UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Establishing methods to screen novel small molecules targeting insulin-like growth factor/insulin-like… Alsabban, Abdulrahman Essam 2013

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2013_fall_alsabban_abdulrahman.pdf [ 4.54MB ]
Metadata
JSON: 24-1.0074271.json
JSON-LD: 24-1.0074271-ld.json
RDF/XML (Pretty): 24-1.0074271-rdf.xml
RDF/JSON: 24-1.0074271-rdf.json
Turtle: 24-1.0074271-turtle.txt
N-Triples: 24-1.0074271-rdf-ntriples.txt
Original Record: 24-1.0074271-source.json
Full Text
24-1.0074271-fulltext.txt
Citation
24-1.0074271.ris

Full Text

	 ?	 ?Establishing	 ?methods	 ?to	 ?screen	 ?novel	 ?small	 ?molecules	 ?targeting	 ?insulin-??like	 ?growth	 ?factor/insulin-??like	 ?growth	 ?factor	 ?binding	 ?protein	 ?interaction	 ?by	 ?Abdulrahman	 ?Essam	 ?Alsabban	 ?	 ?M.B.B.S,	 ?King	 ?Abdul	 ?Aziz	 ?University,	 ?2009	 ?	 ?A	 ?THESIS	 ?SUBMITTED	 ?IN	 ?PARTIAL	 ?FULFILMENT	 ?OF	 ?THE	 ?REQUIREMENTS	 ?FOR	 ?THE	 ?DEGREE	 ?OF	 ?Master	 ?of	 ?Science	 ?in	 ?The	 ?Faculty	 ?of	 ?Graduate	 ?and	 ?Postdoctoral	 ?Studies	 ?	 ?(Surgery)	 ?The	 ?University	 ?Of	 ?British	 ?Columbia	 ?(Vancouver)	 ?August,	 ?2013	 ??	 ?Abdulrahman	 ?Essam	 ?Alsabban,	 ?2013	 ?ii	 ?	 ?Abstract Insulin-like growth factors (IGFs) are important systemic mediators of growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis and metastatic activities in various cancers by activating IGF-IR tyrosine kinase-mediated signaling. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). Increased IGFBP-2 and IGFBP-5 expression observed in castration-resistant prostate cancer is thought to promote tumor progression by enhancing IGF-mediated signaling. IGFBPs have cooperative carboxy-terminal and amino-terminal low and a high affinity IGF binding sites. I hypothesize that blocking the high affinity IGF binding site can affect the bioavailability of IGFs to target tissues and thus be used for treatment of various IGF-responsive diseases including prostate cancer.  I initially characterized immunologic reagents capable of being used in sandwich ELISA formats to detect IGF-I and IGFBP-5 and attempted several configurations to establish an IGF-I/IGFBP-5 ?bridged? sandwich ELISA platform to measure association and dissociation of IGF-I/IGFBP-5 complex formation. The inability of all bridged ELISA formats tested to measure IGF-I/IGFBP-5 binding, lead me to developed a Bio-Layer Interferometry-based assay that measures IGF-I/ IGFBP-5 binding kinetics that will allow for screening of factors that can affect this intermolecular interaction. I demonstrated that biotinylated IGF-I bound to streptavidin-coated biosensors can be used to measure binding of recombinant IGFBP-5 [2.24 nm shift in optical density (Response)]. I also demonstrated that IGF-I could efficiently disrupt this interaction (0.21 nm shift), while the amino-terminal IGF-I mutant, E3R, exhibits an intermediate iii	 ?	 ?competitive activity (1.47 nm shift) and insulin exhibits a low competitive activity (1.83 nm shift). In addition, I demonstrated that IGF-I can competitively disrupted this interaction, resulting in a dissociation rate constant (Kdis 1.5-3 1/s), In contrast, the amino terminal IGF-I mutant, E3R binds with an intermediate affinity (Kdis 5.6-4 1/s), and buffer free sample results in a (Kdis) of 1.5-4 (1/s).  These results demonstrate the capacity of this BLI-based assay to differentiate relative competitive activity of compounds that target the high affinity IGF-I binding site of IGFBPs and establish a platform to screen for factors that might be developed as rationale therapeutics to disrupt sequestration of IGF-I by IGFBPs.             iv	 ?	 ?Preface This dissertation is original, unpublished, independent work by the author, Abdulrahman Essam Alsabban                v	 ?	 ?Table of Contents  Abstract ????????????????????????????????..  ii Preface ....................................................................................................................... iv  Table of Contents ????????????????????????????. v List of Tables ?????????????????????????????... viii List of Figures ?????????????????????????????.. ix List of Abbreviations ????????????????????????..?? xi Acknowledgments ?????????????????????????...?.. xiii Dedication ??????????????????????????...????  xiv 1. Chapter one: Introduction   ??????????????????????.  1 1.1. IGF axis ?????????????????????????????? 1 1.1.1.  IGF receptors ????????????????????????.. 2 1.1.2.  Insulin-like growth factors (IGFs) ????????????????. 3 1.1.3.  IGFBPs ??????????????????????????...  5 1.1.4. IGFBP-rP ???...??????????????????????. 11 1.1.5.  IGFBP proteases ??????????????????????...11 1.2. IGF axis modulations in cancer development and progression ?????? 15 1.3. High IGF level in different diseases and risk of cancer development ??...... 16 1.4. IGF axis in Prostate cancer  ????????????????????? 17 1.5. Therapeutic strategies targeting IGF axis in cancer treatment ??????.. 19 1.6. Molecular interaction between IGFBP-5 and IGF-I ???????????.. 21 1.7. Available methods to investigate protein/protein interactions ??????.... 23 1.8. Specific experimental objectives ???????????????????  24 vi	 ?	 ?2. Chapter two: Building bridged ELISA for measurement and detection of IGF-I/IGFBP-5 binding ................................................................................................ 26 2.1. Materials and Methods ??????????????????????...  26 2.2. ELISA assays development ????????????????????? 28 2.2.1. Characterizing IGFBP-5 ELISA protein and antibodies .?????..... 28 2.2.2. Characterizing IGF-I ELISA protein and antibodies ????????. 29 2.2.3. Characterizing bridged ELISA format to test the binding of IGF-I to     IGFBP-5 ................................................................................................ 29 2.2.4. Characterizing bridged ELISA format using IGFBP-5 capture antibody and biotinylated IGF-I (BIGF-I) ....................................................................... 30  2.3. ELISA assay results ....................................................................................... 31 2.3.1. Assessment of rhIGFBP-5 using sandwich ELISA  .............................. 31 2.3.2. Assessment of rhIGF-I using sandwich ELISA  ................................... 32 2.3.3. Assessment of signal detection using biotinylated detection antibodies in IGF-I/IGFBP-5 bridged sandwich ELISA ................................................. 33 2.3.4. Assessment of signal detection using biotinylated IGF-I in BIGF-I/IGFBP-5 bridged sandwich ELISA ......................................................................... 35 2.4. Conclusions ................................................................................................... 37 3. Chapter three: Bio-Layer interferometry (BLI) technology for studying of IGF-I/IGFBP-5 binding .............................................................................................  48  3.1. Materials and Methods .................................................................................. 49 3.2. BLI assays development ............................................................................... 50 vii	 ?	 ?3.2.1. Techniques to evaluate the competitors in dissociation on the BIGF-I/IGFBP-5 complex ................................................................................. 55 3.2.2. Techniques to evaluate the competitors in association on BIGF-I/IGFBP-5 complex formation .................................................................................. 57 3.3. BLI assay results .........................................................................................   59 3.3.1. Detection of BIGF-I binding to IGFBP-5 is possible using the BLI technique ................................................................................................ 59 3.3.2. Studying the competition in dissociation on BIGF-I/IGFBP-5 complex 63 3.3.3. Studying the competition in association on the BIGF-I/IGFBP-5 complex formation ................................................................................................. 68 3.4. Conclusions ................................................................................................... 72 4. Chapter four: Discussion and Conclusions .................................................... 73  4.1.  Discussion .................................................................................................... 73 4.1.1. Serum and tissue IGFBPs ................................................................... 73  4.1.2. Studying IGF-I/IGFBP-5 interaction using ELISA and BLI ................... 75 4.1.3. Therapies targeting IGF axis ................................................................ 77 4.2. Conclusion ..................................................................................................... 78 Bibliography ............................................................................................................  79 Appendix  ................................................................................................................. 108     viii	 ?	 ?List of Tables  Table 3.1: Kinetic and equilibrium constants for the interaction of IGF-I as a ligand on the biosensor with IGFBP-5 as analyte ????...??????????????? 62            ix	 ?	 ?List of Figures  Figure 1.1: Schematic diagram of IGF axis members around the IGF-IR ????... 14             Figure 2.1: Sandwich ELISA assay to characterize rhIGFBP-5 and its capture and          detection antibodies ???????????????????????????.. 39 Figure 2.2: Sandwich ELISA assay to characterize rhIGF-I and its capture and detection antibodies ???????????????????????????????? 40 Figure 2.3: Bridged ELISA assay to check the detection of IGF-I signal when IGF-I was added on IGFBP-5 fixed to the plate ??????????????????.??. 41 Figure 2.4: Bridged ELISA assay to check the detection of IGFBP-5 signal when IGFBP-5 was added on IGF-I fixed on the plate ??????????????????? 42 Figure 2.5: Bridged ELISA assay to check the detection of BIGF-I signal when BIGF-I was added on IGFBP-5 fixed on the plate ??????????????????. 44 Figure 2.6: Bridged ELISA assay to check the detection of IGFBP-5 signal when IGFBP-5 is added on BIGF-I fixed to the streptavidin-coated plate ??????????.... 45 Figure 2.7: Bridged ELISA assay to check the detection of IGFBP-5 signal, using non-biotinylated IGFBP-5 antibodies, when IGFBP-5 is added on BIGF-I fixed to the streptavidin covered plate ???????????????????????.?... 46 Figure 3.1: Illustration of the streptavidin-coated Octet biosensor ???????...  53 Figure 3.2: Illustration explaining a typical sensogram ???????...????.... 54 Figure 3.3: Illustration of the Octet biosensor tip in the competing assay ????..  56 Figure 3.4: Illustration of the Octet biosensor tip in the blocking assay ??..???. 58 Figure 3.5: This is the raw data showing the sensograms of the binding of BIGF-I to the biosensor, followed by binding of IGFBP-5 ?????????????????... 61 x	 ?	 ?Figure 3.6: The effect of IGF-I, IGF E3R and Insulin on dissociating IGFBP-5 from BIGF on the biosensor ?????????????????????????????. 64 Figure 3.7: Comparing the Kdis value of IGF-I and IGF E3R ??????????.. 67 Figure 3.8: The effect on IGFBP-5 binding to BIGF-I on the biosensor when samples were mixed with IGFBP-5 in the same well ?????????????????? 70 Figure 3.9: The effect of using different concentrations of IGF-I to block IGFBP-5 binding of BIGF-I on biosensor ??????????????????????..... 71              xi	 ?	 ?List of Abbreviations AD                 Androgen-Dependant AKT               Serine/Threonine Protein Kinase ALS               Acid-Labile Subunit ASO              Antisense Oligonucleotides  BAD              Bcl-2 Associated Agonist of Cell Death BIGF-I           Biotinylated Insulin-Like Growth Factor BLI                Bio-Layer Interferometry BSA              Bovine Serum Albumin  CRPC           Castration-Resistant Prostate Cancer DM                Diabetes Mellitus  DPI                Dual Polarisation Interferometry  ECM              Extracellular Matrix ELISA           Enzyme-Linked Immunosorbent Assay GH                Growth Hormone HRP              Horseradish Peroxidase  IGF                Insulin-Like Growth Factor IGF-R            Insulin-Like Growth Factor Receptor IGFBP           Insulin-Like Growth Factor Binding Proteins IGFBP-rP      Insulin-Like Growth Factor Binding Proteins Related Protein IR                  Insulin Receptor IR-A              Insulin Receptor A IR-B              Insulin Receptor B Ka                Constant of Association xii	 ?	 ?KD                Affinity Constant Kdis             Constant of Dissociation mAb             Monoclonal Antibody MAPK          Mitogen-Activated Protein Kinases  MMPs          Matrix Metalloproteinases  OD               Optical Density PAPP-A       Pregnancy-Associated Plasma Protein-A  PBS             Phosphate Buffered Saline  PCa              Prostate Cancer PI-3              Phosphatidylinositol-3 PSA             Prostate-Specific Antigen  RDB             Reagent Diluent Buffer  rhIGF-I         Recombinant Human Inulin-Like Growth Factor 1 rhIGFBP-5   Recombinant Human Inulin-Like Growth Factor Binding Protein 5 rhGH            Recombinant Human Growth Hormone  SA                Streptavidin TGF- ?         Transforming Growth Factor Beta        xiii	 ?	 ?Acknowledgments  Completing this thesis would not have been possible without the tremendous support and guidance I received from several people who helped me along this journey. I owe particular thanks to Dr. Michael E. Cox for all of the time, encouragement and helpful discussions that he so generously provided in order to help me to think and work as a scientist.  Thanks to Mazyar Ghaffari who taught me most of the skills that I acquired during my masters degree, and he became one of my best friends. I also would like to thank the faculty, staff and my fellow students at the Vancouver Prostate Centre, who really became my big family in Vancouver.  I offer my enduring gratitude to Dr. Ben Chew for enlarging my vision of science and providing me with the advice and support to advance in this field and in my career. Special thanks goes to my mother ?Sahar? and my father ?Essam? who are the best parents in the world. Big thanks goes to my bothers ?Abdulelah? and ?Abdulbari?, and to my sisters ?Bedour?, ?Zehour? and ?Nour? and my best friend ?Ahmad? for always being there for me. Finally, I would like to thank my other half, my lovely wife ?Ahilla? whose love and support carried me through along the way since we became a couple. I am very grateful to God that we were blessed with our first child ?Essa? few months after starting my masters. You have been incredibly patient and understanding. Words cannot express my gratitude. 	 ?  xiv	 ?	 ?   Dedication   To ?Khala ?Abeer ?May ?God ?rest ?her ?soul ?  1	 ?	 ?1. Chapter one: Introduction Cancer is the most common cause of death in Canadians. The incidence of cancer is continuously increasing over the years. Every hour, an average of 21 people will be diagnosed with some type of cancer, and nine people will die from cancer in Canada in 2012. The most commonly diagnosed cancer in men is prostate cancer, breast cancer is the most common cancer in women, while lung cancer is the most common cause of cancer mortality in Canada [1]. Different growth pathways contribute to the growth and survival of cancer cells including insulin like growth factor (IGF) axis. Dysregulation of the IGF axis members has been linked to the development and progression of different types of cancers including prostate, lung and, breast cancer [2].  1.1. IGF axis The IGF axis plays a key role in controlling cellular growth, differentiation and apoptosis [3, 4]. As such, these molecules are important in the development and regulation of many tissues and in the pathophysiology of various malignancies [2, 5, 6]. The IGF axis consists of two peptide growth factors, IGF-I and IGF-II [7, 8], cell-surface IGF-I and -II receptors (IGF-IR and IGF-IIR) [9, 10], six IGF-binding proteins (IGFBP)-1?6 [11-16], IGFBP-rP [17], and a group of IGFBP proteases [18]. In this thesis, I will discuss the different components of the IGF axis, and their effect on cells in normal tissue and in cancer, with emphasis on the involvement of the IGFBP-5 in the progression of castration resistant prostate cancer (CRPC).      2	 ?	 ?1.1.1. IGF receptors In the IGF system there are two main receptors (IGF-IR and IGF-IIR). Both IGF-IR and IGF-IIR are trans-membrane glycoproteins that differ completely in their structure and function [19-21]. In contrast to IGF-IIR, IGF-IR induces cell growth and survival.  IGF-IR is a tetramer tyrosine kinase receptor, composed of two ? subunits and two ? subunits [19-22]. There is approximately 84% homology at tyrosine kinase domains between the IGF-I receptor and the insulin receptor (IR) [23], and thus IGF-IR and IR can hybridize to form a heterodimer composed of one ? and one ? subunit of each receptor. This hybrid receptor can bind to IGF-I and IGF-II and Insulin. However, it binds IGF-I with higher affinity than insulin, and tends to have a cell growth promoting effect, similar to IGF-IR, more than the metabolic effect of the insulin receptor, by activating downstream signalling pathways [24, 25]. Binding of IGFs to IGF-IR activates its intrinsic tyrosine kinase activity.  