The Role of Glycosyltransferases in Colorectal Cancer.

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), ß1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-ß) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell-cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.

Insulin-like growth factor binding protein 3 gene of golden pompano (TroIGFBP3) promotes antimicrobial immune defense.

Insulin-like growth factor binding protein 3 (IGFBP3), an important member of the IGFBP family, plays an important biological role in regulating cellular proliferation, differentiation, growth, apoptosis, and innate immunity. However, studies concerning IGFBP3 in teleosts are very limited and IGFBP3 function remains unclear. In this study, we conducted both in vivo and in vitro functional analyses of an IGFBP3 (TroIGFBP3) from the teleost fish golden pompano (Trachinotus ovatus). TroIGFBP3 is composed of 286 amino acid residues and shares a high amino acid sequence similarity (50.18%-93.71%) with other IGFBP3 sequences in humans and teleosts. TroIGFBP3 was widely distributed in various tissues, with the highest expression in the liver. TroIGFBP3 expression was significantly upregulated following Vibrio harveyi infection. The results of in vitro assays showed that TroIGFBP3 could stimulate macrophage activation and promote peripheral blood leukocytes (PBLs) proliferation. Meanwhile, TroIGFBP3 overexpression significantly inhibited bacterial infection in fish tissues, whereas TroIGFBP3 knockdown resulted in increased bacterial dissemination and colonization in golden pompano tissues in vivo. Furthermore, recombinant TroIGFBP3 could inhibit cellular proliferation and promote apoptosis of mouse tumor cells. Taken together, these results indicated that TroIGFBP3 plays a significant role in innate antibacterial immunity and provides a theoretical foundation for investigating the function of IGFBP3 in fish immune response.

Protective Effects of cis-2-Dodecenoic Acid in an Experimental Mouse Model of Vaginal Candidiasis.

OBJECTIVE: To evaluate the efficacy of cis-2-dodecenoic acid (BDSF) in the treatment and prevention of vaginal candidiasis in vivo. METHODS: The activities of different concentrations of BDSF against the virulence factors of Candida albicans (C. albicans) were determined in vitro. An experimental mouse model of Candida vaginitis was treated with 250 µmol/L BDSF. Treatment efficiency was evaluated in accordance with vaginal fungal burden and inflammation symptoms. RESULTS: In vitro experiments indicated that BDSF attenuated the adhesion and damage of C. albicans to epithelial cells by decreasing phospholipase secretion and blocking filament formation. Treatment with 30 µmol/L BDSF reduced the adhesion and damage of C. albicans to epithelial cells by 36.9% and 42.3%, respectively. Treatment with 200 µmol/L BDSF completely inhibited phospholipase activity. In vivo mouse experiments demonstrated that BDSF could effectively eliminate vaginal infection and relieve inflammatory symptoms. Four days of treatment with 250 µmol/L BDSF reduced vaginal fungal loads by 6-fold and depressed inflammation. Moreover, BDSF treatment decreased the expression levels of the inflammatory chemokine-associated genes MCP-1 and IGFBP3 by 2.5- and 2-fold, respectively. CONCLUSION: BDSF is a novel alternative drug that can efficiently control vaginal candidiasis by inhibiting the virulence factors of C. albicans.

Enhancement of mammary tumour growth by IGFBP-3 involves impaired T cell accumulation.

