Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 5 uppsatser.

Insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in circulation. IGFBPs 1-6 bind IGFs with high affinity and can either potentiate or inhibit IGF signaling in a context dependent manner. IGFBP2 is a 36 kDa protein and the second most abundant IGFBP in serum. Numerous studies in the recent past have implied a pro-tumorigenic role of IGFBP2. Elevated expression of IGFBP2 has been observed in multiple malignancies, including glioblastoma multiforme (GBM), ovarian, pancreatic, gastric, prostate, colon, breast, thyroid cancer and leukemia. In addition, increased expression of IGFBP2 in both tissues and serum of patients has been correlated with poor prognosis in prostate, glioblastoma and colon cancers. Pro-tumorigenic actions of IGFBP2 have been supported by in vitro studies, where IGFBP2 increases the tumorigenic potential of adrenocortical tumor cells, epidermoid carcinoma cells, glioma cells and ovarian cancer cells. Further, using xenograft animal models, the role of IGFBP2 in the progression of glioma has been established. In breast cancer, IGFBP2 was found to be over expressed in ductal carcinoma in situ and invasive breast cancer samples. IGFBP2 over expression has been shown to confer drug resistance and an increased expression has been reported to correlate with lymph node metastasis in T1 breast carcinomas. These reports implicate IGFBP2 in breast cancer biology. However, its role in breast cancer progression is not well defined. With this background, the following objectives were set for the current study: Functional characterization of IGFBP2 with respect to its possible role in breast cancer progression. Elucidation of the molecular mechanisms of IGFBP2 actions. Towards this, immunohistochemistry was performed on 132 invasive ductal carcinoma (IDC) grade III tumors using IGFBP2 specific antibody. It was observed that IGFBP2 expression was significantly higher in tumors in comparison to normal tissues that showed no detectable staining for IGFBP2. It was also observed that expression of IGFBP2 significantly correlated with the expression of ER. To understand the functional significance of IGFBP2 over expression in breast cancer, IGFBP2 was characterized with respect to proliferation, survival and tumor forming ability (in vitro and in vivo) in BT474 breast cancer cells. The knockdown of IGFBP2 expression resulted in suppression of colony formation (nearly 70%) in these breast cancer cells, which could be partially reversed upon exogenous addition of IGFBP2 protein. Proliferation assays using stable clones with knockdown of IGFBP2 in BT474 cells showed a significant decrease in proliferation as compared to vector transfected cells in the presence of serum. Culturing of IGFBP2 knockdown breast cancer cells in serum free medium resulted in their growth arrest in G0/G1 phase of cell cycle as compared to control cells, which progressed through the cell cycle. Prolonged culturing of IGFBP2 knockdown cells in serum free condition (up to 72 h) resulted in the increase of cells in sub G1 phase of the cell cycle. Prolonged depletion of growth factors (serum free conditions) could result in apoptosis of these G1 arrested IGFBP2 knockdown cells. When serum starved IGFBP2 knockdown cells were treated with IGFBP2 protein, the cells arrested in G0/G1 phase were able to progress through the cell cycle and concomitant decrease in sub G1 fraction was observed. Knockdown of IGFBP2 resulted in significantly decreased number and visibly smaller colonies in anchorage independent conditions in vitro. Consistent with this observation, in vivo tumor xenograft formation with IGFBP2 knockdown cells also showed significant reduction in tumor weight as compared to vector generated tumors. These results imply that IGFBP2 has potent growth promoting