The post-receptor signal transduction events include phosphorylation of insulin receptor substrate (IRS) family of proteins and activation of phosphatidylinositol-3 (PI-3) and mitogen-activated protein kinases (MAPK) [19, 26]. This will results in a myriad of events, including the up-regulation of cyclin D1 leading to the phosphorylation of retinoblastoma protein, and expression of downstream target genes such as cyclin E. [27, 28] Moreover, IGF-IR activation down-regulates the cell-cycle suppressors like PTEN [29, 30] indicating that multiple pathways are involved in producing its mitogenic effect.  In addition to promoting cellular proliferation, the IGF system has a strong antiapoptotic effect. Cancer cells can often escape the normal programmed cell death 3	 ?	 ?mechanisms through the activation of the AKT pathway, which inhibit proapoptotic proteins such as BAD [31], and activates pro-survival factors such as nuclear factor ?B [32] (Fig. 1). AKT in cancer-related IGF signaling has a major role in tumor invasion and metastasis [33]. Taken together, the IGF-IR will promote cellular proliferation and enhanced survival in IGF-responsive cells.  In contrast, IGF-IIR is a monomeric single large trans-membrane receptor [21, 34] that is completely unrelated to the IGF-IR and insulin receptor (IR). These receptors, however, are clearly multifunctional. Their most well characterized role is as mannose-6-phosphate receptors involved in the targeting of lysosomal enzymes to the lysosomes within the cell. However, they also bind latent transforming growth factor beta (TGF-?) and enable its activation on the cell surface, and also bind to retinoids, urokinase receptors, and many other proteins [35]  It has no known intracellular catalytic or adaptor signalling capacity and selectively binds to IGF-II [34]. In relation to the IGF growth function, it is thought to act as a clearance receptor for IGF-II, by binding IGF-II and decreasing their level in the surrounding environment. The extracellular domain of IGF-IIR dissociates after proteolytic degradation of the receptor upon binding IGF-II, and this extracellular part binds IGF-II in the blood resulting in its further degradation [36-39]. These receptors, in addition to the IGFBPs, appear to provide an additional control on the amount of IGF-II to which a cell is exposed.  1.1.2. Insulin-like growth factors (IGFs) IGF-I and IGF-II are single chain polypeptides [40], they shares around 50% homology to proinsulin, thus the name [21]. Because of the structural similarities 4	 ?	 ?between IGF-I, IGF-II and insulin, IGF-IR may also be activated by IGF-II and insulin. However, the affinity of IGF-IR to IGF-II and insulin is approximately 10-fold and 1000-fold lower than for IGF-I, respectively [41].  IGF-I is composed of 70 amino acids, it is synthesized primarily in the liver and bone marrow [42] under the influence of the growth hormone (GH) [43], but it is also found expressed widely and in many cell types in tissues throughout the body as well [44]. IGF-I levels increases gradually after birth, peaks at puberty, and declines afterwards [45]. It is the main ligand of the IGF-IR, it plays an important role in organogenesis and promotes cellular differentiation [40], plus it was found that it induces tumor progression by promoting antiapoptotic and mitogenic functions on tumor cells that secrete IGF-I by activation of IGF-IR [46]. IGF-II is a 67 amino acid molecule, which is synthesized primarily by the liver, and it is critical for fetal growth and development [47]. It is present in higher plasma levels than IGF-I [48], and is believed to have antiapoptotic activity in some cancers [49]. There are, however, good amount of evidence indicating potential roles for IGF-II in adult human physiology and many of these consistently suggest a role in metabolic regulation. In cases where tumors produces excessive amount of IGF-II, it results in a clinical syndrome called non?islet cell tumor induced hypoglycemia. The systemic symptoms of this condition are mainly severe episodic hypoglycemia, with no cellular proliferative actions.  It has been known for many years that the insulin receptor has two distinct isoforms, IR-A and IR-B, which are derived from alternative splicing of the insulin receptor gene. The two receptor isoforms varied very little in their response to insulin. 5	 ?	 ?Interestingly, recent studies showed that the IR-A isoform has a high affinity for IGF-II, approaching the affinity with which it binds to insulin [33]. And since IGF-II and insulin bind with similar affinities to the IR-A, but IGF-II is present in humans circulation at a 1000-fold higher concentration than insulin, this means that in reality IGF-II could be the predominant ligand activating the IR-A receptor. Also, recent studies have demonstrated that compared with insulin, IGF-II triggers different cell signaling and responses [50, 51]. This could explain why IGF-II has a major role in metabolic regulation via the insulin receptor, which differs from the growth-promoting role of IGF-I via the IGF-IR. This may also help us understand why the IGF-II level is maintained at a high level throughout our life.  1.1.3. IGFBPs  IGFBPs are very important elements in the regulation of the IGF actions. They are a family of 6 proteins that have a high affinity to IGF, each contain 216-289 amino acids forming 3 nearly equally sized domains, with the conserved N- and C- domains and the L-domain joining them in the center ?Linker? [52, 53]. The N- and C- domains of the IGFBPs have the IGF binding sites. Fragmented N- or C- domains have much lower affinity than the full length IGFBP [54-56]. The relative IGF binding affinity of isolated N- or C- domains differs between IGFBPs [56-59]. In serum, IGF is mostly found in a ternary complex bound to IGFBP-3, the most abundant IGFBP in serum. IGFBP-3 binds to another protein called acid-labile subunit (ALS) [60], forming a ternary complex. Because the ALS is a large glycoprotein 85kDa, the formed ternary complex is too large (150 kDa) to cross the vascular epithelial layer, this will result in a large IGF reserve in the circulation [60-62], preventing unwanted 6	 ?	 ?excessive growth signaling , as well as preventing unnecessary metabolic effects of Insulin-like growth factors in different tissues [63]. The ternary complex will also extend the half-life of the IGF from 10 min (when free) to 12h (when bound to the ternary complex) [63, 64]. IGFBP-5 is the only binding protein, other than IGFBP-3, that can bind to ALS and form a ternary complex [65]. Approximately 90% of IGFBP-3 and 55% of IGFBP-5 circulate in these complexes in healthy adults [66]. The other IGFBPs (IGFBP-1,-2,-4,-6) are able to bind to IGF making binary complexes, which are smaller (40-50 kDa) and are able to cross the vascular epithelium, and thus be delivered to the target tissues [26, 67]. The IGFs binds to IGFBPs with higher affinity (KD: 10-10 M) than to the IGF-I receptor (KD:10-8 M) [2], so most of the IGFs in the body are relatively unavailable to activate the receptor when bound to IGFBPs. Because of the large soluble stores of IGFs that are maintained in association with the high affinity IGFBPs, there are many mechanisms for lowering the affinity of these interactions to make the IGF available for cellular actions in the tissues. Studies have shown that most IGFBPs interact and binds to Integrins and/or extra cellular matrix (ECM) components [68], which decrease their affinity to IGFs. Furthermore, IGFBPs can be cleaved, releasing free IGF, and the IGFBP fragments themselves may have their own biological activities [69-71]. There is considerable evidence that IGFBPs, not only can keep IGFs away from cell receptors and restrict activity, but also can enhance activity at the cellular level through different mechanisms [72], and also enhance delivery of IGFs to specific tissue compartments [73]. 7	 ?	 ?In addition to modulating the activity of the IGF-IR by controlling the bioavailability of the IGFs, IGFBPs have shown to affect cell survival and apoptosis in an IGF-independent way, either through interacting with other signaling pathways, or through their own signaling pathway [74]. There is evidence that IGFBPs can promote metastasis through affecting cell motility and adhesion [74]. Post-translational modifications, which occur in the L-domain of the IGFBPs, will influence IGFBPs activities. Thus when phosphorylation occurs, it affects their IGF binding affinity [75], plus makes the IGFBP more prone to be degraded by proteases [76]. And when glycosylation of an IGFBP occurs, it will affect cell interaction with that particular IGFBP [77, 78].  The 6 binding proteins are unrelated to the cell-surface receptors, but are structurally very closely related to each other, although they are each products of distinct genes and they all have very distinct functional properties [72]. There is still limited understanding of the exact physiological role of each of the IGFBPs. IGFBP-1 is a 30 kDa protein which binds to IGF-I and IGF-II with equal affinity (KD ?0.1 nM) [79-82]. It is predominantly derived from the liver where its expression is affected by the insulin level. In the circulation, IGFBP-1 levels undergo a circadian variation as a result of the dynamic insulin regulation, which seems to provide an additional acute control to ensure that IGF activity is modulated appropriate to prevailing metabolic conditions [83]. It can induce or inhibit the IGF actions in many types of cells [26]. As an example of the inhibiting activity of IGFBP-1, it inhibited IGF-I-induced growth in MCF-7 breast cancer cells [84].   8	 ?	 ?IGFBP-2 is a 36 kDa protein that is widely expressed during fetal development and in adults. It is mainly produced by liver, adipocytes, reproductive system and central nervous system [85]. Both inhibitory and stimulatory effects of IGFBP-2 on IGF actions in vitro have been reported [26, 86]. Inhibiting or stimulating IGF signaling depends on the interaction of IGFBP-2 with the extracellular matrix and cell surface. The IGFBP-2 interaction with IGF-I has been appeared to facilitate binding to heparin and extracellular matrix proteins, indicating possible mechanisms for delivery of IGF-I to target tissues [87, 88]. IGFBP-2 level changes were associated in the development of different types of cancer including breast [89] and prostate cancer. In prostate cancer, high level of serum IGFBP-2 was associated with low grade prostate cancer [90]. In addition, IGFBP-2 expression is one of the up-regulated proteins during castration resistance [91]. There is evidence suggests IGF-independent actions of IGFBP-2. In contrast to IGFBP-1, plasma concentrations of IGFBP-2 fluctuate little over a 48-h sampling period in healthy adults, with no notable change after eating or after a glucose challenge. In humans, IGFBP-2 levels increases after a prolonged fasting, which may indicate that IGFBP-2 concentrations are metabolically regulated reflecting long-term alterations in hepatic exposure to insulin, while IGFBP-1 reflect short-term fluctuations in insulin levels [92]. IGFBP-3 has a molecular weight of 29 kDa [93], that is expressed in liver and many other tissues [83]. In humans IGFBP-3 acts as the main circulating carrier protein, but it is also extensively expressed in many tissues and has many additional local functions [83]. IGF-I level stimulates hepatic IGFBP-3 production [94, 95], and in serum, 9	 ?	 ?the majority of IGF is associated with IGFBP-3 and the ALS, creating an IGF reserve in serum. IGFBP-3 has been linked to different types of human cancers. In LNCaP prostate cancer cells, vitamin D showed antiproliferative effect partially through increasing IGFBP-3 expression. Calcitriol, the active form of vitamin D, binds to its receptor making a complex and this complex binds to the IGFBP-3 promoter in the LNCaP cells [96-98]. Even high levels of androgen, which in contrast with low levels of androgens inhibit cellular growth in androgen sensitive prostatic cancer cells [99], showed increased expression of IGFBP-3 in androgen responsive LNCaP cells using the same receptor complex mechanism [100]. Increased levels of IGFBP-3 are actually associated with decreased risk of prostate cancer. IGFBP-3 also has an IGF-independent effect. Studies have shown that IGFBP-3 can interact with TGF-? receptor and it can affect the TGF-? signaling pathways, resulting in cell growth inhibition [101, 102]. In addition, IGFBP-3 can induce apoptosis by increasing the ratio of pro-apoptotic to anti-apoptotic proteins in breast cancer cells [103]. IGFBP-4 is a 24 kDa protein. It is expressed in different tissues including liver, bone tissue. IGFBP-4 showed a strong inhibitory effect on IGF-I by preventing the activation of the IGF-IR when the IGFBP-4 is found in the tissue. Conversely, intravenous administration of IGFBP-4, in the presence of a protease, will promote cellular proliferation [104]. The main protease of the IGFBP-4 is a calcium-dependent serine protease secreted by smooth muscle cells. The protease can cleave the IGFBP-4 into smaller fragments with lower affinity to IGF, which allow IGF to activate the IGF-I receptor [105]. It was found that level of the pregnancy-associated plasma protein-A 10	 ?	 ?(PAPP-A) [106] is high in many tissues including ovarian granulosa cells [107], leading to IGFBP-4 proteolysis and the release of IGF and subsequent dominant follicle development [108, 109]. IGFBP-5 has a molecular weight of 29 kDa. Similar to IGFBP-3, IGFBP-5 is able to make a ternary complex in circulation, but it makes only around 10% of the ternary complex pool in circulation [66]. The production of IGFBP-5 has been detected in different tissues including mammary glands during normal development, where it induced apoptosis [110], and during pregnancy where it appears to affect cellular differentiation [111]. In breast cancer cell lines, IGFBP-5 induced apoptosis and inhibited cellular [112, 113] in an IGF-dependent manner. In prostate, IGFBP-5 expression was increased in benign prostatic hyperplasia [114] and it was also expressed in LNCaP, DU145, and PC-3 cells prostate cancer cell lines [115, 116]. It showed antiapoptotic activity of IGF during the development of castration resistance in LNCaP cell line [117, 118]. More details about the role of IGFBP-5 in prostate cancer will be discussed later in this thesis. IGFBP-6 is a 34 kDa protein that is unique among the IGFBP family. In contrast to other IGFBPs, which have a higher/equal affinity to IGF-I as compared to IGF-II, IGFBP-6 has ?50-fold binding preference for IGF-II over IGF-I. It can inhibit IGF-II activity mediated through the IGF-IR, including proliferation, differentiation, migration, and survival in different cell lines [119, 120].  It is believed that C-terminal domain of IGFBP-6 is responsible for its IGF-II-binding preference [55].  IGFBP-6 also showed IGF-independent actions including promoting cell migration [121, 122], and inhibits angiogenesis [123] in Rhabdomyosarcomas. 11	 ?	 ?1.1.4. IGFBP-rP (related proteins) The IGFBP superfamily includes 6 members (IGFBP-1 to IGFBP-6) with high affinity for IGF-I and IGF-II, and 10 IGFBP-related proteins (IGFBP-rP1 to IGFBP-rP10) with low affinity for these ligands. Remarkably, IGFBP-related protein 1 (IGFBP-rP1), also known as insulin-like growth factor binding protein 7 (IGFBP-7) [17], is identified as a secretory and low-affinity IGFBPs. It is distinct from other low-affinity IGFBP-rPs that it can bind strongly to insulin [124], suggesting that IGFBP-7 is likely to have distinct biological functions from other IGFBPs. IGFBP-related protein-1 (or IGFBP-7) has been found to have an important role in the female reproductive system. It was implicated in human endometrial receptivity, folliculogenesis as well as growth, development, and regression of the corpus luteum in higher mammals [125-127]. Other studies showed that it could induce apoptosis in M12 prostate cancer cell line [128].  1.1.5. IGFBP proteases There are several mechanisms to release IGFs from IGFBPs through decreasing the affinities of IGFBPs for IGFs, and proteolytic cleavage has now been demonstrated for all six IGFBPs and has gained wide acceptance as the predominant mechanism for IGF release from IGFBPs. There different classes of IGFBP proteases, and we will talk briefly about them.  Matrix Metalloproteinases (MMPs) are a family of IGFBP circulating protease which were initially identified to act on IGFBP-3, then it has been described to act on other IGFBPs in different conditions [129]. However, these proteases sometimes show a limited cleavage activity on IGFBP-3. This will result in a cleaved IGFBP-3, which still retains IGF in the ternary complex, but is bound with a lower affinity. Even a small 12	 ?	 ?decrease in affinity could result in a shift in the complex equilibrium that must exist in vivo, with the IGF re-equilibrating to other IGFBPs that are present and which are generally not cleaved by the same protease [129]. These other IGFBPs only form binary complexes and therefore have greater ability to transport the IGFs out into target tissues. Indeed, some of these binary complexes differentially deliver IGFs to specific tissues [73].  