Epidemiological studies show an association between obesity and poor breast cancer prognosis. We previously demonstrated that global IGFBP-3 deficiency, in IGFBP-3-null mice, resulted in a 50% reduction in mammary tumour growth over 3 weeks relative to tumours in wild-type (WT) C57BL/6 mice. This growth reduction was ameliorated by high fat feeding-induced obesity. This study aimed to examine how IGFBP-3 promotes tumour growth by influencing the immune tumour microenvironment in healthy and obese mice. Syngeneic EO771 cells, which lack detectable IGFBP-3 expression, were grown as orthotopic tumours in WT and IGFBP-3-null C57BL/6 mice placed on either a control chow or a high-fat diet (HFD), and examined by quantitative PCR and immunohistochemistry. In WT mice, increased stromal expression of IGFBP-3 was positively associated with tumour growth, supporting the hypothesis that IGFBP-3 in the microenvironment promotes tumour progression. Examining markers of immune cell subsets, gene expression of Ifng, Cd8a, Cd8b1 and Tnf and CD8 measured by immunohistochemistry were elevated in tumours of IGFBP-3-null mice compared to WT, indicating an accumulation of CD8+ T cells, but this increase was absent if the IGFBP-3-null mice had been exposed to HFD. Expression of these genes was negatively associated with tumour growth. Although similar among groups overall, Nkg2d and Tnfsf10 tumoural expression was associated with decreased tumour growth. Overall, the results of this study provide an immune-based mechanism by which host IGFBP-3 may promote breast tumour growth in the EO771 murine breast cancer model, and suggest that targeting IGFBP-3 might make a novel contribution to immune therapy for breast cancer.

A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors.

Injectable hyaluronic acid down-regulates interferon signaling molecules, IGFBP3 and IFIT3 in the bovine intervertebral disc.

Low back pain which is a major cause of disability for people aged between 20 and 50years imposes a serious socio-economic burden. The current focus of regenerative medicine is on identifying molecular markers to facilitate the design of targeted therapeutics. Previously, we have demonstrated that expression of the anti-proliferative interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and pro-apoptotic insulin-like growth factor-binding protein-3 (IGFBP3), are up-regulated as downstream targets of the inflammatory cytokine interferon α (IFNα) signaling pathway in the human annulus fibrosus (AF). Here, we hypothesised that injection of hyaluronic acid (HA) would have an anti-inflammatory and matrix modulatory effect on injured and IFNα2ß inflamed bovine intervertebral discs (IVD). Discs with an AF defect and challenged with IFNα2ß were used in a bovine IVD organ culture model to test the effect of HA on the IFNα2ß pathway, as well as the matrix proteins aggrecan and collagen I. qRT-PCR was used to assess the gene expression of IFNα2ß signaling molecules. Additionally, immunostaining was used to measure protein expression. Our results show that HA treatment significantly down-regulates IFNAR1, IFNAR2, STAT1/2, JAK1, IFIT3 and IGFBP3 mRNA expression in the inflamed groups. Protein analysis confirmed the PCR results. In the extracellular matrix, aggrecan and collagen I were up-regulated while ADAMTS4 was down-regulated upon treatment of the injured and inflamed discs with HA. Hence, HA demonstrates both an anti-inflammatory role, resulting in the down-regulation of IFIT3 and IGFBP3 in the AF, and a matrix modulatory effect by up-regulating aggrecan and collagen I expression. STATEMENT OF SIGNIFICANCE: The pro-inflammatory environment of the degenerated IVD represents a challenge for regenerative therapies. The study demonstrates that hyaluronan acts as an anti-inflammatory molecule by down-regulating IFNAR1 and IFNAR2, the signaling molecules STAT1, STAT2, JAK1 and the downstream apoptotic targets IGFBP3 and IFIT3. We also demonstrated that hyaluronan modulates the disc matrix environment by increasing aggrecan and collagen I synthesis and down-regulating ADAMTS4 that degrades the matrix under inflammatory conditions. The significance of this work lies in the fact that hyaluronan acts as an anti-inflammatory molecule by shifting the disc environment towards a more anabolic state and by promoting native IVD matrix production.

Influence of video-assisted thoracoscopic lobectomy on immunological functions in non-small cell lung cancer patients.

