. All mononuclear cells studied did express IGFBP-2, but the expression level was found to be dependent on the cell type and origin of the cell. T cells showed a higher expression of IGFBP-2 mRNA than did B cells, and CD34+ stem cells expressed IGFBP-2 mRNA at a high level. Expression was highest in bone marrow and thymus. Stimulation of peripheral mononuclear cells resulted in a marked increase of IGFBP-2 mRNA and also intracellular IGFBP-2, as analysed by fluorescence staining. This increase parallels the increase of other known T-cell activation markers. Furthermore, the increase of intracellular IGFBP-2 seems to precede T-cell blast formation and all T cells in active phases of the cell cycle have high levels of IGFBP-2. Our results provide a basis for further investigations on the contribution of the IGF-system to the regulation of T-cell proliferation and differentiation. IGFBP-2, in particular, may have an important influence in the regulation of T-cell activation and proliferation.     

Modulation of extracellular matrix gene expression by heparin and endothelial cell growth factor in human smooth muscle cells

We have previously shown that human vascular smooth muscle cells grown for several passages in the presence of heparin and endothelial cell growth factor (ECGF) exhibit profound alterations in the synthesis of extracellular matrix proteins. In the present study, we demonstrate that the mRNA steady-state levels for various matrix macromolecules were altered in the presence of heparin and ECGF, but not ECGF alone. Specifically, the expression of types I and IV collagens, fibronectin, and decorin proteoglycan genes were markedly inhibited, whereas that of versican proteoglycan and beta-actin genes were unaffected. The effects were fully reversible. The suppression of the collagen gene expression was related to decreased collagen production by cells incubated with heparin and ECGF. Thus, heparin and ECGF synergistically elicit a coordinate and selective inhibition of matrix gene expression in human smooth muscle cells.     

Regenerating skeletal muscle cells express insulin-like growth factor I

The expression of the trophic peptide insulin-like growth factor I (IGF-I; somatomedin C) was investigated in the regenerating soleus muscle of mice after injury by the snake venom taipoxin. No specific IGF-I immunoreactivity was observed in muscle cells during control conditions. Within 2 days after taipoxin injection, IGF-I immunoreactivity could be demonstrated in activated satellite cells. Myoblasts and myotubes expressed high IGF-I immunoreactivity. The IGF-I immunoreactivity was strictly cytoplasmatic and obviously associated with polyribosomes. No vesicular or membraneous IGF-I immunoreactivity could be demonstrated. It is concluded that IGF-I is synthesized in myogenic cells during skeletal muscle regeneration. It is suggested that IGF-I exerts its effects on skeletal muscle mainly by autocrine mechanisms.     

Basic fibroblast growth factor regulates type I collagen and collagenase gene expression in human smooth muscle cells.

Basic fibroblast growth factor (bFGF) is a multifunctional peptide well known for angiogenic, neurotropic, and mesoderm-inducing effects. In the present study, we have investigated the effects of bFGF on collagen and collagenase gene expression in human iliac arterial smooth muscle cells. We report that bFGF inhibits type I collagen gene expression and collagen biosynthesis, with concomitant stimulation of collagenase gene expression. The smooth muscle cells incubated with human recombinant bFGF decreased the mRNA steady state levels of pro-alpha 1(I) type I collagen by as much as 72%. [3H]Hydroxyproline synthesis was also suppressed by 59% compared with untreated control cultures. Indirect immunofluorescence confirmed corresponding changes at the protein level. In contrast to the down-regulation of type I collagen gene expression, collagenase gene expression was found to be up-regulated severalfold by bFGF. The data suggest that bFGF is capable of regulating collagen and collagenase gene expression divergently in human smooth muscle cells and that the effects appear to be mediated at a pretranslational level.     

Immunoreactive insulin-like growth factor I in human follicular fluid

Recent studies in laboratory animals suggest that insulin-likegrowth factor I (IGF-I) plays an important role in the regulationof granulosa cell function. The purpose of the present studywas to investigate the presence of immunoreactive IGF-I in humanfollicular fluid (FF) and compare the levels of follicular IGF-I(64 follicles) with those detectable in serum (n= 19) in hyperstimulatedcycles from 25 infertile patients. Also, the FF IGF-I levelswere correlated to corresponding follicular volume (n= 62) andoocyte maturation (n= 37). Levels of IGF-I were determined usinga specific radioimmunoassay after acidification and extractionby reversed phase chromatography. Levels of IGF-I in serum weresignificantly higher than those in FF (37.1± 10.1 versus24.0± 9.3 nmol/I, n= 19, P< 0.001). A positive correlationwas found between follicular and serum IGF-I concentrations(r= 0.73). No significant differences were found in FF IGF-Ilevels derived from follicles of different size or from follicleshaving oocytes with different grades of maturation. These dataindicate that immunoreactive IGF-I is present in human FF innanomolar concentrations and that FF IGF-I levels correlatewith those detectable in serum. The source of FF IGF-I and itsregulatory role in humans remains to be elucidated.     

Impaired muscle growth and response to insulin-like growth factor 1 in dysferlin-mediated muscular dystrophy

Loss-of-function mutations in dysferlin cause muscular dystrophy, and dysferlin has been implicated in resealing membrane disruption in myofibers. Given the importance of membrane fusion in many aspects of muscle function, we studied the role of dysferlin in muscle growth. We found that dysferlin null myoblasts have a defect in myoblast-myotube fusion, resulting in smaller myotubes in culture. In vivo, dysferlin null muscle was found to have mislocalized nuclei and vacuolation. We found that myoblasts isolated from dysferlin null mice accumulate enlarged, lysosomal-associated membrane protein 2 (LAMP2)-positive lysosomes. Dysferlin null myoblasts accumulate transferrin-488, reflecting abnormal vesicular trafficking. Additionally, dysferlin null myoblasts display abnormal trafficking of the insulin-like growth factor (IGF) receptor, where the receptor is shuttled to LAMP2-positive lysosomes. We studied growth, in vivo, by infusing mice with the growth stimulant IGF1. Control IGF1-treated mice increased myofiber diameter by 30% as expected, whereas dysferlin null muscles had no response to IGF1, indicating a defect in myofiber growth. We also noted that dysferlin null fibroblasts also accumulate acidic vesicles, IGF receptor and transferrin, indicating that dysferlin is important for nonmuscle vesicular trafficking. These data implicate dysferlin in multiple membrane fusion events within the cell and suggest multiple pathways by which loss of dysferlin contributes to muscle disease.