Insulin-like Growth factor-II Protein Research Articles

Interplay between microRNA-17-5p, insulin-like growth factor-II through binding protein-3 in hepatocellular carcinoma.

To investigate the effect of microRNA on insulin-like growth factor binding protein-3 (IGFBP-3) and hence on insulin-like growth factor-II (IGF-II) bioavailability in hepatocellular carcinoma (HCC). Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mirVana miRNA Isolation Kit. microRNA-17-5p (miR-17-5p) expression was mimicked and antagonized in HuH-7 cell lines using HiPerFect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cDNA followed by quantification of miR-17-5p and IGFBP-3 expression using TaqMan real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-II protein was measured in transfected HuH-7 cells using IGF-II ELISA kit. Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where miR-17-5p was extensively underexpressed in HCC tissues (P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients (P = 0.0041) compared to healthy donors. Forcing miR-17-5p expression in HuH-7 cell lines showed a significant downregulation of IGFBP-3 mRNA expression (P = 0.0267) and a significant increase in free IGF-II protein (P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of miR-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone (P = 0.0474). These data suggest that regulating IGF-II bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miRNAs.

Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed "pro" and "big" IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.

Chemical Synthesis of a Fluorescent IGF-II Analogue

Insulin-like growth factor II (IGF-II) is a protein with high structural and sequence similarity to insulin. Unlike insulin, it binds both the type 1 IGF receptor and the exon 11- isoform of the insulin receptor with high affinity. The overexpression and up regulation of IGF-II has been associated with the progression of various forms of cancer. The exact binding mechanism of IGF-II to its high affinity receptors is still not completely understood. Herein we describe the successful synthesis of a novel fluorescent IGF-II protein (F19Cou IGF-II), where residue 19 (phenylalanine) has been replaced by a fluorescent chromophore (coumaryl glycine). This novel coumaryl IGF-II analogue will be a useful tool for analysing the receptor interaction mechanisms in future studies.

Correlation between epigenetic alterations in the insulin growth factor-II gene and hepatocellular carcinoma

To investigate whether epigenetic alterations in the insulin-like growth factor-II (IGF-II) gene that cause differential transcription or expression are correlated with onset and severity of hepatocellular carcinoma (HCC). Patient-matched specimens of HCC, paracancerous, and non-cancerous tissues were collected from 40 primary liver cancer patients. Epigenetic alterations in the promoter (P3) sequence of the IGF-II gene were analyzed by methylation-specific PCR (MSP) and IGF-II transcription was measured by RT-PCR. IGF-II protein expression and clinicopathological features were assessed by immunohistochemistry and microscopic observation. The rate of IGF-II P3 methylation was significantly lower in HCC tissues (0%) than in paracancerous tissues (vs. 47.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 100%; x2 = 80.000, P less than 0.001). IGF-II mRNA expression was significantly higher in HCC tissues (100%) than in paracancerous tissues (vs. 52.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 80.000, P less than 0.001). IGF-II protein expression was significantly higher in HCC tissues (82.5%) than in paracancerous tissues (vs. 45.0%; x2 = 12.170, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 56.170, P less than 0.001). IGF-II overexpression in HCC was significantly associated with degree of differentiation, extent of infiltrated serosa, size of tumor, and HBV-positive infection status. Epigenetic alterations in the IGF-II gene regulate its transcription and expression and are closely associated with HCC development and progression.

Co-encapsulation of bioengineered IGF-II-producing cells and pancreatic islets: effect on beta-cell survival

Insulin-like growth factor-II (IGF-II) has been shown to promote pancreatic β-cell survival. We evaluated the effect of co-encapsulating islets and bioengineered IGF-II-producing cells on islet cell survival. IGF-II or green fast protein (GFP) genes were transferred into TM4 cells, and purified using a neomycin resistance gene, leading to pure cell cultures (TM4-IGF-II or TM4-GFP) with a stable overexpression of the transferred gene. Islets were co-encapsulated with TM4-IGF-II or TM4-GFP, or encapsulated alone in alginate microcapsules. Rat and mouse islet cell survival was studied in vitro and in vivo, respectively. After 12 days in culture, islet viability (dual staining, acridine orange/propidium iodide) was 83% with TM4-IGF-II, compared with 51% (P<0.05) and 41% (P<0.001) with TM4-GFP and islets alone, respectively. The study of islet necrotic centers and the evaluation of islet function, using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, yielded similar results. From 125 days after transplantation, more diabetic mice maintained normoglycemia when they were transplanted with islets co-encapsulated with TM4-IGF-II (4/7). A significant difference for the maintenance of normoglycemia was observed between recipients of islets co-encapsulated with TM4-IGF-II versus islets alone (P=0.023), or with TM4-GFP (P=0.048). In conclusion, the co-encapsulation of islets with bioengineered IGF-II-producing cells promotes islet cell survival.