There have been many studies documenting increased proteolysis of IGFBP-3 in the circulation whereby an increase in the availability of these anabolic IGFs may be an advantage, like in pregnancy [129]. In the human circulation, the balance of the proteases and the proteases inhibitors affects IGFBP-3 cleavage. Proteases capable of cleaving IGFBP-3 are present in both the circulation and extravascular fluids, and in the normal healthy individual there is little detectable IGFBP-3 protease activity in serum, because of the presence of inhibitors which protects the IGFBP-3 from proteolysis [129]. In situations where we have an increases in IGFBP-3 proteolysis, it?s due to a decrease in these inhibitors rather than an increase in levels of proteases [130]. In addition to being a protease for IGFBP-3, MMPs also have shown to have a degrading activity towards IGFBP-5 in human fibroblast conditioned medium[131], and in murine osteoblast cultures [132]. Serine proteinases are a family of proteins that showed proteolytic activity towards IGFBPs. Complement protein 1s (C1s), a component of the complement cascade was purified from human fibroblasts that is responsible for cleavage of IGFBP-5 in these fibroblasts [133]. This protease is commonly found as a pro-enzyme and its 13	 ?	 ?activity is highly regulated by the serpin, C1-inhibitor [134]. Interestingly, prostate-specific antigen (PSA) is a serine proteinase as well [135]. Cathepsins are lysosomal proteinases which has a strong IGFBP cleaving activity under acidic conditions. In human prostatic cancer cell acidified conditioned media, cathepsin D degraded IGFBPs 1?5 [136, 137]. Other studies suggested that the lysosymal degradation of IGFBPs by cathepsins is intracellular, after internalization from the pericellular environment [138, 139]. All of the IGF that is present in the body is effectively present in IGFBP complexes, and there is clearly a very complex system with the 6 IGFBPs, each of which exists in several functionally distinct forms. It has also become clear that each of the IGFBPs interacts with high affinity with many other proteins, especially in the ECM and on the cell surface.  The pattern of expression of these interacting components differs between tissues, and within any tissue differs with developmental stage and pathology. This extremely complex interaction between different components for controlling IGF availability and actions provides an insight into how some specificity could be conferred on this pluripotential system. Because of this sophisticated interplay of multiple components, IGF actions can be very finely controlled in a tissue-specific manner.   14	 ?	 ?  Figure 1: Schematic diagram of IGF axis members around the IGF-IR.  The downstream signaling of IGF-IR and the hybrid receptor (heterodimer of IGF-IR and IR) is shown. Abbreviations: Akt: Serine/threonine protein kinase; BAD: Bcl-2 associated agonist of cell death; Bcl-2: Apoptosis regulator Bcl-2; B-raf: Serine threonine protein kinase B-raf; ECM: Extracellular matrix; IGF-I: insulin-like growth factor I; IGF-II: Insulin-like growth factor II; IGF-IR: Insulin-like growth factor I receptor; IGF-IIR: Insulin-like growth factor II receptor; IKK: I?B kinase; IR: Insulin receptor; IRS: Insulin receptor substrate; Kras: GTPase KRas; P13K: Phosphoinositide 3 kinase.   15	 ?	 ?1.2. IGF axis modulations in cancer development and progression For the last several decades the relationship of GH and IGFs with the tumor progression and development has been studied extensively. They have growth promoting and anabolic actions that affect cell division, cellular differentiation, and metabolism. Evidence of the involvement of this growth axis in the development and progression of tumors comes from different sources including mouse models [140-145], in vitro studies [146-154], and even epidemiological studies [155-158]. Early knockout studies have shown that the IGF axis plays a key role in controlling the rate of mitosis of cells during embryogenesis and postnatal development [6]. Therefore, individuals with higher levels of IGF-I have a higher baseline rate of mitosis occurring concomitant with increased activation of the Ras/Raf and PI3K pathways and increased cell motility signaling. This high rate of cellular division occurring in the backdrop of a microenvironment favoring survival and motility is believed to increase the probability of developing somatic mutations sufficient for carcinogenesis. A study of 228 subjects with defective GH signaling pathway was compared to 338 normal relatives. The cancer risk in the affected group was 0%, while 9% to 24% in the control group [159]. Another large prospective study demonstrated a consistent relation between high IGF-I, low IGFBP-3 and an increase in the incidence of prostate, breast, and colorectal cancer in such individuals [155, 156, 158, 160].  Furthermore, resected tumor specimens have been shown to contain high level of expression of IGF-IR [1]. On the other hand, there are other studies that challenge this hypothesis. For example, patients with acromegaly have only mild increase in 16	 ?	 ?cancer risk compared to normal people [161-163], this is despite the consistent high levels of GH [164]. But because of the increasing risks over time in acromegaly patients for developing diabetes (DM) and cardiovascular disease, the analysis becomes difficult. 1.3. High IGF level in different diseases and the risk of cancer development  Pathological Excess of GH: Acromegaly is a pathological condition where there is excess secretion of GH and IGFs. These patients are giants, and the most common cause of death is from cardiovascular diseases, but because they have high levels of IGFs, they are at increased risk of developing certain cancers, especially colorectal cancer [163, 165-167]. Aetiological excess of GH: The use of GH in children with GH deficiency and other slow growing diseases has been used for many decades. Many studies have been done to study the risk of cancer development in the treated group, especially after a case report in the 1980s of children treated with GH who developed leukemia [168]. However, many children who developed cancer in these cohorts had conditions that predisposed them to cancer, which might have over-estimated the induction of malignancy by GH treatment [169]. This evidence is supported by the national Cooperative Growth study, a large, ongoing cohort study, initiated in 1985, of children treated with GH in the USA, which shows that there is no increased risk of leukemia in the treated group [170]. On the other hand, the incidence of cancer in adults previously treated with rhGH has not been extensively studied, nor has its incidence been assessed in children who continued to 17	 ?	 ?receive rhGH through late adolescence into adulthood. However, data are emerging on cancer incidence in adults with GH deficiency treated with rhGH, although constrained by short follow-up periods and the potential for reporting bias; suggests that rhGH has no substantial role in tumor recurrence or secondary neoplasia in adults [171] Recombinant human insulin-like growth factor I: Recombinant human insulin-like growth factor I (rhIGF-I) therapy is used when the serum level of IGF-I is low and the serum level of GH is normal in the absence of other causes for the treatment of short stature of children [172]. The side effects of the treatment are acceptable, with no increased risk of malignancy [172, 173], although more studies with longer follow up are needed to assure the safety of such treatments.   1.4. IGF axis in Prostate cancer PCa is the second-leading cause of cancer deaths in North American males [174]. Altered expression of insulin-like growth factor (IGF) axis components are strongly implicated in the development, metastasis and progression of prostate cancer [175-178]. Increased circulating IGF-I levels will lead to an increase in the risk of prostate cancer development [177, 179]. Furthermore, metastatic lesions exhibit an increase in the IGF-IR expression compared with primary lesions [178, 180]. These observations and others indicate that IGF-I-mediated signaling plays an important role in PCa growth and progression. Cellular responsiveness to IGFs depends on the bioavailability of IGFs to the target tissue, which is regulated by IGFBPs [26]. Tumor IGFBP levels are significantly altered during CRPC progression [181-184]. Studies on humans showed that in prostate cancer with high Gleason score there is an increase in the expression of IGFBP-2 and 18	 ?	 ?IGFBP-5, and a decrease in the expression of IGFBP-3 [91]. Similar changes in the expression of IGFBP-2, -3 and/or -5 have been observed during castration-induced regression of AD Shionogi carcinoma [182] and LNCaP xenografts [117]. Interestingly, autocrine IGFBP-5 production promoted cellular growth of androgen-deprived LNCaP cells [118]. This demonstrates the important role of IGFBPs in regulating IGF responsiveness during AI progression of prostate cancer. Although IGFBPs, including IGFBP-5, have been reported to exert positive and negative IGF-I independent activities [185], in LNCaP cells subjected to androgen deprivation and cytotoxic stress, IGFBP-5 can suppress apoptosis only in the presence of IGF-I [118].  IGFBP-5 is a critical component of the IGF system in bone. It has a high binding affinity for hydroxyapatite, which ensures that IGFBP-5 and its bound IGFs are fixed within bone matrix [186, 187].  It is the most abundant IGFBP stored in bone [188] and the most up-regulated gene in castrated mouse bone [189]. This may explain why bones are the most common site of prostate cancer metastasis [190]. The stromal-epithelial interactions are a key element in prostate cancer progression and invasion, and during androgen blockade, the bone microenvironment that is rich in IGFs provides an alternative growth pathway for prostate cancer cells. The propensity of PCa for skeletal metastasis and subsequent castration resistance progression is enhanced by IGF-I signaling modulated by IGFBP-5. A study on MG63 cells expressing IGFBP-5 showed that the IGFBP-5 promotes IGF-dependent survival signaling in LNCaP cells.     19	 ?	 ?1.5. Therapeutic strategies targeting IGF axis in cancer treatment Drugs targeting various component of the IGF axis, with varying degree of success, have been developed for treatment of different types of cancer. Description and challenges of each targeting strategy will be explained in this section. Targeting IGF-IR: Monoclonal antibodies (mAb), small molecule inhibitors, and antisense oligonucleotides were developed against IGF-IR. mAb targeting IGF-IR can induce internalization and degradation of the receptor upon binding. In clinical trials, the response to IGF-IR?targeting mAb monotherapy is rare, and it was significant in some cases of Ewing sarcoma [191]. Disease stabilization, defined as lack of progressive disease or objective response at 12 weeks, was the main efficacy signal of IGF-IR mAb, like figitumumab and ganitumumab, in phase I and II studies in patients with different types of sarcoma[191]. The main side adverse effect of these drugs was hyperglycemia. Although these antibodies do not bind to IR, many can partially modulate the activity of IR, including IR-B, by blocking the hybrid receptors.  In contrast, tyrosine kinase inhibitors were used to target IGF-IR. But because of the homology between the IGF-IR and the IRs in the tyrosine kinase domain, these inhibitors resulted in impaired glucose homeostasis and hyperglycemia [192]. linsitinib (OSI-906), an oral small molecule, is currently in a phase III randomized, double-blind, placebo-controlled monotherapy trial in patients with advanced adrenocortical carcinoma. Lastly, there are few antisense oligonucleotide drugs targeting IGF-IR. ATL1101, a second generation antisense oligonucleotides (ASO) selectively suppressing the IGF-IR, was able to inhibit growth and delay tumor progression in vivo 20	 ?	 ?and in vitro in a prostate cancer model [193]. These are preclinical results, which still needs validation in clinical studies to see the effect of this ASO on IR. Targeting IGFs: Only few (mAb) IGF antibodies were developed, MEDI-573 is the only one which was clinically investigated. MEDI-573 is a human monoclonal IgG2l antibody that specifically binds to both free IGF-I and IGF-II at the binding sites of IGF-IR, IR-A and IGFBP-3 [194]. This will prevent the activation of the receptors and the downstream signaling pathways (AKT and Ras/Raf pathways), in addition of short half-life in the serum. Interestingly, this mAb doesn?t affect insulin activation of IRs and their metabolic downstream signaling. The preliminary results of the ongoing phase I clinical trial has shown stabilization of disease > 3 months in 7 of 16 patients with chemo-resistant cancers [195], without inducing hyperglycemia [195]. If confirmed, this will become an important advantage of targeting the ligands of IGF axis instead of the IGF-IR, which shows hyperglycemia consistently. Targeting IGFBPs: Although IGFBPs can prevent IGF from binding to IGF-IR because their higher affinity to IGF than the IGF-IR, it can also induce tumor growth and progression in situations where the IGFBP proteases levels are high and/or when IGFBPs interact with ECM. Thus, modifying IGFBP depends on the targeted tissue and the status of the disease. For example, IGFBP-3 have shown proapoptotic, anti-proliferative and anti-angiogenic functions in in-vitro tumor models [196-198]. On the opposite side, IGFBPs can promote tumor progression in the presence of proteases. IGFBP-2 and IGFBP-5 up-regulation in CRPC is a good example of that. In the presence of PSA and other factors affecting the IGF-I/IGFBP-2,5 binding, it will result in the delivery of the IGFs to the IGF-IR and activation of the downstream signaling 21	 ?	 ?pathway, thus helping the progression to castration resistant disease [182, 184]. Few antisense oligonucleotide drugs are still in their early phases of development. 1.6. Molecular interaction between IGFBP-5 and IGF-I  All IGFBPs share a common protein structure that can be divided into three domains: N- and C-terminal domains which are highly conserved, and the L (linker) domain which is non-conserved and links the two other domains and contains sites of post-transitional modifications and proteolysis [199]. The N- [200] and C- terminals [201] both are cysteine-rich domains containing 12 and 6 cysteine residues (except IGFBP-6 which have 5 cysteine residues in the C-terminus), respectively, and both participate in IGF binding. NMR study was carried out on N-terminal fragment of IGFBP-5 (residues 40?92), and it clearly demonstrated that a major IGF-binding domain comprises Val49, Tyr50, Pro62, and Lys68?Leu75, where the conserved Leu and Val residues localize in a hydrophobic patch on the surface of the IGFBP-5 protein [202]. Substitution of specific hydrophobic residues in this region of IGFBP-5 resulted in about 1000-fold reduction in affinity for IGF-I [203]. However, the N-terminal fragments of IGFBP-5 (or mini IGFBP-5) had a 10?200-fold lower affinity for IGFs than the full-length protein [202]. This indicated that the C-terminus of the IGFBPs is required to add stabilization of IGF complex formation and leads to the high affinity binding.  The C-terminus of IGFBP-5 is rich in basic residues between residues 201-218. Many of these basic residues play an important role in IGFBP-5 binding to a variety of molecules including IGF-I [204], heparin [205, 206], extracellular matrix (ECM) [207], many ECM proteins like osteopontin [208], a putative IGFBP-5 receptor [74], and plasminogen activator inhibitor-I [209]. Mutations of all basic residues in this region 22	 ?	 ?results in a 15-folds reduction in IGFBP-5 affinity towards IGF-I. However, this was less than the 45-folds reduction when the N-terminal was mutated in this study [204]. Thus, targeting the N-terminal with small molecule drugs would be more effective in decreasing IGFBP-5 affinity towards IGF-I.   23	 ?	 ?1.7. Available methods to investigate protein/protein interactions There are many methods to detect protein-protein interaction, each of which has its own specificity and sensitivity, and thus each has their strengths and weaknesses. We can divide them into two categories: Biochemical methods and Biophysical methods. I will give two examples of each category. Biochemical methods:  - Co-immunoprecipitation is considered to be the gold standard assay for protein/protein interactions. The protein of interest (ligand) is isolated with an antibody. The interacting partners (analytes) which binds to the ligand protein can be identified by Western blotting [210]. Interactions detected by this approach are considered to be real. However, the problem with this approach is that it can only verify interactions between proteins, whether the interactions were direct or indirect through other bridging molecules. Thus, it is not a screening approach.  - Enzyme-linked immunosorbent assays (ELISAs) are plate-based assays designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. The main principle of an ELISA is that an antigen must be immobilized to a solid surface and then make a complex with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product. A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It also can be linked to a protein such as streptavidin if the antibody is 24	 ?	 ?biotin labeled. The most commonly used enzyme labels are horseradish peroxidase (HRP) [211]. Biophysical methods: - Bio-Layer Interferometry is a label-free technology for measuring bio-molecular interactions [212, 213] (protein/protein or protein/small molecule). It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time, providing detailed information regarding the kinetics of association and dissociation of the two molecules as well as the affinity constant for the protein interaction (ka, kdis and KD). The detection method can also be used to determine the molar concentration of analytes. - Dual polarisation interferometry (DPI) can also be used to measure protein?protein interactions. DPI provides real-time measurements of molecular size, density and mass. One protein is immobilized on the surface of a waveguide. Conformational changes during interaction can be quantified in addition to kinetics and affinity [214]. 