effects on breast cancer and acts as a mitogen/survival factor for breast cancer cells. To elucidate the molecular mechanisms underlying the pro-tumorigenic effects of IGFBP2, the transcriptome profile following IGFBP2 perturbation in breast cancer cells was determined. IGFBP2 knockdown resulted in significant changes in the expression of genes associated with cellular proliferation and tumorigenicity. The down regulated genes were found to be associated with several events, notably cell cycle, p53 and Wnt signaling, as revealed by Gene Set Enrichment Analysis (GSEA). To further validate these results in breast cancer tissues, whole genome expression analysis was performed in 19 breast tumor samples which were categorized as IGFBP2 positive or negative based on immunohistochemical staining pattern. In comparison to IGFBP2 negative tumors, IGFBP2 positive tumors showed increased expression of genes belonging to MAPK, focal adhesion and Wnt signaling pathway. In order to identify the genes commonly regulated by IGFBP2 in cell lines and tumors, the gene expression profiles of IGFBP2 positive versus IGFBP2 negative tumors and IGFBP2 knockdown breast cancer cells were compared. 347 genes were found to be common among IGFBP2 regulated genes in tumors and cell line. The most significant networks representing the web of interactions among these genes were found to be associated with cellular growth and proliferation, cellular movement and nucleic acid metabolism, indicating an association of IGFBP2 expression phenotype to the distinct changes in expression of genes associated with the regulation of cellular growth and migration. Silencing of IGFBP2 in BT474 cells resulted in a reduced IGF signaling as evidenced by the reduced phosphorylation of IGF1R and concomitantly that of ERK. This effect could be reversed upon addition of the IGFBP2 protein, implying that IGFBP2 potentiates IGF signaling in breast cancer cells. Besides IGF ligand and their receptors, regulation of proliferation associated genes like CENPF, TOP2A, CCND1 and FOXM1 by IGFBP2 was observed, thus providing a molecular basis for the pro-proliferative effects of IGFBP2 on breast cancer cells. Addition of IGFBP2 to immortal breast cells resulted in reduced IGF1R signaling and reduced pERK and pAKT signaling. Additionally, the genes involved in cellular proliferation were down regulated upon IGFBP2 treatment in immortal cells. IGFBP2 knockdown clones had reduced expression of FOXM1, a key regulator of cell cycle for G1/S and G2/M transition, and M phase progression. The regulation of CENPF and CCND1 genes was established following over expression of FOXM1 in IGFBP2 knockdown cells. One of the important and novel finding of this study is the regulation of Wnt signaling pathway genes such as CCND1, MMP7, FGF18, MYCBP, FN1 and survivin by IGFBP2. In support of this, β-catenin protein was found to be regulated by IGFBP2 in breast cancer and GBM cells, as evidenced by knockdown and over expression studies. Furthermore, regulation of β-catenin by IGFBP2 was found to involve integrin-FAK and IGF1R signaling. Another important finding of this study is the correlation of IGFBP2 over expression with elevated β-catenin levels in breast tumors. When expression of both IGFBP2 and β-catenin was correlated with the lymph node status of breast cancers, a significant association of IGFBP2 and β-catenin staining with increased lymph node metastasis was observed in comparison with tumors that did not show staining for either protein. Altogether, in this study employing genomic, cellular and molecular approaches, a pro- tumorigenic role for IGFBP2 in breast cancer has been established. Furthermore, this study provides novel insights into the molecular mechanisms employed by IGFBP2 involving IGF1R, FAK and Wnt signaling pathways during breast cancer progression.