In this study, we compared the effects of video-assisted thoracic surgery (VATS) and traditional open surgery (TOS) on immune system functioning in non-small cell lung cancer (NSCLC) patients. We enrolled 122 NSCLC patients in this study. The patients were randomly divided into VATS group (n = 61) and TOS group (n = 61). Plasma DNA concentration was analyzed by fluorescence quantitative PCR. Automatic blood analyzer was used to measure WBC-C, and immune nephelometry was employed to assess hs-CRP concentrations. The number of CD3+T, CD4+T and CD8+T lymphocytes in peripheral blood was estimated by flow cytometry. ELISA was used to quantify the levels of IGFBP-3, VEGF and IL-6. Compared to the TOS group, surgery-related blood loss and pain score on day 1 after surgery were lower in VATS group. After surgery, the out-of-bed activity occurred earlier and in-hospital stays were shorter in the VATS group compared to the TOS group. Plasma free DNA concentration of VATS group patients at first, third and fifth days after surgery was lower than that of the TOS group. WBC-C and hs-CRP levels were lower in the VATS group at each time point after surgery. The number of CD3+T, CD4+T, CD8+T lymphocytes and CD4+/CD8+ was lower in the TOS group compared to VATS group. Plasma IGFBP-3, VEGF and IL-6 levels were significantly lower in VATS group compared to the TOS group. Finally, incidence of complications in the VATS group was dramatically lower than the TOS group (all P < 0.05). Based on our findings, compared to TOS, VATS significantly decreased the incidence of acute-phase reaction and lowered the inhibition of immune functions after surgery.

Circulating IGF1 and IGFBP3 in relation to the development of ß-cell autoimmunity in young children.

OBJECTIVE: This study aimed at investigating the role of IGF1 and IGF binding protein 3 (IGFBP3) in the development of ß-cell autoimmunity. METHODS: Five hundred and sixty-three subjects with HLA-conferred susceptibility to type 1 diabetes (T1D) were monitored for signs of seroconversion to positivity for insulin and/or GAD, IA2, and zinc transporter 8 autoantibodies by the age of 3 years. In 40 subjects who developed at least one autoantibody, IGF1 and IGFBP3 plasma concentrations were measured and compared with 80 control subjects who remained negative for autoantibodies, and were matched for age, sex, country of origin, and HLA genotype. The increments of IGF1, IGFBP3, and IGF1/IGFBP3 molar ratio before and after seroconverison were compared with corresponding time intervals in controls. RESULTS: The IGF1 concentrations at the age of 12 months and the IGF1/IGFBP3 ratio at the age of 24 months were lower in the autoantibody-positive children (P<0.05). The increase in circulating IGFBP3 was significantly higher in the autoantibody-positive children before seroconversion than in the corresponding time intervals in controls (0.43 mg/l; 95% CI 0.29-0.56 vs 0.22 mg/l; 95% CI 0.10-0.34 mg/l; P<0.01). Children carrying the high-risk HLA genotype had lower plasma IGF1 and IGFBP3 concentrations at the age of 24 months than those with low-risk genotypes (P<0.05 and < 0.01 respectively). CONCLUSIONS: Circulating IGF1 and IGFBP3 appear to have a role in early development of ß-cell autoimmunity. The decreased IGF1 concentrations in children with the high-risk HLA genotype may contribute to the reduced growth previously described in such children.

Antitumor activity and immunogenicity of recombinant vaccinia virus expressing HPV 16 E7 protein SigE7LAMP is enhanced by high-level coexpression of IGFBP-3.

We constructed recombinant vaccinia viruses (VACVs) coexpressing the insulin-like growth factor-binding protein-3 (IGFBP-3) gene and the fusion gene encoding the SigE7Lamp antigen. The expression of the IGFBP-3 transgene was regulated either by the early H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that IGFBP-3 expression regulated by the H5 promoter yielded higher amount of IGFBP-3 protein when compared with the E/L promoter. The immunization with P13-SigE7Lamp-H5-IGFBP-3 virus was more effective in inhibiting the growth of TC-1 tumors in mice and elicited higher T-cell response against VACV-encoded antigen than the P13-SigE7Lamp-TK(-) control virus. We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in more profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7Lamp-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7Lamp-TK(-). Intracellular mature virions (IMVs) of the IGFBP-3-expressing virus P13-SigE7Lamp-H5-IGFBP-3 have two structural differences: they incorporate the IGFBP-3 protein and they have elevated phosphatidylserine (PS) exposure on outer membrane that could result in increased uptake of IMVs by macropinocytosis. The IMV PS content was measured by flow cytometry using microbeads covered with immobilized purified VACV virions.