1.8. Specific Experimental objectives Most of the IGF axis components have been linked to the development and progression of various types of cancer. IGFBPs binds to IGF-I and IGF-II with high affinity, and thus it control?s their bioavailability, depending on the presence of many factors, by inducing or antagonizing the activation of IGF-IR tyrosine kinase, and thus 25	 ?	 ?affecting cancer cell proliferation, survival, metastasis and angiogenesis. In CRPC, IGFBP-2 and IGFBP-5 were one of the highest up-regulated proteins in prostate cancer cells.  These changes enhanced IGF-I mediated prostate cell survival. Many IGF axis targeted drugs were developed for the treatment of cancer. Since the IGFBPs are considered a key factor in this system, and because IGFBP-5 has a well characterised high-affinity binding pocket for IGF-I, as well as it has significant importance in the progression of CRPC.  My objective during my two years of work at the Vancouver Prostate Centre, was to develop methods to study IGF-I/IGFBP-5 binding kinetics. I studied this molecular interaction using ELISA, as well as bio-layer interferometry (BLI) method. This method can be used to test drugs targeting IGFBPs.    26	 ?	 ?2. Chapter two: Building bridged ELISA for measurement and detection of IGF-I/IGFBP-5 binding  We originally planned to develop an ELISA technique to detect the binding of IGFBP-5 and IGF-I and the ability of several proof-of-principle approaches to either block formation of IGF-I/IGFBP-5 complexes or dissociate preformed IGF-I/IGFBP-5 complexes. The premise eas that we could use either an IGF-I or an IGFBP-5 capture antibody to immobilize the respective ligand to a microtitre plate. We would then be able to bind the reciprocal ligand to the immobilized ligand and detect it with the respective biotin-conjugated detection antibody.   We started by characterizing commercially available IGF-I and IGFBP-5 ELISA assays that utilize antibody capture to immobilize the respective ligand to the plate, and a detection antibody to detect this protein, in an effort to determine potential dynamic range of the assays. Using the ligand dosing conditions described below, we predicted we would have insight into how to best construct a ?bridged? ELISA assay in which either IGF-I or IGFBP-5 would be antibody-captured on the plate, the reciprocal ligand would then bind to the captured ligand and detected with the respective detection antibody.  2.1. Materials and Methods Phosphate buffered saline (PBS) was purchased from Thermo Scientific (Rockford, IL, USA; Catalogue #BP399-20); Bovine serum albumin (BSA) was purchased from (Thermo Scientific; Catalogue #23209). Antibodies and recombinant proteins were diluted into Reagent Diluent Buffer (RDB; 5% Tween 20 in PBS).  27	 ?	 ?Blocking buffer was composed of RDB with 0.05% NaN3 and washing buffer was composed of 0.05% Tween 20 in PBS.  All recombinant proteins and antibodies were purchased from R&D Systems (Minneapolis, MN).  Recombinant human IGFBP5 (Catalogue #875-B5-025) was reconstituted from lyophilized powder in PBS to a stock of 480 ng/ml, aliquoted and stored at -20 C.  Recombinant human IGF-I (Catalogue #291-G1-200) reconstituted from lyophilized powder in PBS to a stock of 110 ng/ml, aliquoted and stored at -20 C. Aliquots were thawed and diluted as needed in RDB and stored on ice for immediate use. Mouse anti-human IGFBP-5 monoclonal antibody (Catalogue #MAB8751) was used as the IGFBP-5 capture antibody. It was purchased as a 720 ?g/ml stock and diluted to a working concentration of 4 ?g/ml in PBS. Biotinylated goat anti-human IGFBP-5 polyclonal antibody (Catalogue #BAF875) was used as IGFBP-5 detection antibody.  It was purchased as a 36 ?g/ml stock and diluted to a working concentration of 200 ng/mL in RDB. Mouse anti-human IGF-I antibody (Catalogue #MAB291) was used as an IGF-I capture antibody.  It was purchased as a 720 ?g/ml stock and diluted to a working concentration of 4 ?g/ml. Biotinylated goat anti-human IGF-I antibody (Catalogue #BAF291) was used as an IGF-I detection antibody.  It was purchased as a 27 ?g/ml stock and diluted to a working concentration of 150 ng/ml. All antibodies were provided as stocks and were stored at -20 C.	 ?The reagents used for colorimetric signal detection in the ELISA assays were purchased from R&D Systems and included: Streptavidin-conjugated horseradish peroxidase (HRP; Catalog # DY994); Color Reagent A (H2O2) and Color Reagent B 28	 ?	 ?(Tetramethylbenzidine) from (Catalogue #DY999) a 1:1 mixture of these two reagents forms Substrate Solution; and Stop Solution (2N H2SO4; Catalogue #DY994). All of these reagents were stored at 4 C. The plates used were either Pierce 8-Well Strip Plates from (Thermo Scientific; Catalogue #15031), or 96 wells clear bottom streptavidin-coated plates from (Thermo Scientific; Catalogue #PI-15118). The optical density of each well was determined using a microplate reader set to 450 nm, with a reference wavelength of 540 nm. All ligand dose determination ELISA assays were performed in triplicate, and all incubations were performed at room temperature.  Two tail Student?s T test was used to determine the statistically different values of samples with the significance level set at p<0.05. 2.2. ELISA assays development 2.2.1. Characterizing IGFBP-5 ELISA protein and antibodies Pierce 8-Well Strip plates, which have a negatively charged bottom, were coated with mouse anti-human IGFBP5 antibody (100 ?l, 4 ?g/ml), sealed and kept overnight at room temperature. Wells were then washed 3x with 400 ?l of the ELISA washing buffer (wash cycle), blocked with 300 ?l of blocking buffer for one hour, followed by a wash cycle.  Doubling dilutions [from 1.25 to 40 ng/ml] of rhIGFBP-5 in a final volume of 100 ?l/well of RDB were added to wells in duplicate to obtain a standard curve. After a 1 hr incubation, the wells were subjected to a wash cycle before incubation with 100 ?l of biotinylated goat anti-human IGFBP-5 detection antibody at 200 ng/ml in RDB containing 2% BSA for 2 hrs.  After a wash cycle, 100 ?l of Streptavidin-HRP was added to each well and incubated in the dark for 20 min.  After another wash cycle the relative 29	 ?	 ?amount of detection antibody/streptavidin-HRP retained in the wells was determined by incubation with 100 ?l of a 1:1 mixture of Color Reagent A and Color Reagent B for 20 min.   Development of the color reaction was stopped by adding 50 ?l Stop solution to each well while shacking the plate, to ensure proper mixture, and the optical density of each well was immediately determining at 450 nm with a wavelength correction measurement at 540 nm using a microplate reader (Fig.2.1 A). 2.2.2. Characterizing IGF-I ELISA protein and antibodies An IGF-I sandwich ELISA was also assessed using the same methodology as above with the following modifications:  mouse anti-human IGF-I antibody was used as a capture antibody, the ligand test was doubling dilutions of rhIGF-I [from 0.0625 to 2 ng/ml] and a biotinylated goat anti-human IGF-I antibody was used as a detection antibody at a concentration of 150 ng/mL in RDB (Fig. 2.2 A). Based on the predicted detection range of the basic ELISAs for IGF-I and IGFBP-5, we expected that such an assay would provide a dynamic range to detect changes in IGF-I/IGFBP5 binding affinity when agents targeting IGF-I binding site on IGFBP5 were introduced.    2.2.3. Characterizing bridged ELISA formats to test the binding of IGF-I to IGFBP-5  The first bridged ELISA format tested was to use IGFBP-5 capture antibody to immobilize rhIGFBP5, to then attempt to detect IGF-I binding to IGFBP5 by adding rhIGF-I and detecting immobilized IGF-I using the biotinylated IGF-I detection antibody using the basic ELISA methods detailed above with the following modifications. 30	 ?	 ?Microtitre plate wells were coated with IGFBP-5 mouse anti-human antibody, washed, blocked and incubated with 10 ?g/ml rhIGFBP-5 as in 2.1.1.  After a wash cycle, wells were incubated with 100 ?l rhIGF-I at [0, 0.5, 1, 2 ng/ml] for 2 hours. After a wash cycle, assays were developed using the biotinylated goat anti-human IGF-I detection antibody, streptavidin-HRP and color development as in 2.1.2 (Fig. 2.3 A)  In second bridged ELISA format tested, we used IGF-I capture antibody to immobilize rhIGF-I [2 ng/ml], and then to attempt to detect IGFBP-5 binding to IGF-I by adding rhIGFBP-5 [2.5, 5, 10, 20 ng/ml] and detecting it using biotinylated IGFBP-5 detection antibody as in 2.1.1 (Fig 2.4 A). 2.2.4. Characterizing bridged ELISA format using IGFBP-5 capture antibody and biotinylated IGF-I (BIGF-I)  To minimize the possible effects of antibodies interfering with IGF-I/IGFBP-5 interaction and/or detection we assessed approaches that relied on only one antibody in the ELISA format. In the first approach, 100 ?l/well of rhIGFBP-5 [10 ?g/ml] was captured using mouse anti-human IGFBP5 antibody as in 2.2.1. After the wash cycle and blocking, wells were incubated with a serial dilution of 100 ?l/well of BIGF-I [0, 0.25, 0.5, 1, 2, 4, 8, 16 ng/ml] for 2 hours, wells were then washed and bound BIGF-I was detected using streptavidin-HRP as in 2.1.1 (Fig. 2.5 A). We also tried an alternative approach using Streptavidin-coated plates to capture BIGF-I.  After a wash cycle and blocking, wells were incubated with 100 ?l/well of rhIGFBP-5 [10 ?g/ml], washed and detected using the biotinylated IGFBP-5 goat anti-human antibody and streptavidin-HRP as in 2.1.1 (Fig.2.6 A). 31	 ?	 ?In addition, we attempted to use non-biotinylated IGFBP-5 antibodies to prevent biotin cross detection by the streptavidin-HRP complex from the bottom of the plate as well as the IGFBP-5 detection antibody. For this approach, we substituted IGFBP-5 biotinylated antibody with the non-biotinylated mouse anti-human IGFBP-5 antibody, previously used as a capture antibody, and a secondary HRP-conjugated sheep anti-mouse IgG antibody, purchased from Amersham (Buckinghamshire, England; Catalogue #RPN2401), were used for detection (Fig.2.7 A). 2.3. ELISA assay results: 2.3.1. Assessment of rhIGFBP-5 using sandwich ELISA  To test the dynamic range for detection of rhIGFBP-5, a sandwich ELISA was made using its capture and detection antibodies to create a 7 points standard curve. Mouse anti-human IGFBP-5 antibody was used to immobilize IGFBP-5 to the plate. Then, doubling dilutions of rhIGFBP-5 [from 1.25 to 40 ng/ml] were added, followed by detection of biotinylated goat anti-human antibody using streptavidin-HRP and color development as in 2.1.2. A dose responsive curve, as detected by optical density measurement, is shown in (Fig. 2.1). Dilutions of rhIGFBP-5 in the range of 1.25 ? 40 ng/ml in RDB are shown; demonstrating that the lowest rhIGFBP-5 signal detected was 0.38 OD450, when we used the lowest concentration of 1.25 ng/ml. The signal was statistically significant (P=0.004) from the signal detected in the absence of rhIGFBP-5 (0.15 OD450). The signal of highest concentration of rhIGFBP-5 used [40 ng/ml] was 2.01 OD450. This resulted in a dynamic range for the developed sandwich ELISA of 0.38 ? 2 optical density. The signals detected from all rhIGFBP-5	 ?concentrations were statistically 32	 ?	 ?distinguishable from each other. Based on this experiment, we have chosen a concentration of 10 ng/ml rhIGFBP-5 for antibody immobilization in the first bridged ELISA format, because the signal detected at this concentration 1.21 OD450 will still allow a good dynamic range, it should bind all IGF-I at its highest concentration used in ELISA, and also for cost effectiveness.  Although the epitopes of the respective capture and detection antibodies are not reported, this demonstration that mouse anti-human IGFBP-5 antibody (IGFBP-5 capture antibody), and biotinylated goat anti-human antibody (IGFBP-5 detection antibody) can both bind to rhIGFBP-5 at the same time indicates that they have distinct epitopes. Since epitopes tend to be on the order of 5-8 amino acids, then we anticipated that at least one of these anti-IGFBP-5 antibodies would not directly interfere with IGF-I binding. 2.3.2. Assessment of rhIGF-I using sandwich ELISA  We also characterized an IGF-I sandwich ELISA that used a mouse anti-human IGF-I antibody to immobilize IGF-I to the plate, and biotinylated goat anti-human IGF-I antibody, and a 7 points standard curve to determine the dynamic range of rhIGF-I detection. Doubling dilutions of rhIGF-I [from 0.0625 to 2 ng/ml] were used. A dose-dependent response, as detected by optical density measurement, is shown in (Fig. 2.2).  rhIGF-I resulted in a signal of 0.49 OD450 when the lowest concentration was used [0.0625], which is indistinguishably different (P=0.09) from the signal detected in the absence of rhIGF-I (0.45 OD450). As a result, the lowest concentration of rhIGF-I significantly different from signal detected from blank was [1.25 ng/ml], with a signal of 0.55 OD450 (P=0.004). The signal of highest concentration of rhIGFBP-5 used [2 ng/ml] 33	 ?	 ?was 1.4 OD450. Except of a concentration of [0.0625], all rhIGF-I	 ?concentrations resulted in a signal detection that is statistically distinguishable from each other. This resulted in a dynamic range for the developed sandwich ELISA of 0.55 ? 1.4 optical density. It is possible that this narrow dynamic range is due to the small size of IGF-I (70 a.a) compared to the antibodies used (150 a.a. each), which may lead the antibodies to compete for their epitopes on rhIGF-I. We can conclude that mouse anti-human IGF-I antibody and biotinylated goat anti-human IGF-I antibody have different epitopes in rhIGF-I, and both can bind in the same time. We?ve chosen a concentration [2 ng/ml] of rhIGF-I to be used for the bridged ELISA for cost effectives and because based on molarity binding, 1 mole of IGF-I should bind to 3 moles of IGFBP-5, so rhIGF-I should be totally bound to [10 ng/ml] of IGFBP-5.   2.3.3. Assessment of signal detection using biotinylated detection antibodies in IGF-I/IGFBP-5 bridged sandwich ELISA After validating the activity of rhIGF-I and rhIGFBP-5 and their capture and detection antibodies, we attempted to create a bridged ELISA, in which either IGF-I or IGFBP-5 would be antibody-captured on the plate, or the reciprocal ligand would bind to the captured ligand and detected with the respective detection antibody. Based on the dynamic range provided from the previous experiments, different formats of bridged ELISA were made. In the first experiment, rhIGFBP-5 at a concentration of [10 ng/ml] was immobilized to the plate using IGFBP-5 capture antibody, followed by adding rhIGF-I at concentration [0, 0.5, 1, 2 ng/ml] and allowed for incubation for 2 hours at room temperature. The signal was detected using biotinylated 34	 ?	 ?IGF-I detection antibody. Based on the results shown in Fig 2.2, IGF-I detection antibody system can detect antibody-immobilized IGF-I when as little as 100 ?l of [0.125 ng/ml] was added to the plate.   Since we detected no more than 0.03 OD450 for any IGF-I concentration added and since we knew that adding IGFBP-5 at this concentration [10 ng/ml] resulted in a robust detection indicative of efficient capture of IGFBP-5,  we concluded that there was no IGF-I detected using this approach.  As a result of the previous experiment, an alternative configuration was created, where rhIGF-I at a concentration of [2 ng/ml] was immobilized to the plate using IGF-I capture antibody, and then rhIGFBP-5 at concentrations of [2.5, 5, 10, 20 ng/ml] was added, and allowed to incubate in room temperature for 2 hours, followed by detection using biotinylated IGFBP-5 detection antibody and color development as in Fig. 2.1.2. Based on the results shown in Fig 2.1, IGFBP-5 detection antibody system can result in a signal detection of 0.39 OD450 when 100 ?l of antibody-immobilized IGFBP-5 [1.25 ng/ml] was added to the plate. Since we detected a signal of 0.16 OD450 and 0.23 OD450 with the lowest and highest concentrations of IGFBP-5 used respectively, and since we previously demonstrated that adding IGF-I at this concentration [2 ng/ml] resulted in a robust detection indicative of efficient capture of IGF-I, we concluded that if the signal detected could be due to the detection of IGFBP-5 in a concentration lower than 1.25 ng/ml.  In order to test if this signal detected was due to IGF-I/IGFBP-5 binding, we repeated this experiment in the absence of rhIGF-I. Wells of the plate were covered with IGF-I capture antibody, followed directly by adding rhIGFBP-5, and then detection using biotinylated IGFBP-5 detection antibody and color development as in 2.1.2. In the 35	 ?	 ?absence of rhIGF-I, a signal detection of 0.19 OD450 and 0.33 OD450 with the lowest and highest concentrations of IGFBP-5 used respectively (Fig. 2.4). These signals are indistinguishable from the signal detected in the presence of rhIGF-I, so we concluded that this signal is not a result of the biding of rhIGF-I and rhIGFBP-5.  While we knew that we could readily capture and detect either ligand with the respective antibodies, neither combination of capture or detection antibodies could detect a putative IGFBP-5/IGF-I complex. This could possibly be due to conformational changes of IGFBP-5 and/or IGF-I when bound to their capture and/or detection antibodies, which can result in masking their IGF-I/IGFBP-5 binding site. Also it is possible that any of these antibodies shares IGF-I binding site on IGFBP-5 with IGF-I, and vice versa. 