Understanding how insulin-like growth factor-I (IGF-I) signaling in mammary epithelial cells may be modified or interrupted by modifications in the cellular environment may lead to 1) methods to increase the growth and proliferation of normal mammary epithelial cells for an increase in the amount of milk produced on a per animal basis or to 2) the development of medical interventions to disrupt the growth and proliferation of cancerous mammary epithelial cells. IGF-I, a signaling protein provided by stromal cells and through the bloodstream, stimulates the proliferation of mammary epithelial cells and is crucial for mammary development. Collagen I is an extracellular matrix protein (ECM) found in skin and in other connective tissues throughout the body. The guiding question in this dissertation was how IGF-I signaling and how binding protein profile were influenced by autocrine IGF-I and by collagen I. The MAC-T cell line was chosen as the cell model utilized in these investigations because it is an immortalized bovine mammary epithelial cell line known to retain hormonal responsiveness to IGF-I. It was hypothesized that the production of IGF-I by mammary epithelial cells (autocrine secretion) would alter the response of these cells to additional IGF-I by de-sensitizing the IGF-I receptor on the cell surface. The normal mammary epithelial cell does not produce IGF-I and responds to IGF-I supplied either by stromal cells (paracrine pathway) or through the bloodstream (endocrine pathway). The IGF-I secreting bovine mammary epithelial cell line was investigated for the response of the cells to autocrine IGF-I, and the response was compared to the normal, parental cell line. To examine the effect of autocrine IGF-I on the cells, IGF-I was added both to MAC-T cells and to cells transfected to secrete IGF-I (SV40-IGF-I). The cell response of the two cell lines was compared using microphysiometry, a tool that measures IGF-IR stimulation by detecting resultant extracellular acidification. It was found that the SV40-IGF-I cell line retains IGF-I receptor sensitivity, yet, unlike the parental cell line, does not proliferate in response to IGF-I. Both cell lines exhibited increased protein synthesis in response to IGF-I as measured by amino acid uptake (AIB incorporation), but the lack of a proliferation response to additional IGF-I in the SV40-IGF-I cell line suggested that the autocrine cell line exhibited an un-coupling of IGF-IR stimulation with downstream cell proliferation. Both autocrine IGF-I and added IGF-I increased the amount of IGFBP-3 secreted by the cells into growth media. Additionally, it was hypothesized that the presence of collagen I, an important ECM protein, would alter the cell production of insulin-like binding protein-3 (IGFBP-3), a protein that modulates IGF-I interaction with the IGF-I receptor (IGF-IR). The literature reports that surface substrate can affect the phenotypic expression of cells, presumably via interaction with integrins, the cell surface receptors that connect cells to ECM proteins and that are responsible for cell adhesion and for cell migration. It was hypothesized that the MAC-T cells would interact with a collagen I surface (possibly via the a2b1 integrin) and that the stimulation of this transmembrane signaling molecule would in turn impact the IGF-I signaling pathway. Comparison studies on tissue culture plastic, collagen I BIOCOAT, and collagen I gel were performed. It was found that collagen I gel increased IGFBP-3 secretion and decreased insulin-like binding protein-2 (IGFBP-2) secretion in MAC-T cells. The collagen I BIOCOAT did not induce this response. Additional studies were performed to determine if there were differences in IGF-IR phosphorylation, exogenous IGF-I utilization, and IGFBP mRNA production by cells cultured on the three different substrates. IGF-IR phosphorylation was only evident following the addition of IGF-I to MAC-T cells on all three substrates. Measurement of residual IGF-I present in the cultured media of cells on all three substrates by radioimmunoassay did not reveal any differences in the amount of IGF-I present. Northern blot analysis revealed that the addition of IGF-I caused an increase in detected IGFBP-3 mRNA and a decrease in detected IGFBP-2 mRNA across all three surfaces. As measured by ligand blot analysis, cells cultured on all three surfaces showed an increase in IGFBP-3 protein in the media with IGF-I addition, and the collagen I gel showed more IGFBP-3 protein than the other two surfaces. However, cells cultured on collagen I gel showed a decrease in IGFBP-2 protein expression compared to cells cultured on tissue culture both with and without the addition of IGF-I. Cells cultured on tissue culture plastic and on collagen I BIOCOAT did not show a decrease in IGFBP-2 to correspond with the decreased IGFBP-2 mRNA detected in the presence of IGF-I on all three substrates. DNA assays to detect cell proliferation revealed no differences in cell DNA content in the absence of exogenous IGF-I and revealed similar increases in response to IGF-I addition on all three substrates. In conclusion, it was found that autocrine IGF-I un-couples increased IGF-IR stimulation by exogenous IGF-I from a downstream cell proliferation response. IGFBP-3 inhibits the ability of IGF-I to interact with the IGF-IR in MAC-T cells and inhibits subsequent cell proliferation. Collagen I gel increases IGFBP-3 secretion and decreases IGFBP-2 secretion by MAC-T cells. The relevance of this work is that it adds to the body of knowledge in understanding cellular function in mammary epithelial cells. It is known that the growth and the maintenance of living tissue are dependent on an intricate system of intercellular and intracellular responses which are orchestrated by the movement and secretion of proteins and other molecules. Goals of understanding mammary epithelial cell function include having the means to find ways to increase cell functionality via bioengineering and having the means to find ways to restore cells to normal function in disease processes such as cancer. / Ph. D.