Abstract 281: Insulin-like growth factor II differential activation of the IGF-1 and insulin receptors in African-American and Caucasian breast cancer tissues

Abstract African-American (AA) women with breast cancer are more likely to have advanced disease at diagnosis, higher risk of recurrence and poorer prognosis than Caucasian (CA) females. Insulin-like growth factor- II (IGF-II) is a potent mitogen that induces cell proliferation and survival signals through activation of the IGF-I (IGF-IR), insulin (INSR) and hybrid receptors. We hypothesize that differential expression of the IGF1R and INSR isoforms (IR-A, IR-B) between AA and CA women potentiates IGF-II mitogenic effects, thus contributing to the health disparity observed between these ethnic groups. We examined IGF-II, IGF1R and INSR isoforms protein expression and phosphorylation in paired breast tissue samples from AA and CA women by western blot analysis, immunohistochemistry and ELISA techniques. IGF-II expression was significantly higher in AA cell lines and tissue samples when compared to Caucasians. Furthermore, significantly increased expression of IGF1R was observed in AA normal tissues as compared to CA normal tissues, while IGF1R expression was similar between AA normal and malignant tissues. Moreover, AA tumor samples expressed significantly lower levels of IR-B protein than CA tumor samples. IGF1R, INSR, IRS-1 and Shc phosphorylation were significantly higher in AA tumor samples. We conclude that the differential expression and phosphorylation of IGF-II, IGF1R and INSR between AA and CA females coupled with differential activation of downstream signaling pathways may contribute to the increased risk of malignant transformation seen in young AA females, and the more aggressive breast cancer phenotype observed among AA breast cancer patients and represent, along with IGF-II, potential therapeutic targets against health disparities in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 281.

Insulin-like growth factor-II: a novel autocrine growth factor modulating the apoptosis and maturation of umbilical cord blood erythroid progenitors

To search a novel function of erythroid progenitor cells circulating as the major nucleated cell population in umbilical cord blood (CB) cells. Human CB-derived CD36(+) erythroid progenitors were subjected to cDNA microarray. Gene expression and biological property of CB-erythroid progenitors and adult peripheral blood (PB)-erythroid progenitors were compared by using real-time polymerase chain reaction (PCR) and serum-free culture system with erythropoietin (EPO). The microarray revealed 124-fold higher levels of insulin-like growth factor-II (IGF-II) gene expression in CB-CD36(+) erythroid progenitors than in stimulated lymphocytes of adult PB. Real-time PCR verified that IGF-II mRNA levels were highest in CB-CD36(+) erythroid progenitors compared to other CB- or adult PB-fractionated cells. When CB-CD36(+) erythroid progenitors were cultured with EPO in serum-free medium, anti-IGF-II-antibody (Ab) reduced the number of erythroid colonies. When CB- and adult PB-derived erythroid colony-forming cells (ECFCs) were cultured with interleukin-3, stem cell factor, and EPO, mRNA levels per cells of IGF-II peaked on day 12, but those of type 1 and type 2 receptors did not increase with ECFCs maturation. The maturation rate by IGF-II was higher in CB-ECFCs than in adult PB-ECFCs. The majority of CB-ECFCs expressed IGF-II protein. Anti-IGF-II-Ab, but not anti-IGF-I-Ab, reduced the number of CB-ECFCs in liquid culture with EPO. Anti-IGF-II-Ab accelerated apoptosis of ECFCs, assessed by dimethylthiazole tetrazolium bromide, bromodeoxyuridine, and flow cytometric analyses. ECFCs failed to attain full maturity in the presence of anti-IGF-II-Ab. These results suggest that IGF-II is produced by erythroid progenitors themselves, and has a crucial role in fetal erythropoiesis by modulating apoptosis and maturation in an autocrine fashion.