2.3.4. Assessment of signal detection using biotinylated IGF-I in  BIGF-I/IGFBP-5 bridged sandwich ELISA After the inability of signal detection of bridged IGF-I/IGFBP-5 ELISA assay using both capture and detection antibodies, possibly due to cross-reactivity, we tried to minimize the usage of antibodies. In an attempt to eliminate the effect of IGF-I biotinylated detection antibody, BIGF-I was used with Streptavidin-HRP complex for detection.  In this experiment, rhIGFBP-5 at a concentration of [10 ng/ml] was fixed to the plate using IGFBP-5 capture antibody, and then different concentrations of BIGF-I [0, 0.5, 1, 2, 4, 8, 16 ng/ml] were added, followed by Streptavidin-HRP complex for detection. The signal detected was very low with all BIGF-I concentrations, with an average signal of 0.01 OD450 (Fig. 2.5). It is possible that this low signal is due to the 36	 ?	 ?high affinity between biotin on IGF and streptavidin-HRP complex, which can result in the detachment of BIGF from the plate. In order to exclude the possibility of IGF-I capture antibody cross-reactivity; streptavidin-coated plate was used to immobilize BIGF-I to the plate. In this experiment, streptavidin-coated plate was used to capture BIGF-I at a concentration of [2 ng/ml], followed by blocking using ELISA blocking buffer and 5% milk. rhIGFBP-5 was added at a concentration of [0, 1.25, 2.5, 5, 10, 20 ng/ml], followed by adding biotinylated IGFBP-5 detection antibody and streptavidin-HRP complex for detection. The signal detected from this experiment was extremely high (an average of 3.27 OD450) across different concentrations of IGFBP-5, even when no IGFBP-5 was added (Fig. 2.6).  This high background signal detection may be due to cross detection of the biotin on BIGF-I on the bottom of the plate by HRP-streptavidin complex, which is originally used for detection of biotinylated IGFBP-5 detection antibody. Although this is apparently a blocking issue, and because of the different difficulties we experienced while trying different configurations, we decided to use a different approach to detect the IGF-I/IGFBP-5 binding.   In our last attempt to detect IGF-I/IGFBP-5 binding using ELISA, we tried to avoid the cross-reactivity of biotin-conjugated IGF-I and biotinylated IGFBP-5 detection antibody by the streptavidin-HRP complex used, we used the non- biotinylated mouse anti-human IGFBP-5 antibody to detect IGFBP-5, and signal detection was done using non-biotin-conjugated secondary anti-mouse antibody (which is conjugated with HRP). In this experiment, BIGF-I was captured to the streptavidin-coated plate, followed by adding IGFBP-5 [0, 1.25, 2.5, 5, 10, 20 ng/ml] with an incubation of 2 hours at room 37	 ?	 ?temperature. Mouse anti-human IGFBP-5 antibody was used to detect IGFBP-5, and signal detection can be made using HRP-conjugated sheep anti-mouse secondary antibody. A signal detection of 0.27 OD450 and 0.42 OD450 with the lowest and highest concentrations of IGFBP-5 used respectively. In order to check if this signal detected was a result of IGF-I/IGFBP-5 binding, we repeated the experiment in the absence of BIGF-I. rhIGFBP-5 was added directly to the streptavidin covered plate, followed by mouse anti-human IGFBP-5 antibody and the secondary anti-mouse antibody. This resulted in a signal detection of 0.21 OD450 and 0.33 OD450 with the lowest and highest concentrations of IGFBP-5 used respectively, which is indistinguishably different from the signal detected in the presence of BIGF-I (P=0.16) (Fig 2.7). 2.4. Conclusions Different ELISA constructs were used in an attempt to detect and study IGF-I/IGFBP-5 binding. Although sandwich ELISA assay of IGF-I and IGFBP-5 showed dose-dependent signal detection, we were unable to build a bridged ELISA assay to detect the binding of the two proteins.  While the sandwich ELISAs indicate that the respective ligands can be bound to two antibodies simultaneously, whether the ligand/antibody interactions alter physiologic properties of the ligands is not determined.  In each configuration of the bridged ELISA formats, it remains a formal possibility that one antibody or the other binds its respective ligand in such a way as to impair IGF-I/IGFBP-5 interactions.  This is not too surprising since about half of IGF-I is in contact with IGFBP-5 when bound in its high affinity configuration.  The final attempt to immobilize IGFBP-5 to plates with the capture antibody and detect BIGF-I directly 38	 ?	 ?indicated that the most likely issue faced is that antibody binding to IGFBP-5 disrupts the ability of IGFBP-5 to bind IGF-I with high affinity. It is possible that the antibodies caused conformational changes to IGF-I and/or IGFBP-5, which lead to masking of the binding site. It is also possible that the antibody shares IGF-I or IGFBP-5 binding sites, which prevents the binding between the two proteins. We therefor took an alternative, antibody-independent approach to assay development.    39	 ?	 ?  Figure 2.1: Sandwich ELISA assay to characterize rhIGFBP-5 and its capture and detection antibodies. (A) A schematic graph of the components of this ELISA, where the capture antibody is mouse anti-human IGFBP-5 antibody. Detection antibody is biotinylated goat anti-human IGFBP-5 antibody. (B) The Y-axis is the optical density, and the X-axis is the concentration of IGFBP-5. A 7 points standard curve was created using 2-fold serial dilutions in Reagent diluent buffer (RDB). Starting at a concentration of 40 ng/ml. A B 40	 ?	 ?  Figure 2.2: Sandwich ELISA assay to characterize rhIGF-I and its capture and detection antibodies. (A) A schematic graph of the components of this ELISA, where the capture antibody is mouse anti-human IGF-I antibody. Biotinylated goat anti-human IGF-I antibody was used for detection. (B) The Y-axis is the optical density, and the X-axis is the concentration of IGF-I. A 7 points standard curve was created using 2-fold serial dilutions in Reagent diluent buffer (RDB). Starting at a concentration of 2 ng/ml. A B 41	 ?	 ?  Figure 2.3: Bridged ELISA assay to check the detection of IGF-I signal when IGF-I was added on IGFBP-5 fixed to the plate. (A) A schematic graph of the components of this ELISA, where IGFBP-5 capture antibody (mouse anti-human IGFBP-5 antibody) was used to fix IGFBP-5 [10 ng/ml] on the plate. IGF-I was added on IGFBP-5, followed by adding IGF-I detection antibody (biotinylated goat anti-human IGF-I antibody). (B) The Y-axis is the optical density, and the X-axis is the concentration of IGF-I. (Black line) represents the typical level of detection of IGF-I when it is loaded on IGF-I capture antibody. (Red line) represents the level of detection of IGF-I when it was added to IGFBP-5 to make a bridged ELISA. A B 42	 ?	 ?  Figure 2.4: Bridged ELISA assay to check the detection of IGFBP-5 signal when IGFBP-5 was added on IGF-I fixed on the plate. (A) A schematic graph of the components of this ELISA, where IGF-I capture antibody (mouse anti-human IGF-I antibody) was used to fix IGF-I [2 ng/ml] on the bottom of the plate. IGFBP-5 was added on IGF-I, followed by adding IGFBP-5 detection antibody (biotinylated goat anti-human IGFBP-5 antibody). (B) The Y-axis is the optical density, and the X-axis is the concentration of IGFBP-5. (Black line) represents the typical level of detection of IGFBP-5 when it is loaded on IGFBP-5 capture antibody. (Red line) represents the level of detection of IGFBP-5 when it was added to IGF-I to make a bridged ELISA. This A B 43	 ?	 ?weak signal was not specific, because we got the same signal when we added IGFBP-5 and its detection antibody in the absence of IGF-I (grey line).   44	 ?	 ?        Figure 2.5: Bridged ELISA assay to check the detection of BIGF-I signal when BIGF-I was added on IGFBP-5 fixed on the plate. (A) A schematic graph of the components of this ELISA, where IGFBP-5 capture antibody (mouse anti-human IGFBP-5 antibody) was used to fix IGFBP-5 [10 ng/ml] on the bottom of the plate.  BIGF-I was added on IGFBP-5. (B) A table showing the optical density signal detected using various concentrations of BIGF-I.   A B 45	 ?	 ?  Figure 2.6: Bridged ELISA assay to check the detection of IGFBP-5 signal when IGFBP-5 is added on BIGF-I fixed to the streptavidin-coated plate.                        (A) A schematic graph of the components of this ELISA. BIGF-I [1 ng/ml] was fixed to the streptavidin-coated plate. IGFBP-5 was added on BIGF-I, followed by adding IGFBP-5 detection antibody (biotinylated goat anti-human IGFBP-5 antibody). (B) The Y-axis is the optical density, and the X-axis is the concentration of IGFBP-5. (Black line) represents the typical level of detection of IGFBP-5 when it is loaded on IGFBP-5 capture antibody. (Red line) represents the level of detection of IGFBP-5 when it was added to BIGF-I to make a bridged ELISA. A B 46	 ?	 ?  Figure 2.7: Bridged ELISA assay to check the detection of IGFBP-5 signal, using non-biotinylated IGFBP-5 antibodies, when IGFBP-5 is added on BIGF-I fixed to the bottom of streptavidin covered plate. (A) A schematic graph of the components of this ELISA. BIGF-I [2 ng/ml] was fixed to the bottom of streptavidin-coated plate. IGFBP-5 was added on BIGF-I, followed by adding IGFBP-5 non-biotinylated mouse anti-human IGFBP-5 antibody, followed by adding secondary anti-mouse antibody for detection. (B) The Y-axis is the optical density, and the X-axis is the concentration of IGFBP-5. (Red line) represents the level of detection of IGFBP-5 when it is added on BIGF-I and detected using non-biotinylated detection antibodies. (Blue line) represents A B 47	 ?	 ?the signal when IGFBP-5 was added directly on the streptavidin-coated plate in the absence of BIGF-I.    48	 ?	 ?3. Chapter three: Bio-Layer interferometry (BLI) technology for studying of IGF-I/IGFBP-5 binding  After the inability to establish a bridged ELISA assay for detection and studying of the IGF-I/IGFBP-5 binding, it was apparent that a different technique was needed. We decided to use a BLI-based technique, which would allow for a label-free technology that can be to characterize kinetic of proteins/protein interactions. It is an optical technique that analyzes the interference pattern of white light that is reflected from two different surfaces. The technique uses fibers (biosensors) with two surfaces: an upper surface and a lower surface. The lower surface of the tip is coated with special material, making an optical layer that can bind to different molecules. A white light is directed into the biosensor tip, and wavelengths of the reflected beam from the upper and lower surfaces are measured. If a molecule is bound to the lower surface (optical layer), it causes a shift in the interference pattern that can be measure in real-time (Fig 3.1).  Based on the material used at the tip, a capturing molecule (or a ligand) can attach to the tip, increasing the thickness of the optical layer, which will result in a wavelength shift that can be detected by the system. This wavelength shift is directly related to change of the optical layer thickness. Biosensors are then dipped into samples containing the analyte, resulting in a further thickening of the optical layer of the biosensor tip, and a larger wavelength shift (Fig. 3.1).  In a typical experiment, a baseline is obtained in assay buffer in a microtiter plate well. Next, biosensors are incubated in ligand to ?load? the tips and a ligand-bound baseline is obtained. Then, the instrument transfers biosensors, with ligand attached to the tip, to wells containing analyte. This is called the association phase. If binding 49	 ?	 ?occurs, the instrument detects the difference in light reflected from the target surface relative to the response in buffer and reports response as a change in wavelength shift for the interference pattern. The sensor is transferred back to a well containing buffer and the release of the analyte from the biosensor is monitored [215]. This is called the dissociation phase (Fig. 3.2). When a typical experiment is done, as described above, with the association into analyte containing wells and dissociation into sample-free buffer, binding kinetics of the ligand/analyte binding, including association rate constant (Ka), dissociation rate constant (Kdis), and affinity constant (KD) values can be precisely calculated using the BLI technology.  For our purposes, we want to measure the binding kinetics of IGF-I/IGFBP-5 interaction. In addition, we want to study the effect of different samples (competitors) on IGF-I/IGFBP-5 binding, in both association and dissociation phases. These additional modulations of the experiment will affect the accuracy of the apparent KD value, but it allows the comparison between competitors using rates of association, and rates of dissociation measurements are calculated by the Octet red system.     3.1. Materials and Methods: An Octet Red system (Fort?Bio, Inc., Menlo Park, CA, USA) at the Vancouver Prostate Centre, Streptavidin-coated biosensors purchased from Fort?Bio (Catalogue #18-5020), and Black 96-wells plates purchased from (Greiner Bio-One, Germany; Catalogue #655209) were used for BLI assay to avoid light scattering. BLI assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mg/ml fractionated BSA, 0.002% Tween 20) was used throughout the experiment.  50	 ?	 ?Biotinylated IGF-I (BIGF-I; Cell Sciences, Canton, MA, USA; Catalogue #AQU500) was diluted in BLI assay buffer to a working concentration of 2.5 or 5 ?g/ml; Recombinant human IGFBP-5 (IGFBP-5; R&D Systems) stored at a concentration of 100 ?g/ml, was diluted in BLI assay buffer as needed; Recombinant human IGF-I (IGF-I; R&D Systems; Catalogue #291-G1-200) stored at a concentration of 200 ?g/ml, and diluted in BLI assay buffer as needed. Insulin-like growth factor E3R (IGF-I with mutation replacing Arg for a Glu at the 3 position of the protein sequence, which results in a lower affinity towards IGFBPs [216, 217]) purchased from Sigma-Aldrich (Catalogue #I2656) was stored at a concentration of 100 ?g/ml, and diluted in BLI assay buffer. All of the previous proteins were stored at -20 C. Insulin (Sigma-Aldrich; Catalogue #I9278) was stored at 4? C, and diluted as needed in BLI assay buffer. Biocytin from Sigma-Aldrich (Catalogue #B4261) was used for blocking of biosensors, was diluted to a working concentration of 10 ?g/ml in BLI assay buffer. 3.2. BLI assay development The Octet Red (Fort?Bio?) system enable, a real time measurement of the wavelength shift (response) over time (seconds), creating a graph for each biosensor called ?sensogram?. The Octet Red software can also measure the binding kinetics of protein/protein interaction including the dissociation rate constant (Kdis), the fraction of complexes that decays per second; and the association rate constant (Ka), the rate of complex formation. Using the equation KD=Kdis/Ka, the software also enable us to calculate the Affinity constant. Using streptavidin-coated biosensors, BIGF-I can be loaded as a ligand to the biosensor tip. If binding occurs, the interference pattern shifts to a higher wavelength 51	 ?	 ?(response) (Fig. 3.1). Knowing the fact that the binding of biotin to streptavidin have an extremely slow off rate [218], we are confident that if binding occurs, BIGF-I/biosensor complex is stable and can be used for multiple cycles without significantly affecting BIGF levels on biosensors. Blocking of non-specific binding to biosensor tip was done by dipping the biosensor into biocytin (as a negative control). After that, BIGF-I immobilized on streptavidin-coated biosensors are dipped into BLI buffer for equilibrium and for establishment of a baseline, followed by dipping them into IGFBP-5 (analyte) for association. A further wavelength shift is expected if IGFBP-5 binds to BIGF-I by increasing the thickness of the optical layer on the biosensor tip.  Two approaches were used to test the effectiveness of our samples (competitors): competing in dissociation phase, and competing in association phase. If competing in the dissociation phase assay, BIGF-I immobilized biosensor is dipped into IGFBP-5 for association, and the BIGF-I/IGFBP-5 complex on biosensor is dipped into samples of different competing molecules for dissociation. The effectiveness of the competing sample was evaluated by measuring the rate of BIGF-I/IGFBP-5 complex decay. Conversely, when competing in the association phase assay, BIGF-I immobilized biosensor is dipped into IGFBP-5 mixed with competing samples for association. The competitive effectiveness of samples was evaluated by measuring the amount of response (wavelength change), which directly represents the thickness of the optical layer resulting from IGFBP-5 binding to BIGF. Competing samples that are able to bind to IGFBP-5 will decrease IGFBP-5 ability to bind to BIGF-I, proportional to their affinity for IGFBP-5 at the IGF-I binding site, and thus lower response.   52	 ?	 ?Before the experiment starts, two plates are placed inside the Octet Red machine, the Biosensor plate and the sample plate. Biosensors are placed on the biosensor plate first, which contains the BLI assay buffer. The Octet Red machine then transfers the biosensors into different wells of the sample plate, which contains different samples and proteins, as well as the BLI assay buffer. The sample plate, where the actual experiment occurs, is continuously shaking to ensure proper mixture of samples with the biosensor tip at 1000 RPM. Black 96-wells plates were used for the BLI assay, and each well has 200 ?l of buffer/protein. All incubations were performed at 30 C.  