Role of insulin-like growth factor binding protein-3 in allergic airway remodeling.

RATIONALE: The hallmarks of allergic asthma are airway inflammation, obstruction, and remodeling. Airway remodeling may lead to irreversible airflow obstruction with increased morbidity and mortality. Despite advances in the treatment of asthma, the mechanisms underlying airway remodeling are still poorly understood. We reported that insulin-like growth factor (IGF) binding proteins (IGFBPs) contribute to extracellular matrix deposition in idiopathic pulmonary fibrosis; however, their contribution to airway remodeling in asthma has not been established. OBJECTIVES: We hypothesized that IGFBP-3 is overexpressed in asthma and contributes to airway remodeling. METHODS: We evaluated levels of IGFBP-3 in tissues and bronchoalveolar lavage fluid from patients with asthma at baseline and 48 hours after allergen challenge, in reparative epithelium in an in vitro wounding assay, and in conditioned media from cytokine- and growth factor-stimulated primary epithelial cells. MEASUREMENTS AND MAIN RESULTS: IGFBP-3 levels and distribution were evaluated by Western blot, ELISA, and immunofluorescence. IGFBP-3 is increased in vivo in the airway epithelium of patients with asthma compared with normal control subjects. The concentration of IGFBP-3 is increased in the bronchoalveolar lavage fluid of patients with asthma after allergen challenge, its levels are increased in reparative epithelium in an in vitro wounding assay and in the conditioned medium of primary airway epithelial cell cultures stimulated with IGF-I. CONCLUSIONS: Our results suggest that one mechanism of allergic airway remodeling is through the secretion of the profibrotic IGFBP-3 from IGF-I-stimulated airway epithelial cells during allergic inflammation.

Depletion of circulating insulin-like growth factor binding protein 3 after open surgery is associated with high interleukin-6 levels.

PURPOSE: We have previously shown that plasma from open, but not laparoscopic-assisted, surgery patients has increased mitogenic activity for colon cancer cells. Decreased insulin-like growth factor binding protein 3 levels, most likely the result of an open surgery-induced proteolytic activity, may account for this finding. Plasma proteases are activated by interleukin-6. This study was designed to investigate plasma insulin-like growth factor binding protein 3 and interleukin-6 levels after major open or laparoscopic-assisted surgery. METHODS: EDTA plasma was obtained from 24 patients undergoing resection for colonic adenocarcinoma. Insulin-like growth factor binding protein 3 was detected by Western blot analysis and enzyme-linked immunosorbent assay. Interleukin-6 levels were determined by enzyme-linked immunosorbent assay. The effect of insulin-like growth factor binding protein 3 on tumor growth was tested using HCT116 cells. RESULTS: In patients undergoing open surgery, enzyme-linked immunosorbent assay revealed a significant decrease in total insulin-like growth factor binding protein 3 levels on postoperative Day 1 (915.6 +/- 378.5 ng/ml) compared with preoperative levels (1267.5 +/- 407.9 ng/ml; P < 0.001). Western blots revealed a decrease in the levels of intact insulin-like growth factor binding protein 3. In patients undergoing laparoscopic-assisted surgery, levels of total and intact insulin-like growth factor binding protein 3 before surgery (1088.9 +/- 232.5 ng/ml) and on postoperative Day 1 (1,202.3 +/- 285.6 ng/ml) were comparable with no significant changes in Western blot analysis. Interleukin-6 levels were undetectable preoperatively. On postoperative Day 1, interleukin-6 concentration was higher in open surgery group (434.8 +/- 506.6 pg/ml) compared with laparoscopic-assisted surgery group (100.9 +/- 60.2 pg/ml; P < 0.0001), and correlated significantly with a decrease in plasma insulin-like growth factor binding protein 3 after open surgery (r = 0.81; P < 0.0001). CONCLUSIONS: A significant decrease in both total and free insulin-like growth factor binding protein 3 occurs after open but not laparoscopic colectomy. There is an associated increase in the levels of interleukin-6. It remains to be proven that the interleukin-6 elevations are responsible for the low insulin-like growth factor binding protein 3 level seen after open surgery.