Thesis (Ph. D.)--University of Washington, 2003. / Includes bibliographical references (leaves 101-113).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

München, Techn. University, Diss., 2005.

Doctor of Philosophy / Department of Animal Sciences and Industry / Bradley J. Johnson / We used a total of fifty-nine gilts (BW=137.7 kg) from three different breeding groups were used to assess the effects of feeding L-carnitine during gestation on gilt growth characteristics, gilt and fetal blood metabolites, litter characteristics, and IGF axis components in fetal hepatic and skeletal muscle, maternal uterine and chorioallantois tissues, and porcine embryonic myoblasts collected from fetuses. Experimental treatments were arranged in a 2 × 3 factorial arrangement with main effects of L-carnitine and day of gestation. Gilts were fed a constant feed allowance of 1.75 kg/d and a top-dress containing either 0 or 50 ppm of L-carnitine starting on the first day of breeding through the allotted gestation length (40, 55, or 70). No differences (P > 0.16) were observed for BW or estimated protein or fat mass at any gestation length. Gilts fed L-carnitine tended to have greater (P = 0.10) backfat at d 40 and were numerically heavier at d 70 compared to control gilts. No differences (P > 0.77) were observed in circulating total and free carnitine at breeding, but concentrations increased (P < 0.01) as gestation length increased for the gilts fed L-carnitine compared to those fed the control diet. Fetuses from the gilts fed L-carnitine tended to be heavier (P = 0.06) and fetal circulating IGF-II lower (P = 0.09) at day 70 compared to the fetuses from the control gilts. Insulin-like growth factor-I (IGF-I) mRNA was lower (P = 0.05) in fetal hepatic tissue in fetuses collected from gilts fed supplemental L-carnitine. Insulin-like growth factor binding protein-3 (IGFBP-3; P = 0.05) and IGFBP-5 mRNA increased (P = 0.01) and IGF-I mRNA numerically increased (P = 0.16) in the endometrium of gilts supplemented with L-carnitine. At d 55 or 70 gestation, fetuses were removed for collection of porcine embryonic myoblasts (PEM) from the semitendinosus. There were no treatment differences (P > 0.10) for the expression of IGF-I, IGF-II or IGFBP-5 mRNA levels. However, PEM isolated from fetuses collected from gilts fed L-carnitine had lower (P = 0.08) IGFBP-3 mRNA levels compared to the controls. Myoblasts isolated from fetuses obtained from gilts fed L-carnitine had greater (P = 0.09; 8.8 %) 5.1H11 monoclonal antibody attachment after 72 h in culture. Although not significant (P = 0.20), the total number of PEM in the S phase of the cell cycle was 4.7 % greater in PEM collected from fetuses from gilts fed L-carnitine compared to the controls. This study shows L-carnitine supplementation to gestating gilts has beneficial effects on average fetal weight, due in part to changes in the expression of the IGF axis at the fetal-maternal interface in swine. These changes in the IGF axis play a fundamental role in porcine fetal growth and development due to enhanced proliferation and delayed differentiation of PEM.

Astronauts at are risk of losing 1.0-1.5% of their bone mass for every month they spend in space despite their adherence to high impact exercise training programs and diets high in nutrients, potassium, calcium, and vitamin D, all designed to preserve the skeletal system. This article reviews the basics of bone formation and resorption and details how exposure to microgravity or simulated microgravity affects the structure and function of osteoblasts, osteocytes, osteoclasts, and their mesenchymal and hematologic stem cell precursors. It details the critical roles that insulin-like growth factor-1 and its receptor insulin-like growth factor-1 receptor (GFR1) play in maintaining bone homeostasis and how exposure of bone cells to microgravity affects the function of these growth factors. Lastly, it discusses the potential of tumor necrosis factor-related apoptosis-inducing ligand, syncytin-A, sclerostin inhibitors and recombinant IGF-1 as a bone-saving treatment for astronauts in space and during their colonization of the Moon.

Insulin-like growth factor-I is a small growth factor known to signal in a variety of mammalian cells through the IGF-I cell surface receptor (IGF-IR). A unique feature of the IGF-I system is the regulation of this binding by soluble IGF binding proteins. Recent studies from our laboratory show that there is a pH dependence in the association of IGF-I with the cell surface in the presence of IGFBP-3 which suggested increased association of IGF-I with IGFBP-3 at low pH. We studied cell free interaction of IGF-I and IGFBP-3 as a function of pH using surface plasmon resonance (SPR) in order to understand the mechanism that causes the increased association. In our studies three different SPR instruments with different surfaces for immobilization of one of the binding partners were used: a Leica Bio-SPR 9000 with a low molecular weight carboxymethylated dextran (CMD) surface, a BIAcore 2000 with a high molecular weight CMD surface and a Leica SPR 2001 Alpha with a planar mixed self-assembled monolayer (mSAM) surface. Since the experimental system we used was transport sensitive, only the mSAM surface, under optimized conditions, produced results that fit to a single site model. Results suggest that use of CMD layers for immobilization of one partner of a high-affinity binding complex can result in transport limited binding for which simple analysis is inappropriate. Future studies are planned to expand the work with the mSAM surface to elucidate whether a significant difference between the binding parameters as a function of pH exists. / Master of Science