Expression of osteotropic growth factors and growth hormone receptor in a canine distraction osteogenesis model

Osteotropic growth factors play an important role in bone metabolism. Nevertheless, knowledge about their expression in relation to distraction osteogenesis remains limited. The aim of the present study was to determine the expression of growth hormone (GH), growth hormone receptor (GHR), insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), and bone morphogenetic protein 2 (BMP-2) in distraction-induced bone regeneration. Expression of these factors was assessed during the consolidation phase, comparing distraction osteogenesis with osteotomy-induced bone formation. Real-time PCR was performed as a semiquantitative measurement of mRNA, and the relative expression levels of these factors were determined. In addition, plasma GH profiles and plasma concentrations of IGF-I, IGF-II, and insulin-like growth factor-binding protein 4 and -6 (IGFBP-4 and -6) were measured to assess their potential systemic role during bone formation. Expression of GHR, IGF-I, and BMP-2 had significantly increased in comparison with the expression of these factors in mature bone. Expression of GHR was significantly higher in distraction-induced bone regenerate than in osteotomy-induced bone. No significant differences were found for the expression of IGF-I and BMP-2 between distraction and osteotomy. Plasma concentrations of GH, IGF-I, IGF-II, IGFBP-4, and IGFBP-6 did not demonstrate any significant differences between treatment groups and controls. Upregulation of GHR expression in distraction osteogenesis may enhance sensitivity to endogenous systemic GH and thus promote consolidation of the regenerated bone. Changes in the systemic osteotropic growth factors GH, IGF-I, IGF-II, IGFBP-4, and IGFBP-6 do not seem to be of importance during distraction osteogenesis.

Serum vascular endothelial growth factor C (VEGF-C) as a specific biomarker for advanced cervical cancer: Relationship to insulin-like growth factor II (IGF-II), IGF binding protein 3 (IGF-BP3) and VEGF-B

An early non-invasive diagnosis of cervical cancer and its metastasis can save lives. We have shown that serum IGF-II levels can be effectively used for a specific early diagnosis of cervical cancer. Here, we shall determine if serum levels of vascular endothelial growth factors B and C (VEGF-A [corrected] VEGF-C) associated with vasculogenic and lymphogenic metastasis may be used for an early diagnosis of advanced metastatic cervical cancer and compare these levels with those of the serum IGF-II and IGF-binding protein 3 (IGF-BP3). (a) Serum levels of IGF-II, IGF-BP3, VEGF-A [corrected] (VEGF(165)) and VEGF-C (ELISA kits) were determined in: 82 controls with normal Pap smears; 29 women with atypical squamous cells of undetermined significance (ASCUS) and normal cervical biopsy; 46 ASCUS and cervical intraepithelial neoplasia (CIN) on biopsy; 8 pre-therapy CIN-I; 23 successfully treated CIN-I; 75 persistent CIN-I; 14 CIN-II/III pre-therapy; 14 successfully treated CIN-II/III; 70 persistent CIN-II/III; 86 pre-therapy cervical cancer; 26 in early grades of cervical cancer; 21 in late grades of cervical cancer; 22 cervical cancer patients in remission; 50 persistent cervical cancer; 18 with ovarian cancer; and 57 with endometrial cancer. (b) Serial serum samples collected over 5 years in 5 women with progressing cervical cancer were also tested. (c) Serum and tissue VEGF-C were enumerated in 20 matched serum (ELISA) and tissue (semi-quantitative immunofluorescent antibody assay) samples from controls, early cervical cancer, late cervical cancer, ovarian cancer and endometrial cancer patients. Student's t test, chi-square analysis and linear regression analysis were used. (a) As anticipated, serum IGF-II levels were elevated as early as ASCUS with CIN on biopsy and continued to be elevated in CIN (all grades; pre-therapy and persistent) and cervical cancer (pre-therapy, early, late and persistent). Serum IGF-II levels were normal in ASCUS with normal biopsy, successfully treated CIN-I, II/III, cervical cancer as well as pre-therapy ovarian and endometrial cancers (therapy efficacy: P < 0.0001 by chi-square analysis). Serum IGF-BP3 showed a significant decrease with advancing disease. Serum VEGF-A [corrected] levels were the highest in pre-therapy, early, advanced and persistent cervical cancer, as well as in ovarian and endometrial cancers. Serum VEGF-C levels, on the other hand, were the highest in late and persistent cervical cancers, but not in ovarian or endometrial cancers. (b) In the 5 women with serial samples, the serum levels of the growth factors showed similar trends. (c) VEGF-C levels in serum and tissue were elevated in cervical cancers especially in advanced grades, while they were normal in serum and tissue from the controls and women with ovarian and endometrial cancers. There was a highly significant positive correlation between VEGF-C and IGF-II and a negative correlation between IGF-BP3 and VEGF-C (P < 0.0001). Serum IGF-II up-regulation is specific to cervical cancer and helps in the early diagnosis of malignant proliferation, while serum VEGF-C up-regulation appears to be a unique marker for an early diagnosis of cervical cancer metastasis. VEGF-C and IGF-II systems appear to be interrelated in cervical cancer, contributing to the early malignant cell proliferation and lympho-vascular metastasis. Serum IGF-BP3 and VEGF-A [corrected] appear to be common markers for all gynecological cancers.