Endocrinologic and immunologic factors associated with recovery of growth in children with human immunodeficiency virus type 1 infection treated with protease inhibitors.

INTRODUCTION: Growth failure is a common presenting sign in children with HIV disease and is a sensitive indicator of disease progression in children with AIDS. Highly active antiretroviral therapy (HAART) is associated with a significant decrease in viral load and a subsequent rise in CD4+ T cell counts in HIV-1-infected children and also with increased height and weight. The underlying mechanisms of catch-up growth during HAART are yet unknown. METHODS: Height and weight measurements, blood sample analyses for HIV-1 RNA and peripheral blood CD4+ T cell counts were obtained twice within 1 month before the start of HAART and after 12, 24, 36 and 48 weeks of treatment. Serum concentrations of insulin-like growth factor I (IGF-1), IGFs complexed to specific, structurally homologous binding proteins (IGFBP-3), cortisol, free thyroxine and tumor necrosis factor alpha (TNF-alpha) were measured before the start of therapy and after 24 weeks. In addition serum IGF-1 and IGFBP-3 values were determined after 48 weeks. RESULTS: Twenty-seven HIV-1-infected children with a median age of 5.5 years (range, 0.3 to 14.9 years) were included. Overall no significant changes in height and body mass index (BMI) z scores were observed. The median baseline plasma viral load of 68,800 copies/ml decreased to less than the detection limit of 500 copies/ml in 80% of the children after 48 weeks. TNF-alpha values were elevated (44 pg/ml) at baseline and decreased significantly to 37 pg/ml after 24 weeks. At baseline elevated TNF-alpha was observed in 78%, which decreased to 55% after 24 weeks. Baseline free thyroxine and cortisol values were normal and did not change during therapy. Baseline serum of IGF-1 and IGFBP-3 concentrations were normal, but IGF-1 tended to be lower than IGFBP-3. Both values increased significantly after the initiation of therapy. IGFBP-3 decreased after 48 weeks whereas IGF-1 stabilized. The increase in IGF-1 was significantly higher in children in whom the BMI and length (after correction for age and sex) increased the most. CONCLUSION: Hypothyroidism and adrenal axis abnormalities are not associated with restoration of growth after the initiation of antiretroviral therapy in HIV-1-infected children. The combination of relatively high serum IGFBP-3 concentration and relatively lower serum IGF-1 suggests the presence of a growth hormone-resistant state. During treatment with a protease inhibitor-containing regimen, decreased serum IGFBP-3 and stabilization of IGF-1 after a significant initial increase suggest restoration of normal sensitivity to growth hormone and recovery to an anabolic condition.

Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I.

IGF-binding protein (IGFBP)-3 is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. Levels of secreted IGFBP-3 and IGFBP-3 mRNA are significantly reduced during differentiation and increase after differentiation is complete, suggesting that IGFBP-3 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-3 reportedly may either suppress or stimulate proliferation of cultured cells depending on cell type. Additionally, IGFBP-3 has been shown to affect proliferation via both IGF-dependent and IGF-independent mechanisms in some cell types but not all. Currently, the effect, if any, of IGFBP-3 on myogenic cell proliferation is not known. Consequently, the goal of this study was to assess the IGF-I-dependent and IGF-I-independent actions of recombinant porcine IGFBP-3 on proliferation of cultured porcine myogenic cells. To facilitate these investigations, we have expressed porcine IGFBP-3 in the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-3 (rpIGFBP-3), and produced and characterized an anti-porcine IGFBP-3 antibody that neutralizes the biological activity of porcine IGFBP-3. rpIGFBP-3 suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner with equimolar concentrations of IGF-I and rpIGFBP-3, resulting in complete suppression of IGF-I-stimulated proliferation. rpIGFBP-3 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMC, indicating that rpIGFBP-3 possesses IGF-independent activity in this cell system. These data have established that IGFBP-3 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact ultimate muscle mass achievable postnatally.