References include biotin-protein surface that is monitored in assay buffer throughout the run and the streptavidin surface blocked with biocytin (with and without compound). Reference subtractions were performed in Fort?Bio software, and kinetic analysis was performed with the Octet Fort?Bio Analysis 6.4 software. We used 1:1 fitting model was used with full local fitting of the curves. Two tail Student?s T tests were performed to determine the statistically different values of samples at 95% confidence. 53	 ?	 ?  Figure 3.1: Illustration of the streptavidin-coated Octet biosensor. The tip of the biosensor, which is coated with a special optical layer, is shown. A white light is directed into the biosensor tip. Two beams will be reflected to the back end. The first reflected beam comes from the up part of the optical layer as a reference. The second reflected beam comes from the streptavidin or molecular layer.  When BIGF-I is attached to the tip, the wavelength change of the reflected beam results in a change in the interference pattern, which is detected by the BLI system (response). When the biosensor tip is dipped into the well containing target molecule (e.g. IGFBP-5), the target molecule binds to the capture molecule (BIGF-I) and the two forms a molecular layer. A further shift in the wavelength will result causing a change in the spectrum color pattern.  (The figure was used with permission form Fort?Bio, see appendix)  54	 ?	 ?  Figure 3.2: Illustration explaining a typical sensogram. (1) Biosensors are pre-witted into buffer to establish a baseline (this step can be done online or offline). (2) Loading of a biotinylated ligand, results in a change in the optical layer thickness, detected as a nm shift (response). (3) Then, biosensors dipped into buffer again to establish a new baseline prior to association. (4) Biosensors are dipped into analyte for association, followed by dipping into buffer for dissociation (5).  (The figure was used with permission form Fort?Bio, see appendix)    55	 ?	 ?3.2.1. Techniques to evaluate the competitors in dissociation on the BIGF-I/IGFBP-5 complex: In the assay, biosensors were pre-wetted and equilibrated into BLI assay buffer for 10-15 min, then loaded with of BIGF-I by incubation for 10-15 min. Excess streptavidin on biosensors tip was blocked by incubation in 10 ?g/ml of biocytin for 3 min. Biosensors were dipped into BLI buffer to establish a baseline for 1 min, followed by the association with IGFBP-5 for 7 min, and dissociation into either BLI buffer (Fig 3.3 A), IGF-I (Fig 3.3 B), IGF E3R (Fig 3.3 C), or insulin (Fig 3.3 D) for 10 min.  Every cycle of baseline, association and dissociation were followed by 3 cycles of biosensor regeneration. The biosensors were regenerated after every dissociation step into acid with pH of 1.25 for 60 sec and then neutralized into assay buffer with pH of 7.4 for 120 sec. These regeneration cycles were aiming to remove the remaining IGFBP-5 from the BIGF-I/biosensor complex after dissociation into buffer, and to ensure equal IGFBP-5 loading across biosensors before the next cycle. In this assay, we are evaluating the ability of each sample to compete IGFBP-5 off the BIGF-I/biosensor complex during dissociation. The dissociation constant or Kdis (the fraction of complexes that decays per second; the larger the number, the faster the dissociation process of IGFBP-5 from biosensor/BIGF-I) was compared between different samples (n=2).   56	 ?	 ? Figure 3.3: Illustration of the Octet biosensor tip in the competing assay. (A) During the dissociation phase, biosensors loaded with BIGF-I/IGFBP-5 were dipped into wells containing either (A) BLI assay buffer, (B) IGF-I, (C) IGF-E3R or (D) Insulin.     57	 ?	 ?3.2.2.  Techniques to evaluate the competitors in association on the BIGF-I/IGFBP-5 complex formation Biosensors were pre-wetted and equilibrated into BLI assay buffer, and loaded with of BIGF-I by incubation as in 3.2.1. Blocking of the biosensor tip was done in 10 ?g/ml of biocytin as well. In contrast with 3.2.1, after establishing a baseline, biosensors were dipped into IGFBP-5 pre-incubated with either BLI assay buffer (Fig 3.4 A), IGFBP-5 with IGF-I (Fig 3.4 B), IGFBP-5 with IGF E3R (Fig 3.4 C), or IGFBP-5 with insulin (Fig 3.4 D) for association (n=3).  The wavelength shift (response) over time (seconds), resulting from IGFBP-5 binding to BIGF-I on the biosensor was measured in real time during the association step, and it was compared between different samples (Fig. 3.4). We would expect that IGF-I would result in the lowest response, because it can bind to most of the IGFBP-5 in the well, and thus preventing BIGF-I on the biosensor from binding to IGFBP-5. IGF E3R should have an intermediate blocking effect, and Insulin should show the least blocking effect.   58	 ?	 ? Figure 3.4: Illustration of the Octet biosensor tip in the blocking assay. For association, the biosensors were dipped into either IGFBP-5 alone (A), or into a mixture of IGFBP-5 (at a constant concentration) with IGF-I (B), or IGF E3R (C) or Insulin (D). The dissociation step was done in a sample-free assay buffer.    59	 ?	 ?3.3. BLI assay results 3.3.1. Detection of BIGF-I binding to IGFBP-5 is possible using the BLI technique The first experiment done using the BLI technology aimed to establish the ability of BIGF-I to bind to the streptavidin-coated biosensor tip, and then to test if IGFBP-5 can bind to BIGF-I on biosensor. We also wanted to choose the ideal concentration of ligand and analyte to be used in subsequent competition experiments. Streptavidin-coated biosensors were loaded with different concentrations of BIGF-I diluted in BLI assay buffer [66, 132, 329, 658, 1316, 2632 nM] for 10 min. Blocking was done with 10 ?g/ml of biocytin, followed by dipping biosensors into IGFBP-5 wells at concentrations ranging between [36, 89, 179, 357 nM] for association for 7 min. The dissociation step was in BLI assay buffer containing wells for 10 min.   We were able to see a dose-dependent response as a result of BIGF-I binding to streptavidin-coated biosensor. A response range from 0.4 to 1.57 nm was seen when BIGF-I binds at a concentration of 66 nM and 2632 nM respectively (Fig 3.5 1). The binding of BIGF-I to streptavidin-coated biosensor was very rapid. 90% of BIGF [658, 1316, 2632 nM] was bound to the biosensor within 63 seconds, while with the lower concentration of BIGF [66, 132, 329], around 85% was bound within 109 seconds (Fig 3.5 1).  Blocking was done by placing biosensors into biocytin had a minimal effect on BIGF-I level on the biosensor (<4% change in response) (Fig 3.5 3).  A dose-dependent binding of IGFBP-5 to BIGF-I was detected as well. When the highest concentration of BIGF-I [2632 nM] was loaded on biosensors, a response ranging from 0.67 to 0.96 nm was detected when IGFBP-5 was used at a concentration 60	 ?	 ?ranging from [1.25 to 10 ?g/ml] (Fig 3.5 9,12,14,15). While when the lowest concentration of BIGF-I [66 nM] was immobilized on the biosensor, a response ranging from 0.07 to 0.09 nm was detected when IGFBP-5 was used at a concentration ranging from [1.25 to 10 ?g/ml].  We can conclude from this experiment that a concentration of [66 nm] of BIGF-I is too low to show a good dynamic range when bound to IGFBP-5 at any concentration. It also shows that the higher concentration of BIGF-I used, the better the dynamic range we get at different concentrations of IGFBP-5. The KD value for IGFBP-5 biding to BIGF-I, across the different concentrations was in the range of 7.76 -09 to 6.04 -10 nM (Table 1). As a result of this experiment, a concentration of 658 nM of BIGF-I and a concentration of 179 nM and 357 nM of IGFBP-5 were used in the following experiments because these concentrations showed a wide dynamic range that can detect minor changes in the amount of IGFBP-5 attached to the biosensor. In addition, we believe that we reach IGFBP-5 saturation on biosensor using these concentrations.   In order to test if the wavelength shift (response) change was a result of BIGF-I binding to IGFBP-5, we loaded other set of biosensors into biocytin, instead of BIGF-I, followed by placing them into IGFBP-5 wells and the remaining of the experiment was the same as above, and no binding was detected, with a response change indistinguishable from blank (Fig. 3.5 A2 to F2). In conclusion, unlike the results obtained in the ELISA assay, using this BLI-based method we were able to detect IGF-I/IGFBP-5 binding.  61	 ?	 ? Figure 3.5: This is the raw data showing the sensograms of the binding of BIGF-I to the biosensor, followed by binding of IGFBP-5. The Y-axis is the (response) in nanometer, and the X-axis is the time in seconds. We used BIGF-I at concentrations of 66 nM (Blue), 132 nM (Red), 329 nM (Light blue), 658 nM (Green), 1316 nM (Orange), 2632 nM (Purple). Sequential dipping of all biosensors into IGFBP-5 at concentrations ranging from (35 - 357 nM) during the association phase is shown in this figure. As a negative control, biosensors loaded with biocytin instead of BIGF-I was used (A2-F2) and underwent the same experiment steps, and showed no binding to IGFBP-5. They were subtracted from all sensograms during the analysis.    62	 ?	 ?Table 1: Kinetic and equilibrium constants for the interaction of IGF-I as a ligand on the biosensor with IGFBP-5 as analyte     * Poor fitting in 1:1 curve fitting model (http://www.fortebio.com/documents/ForteBio_App_Note_14.pdf)      63	 ?	 ?3.3.2. Studying the competition in dissociation on the BIGF-I/IGFBP-5 complex  In order to create a system to screen small molecules targeting the IGF binding site on IGFBP-5, we wanted to build an assay to test the dissociating abilities of these drugs of the BIGF-I/IGFBP-5 complex compared to high and low affinity antagonists. In order to define upper and lower limits of competitors for BIGF-I binding to IGFBP-5 we compared the dissociation rate constant (Kdis) of BIGF-I/IGFBP-5 complex when biosensors were dipped into either: BLI assay buffer, IGF-I, IGF E3R and insulin during the dissociation phase (n=2). BIGF-I at a concentration of 658 nM was loaded on biosensors, followed by loading of IGFBP-5 at concentrations, 179 or 357 nM, for association. During the dissociation step, biosensors were dipped into wells containing BLI assay buffer, IGF-I [329 nM], IGF E3R [329 nM] or Insulin [329 nM]. The best sample in dissociating BIGF-I/IGFBP-5 complex was IGF-I, which resulted in a Kdis value of 1.5x10-3. A Kdis value of 5.51x10-4 and 5.31x10-4 resulted when dissociation was done into IGF E3R and Insulin [329 nM] respectively. While dipping biosensors into BLI assay buffer resulted in a Kdis value of 1.21x10-4. This means that IGF-I was significantly better in dissociating IGFBP-5 from BIGF-I on the biosensor than the IGF E3R and Insulin (Fig. 3.6).   64	 ?	 ?    A B C 65	 ?	 ? Figure 3.6: The effect of IGF-I, IGF E3R and Insulin on dissociating IGFBP-5 from BIGF on the biosensor. (A) The raw data of the experiment is shown. The binding of IGFBP-5, at two concentrations [179 nM (F4) and 357 nM (G4)] to the BIGF-I on the biosensor showed an average response (nm shift in the optical density) of 1.69 and 2.1 nm respectively. The dissociation rate constant of IGFBP-5 from BIGF-I on the biosensor (Kdis) was compared between sample free buffer, IGF-I [329 nM], IGF E3R [329 nM], and Insulin [329 nM]. Regeneration of the biosensor was done in acid (pH of 1.25) after every dissociation step to ensure equal IGFBP-5 binding before testing each sample.  The difference in the Kdis values between samples, when we used IGFBP-5 at 179 nM (B) and 357 nM (C), is shown. The Y-axis is Kdis value, and the X-axis is the concentration of samples (competitors). IGF-I showed the highest Kdis value, which means that when we place the biosensor (with IGFBP-5 bound to BIGF-I) for dissociation into IGF-I, it results in the fastest decay of the IGFBP-5/BIGF-I complex on the biosensor. IGF E3R showed a Kdis value similar to insulin, and both were higher than Kdis value of sample free buffer (buffer).        66	 ?	 ?Although we can see a difference in the Kdis value between samples, the usage of very high concentrations leads to the detection of non-specific bindings and poor fitting during the analysis of the data, which may affect the results. In an attempt to eliminate non-specific binding, and to investigate the dissociating effect further, we used lower concentrations of BIGF-I [329 nM] and IGFBP-5 [45 nM]. We compared the dissociation effect of IGF-I at concentrations of 8, 33 and 132 nM, and compared to IGF E3R at the same concentrations (Fig. 3.7). This experiment showed a better curve fitting in the analysis. Kdis value ranging from 3.05x10-4 to 1.39x10-3 resulted when IGF-I was used at the lowest and highest concentration respectively, compared to a Kdis value of 4.85x10-5 to 3.07x10-4 when the lowest and highest concentration of IGF E3R was used respectively. This difference in the dissociation rate constant (Kdis) between IGF E3R and IGF-I was statistically indistinguishable (P >0.05) mainly due to the small sample number (n=2).           67	 ?	 ?    Figure 3.7: Comparing the Kdis value of IGF-I (black) and IGF E3R (grey). The Y-axis is Kdis value, and the X-axis is the concentration of IGF-I and IGF E3R. Using lower concentration of BIGF-I [329 nM] on the biosensor, and IGFBP-5 [45 nM] in this experiment resulted in a better curve fitting during the analysis of the data. Here we compared the effect of different concentrations of IGF-I (Black) and IGF E3R (Grey) [8, 33 and 132 nM] on the dissociation rate.       68	 ?	 ?3.3.3. Studying the competition in association on the BIGF-I/IGFBP-5 complex formation Since we are building a system to test small molecules targeting the IGF-I binding site on IGFBP-5, we also considered testing competition in the association phase by blocking IGFBP-5 ability to bind to BIGF/biosensor complex. We compared the wavelength shift (response) when BIGF-I on biosensor, after blocking with biocytin, is dipped into wells containing either: IGFBP-5 alone, IGFBP-5 mixed with IGF-I, IGFBP-5 mixed with IGF E3R, and IGFBP-5 mixed with insulin during the association step. IGFBP-5 was used at a concentration of 179 nM across the wells. IGF-I, IGF E3R and Insulin were pre-incubated with IGFBP-5 at concentration of 263 and 1316 nM at a molar ratio of 1:1.5 and 1:7 respectively. Cycles of baseline, association, and dissociation were repeated 3 times for each biosensor, and biosensors were regenerated after each cycle. We would expect that the highest wavelength shift (response) will result when biosensors are dipped into IGFBP-5 alone, followed by insulin and IGF E3R, and the lowest response with IGF-I.  In these experiments, after loading the biosensors with BIGF-I [658 nM], the response detected during the association step when placing biosensors into the wells was as follow: IGFBP-5 alone, an average response of 2.24 nm; IGFBP-5 with IGF-I [263 nM], an average response of 1.00 nm; IGFBP-5 with IGF-I [1316 nM] , an average response of 0.21 nm;  IGFBP-5 with IGF E3R [263 nM] , an average response of 1.79 nm;  IGFBP-5 with IGF E3R [1316 nM] , an average response of 1.47 nm;  IGFBP-5 with Insulin [263 nM] , an average response of 2.08 nm;  IGFBP-5 with Insulin [1316 nM] , an average response of 1.83 nm (Fig. 3.8). 69	 ?	 ?These observation demonstrate that IGF-I (non-biotinylated) was the protein with the highest affinity by binding most of the IGFBP-5 in the well, and thus preventing IGFBP-5 from binding to BIGF-I on the biosensor (low response) in a dose-dependent manner. IGF E3R was better in binding to IGFBP-5 than Insulin, but less effective than non-biotinylated IGF-I (Fig. 3.8).  Because the usage of high concentrations of BIGF-I and IGFBP-5, the results are likely to be affected by the appearance of non-specific binding and poor fitting during the analysis of the curves. And in order to minimize the non-specific binding and to test the dose-dependent blocking effect of IGF-I, this experiment was repeated using lower concentration of BIGF-I and lower concentration of IGFBP-5.  We next used BIGF-I at a concentration of [164 nM] and IGFBP-5 [45 nM] and compared the blocking effect of IGF-I at a concentration of [82, 164, 329, 658, 1316 nM] on the binding of IGFBP-5 to BIGF-I attached to the biosensor, with a molar ratio of 1:0.5, 1:1, 1:2, 1:4, and 1:8 respectively. A dose-dependent blocking effect of IGF-I on IGFBP-5 was noticed, with an average response of 0.67 nm compared to a response of 0.63 and 0.08 when the lowest and highest concentrations were used respectively (Fig. 3.9). A response of 1.29 was detected when with IGFBP-5 alone, showing that at a molar ratio of 2:1 IGFBP-5 to IGF-I, IGF-I [82 nM] decreased IGFBP-5 binding to BIGF-I/biosensor complex by 50% (Fig. 3.9). All responses were statistically different between every concentration of IGF-I used (P <0.05).     70	 ?	 ?  Figure 3.8: The effect on IGFBP-5 binding to BIGF-I on the biosensor when samples were mixed with IGFBP-5 in the same well. (A) Sensogram showing the response (nm shift) over time, when IGFBP-5 was pre-complexed with different samples at concentrations of [263 and 1316 nM]. The samples were IGF-I (C4 and D4), IGF E3R (E4 and F4), and insulin (G4 and H4). The response of IGFBP-5 with no samples (A4) was measured as well. (B) Comparing response between samples, The Y-axis is response, and the X-axis is the sample. A B 71	 ?	 ?  