The RNA-binding Protein IMP-3 Is a Translational Activator of Insulin-like Growth Factor II Leader-3 mRNA during Proliferation of Human K562 Leukemia Cells

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and beta-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or beta-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.

Targeted Knockdown of the RNA-binding Protein CRD-BP Promotes Cell Proliferation via an Insulin-like Growth Factor II-dependent Pathway in Human K562 Leukemia Cells

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.

Circulating levels of insulin-like growth factor-II and IGF-binding protein 3 in cervical cancer

Objective We aimed to further document that elevated levels of circulating insulin-like growth factor II (IGF-II) are associated with cervical cancer and to test the hypothesis that there may be an inverse association between IGF-II and IGF-binding protein 3 (IGF-BP3). Method Serum IGF-II and IGF-BP3 levels were measured, using ELISA kits (Diagnostic Systems Laboratories), in 23 controls; 16 ASC-US with normal biopsies; 14 ASC-US with advanced CIN; 2 pretherapy CIN-I; 8 successfully treated CIN-I; 24 persistent CIN I; 14 pretherapy CIN II/III; 10 posttherapy CIN II/III with normal biopsies; 18 persistent CIN-II/III; 7 with pretherapy cervical cancer; 19 with posttherapy cervical cancer under remission; 15 with posttherapy persistent/recurrent cervical cancer; 10 with persistent ovarian or endometrial cancer; and 3 with endometrial or vulvar with cervical cancer. Student's t test and linear regression analysis were used. Results Compared to controls (493 ± 90 ng/ml) and women with other gynecological cancers, serum IGF-II levels were significantly increased in women with ASC-US, with advanced CIN on biopsy ( P < 0.0001), persistent CIN-I (993 ± 262 ng/ml; P < 0.0001), pretherapy advanced CIN (1086 ± 240; P < 0.0001), pretherapy cervical cancer patients (1746 ± 318 ng/ml; P < 0.0001) and posttherapy persistent/recurrent CIN (1094 ± 300; P < 0.0001); and cervical cancer (1395 ± 189; P < 0.0001). After therapy, the IGF-II levels returned to normal in both CIN and cervical cancer patients under remission. Elevated serum IGF-II levels had 100% sensitivity and 87% specificity for cervical cancer and 81% sensitivity and 82% specificity for CIN. The levels of IGF-BP3 were significantly reduced in women with CIN before and after therapy ( P < 0.0001) and in cervical cancer patients before and after therapy ( P < 0.001). There was an inverse relationship between serum IGF-II and BP-3 levels ( P < 0.01). Decreased serum IGF-BP3 levels had a sensitivity of 72% and specificity of 75% for cervical cancer and 81% sensitivity and 83% specificity for CIN. When both markers were considered together the sensitivity was 72% and specificity 84% for cervical cancer, while for CIN, the sensitivity was 57% and specificity 81%. Conclusion Serum IGF-II may be a reliable marker for early diagnosis and monitoring therapy efficacy (sensitivity and specificity of 100% versus normal controls), while IGF-BP3 levels can be reliably used to predict prognosis.

Expression of insulin-like growth factor-II and insulin-like growth factor binding protein-1 in the placental basal plate from pre-eclamptic pregnancies