Plasma from patients undergoing major open surgery stimulates in vitro tumor growth: Lower insulin-like growth factor binding protein 3 levels may, in part, account for this change.

BACKGROUND: Plasma from laparotomized mice has been shown to stimulate in vitro tumor growth when compared to results with preoperative plasma. This study assessed the effect of plasma from patients who underwent major open (OS) or laparoscopic surgery (LS) on in vitro tumor cell growth. METHODS: Eighty-four patients undergoing major abdominal surgery were studied (45 OS, 39 LS). Peripheral blood was collected preoperatively (PreOP) and on days 1 (POD1) and 3 (POD3) after operation. HT29 human colon cancer cells were plated with samples of the plasma. Proliferation was assessed by cell counts and the bromodeoxyuridine incorporation assay. Insulin-like growth factor binding protein 3 was detected in plasma by Western blot analysis. RESULTS: Increased mitogenic activity was noted in POD1 OS plasma when compared to PreOP OS plasma results (P <.005). This increase correlated with the length of incision (r = 0.58, P <.01). No differences were noted when the PreOP LS and POD1 LS results were compared or for any of the POD3 versus PreOP comparisons. CONCLUSIONS: Major OS is associated with alterations in plasma composition that promote HT29 tumor cell proliferation in vitro. As shown, this effect was due, at least in part, to surgery-related depletion of insulin-like growth factor binding protein 3 in peripheral blood.

Regulation of proliferation of prostate epithelial cells by 1,25-dihydroxyvitamin D3 is accompanied by an increase in insulin-like growth factor binding protein-3.

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to regulate the proliferation of human prostate epithelial cell lines. Since the insulin-like growth factor (IGF) system is involved in the transformation process of epithelial cells, the following study was undertaken to determine if the IGF system, in particular IGF binding protein-3 (IGFBP-3), is altered by 1,25-(OH)2D3 in normal prostate epithelial cells as part of a mechanism for inhibition of transformation. Two cell systems were used in this study: (1) primary cultures of benign human prostate epithelial cells (PECs) and (2) an SV40-T immortalized prostate epithelial cell line (P153) that is non-tumorigenic. 1,25-(OH)2D3 was added to parallel sets of PECs and P153 cells in addition to the presence or absence of IGF-I or des(1-3)IGF-I. Treatment with 1,25-(OH)2D3 resulted in significant growth inhibition of both PECs and P153 cells. Furthermore, 1,25-(OH)2D3 inhibited IGF-induced proliferation, but this was partially reversed by high concentrations of IGF-I. Western ligand blots of condition media demonstrated a significant increase in IGFBP-3; likewise Northern blots demonstrated an increase in mRNA for IGFBP-3. Proliferation assays using an antibody designed to block the IGF-independent effects of IGFBP-3 failed to reverse the inhibitory effect of 1,25-(OH)2D3. Thus, IGFBP-3 acts in an IGF-dependent manner to inhibit cell growth of benign prostate epithelial cells.

Generation of antisera to mouse insulin-like growth factor binding proteins (IGFBP)-1 to -6: comparison of IGFBP protein and messenger ribonucleic acid localization in the mouse embryo.

The insulin-like growth factor (IGF) system is an important regulator of fetal growth and differentiation. IGF bioavailability is modulated by IGF binding proteins (IGFBPs). We have generated six different antisera, directed to synthetic peptide fragments of mouse IGFBP-1 through -6. The specificity of the produced antisera was demonstrated by enzyme-linked immunosorbent assay, Western blotting, and by immunohistochemistry on sections of mouse embryos of 13.5 days post coitum. Specificity for the IGFBP-2 through -6 antisera also was confirmed immunohistochemically in liver and lung of corresponding gene deletion (knock-out) mutant mice and wild-type litter mates. Immunohistochemistry and messenger RNA (mRNA) in situ hybridization on sections of mouse embryos of 13.5 days post coitum revealed tissue-specific expression patterns for the six IGFBPs. The only site of IGFBP-1 protein and mRNA production was the liver. IGFBP-2, -4, and -5 protein and mRNA were detected in various organs and tissues. IGFBP-3 and -6 protein and mRNA levels were low. In several tissues, such as lung, liver, kidney, and tongue, more than one IGFBP (protein and mRNA) could be detected. Differences between mRNA and protein localization were extensive for IGFBP-3, -5, and -6, suggesting that these IGFBPs are secreted and transported. These results confirm the different spatial localization of the IGFBPs, on the mRNA and protein level. The overlapping mRNA and protein localization for IGFBP-2 and -4, on the other hand, may indicate that these IGFBPs also function in an auto- or paracrine manner.