Figure 3.9: The effect of using different concentrations of IGF-I to block IGFBP-5 binding of BIGF-I on biosensor. The Y-axis is response, and the X-axis is the concentration of IGF-I. Biosensors were loaded with BIGF-I [164 nM], and measured the response of IGFBP-5 [45 nM] when pre-incubated with IGF-I at different concentrations [0, 82, 164, 329, 658, 1316 nM]. A dose-dependent blocking effect of IGF-I on IGFBP-5 is noticed. This experiment showed better curve fitting in the analysis due to the usage of lower concentration of ligand (BIGF-I) and analyte (IGFBP-5). (   = P<0.05)         * 72	 ?	 ?3.4. Conclusions We have developed a Bio-Layer Interferometry-based assay that precisely measures IGFBP-5/IGF-I binding kinetics that will allow for screening of molecules affecting this intermolecular interaction. The assay uses biotinylated IGF-I bound to strepavidin-coated biosensors that, in optimized buffer conditions, bind recombinant IGFBP-5 with high affinity.  We have demonstrated that recombinant IGF-I can efficiently disrupt this interaction, while the amino-terminal IGF-I mutant, E3R, exhibits an intermediate competitive activity, and insulin exhibits a low competitive activity, when they were used as competitors in association. These results demonstrate the capacity to differentiate relative competitive activity of compounds that target the high affinity IGF-I binding site of IGFBPs and establish a platform to screen for factors that might be developed as rationale therapeutics to disrupt sequestration of IGF-I by IGFBPs. 	 ?          73	 ?	 ?4. Chapter four: Discussion and Conclusions  4.1. Discussion Two in five Canadians will be diagnosed with cancer during their lifetime. The most common cancer in men is prostate cancer, and the most common cancer in women is breast cancer [1]. IGF axis has been implicated in the pathophysiology of both prostate [115, 116] and breast cancer [219]. IGFBPs have many IGF-dependent activities including growth induction or apoptosis based on presence or absence of factors affecting IGFBP affinity towards IGF-I in the target tissue. IGFBP-5 has shown to have an apoptotic activity on breast cancer cell line [112, 113]. In contrast, increased expression of IGFBP-5 in prostate cancer cells resulted in prostate cancer cells survival and the progression to CRPC [117, 118]. It is believed that this difference in activity of IGFBP-5 is, at least partly, due to the effect of PSA in prostate cancer, which is a protease of IGFBPs [135], resulting in the degradation of IGFBP-5 and the release of IGF-I to their receptors on prostate cancer cells. 4.1.1. Serum and tissue IGFBPs In serum, almost all IGFs are bound to IGFBPs, 75% to form a ternary complex with ALS and IGFBP-3,-5 (150 kDa), and 25 % forms binary complex with IGFBP-1, 2, 4, 6 (50 kDa). The binary complexes are smaller, and are able to cross the endothelium layer to different tissues [220], while the large ternary complexes are trapped within the circulation [221]. Miyakoshi et al. has shown that IGFBP-4 inhibits growth-promoting effects of IGF-I on bone tissue in vitro. But systemic administration of IGFBP-4 resulted in a significant increase of IGF-I in the 50-kDa pool with corresponding decrease in IGF-I in the 150-kDa pool [222]. These high systemic levels of IGFBP-4 led to an increase in 74	 ?	 ?different bone formation parameters in mice by a mechanism involving increased IGF bioavailability via proteolysis of IGFBP-4 [104].  In prostate cancer, a recent case-control study (1,652 cases/1,543 controls) on the effect of finasteride for prostate cancer prevention, showed that serum IGFBP-2 is positively associated with low grade prostate cancer [90]. This could be explained by the shift of IGF-I to the binary complex pool with IGFBP-2, from the ternary complex with IGFBP-3 and ALS, which allow IGF-I to escape from the circulation to the prostate tissue, resulting in the initiation and development of low grade prostate cancer, especially that the prostate tissue produces PSA, which is an IGFBP protease.  IGF bioactivity is largely under the control of IGFBPs in the local cellular environment. When intact, IGFBPs can prevent the IGF-IR activation because they have higher affinity than the IGF-IR, as with IGFBP-5 in breast cancer cell lines. In contrast, in prostate cancer tissue, up-regulation of IGFBP-5 accelerated the progression to androgen-independence status in LNCaP cell line, which produces PSA, through activation of IGF-IR [118]. It is possible that, in the presence of IGFBP proteases, IGFBPs can induce IGF-dependent activation of IGF-IR in local cancer environment by sequestering free circulating IGFs to the cancer tissue which expresses high levels of IGFBPs. This continuous sequestration, IGFBP degradation cycles around the cancer tissue will eventually results in the accumulation of free bioactive IGF to the IGF-IR, which can be highly expressed on cancer cells, as during the progression of CRPC.    75	 ?	 ?4.1.2. Studying IGF-I/IGFBP-5 interaction using ELISA and BLI Although the C-terminus of IGFBP-5 participates in the IGF-I binding, the IGF-I binding site on the C-terminal is a common binding site for other proteins, like heparin (see above). Heparin has been shown to be able to compete with IGF-I for the IGF-I binding site on the C-terminus of IGFBP-5 [204]. In this study, they also showed that ECM proteins can decrease the affinity of IGFBP-5 towards IGF-I by interacting with the C-terminus of IGFBP-5 [204]. Thus, the high affinity binding site of IGF-I on the N-terminus of IGFBP-5 is a better target for drugs design.  In my thesis, we started studying IGF-I/IGFBP-5 binding by using the ELISA technique. We tried to build different constructs of complex bridged ELISA of IGF-I and IGFBP-5 including the usage of capture antibody of one ligand, and detection antibody of the other, but no signal was detected. Since sandwich ELISA assay of both proteins showed dose-dependent signal detection, it?s possible that antibodies used can interfere with the IGF-I/IGFBP-5 interaction. As a result, an alternative non-antibody based method was needed, and we?ve decided to use BLI technology.   Using BLI technique, I was able to detect and study the binding of the IGFBP-5 to BIGF-I on the biosensor. There is evidence that using biotinylated IGFBP-5 as a ligand on the biosensor can change the 3D structure of the protein during the regeneration step of the biosensor (in low pH environment), which affect its binding to IGF-I [223]. Thus, BIGF-I was used as a ligand. In addition, using the smaller BIGF-I (7.6 kDa) on biosensor will show a bigger response (nm shift) on sensogram when IGFBP-5 (29 kDa) binds to BIGF-I. This will result in a wider dynamic range allowing us to monitor small changes when small molecule drugs targeting IGFBP-5 are tested.  76	 ?	 ?The BLI technique provides the advantage of detecting the binding kinetics of protein-protein interaction. We started our BLI experiments using high concentration of BIGF-I and IGFBP-5 at ratios which are hundreds of fold higher than the KD value of the binding, which according to the manufacturer will cause the detection of non-specific binding by the octet red machine and poor fitting of the curves during the analysis of the experiments. The curve-fitting problem was noticed by Beattie et al., who used IGF-I as a ligand in surface plasmon resonance (SPR) biosensor technique [223].  The KD value measured by the software as the ratio of the dissociation constant divided by the association constant (KD= Kdis / Ka). Ideally, the machine measures these values when an analyte binds to a ligand on the biosensor during the association phase (Ka), and the Kdis value is measured when the biosensor is dipped into a sample-free buffer during the dissociation phase. Nevertheless, the usage of randomly biotinylated BIGF-I may affect the accuracy of the real kinetics of the BIGF-I/IGFBP-5 binding in our experiment. It?s possible that a biotin is attached to one of the IGF-I residues involved in the binding of IGFBP-5. Thus, the usage of targeted biotinlyation of the IGF-I in the future may show more accurate results. In the competition in dissociation phase assay, we dipped the biosensor during the dissociation phase into different samples (IGF-I, IGF E3R, Insulin), and we compared the Kdis value with each sample, as a measurement of how effective each sample was at dissociating IGFBP-5 from BIGF-I/biosensor complex. The octet red machine can accurately measures the KD value of BIGF-I/IGFBP-5 binding only when the dissociation was performed in a buffer free of analyte competitors. And since KD= Kdis / Ka, and Kdis is affected by the proteins competing IGFBP-5 off the BIGF-77	 ?	 ?I/biosensor complex, then the KD values are not accurate, and Kdis values were used instead.  While in the competition in association phase assay, we used the (response), which is the nm shift recorded at the end of the association step of each concentration of the analyte (IGFBP-5) to the ligand (BIGF-I) on the biosensor. And since the blocking assay is based on the ability of the sample (IGF-I, IGF E3R, Insulin) to block IGF-I binding site on IGFBP-5, preventing its binding to BIGF-I on the biosensor, thus the lower response means better blocking. And since the interaction of the sample (IGF-I, IGF E3R, Insulin) with IGFBP-5 will affect the concentration of IGFBP-5, it will affect both KD and Ka, and thus response was used instead.  Although other groups used SPR Biacore to study the IGF-I/IGFBP-5 interaction [223], Octet red BLI technique has many advantages over the SPR Biacore. Its ability to measure samples interactions without any microfluidics, plus it enables us to reuse the samples for the same analysis or for another assay, and thus consuming less amount of samples compared to Biacore [224]. 4.1.3. Therapies targeting IGF axis Targeting different aspects of the IGF axis has been used for the management of different carcinomas as mentioned above. Targeting IGF-IR using monoclonal antibodies [191], small molecule inhibitors [192], and antisense oligonucleotides [193] didn?t show the desired result because the hybrid receptor is not affected using this approach. Also monoclonal antibodies against IGF were developed with promising results in phase one studies [195].  78	 ?	 ?Although IGFBPs plays an important role in the pathophysiology of many cancer types, there is only small effort in targeting them. We?ve developed a system that can be used to test drugs affecting the binding of IGFs to IGFBP-5. This system can be modified to be used to test the binding kinetics of IGF-I and IGF-II binding to different IGFBP, which can allow screening of small molecule drugs that can be used in the management of various diseases, where free IGF is needed as in brain ischemic injuries [225], or unwarranted as in the progression of CRPC. 4.2. Conclusion: Modulation of IGF axis components has been implicated in the pathophysiology of the initiation and progression of different types of cancer, including prostate cancer. It was hypothesized that IGFBP-5 and -2 sequester IGFs from circulation to the local environment of the prostate cancer, and with the effect of IGFBP protease (like PSA) and other factors results in the release of IGF-I in proximity of IGF-IR and leads to cell survival. Thus targeting IGFBP-5 and -2 can prevent the sequestration of IGFs around PCa, and thus sensitizing the tumor to various stressors. We have established a BLI-based assay system to detect and study antagonism of IGF-I/IGFBP-5 binding that can be used to assess both IGF-I/IGFBP-5 association and dissociation kinetics when presented with proof of principle high and low affinity antagonists. This assay system will facilitate screening of a panel of small molecules modeled in silica to target the IGF binding to the high affinity site on the N-terminal of IGFBP-5, to identify lead compounds that can disrupt the ability of IGFBPs to function as chaperones for IGF-I for treatment of diseases in which the IGF axis is dysregulated.  79	 ?	 ?Bibliography  1. Canadian Cancer Statistics 2012. 2012. 2. Khandwala, H.M., et al., The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocr Rev, 2000. 21(3): p. 215-44. 3. Valentinis, B. and R. Baserga, IGF-I receptor signalling in transformation and differentiation. Mol Pathol, 2001. 54(3): p. 133-7. 4. Gluckman, P., et al., A role for IGF-1 in the rescue of CNS neurons following hypoxic-ischemic injury. Biochem Biophys Res Commun, 1992. 182(2): p. 593-9. 5. Hadsell, D.L., S.G. Bonnette, and A.V. Lee, Genetic manipulation of the IGF-I axis to regulate mammary gland development and function. J Dairy Sci, 2002. 85(2): p. 365-77. 6. Accili, D., et al., Targeted gene mutations define the roles of insulin and IGF-I receptors in mouse embryonic development. J Pediatr Endocrinol Metab, 1999. 12(4): p. 475-85. 7. Daughaday, W.H., et al., Measurement of somatomedin-related peptides in fetal, neonatal, and maternal rat serum by insulin-like growth factor (IGF) I radioimmunoassay, IGF-II radioreceptor assay (RRA), and multiplication-stimulating activity RRA after acid-ethanol extraction. Endocrinology, 1982. 110(2): p. 575-81. 8. Salmon, W.D., Jr. and W.H. Daughaday, A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med, 1957. 49(6): p. 825-36. 80	 ?	 ?9. Rosenfeld, R.G. and R.L. Hintz, Characterization of a specific receptor for somatomedin C (SM-C) on cultured human lymphocytes: evidence that SM-C modulates homologous receptor concentration. Endocrinology, 1980. 107(6): p. 1841-8. 10. Rechler, M.M., et al., Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: relation to human somatomedins and insulin. J Supramol Struct Cell Biochem, 1981. 15(3): p. 253-86. 11. Baxter, R.C. and J.L. Martin, Binding proteins for insulin-like growth factors in adult rat serum. Comparison with other human and rat binding proteins. Biochem Biophys Res Commun, 1987. 147(1): p. 408-15. 12. Binkert, C., et al., Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2). EMBO J, 1989. 8(9): p. 2497-502. 13. Brinkman, A., et al., Isolation and characterization of a cDNA encoding the low molecular weight insulin-like growth factor binding protein (IBP-1). EMBO J, 1988. 7(8): p. 2417-23. 14. Martin, J.L., K.E. Willetts, and R.C. Baxter, Purification and properties of a novel insulin-like growth factor-II binding protein from transformed human fibroblasts. J Biol Chem, 1990. 265(7): p. 4124-30. 15. Shimasaki, S., et al., Identification of five different insulin-like growth factor binding proteins (IGFBPs) from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human. J Biol Chem, 1991. 266(16): p. 10646-53. 81	 ?	 ?16. Shimonaka, M., et al., Identification of a novel binding protein for insulin-like growth factors in adult rat serum. Biochem Biophys Res Commun, 1989. 165(1): p. 189-95. 17. Oh, Y., et al., Synthesis and characterization of insulin-like growth factor-binding protein (IGFBP)-7. Recombinant human mac25 protein specifically binds IGF-I and -II. J Biol Chem, 1996. 271(48): p. 30322-5. 18. Rajah, R., et al., Insulin-like growth factor binding protein (IGFBP) proteases: functional regulators of cell growth. Prog Growth Factor Res, 1995. 6(2-4): p. 273-84. 19. LEROITH, D., et al., Molecular and Cellular Aspects of the Insulin-Like Growth Factor I Receptor. Endocr Rev, 1995. 16(2): p. 143-163. 20. Yu, H. and T. Rohan, Role of the Insulin-Like Growth Factor Family in Cancer Development and Progression. J. Natl. Cancer Inst., 2000. 92(18): p. 1472-1489. 21. Stewart, C.E. and P. Rotwein, Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol. Rev., 1996. 76(4): p. 1005-1026. 22. Sepp-Lorenzino, L., Structure and function of the insulin-like growth factor I receptor. Breast Cancer Research and Treatment, 1998. 47(3): p. 235-253. 23. Ullrich, A., et al., Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J, 1986. 5(10): p. 2503-12. 24. Benyoucef, S., et al., Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively 82	 ?	 ?spliced exon 11 sequence to ligand binding and receptor activation. Biochem J, 2007. 403(3): p. 603-13. 25. Cox, M.E., et al., Insulin receptor expression by human prostate cancers. Prostate, 2009. 69(1): p. 33-40. 26. Jones, J.I. and D.R. Clemmons, Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev, 1995. 16(1): p. 3-34. 27. Lavoie, J.N., et al., Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem, 1996. 271(34): p. 20608-16. 28. Hamelers, I.H., et al., Insulin-like growth factor I triggers nuclear accumulation of cyclin D1 in MCF-7S breast cancer cells. J Biol Chem, 2002. 277(49): p. 47645-52. 29. Mairet-Coello, G., A. Tury, and E. DiCicco-Bloom, Insulin-like growth factor-1 promotes G(1)/S cell cycle progression through bidirectional regulation of cyclins and cyclin-dependent kinase inhibitors via the phosphatidylinositol 3-kinase/Akt pathway in developing rat cerebral cortex. J Neurosci, 2009. 29(3): p. 775-88. 30. Ma, J., et al., IGF-1 mediates PTEN suppression and enhances cell invasion and proliferation via activation of the IGF-1/PI3K/Akt signaling pathway in pancreatic cancer cells. J Surg Res, 2010. 160(1): p. 90-101. 31. Datta, S.R., et al., Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 1997. 91(2): p. 231-41. 32. Kane, L.P., et al., Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol, 1999. 9(11): p. 601-4. 83	 ?	 ?33. Zhang, D. and P. Brodt, Type 1 insulin-like growth factor regulates MT1-MMP synthesis and tumor invasion via PI 3-kinase/Akt signaling. Oncogene, 2003. 22(7): p. 974-82. 