Objectives: To determine whether expressions of insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) are altered in pre-eclamptic placenta and to elucidate the possible relationship between their expressions and a mechanism for inadequate trophoblast invasion in pre-eclampsia. Methods: Placental tissues were obtained at cesarean delivery from five normotensive, nine mild pre-eclamptic and five severe pre-eclamptic women at 33–39 completed weeks of gestation. After total ribonucleic acid was extracted, reverse transcriptase-polymerase chain reaction was performed to determine IGF-II and IGFBP-1 mRNA expression. Product bands were quantitated by scanning densitometry and results were expressed as ratio of cytokines/β-actin. Western blot analysis was also done to determine IGF-II and IGFBP-1 protein expression. Statistical analysis was determined by Kruskal–Wallis analysis of variance with the Scheffe multiple post-hoc test. Results: The IGF-II mRNA levels of mild and severe pre-eclamptic placenta were significantly lower than that of uncomplicated placenta (P<0.005, P<0.001, respectively), with the level of severe pre-eclamptic placenta being significantly lower than that of mild pre-eclamptic placenta (P<0.05). As for the IGF-II protein expression, a significant decrease was found among the three groups (P<0.001), correlating with the IGF-II mRNA results. However, the mean IGFBP-1 mRNA levels of mild and severe pre-eclamptic placenta were significantly higher than that of uncomplicated placenta (P<0.05, P<0.005, respectively), with the level of severe pre-eclamptic placenta being significantly raised compared with that of mild pre-eclamptic placenta (P<0.05). Finally, a significant increase of IGFBP-1 protein expression was noted among the three groups (P<0.001), correlating with the IGFBP-1 mRNA results. Conclusions: This study suggests that IGF-II and IGFBP-1 might be associated with the impaired trophoblastic invasion that may lead to pathogenesis of pre-eclampsia.

Kinetics and regulation of site-specific endonucleolytic cleavage of human IGF-II mRNAs.

Human insulin-like growth factor II (IGF-II) mRNA can be cleaved at a specific site in its 4 kb long 3'-UTR. This yields a stable 3' cleavage product of 1.8 kb consisting of a 3'-UTR and a poly(A) tail and an unstable 5' cleavage product containing the IGF-II coding region. After cleavage, the 5' cleavage product is targeted to rapid degradation and consequently is no longer involved in IGF-II protein synthesis. Cleavage is therefore thought to provide an additional way to control IGF-II gene expression. In this paper the kinetics and the efficiency of cleavage of IGF-II mRNAs are examined. The cleavage efficiency of IGF-II mRNAs carrying four different leaders (L1-L4) is enhanced in the highly structured leaders L1 and L3. Additionally, under standard cell culture conditions cleavage is a slow process that only plays a limited role in destabilisation and translation of the IGF-II mRNAs. However, in human Hep3B cells and CaCo2 cells which express IGF-II endogenously, cleavage is upregulated 3-5-fold at high cell densities. Regulated endonucleolytic cleavage of IGF-II mRNAs is restricted to cells in which IGF-II expression is related to specific cell processes.

Study on the Expression of Insulin-like Growth Factor II (IGF- II) in Hepatocellular Carcinoma Cells and Developing Rat Embryos.

The insulin-like growth factor II (IGF-II) gene expresses a family of transcripts in embryonic/fetal tissue, and also highly was expressed during hepatocellular carcinogenesis. In this study, we showed that IGF-II mRNA and protein levels are detected in rat embryo, HepG2 human hepatoma cells and Chang liver cells. This study included sections of rat embryos 7~17 days post coitum (d.p.c), HepG2 cells and Chang liver cells. Using immunohistochemistry, Northern blotting and Western blotting, we observed the expression of IGF-II in the rat embryo, HepG2 cells and Chang liver cells. We localized IGF-II gene products in sections of rat embryo 7~17 d.p.c by performing immunohistochemistry. The IGF-II was mainly expressed in the proximal endoderm and ectoplacental cone between 7 and 9 d.p.c. At 10 d.p.c. the expression was localized at the heart primodium as well as the proximal endoderm, and at 11 d.p.c. the IGF-II was expressed in the liver and heart. After 12 d.p.c. and 14 d.p.c., the expression was also detected in the brain, muscle and bone, and head mesenchyme, respectively. While the expression of IGF-II protein was not detected in the normal adult liver, intense staining was detected in the heart, liver and choroids plexus at 17 d.p.c. These results suggest that IGF-II may act as an oncofetal protein during hepatocellular carcinogenesis and embryogenesis.

Ionic Products of Bioactive Glass Dissolution Increase Proliferation of Human Osteoblasts and Induce Insulin-like Growth Factor II mRNA Expression and Protein Synthesis

Bioglass 45S5 is an osteoproductive material, which resorbs by releasing its constitutive ions into solution. Treatment with the ionic products of Bioglass 45S5 dissolution in DMEM for 4 days increased human osteoblast proliferation to 155% of control. Two days after treatment, differential gene expression was analyzed by cDNA microarrays. Expression of a potent osteoblast mitogenic growth factor, insulin-like growth factor II (IGF-II), was increased to 290%. Additionally, there was a 168% increase in the concentration of unbound IGF-II protein in the conditioned media of treated osteoblasts. Expression levels of IGFBP-3, an IGF-II carrier protein, metalloproteinase-2 and cathepsin-D were also increased to 200, 340, and 310% of control levels, respectively. Metalloproteinase-2 and cathepsin-D are proteases that cleave IGF-II from its carrier proteins, resulting in the release of the unbound biologically active IGF-II. We suggest that the stimulatory effect of the ionic products of Bioglass 45S5 dissolution on osteoblast proliferation may be mediated by IGF-II.

Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes.

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in beta cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in beta-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease.

Neoplasms Causing Nonhyperinsulinemic Hypoglycemia.

Non-islet cell tumor hypoglycemia (NICTH) is uncommon. Many of the tumors associated with NICTH are mesenchymal tumors, although carcinomas are also involved in some cases. High serum levels of insulin-like growth factor II (IGF-II) have been associated with NICTH. Analysis of 4 pituitary tumors, 2 adrenocortical tumors, 42 solitary fibrous tumors, and 23 other mesenchymal tumors for IGF-II protein and mRNA showed that most mesenchymal tumors expressed IGF-II protein and mRNA, although only 4 of 48 patients had associated hypoglycemia. Tumor size was related to IGF-II production. Tumors less than 5 cm were usually negative for IGF-II mRNA, whereas 92.3% of tumors greater than 9 cm were positive for IGF-II mRNA. These results show that IGF-II mRNA and protein can be readily detected in many tumors, even when the tumors are not associated with clinical hypoglycemia. The expression and production of this growth factor cannot accurately predict patients with clinical evidence of hypoglycemia.

Overexpression of insulin-like growth factor II (IGFII) in ZR-75-1 human breast cancer cells: higher threshold levels of receptor (IGFIR) are required for a proliferative response than for effects on specific gene expression.

Previous transfection experiments using a zinc-inducible expression vector have shown that overexpression of insulin-like growth factor II (IGFII) in MCF7 human breast cancer cells can reduce dependence on oestrogen for cell growth in vitro (DALY RJ, HARRIS WH, WANG DY, DARBRE PD. (1991) Cell Growth Differentiation 2, 457-464.). Parallel transfections now performed into another oestrogen-dependent human breast cancer cell line (ZR-75-1) yielded three clones of transfected ZR-75-1 cells that produced levels of zinc-inducible IGFII mRNA and secreted mature IGFII protein similar to those found in the transfected MCF7 cells. However, unlike in MCF7 cells, no resulting effects were found on cell growth in the ZR-75-1 clones, even though the ZR-75-1 clones possessed receptors capable of binding 125I-IGFI and showed a growth response to exogenously added IGFII. Medium conditioned by the ZR-75-1 clones could stimulate growth of untransfected MCF7 cells, indicating that the secreted IGFII protein was bioactive. Furthermore, zinc-induced IGFII was capable of increasing both pS2 mRNA levels and CAT activity from a transiently transfected AP1-CAT gene in the ZR-75-1 clones. Constitutive co-overexpression of the protein processing enzyme PC2 resulted in reduced levels of large forms of zinc-inducible IGFII, but zinc treatment still produced no effect on cell growth rate. Finally, however, constitutive co-overexpression of the type I IGF receptor (IGFIR) did result in zinc-inducible increased basal cell growth and reduced dependence on oestrogen for cell growth. These results demonstrate that while overexpression of IGFII per se was sufficient to deregulate MCF7 cell growth, the ZR-75-1 cells are limited in their proliferative response by their intrinsic receptor levels. However, although the proliferative response was limited, molecular responses (expression of pS2 and AP1-CAT) were not limited, indicating that different cellular responses can have different threshold receptor level requirements.

Insulin-like growth factor II (IGF-II) secreted from HepG2 human hepatocellular carcinoma cells shows angiogenic activity

Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. Since insulin-like growth factor II (IGF-II) has been reported to play a significant role in liver regeneration and hepatocarcinogenesis, we initially examined its angiogenic effect on the chorioallantoic membrane (CAM) of 9-day-old chick embryos. We also investigated whether IGF-II secreted from HepG2 human hepatocellular carcinoma cells induces vascularization using the chick embryo CAM. We found that the concentrated conditioned media (CCM) of HepG2 cell culture induced angiogenesis on the CAM. We also identified IGF-II protein in the CCM from HepG2 cells by Western blot analysis. However, CCM from Chang liver cells, which are normal human liver cells and were free of IGF-II, did not induce angiogenesis in the CAM. These results suggest that IGF-II secreted from hepatocellular carcinoma cells may act as an angiogenic factor for the hypervascularization of HCC.