The high molecular weight insulin-like growth factor-binding protein complex: epitope mapping, immunoassay, and preliminary clinical evaluation.

Measurements of insulin-like growth factor I(IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) are important in assessing the GH-IGF axis. As nearly all IGF-I, IGFBP-3, and ALS circulate in a GH-dependent ternary protein complex, direct determination of the complex may be of significant analytical and clinical importance. We evaluated a panel of monoclonal antibodies (mAb) to human IGFBP-3 and classified them into four groups (G-1 to G-4). G-1 antibodies recognized epitopes that mapped at or near IGFBP-3 ligand (IGF)-binding site. This region overlapped with the G-2 defined region, which, in turn, overlapped with G-3 epitopes defined by one antibody (mAb 3). Only G-1 and G-3 antibodies paired without interference. mAb 9 recognized a conformational epitope (G-4), and mAb 10 was nonreactive. In pairwise mixed antibody evaluation, mAbs in G-2 and G-3 showed simultaneous binding to serum IGFBP-3 complexes in combination with an anti-IGF-I or an anti-ALS antibody. On this basis, two novel enzyme-linked immunosorbent assays (ELISAs) involving IGFBP-3/IGF-I (ELISA-1) and IGFBP-3/ALS (ELISA-2) recognition partners were developed, both demonstrating acceptable analytical performance characteristics. IGFBP-3 complexes measured by ELISA-1 and -2 in samples from normal individuals and subjects with GH deficiency, acromegaly, and GH receptor deficiency more tightly correlated with IGF-I, IGFBP-3, and ALS than IGF-II. ELISA-1 determinations were comparatively more age dependent and, in comparison to ELISA-2, showed better discriminations among the various sample groups, particularly among GH receptor deficiency, normal, and GH deficiency subjects. The development of IGFBP-3 complex ELISAs may simplify diagnostic applications and facilitate investigations of the physiological relevance of the ternary complex formation.

Contribution of insulin-like growth factor (IGF)-I and IGF-binding protein-3 to mitogenic activity in bovine mammary extracts and serum.

Peripubertal development of the mammary gland is probably mediated by locally produced growth factors acting in concert with circulating mitogens. Our objective was to investigate the effect of recombinant human insulin-like growth factor-binding protein-3 (rhIGFBP-3) or insulin-like growth factor-I (IGF-I) antibodies on the IGF-I-related mitogenic activity of bovine serum and of mammary tissue extracts in primary mammary epithelial cell cultures. Cells were obtained from prepubertal female calf mammary tissue and cultured in three-dimensional collagen gels. An aqueous mammary parenchymal tissue extract (pooled from 20 prepubertal heifers) or serum (pooled from 3 heifers) at a concentration of 5% was added to the medium containing either rhIGFBP-3 or monoclonal or polyclonal antibodies to human IGF-I. Cell proliferation was evaluated using [methyl-3H]thymidine incorporation as a measure of DNA synthesis. Addition of mammary extracts stimulated DNA synthesis 545% compared with basal medium. Addition of serum stimulated DNA synthesis by 28%. Mitogenic activity of serum and added IGF-I was abolished by addition of rhIGFBP-3 in equimolar concentrations with IGF-I. For mammary extracts, mitogenic activity was inhibited by 35%, 50%, and 82% by the addition of rhIGFBP-3 at, respectively, 1, 2 and 4 times the molar IGF-I concentration in the extract. Addition of rhIGFBP-3 to basal medium reduced DNA synthesis by 26%, whereas IGF-I antibodies had no consistent effect. These results indicate that circulating and mammary-synthesized IGF-I and IGFBPs probably play a critical role in prepubertal development of the bovine mammary gland.

Binding properties and distribution of insulin-like growth factor binding protein-related protein 3 (IGFBP-rP3/NovH), an additional member of the IGFBP Superfamily.

The protein product of the novH oncogene, a member of the CCN family, is structurally related to the insulin-like growth factor (IGF) binding proteins (IGFBPs). We have characterized aspects of structure, function, and distribution of this protein, which, as IGFBP-related protein 3 (IGFBP-rP3), is a proposed member of the IGFBP Superfamily. Affinity cross-linking experiments performed with baculovirus synthesized recombinant human IGFBP-rP3 established that rhIGFBP-rP3 binds IGF-I, IGF-II, and insulin with low affinity. Specificity of binding was shown by competitive cross-linking experiments; binding to IGF-I and -II was also demonstrated by nondenaturing Western ligand blots. Northern blot analysis indicated the presence of IGFBP-rP3 messenger RNA (mRNA) in a broad range of human tissues. Western immunoblotting studies, using a polyclonal rabbit anti-rhIGFBP-rP3 antibody, demonstrated that IGFBP-rP3 protein is synthesized in vitro by several breast and prostate cancer cell lines: Hs578T, PC3, P69, and LNCaP cells. Western immunoblotting studies of human biological fluids identified that IGFBP-rP3 was present in normal serum, pregnancy serum, serum from patients with growth hormone receptor deficiency, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and follicular fluid, while IGFBP-rP3 fragments were identified in cerebrospinal fluid, amniotic fluid, and prepubertal and pubertal urine samples. Our studies demonstrate that IGFBP-rP3 exhibits IGF binding, albeit at low affinity, and IGFBP-rP3 thus merits inclusion in the IGFBP Superfamily. The low affinity IGF binding suggests that IGFBP-rP3 may act primarily independently of the IGFs. The synthesis of IGFBP-rP3 by several malignant cell lines and its presence in human biological fluids suggest that this protein possesses other interesting roles, potentially in cell growth regulation.

Synthesis of IGFBP-3 fragments in a baculovirus system and characterization of monoclonal anti-IGFBP-3 antibodies.

IGFBPs play an important role in IGF biological actions by modulating IGF binding to its receptors. The major IGFBP in serum is IGFBP-3, which transports 70-90% of the circulating IGFs. In target cell systems, it sequesters IGFs and inhibits their hormonal actions, but may potentiate IGF activity or exert IGF-independent effects under specific conditions. IGFBP-3 can be modified by IGFBP-3 proteases, which degrade it into smaller fragments. IGFBP-3 fragments generated by proteolysis have reduced affinity for IGFs, thereby modifying IGF action. To study IGFBP-3 fragments in vivo and in vitro, we constructed six different IGFBP-3 fragments by use of a baculovirus expression system and generated 8 different monoclonal IGFBP-3 antibodies. Based on the known cleavage sites of IGFBP-3 for PSA, MMPs, and the predicted plasmin cleavage sites, we expressed a N-terminal IGFBP-3(1-97) fragment and a C-terminal IGFBP-3(98-264) fragment. By stepwise truncation from the C-terminal end, we created IGFBP-3(98-232), IGFBP-3(98-206), IGFBP-3(98-179), and IGFBP-3(98-159). A strong recognition of the C-terminus and the intermediate parts of IGFBP-3 by six antibodies was found. Four of these mAbs were able to recognize the intermediate fragment alone. Two mAbs were found to immunoreact only with the N-terminal IGFBP-3 fragment and two additional mAbs recognized the N- as well as the C-terminal parts and lacked immunoreactivity for the intermediate part of IGFBP-3. The 15 kDa IGFBP-3 fragment resulting from plasmin digestion was found to only react with N-terminal antibodies, while the 29 kDa fragment in pregnancy serum reacted with both N- and C-terminal antibodies. Thus, these mAbs will be useful tools to determine whether IGFBP-3 fragments found in vivo derive from either the N- or C-terminal domains of IGFBP-3.