34. Oates, A.J., et al., The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), a putative breast tumor suppressor gene. Breast Cancer Res Treat, 1998. 47(3): p. 269-81. 35. Ghosh, P., N.M. Dahms, and S. Kornfeld, Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol, 2003. 4(3): p. 202-12. 36. Kiess, W., et al., Type II insulin-like growth factor receptor is present in rat serum. Proceedings of the National Academy of Sciences of the United States of America, 1987. 84(21): p. 7720-7724. 37. MacDonald, R.G., et al., Serum form of the rat insulin-like growth factor II/mannose 6-phosphate receptor is truncated in the carboxyl-terminal domain. Journal of Biological Chemistry, 1989. 264(6): p. 3256-3261. 38. Xu, Y., A. Papageorgiou, and C. Polychronakos, Developmental Regulation of the Soluble Form of Insulin-Like Growth Factor-II/Mannose 6-Phosphate Receptor in Human Serum and Amniotic Fluid. J Clin Endocrinol Metab, 1998. 83(2): p. 437-442. 39. Costello, M., R.C. Baxter, and C.D. Scott, Regulation of Soluble Insulin-Like Growth Factor II/Mannose 6-Phosphate Receptor in Human Serum: Measurement by Enzyme-Linked Immunosorbent Assay. J Clin Endocrinol Metab, 1999. 84(2): p. 611-617. 84	 ?	 ?40. Sara, V.R. and K. Hall, Insulin-like growth factors and their binding proteins. Physiol. Rev., 1990. 70(3): p. 591-614. 41. Siddle, K., et al., Specificity in ligand binding and intracellular signalling by insulin and insulin-like growth factor receptors. Biochem Soc Trans, 2001. 29(Pt 4): p. 513-25. 42. Rinderknecht, E. and R.E. Humbel, Primary structure of human insulin-like growth factor II. FEBS Lett, 1978. 89(2): p. 283-6. 43. Herington, A.C., H.J. Cornell, and A.D. Kuffer, Recent advances in the biochemistry and physiology of the insulin-like growth factor/somatomedin family. Int J Biochem, 1983. 15(10): p. 1201-10. 44. Xu, S., et al., Insulin-like growth factors (IGFs) and IGF-binding proteins in human skin interstitial fluid. J Clin Endocrinol Metab, 1995. 80(10): p. 2940-5. 45. Tannenbaum, G., H. Guyda, and B. Posner, Insulin-like growth factors: a role in growth hormone negative feedback and body weight regulation via brain. Science, 1983. 220(4592): p. 77-79. 46. Kulik, G. and M.J. Weber, Akt-dependent and -independent survival signaling pathways utilized by insulin-like growth factor I. Mol Cell Biol, 1998. 18(11): p. 6711-8. 47. Baker, J., et al., Role of insulin-like growth factors in embryonic and postnatal growth. Cell, 1993. 75(1): p. 73-82. 48. O'Dell, S.D. and I.N. Day, Insulin-like growth factor II (IGF-II). Int J Biochem Cell Biol, 1998. 30(7): p. 767-71. 85	 ?	 ?49. Pavelic, K., D. Bukovic, and J. Pavelic, The role of insulin-like growth factor 2 and its receptors in human tumors. Mol Med, 2002. 8(12): p. 771-80. 50. Pandini, G., et al., IGF-II binding to insulin receptor isoform A induces a partially different gene expression profile from insulin binding. Ann N Y Acad Sci, 2004. 1028: p. 450-6. 51. Marcellus, R.C., et al., The early region 4 orf4 protein of human adenovirus type 5 induces p53-independent cell death by apoptosis. J Virol, 1998. 72(9): p. 7144-53. 52. Firth, S.M. and R.C. Baxter, Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev, 2002. 23(6): p. 824-54. 53. Bach, L.A.a.R., M.M. , Insulin-like growth factor binding proteins. Diabetes Rev. , 1995(3): p. 38?61. 54. Payet, L.D., et al., Amino- and carboxyl-terminal fragments of insulin-like growth factor (IGF) binding protein-3 cooperate to bind IGFs with high affinity and inhibit IGF receptor interactions. Endocrinology, 2003. 144(7): p. 2797-806. 55. Headey, S.J., et al., Contributions of the N- and C-terminal domains of IGF binding protein-6 to IGF binding. J Mol Endocrinol, 2004. 33(2): p. 377-86. 56. Vorwerk, P., et al., Binding properties of insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-3 N- and C-terminal fragments, and structurally related proteins mac25 and connective tissue growth factor measured using a biosensor. Endocrinology, 2002. 143(5): p. 1677-85. 57. Carrick, F.E., B.E. Forbes, and J.C. Wallace, BIAcore analysis of bovine insulin-like growth factor (IGF)-binding protein-2 identifies major IGF binding site 86	 ?	 ?determinants in both the amino- and carboxyl-terminal domains. J Biol Chem, 2001. 276(29): p. 27120-8. 58. Galanis, M., et al., Ligand-binding characteristics of recombinant amino- and carboxyl-terminal fragments of human insulin-like growth factor-binding protein-3. J Endocrinol, 2001. 169(1): p. 123-33. 59. Headey, S.J., et al., C-terminal domain of insulin-like growth factor (IGF) binding protein-6: structure and interaction with IGF-II. Mol Endocrinol, 2004. 18(11): p. 2740-50. 60. Boisclair, Y.R., et al., The acid-labile subunit (ALS) of the 150 kDa IGF-binding protein complex: an important but forgotten component of the circulating IGF system. J Endocrinol, 2001. 170(1): p. 63-70. 61. Baxter, R.C., Insulin-like growth factor binding proteins in the human circulation: a review. Horm Res, 1994. 42(4-5): p. 140-4. 62. Rechler, M.M., Insulin-like growth factor binding proteins. Vitam Horm, 1993. 47: p. 1-114. 63. Zapf, J., et al., Intravenously injected insulin-like growth factor (IGF) I/IGF binding protein-3 complex exerts insulin-like effects in hypophysectomized, but not in normal rats. J Clin Invest, 1995. 95(1): p. 179-86. 64. Guler, H.P., et al., Insulin-like growth factors I and II in healthy man. Estimations of half-lives and production rates. Acta Endocrinol (Copenh), 1989. 121(6): p. 753-8. 87	 ?	 ?65. Twigg, S.M. and R.C. Baxter, Insulin-like growth factor (IGF)-binding protein 5 forms an alternative ternary complex with IGFs and the acid-labile subunit. J Biol Chem, 1998. 273(11): p. 6074-9. 66. Baxter, R.C., S. Meka, and S.M. Firth, Molecular distribution of IGF binding protein-5 in human serum. J Clin Endocrinol Metab, 2002. 87(1): p. 271-6. 67. Stewart, C.E. and P. Rotwein, Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol Rev, 1996. 76(4): p. 1005-26. 68. Beattie, J., L. McIntosh, and C.F. van der Walle, Cross-talk between the insulin-like growth factor (IGF) axis and membrane integrins to regulate cell physiology. J Cell Physiol, 2010. 224(3): p. 605-11. 69. Bilodeau, N., et al., Insulin-dependent phosphorylation of DPP IV in liver. Evidence for a role of compartmentalized c-Src. FEBS J, 2006. 273(5): p. 992-1003. 70. Frost, V.J., et al., Proteolytic modification of insulin-like growth factor-binding proteins: comparison of conditioned media from human cell lines, circulating proteases and characterized enzymes. J Endocrinol, 1993. 138(3): p. 545-54. 71. Schmid, C., et al., Intact but not truncated insulin-like growth factor binding protein-3 (IGFBP-3) blocks IGF I-induced stimulation of osteoblasts: control of IGF signalling to bone cells by IGFBP-3-specific proteolysis? Biochem Biophys Res Commun, 1991. 179(1): p. 579-85. 72. Imberg, A.J., et al., Maximizing medication therapy management services through a referral initiative. Am J Health Syst Pharm, 2012. 69(14): p. 1234-9. 88	 ?	 ?73. Bar, R.S., et al., Transcapillary permeability and subendothelial distribution of endothelial and amniotic fluid insulin-like growth factor binding proteins in the rat heart. Endocrinology, 1990. 127(3): p. 1078-86. 74. Andress, D.L., Insulin-like growth factor-binding protein-5 (IGFBP-5) stimulates phosphorylation of the IGFBP-5 receptor. Am J Physiol, 1998. 274(4 Pt 1): p. E744-50. 75. Jones, J.I., et al., Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I. Proc Natl Acad Sci U S A, 1991. 88(17): p. 7481-5. 76. Coverley, J.A., J.L. Martin, and R.C. Baxter, The effect of phosphorylation by casein kinase 2 on the activity of insulin-like growth factor-binding protein-3. Endocrinology, 2000. 141(2): p. 564-70. 77. Firth, S.M. and R.C. Baxter, Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3. J Endocrinol, 1999. 160(3): p. 379-87. 78. Marinaro, J.A., et al., O-glycosylation of insulin-like growth factor (IGF) binding protein-6 maintains high IGF-II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis. Eur J Biochem, 2000. 267(17): p. 5378-86. 79. Lee PD, C.C., Powell DR, Regulation and function of insulin-like growth factor-binding protein-1. Proc Soc Exp Biol Med, 1993(1): p. 2-29. 89	 ?	 ?80. Lee PD, G.L., Conover CA, Powell DR, Insulin-like growth factor binding protein-1: recent findings and new directions. Proc Soc Exp Biol Med, 1997. 216(3): p. 319-57. 81. Wheatcroft, S.B. and M.T. Kearney, IGF-dependent and IGF-independent actions of IGF-binding protein-1 and -2: implications for metabolic homeostasis. Trends in Endocrinology & Metabolism, 2009. 20(4): p. 153-162. 82. Beattie, J., et al., Insulin-like growth factor-binding protein-5 (IGFBP-5): a critical member of the IGF axis. Biochem J, 2006. 395(1): p. 1-19. 83. Holly, J.M., The physiological role of IGFBP-1. Acta Endocrinol (Copenh), 1991. 124 Suppl 2: p. 55-62. 84. Yee, D., et al., Insulin-like growth factor binding protein 1 expression inhibits insulin-like growth factor I action in MCF-7 breast cancer cells. Cell Growth Differ, 1994. 5(1): p. 73-7. 85. Shimasaki, S. and N. Ling, Identification and molecular characterization of insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6). Prog Growth Factor Res, 1991. 3(4): p. 243-66. 86. Clemmons, D.R., Role of insulin-like growth factor binding proteins in controlling IGF actions. Mol Cell Endocrinol, 1998. 140(1-2): p. 19-24. 87. Russo, V.C., et al., Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation, migration, and invasion. Endocrinology, 2005. 146(10): p. 4445-55. 90	 ?	 ?88. Arai, T., W. Busby, Jr., and D.R. Clemmons, Binding of insulin-like growth factor (IGF) I or II to IGF-binding protein-2 enables it to bind to heparin and extracellular matrix. Endocrinology, 1996. 137(11): p. 4571-5. 89. Busund, L.T., et al., Significant expression of IGFBP2 in breast cancer compared with benign lesions. J Clin Pathol, 2005. 58(4): p. 361-6. 90. Neuhouser, M.L., et al., Insulin-like growth factors and insulin-like growth factor-binding proteins and prostate cancer risk: results from the prostate cancer prevention trial. Cancer Prev Res (Phila), 2013. 6(2): p. 91-9. 91. Figueroa, J.A., et al., Differential expression of insulin-like growth factor binding proteins in high versus low Gleason score prostate cancer. J Urol, 1998. 159(4): p. 1379-83. 92. Clemmons, D.R., D.K. Snyder, and W.H. Busby, Jr., Variables controlling the secretion of insulin-like growth factor binding protein-2 in normal human subjects. J Clin Endocrinol Metab, 1991. 73(4): p. 727-33. 93. Kelley, K.M., et al., Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics. Int J Biochem Cell Biol, 1996. 28(6): p. 619-37. 94. DiGirolamo, D.J., et al., Mode of growth hormone action in osteoblasts. J Biol Chem, 2007. 282(43): p. 31666-74. 95. Liao, L., et al., Liver-specific overexpression of the insulin-like growth factor-I enhances somatic growth and partially prevents the effects of growth hormone deficiency. Endocrinology, 2006. 147(8): p. 3877-88. 91	 ?	 ?96. Krishnan, A.V., et al., Novel pathways that contribute to the anti-proliferative and chemopreventive activities of calcitriol in prostate cancer. J Steroid Biochem Mol Biol, 2007. 103(3-5): p. 694-702. 97. Peng, L., P.J. Malloy, and D. Feldman, Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter. Mol Endocrinol, 2004. 18(5): p. 1109-19. 98. Boyle, B.J., et al., Insulin-like growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin d(3) growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1. J Urol, 2001. 165(4): p. 1319-24. 99. Sonnenschein, C., et al., Negative controls of cell proliferation: human prostate cancer cells and androgens. Cancer Res, 1989. 49(13): p. 3474-81. 100. Peng, L., et al., Growth inhibitory concentrations of androgens up-regulate insulin-like growth factor binding protein-3 expression via an androgen response element in LNCaP human prostate cancer cells. Endocrinology, 2006. 147(10): p. 4599-607. 101. Leal, S.M., S.S. Huang, and J.S. Huang, Interactions of high affinity insulin-like growth factor-binding proteins with the type V transforming growth factor-beta receptor in mink lung epithelial cells. J Biol Chem, 1999. 274(10): p. 6711-7. 102. Leal, S.M., et al., The type V transforming growth factor beta receptor is the putative insulin-like growth factor-binding protein 3 receptor. J Biol Chem, 1997. 272(33): p. 20572-6. 92	 ?	 ?103. Butt, A.J., et al., Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells. J Biol Chem, 2000. 275(50): p. 39174-81. 104. Miyakoshi, N., et al., Systemic administration of insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) increases bone formation parameters in mice by increasing IGF bioavailability via an IGFBP-4 protease-dependent mechanism. Endocrinology, 2001. 142(6): p. 2641-8. 105. Parker, A., et al., Properties of an insulin-like growth factor-binding protein-4 protease that is secreted by smooth muscle cells. Endocrinology, 1995. 136(6): p. 2470-6. 106. Lawrence, J.B., et al., The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A. Proc Natl Acad Sci U S A, 1999. 96(6): p. 3149-53. 107. Iwashita, M., Y. Kudo, and Y. Takeda, Effect of follicle stimulating hormone and insulin-like growth factors on proteolysis of insulin-like growth factor binding protein-4 in human granulosa cells. Mol Hum Reprod, 1998. 4(4): p. 401-5. 108. Conover, C.A., et al., Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum. Endocrinology, 2001. 142(5): p. 2155. 109. Mazerbourg, S., et al., Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding 93	 ?	 ?protein-4 proteolytic degradation and mRNA expression during follicular development. Endocrinology, 2001. 142(12): p. 5243-53. 110. Marshman, E., et al., Insulin-like growth factor binding protein 5 and apoptosis in mammary epithelial cells. J Cell Sci, 2003. 116(Pt 4): p. 675-82. 111. Boonyaratanakornkit, V., et al., Progesterone stimulation of human insulin-like growth factor-binding protein-5 gene transcription in human osteoblasts is mediated by a CACCC sequence in the proximal promoter. J Biol Chem, 1999. 274(37): p. 26431-8. 112. Swami, S., et al., Vitamin D growth inhibition of breast cancer cells: gene expression patterns assessed by cDNA microarray. Breast Cancer Res Treat, 2003. 80(1): p. 49-62. 113. Huynh, H., X.F. Yang, and M. Pollak, A role for insulin-like growth factor binding protein 5 in the antiproliferative action of the antiestrogen ICI 182780. Cell Growth Differ, 1996. 7(11): p. 1501-6. 114. Tennant, M.K., et al., Insulin-like growth factor-binding proteins (IGFBP)-4, -5, and -6 in the benign and malignant human prostate: IGFBP-5 messenger ribonucleic acid localization differs from IGFBP-5 protein localization. J Clin Endocrinol Metab, 1996. 81(10): p. 3783-92. 115. Kimura, G., et al., Insulin-like growth factor (IGF) system components in human prostatic cancer cell-lines: LNCaP, DU145, and PC-3 cells. Int J Urol, 1996. 3(1): p. 39-46. 94	 ?	 ?116. Conover, C.A., J.E. Perry, and D.J. Tindall, Endogenous cathepsin D-mediated hydrolysis of insulin-like growth factor-binding proteins in cultured human prostatic carcinoma cells. J Clin Endocrinol Metab, 1995. 80(3): p. 987-93. 117. Miyake, H., M. Pollak, and M.E. Gleave, Castration-induced up-regulation of insulin-like growth factor binding protein-5 potentiates insulin-like growth factor-I activity and accelerates progression to androgen independence in prostate cancer models. Cancer Res, 2000. 60(11): p. 3058-64. 118. Miyake, H., et al., Overexpression of insulin-like growth factor binding protein-5 helps accelerate progression to androgen-independence in the human prostate LNCaP tumor model through activation of phosphatidylinositol 3'-kinase pathway. Endocrinology, 2000. 141(6): p. 2257-65. 119. Bach, L.A., Insulin-like growth factor binding protein-6: the "forgotten" binding protein? Horm Metab Res, 1999. 31(2-3): p. 226-34. 120. Bach, L.A., IGFBP-6 five years on; not so 'forgotten'? Growth Horm IGF Res, 2005. 15(3): p. 185-92. 121. Fu, P., J.A. Thompson, and L.A. Bach, Promotion of cancer cell migration: an insulin-like growth factor (IGF)-independent action of IGF-binding protein-6. J Biol Chem, 2007. 282(31): p. 22298-306. 122. Fu, P., et al., Cross-talk between MAP kinase pathways is involved in IGF-independent, IGFBP-6-induced Rh30 rhabdomyosarcoma cell migration. J Cell Physiol, 2010. 224(3): p. 636-43. 95	 ?	 ?123. Zhang, C., et al., IGF binding protein-6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis. Int J Cancer, 2012. 130(9): p. 2003-12. 124. Corkins, M.R., et al., Growth stimulation by transfection of intestinal epithelial cells with an antisense insulin-like growth factor binding protein-2 construct. Biochem Biophys Res Commun, 1995. 211(3): p. 707-13. 125. Casey, O.M., et al., Analysis of gene expression in the bovine corpus luteum through generation and characterisation of 960 ESTs. Biochim Biophys Acta, 2004. 1679(1): p. 10-7. 126. Dominguez, F., et al., A combined approach for gene discovery identifies insulin-like growth factor-binding protein-related protein 1 as a new gene implicated in human endometrial receptivity. J Clin Endocrinol Metab, 2003. 88(4): p. 1849-57. 127. Wandji, S.A., et al., Messenger ribonucleic acids for MAC25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis. Endocrinology, 2000. 141(7): p. 2648-57. 128. Sprenger, C.C., et al., Over-expression of insulin-like growth factor binding protein-related protein-1(IGFBP-rP1/mac25) in the M12 prostate cancer cell line alters tumor growth by a delay in G1 and cyclin A associated apoptosis. Oncogene, 2002. 21(1): p. 140-7. 129. Maile, L.A. and J.M. Holly, Insulin-like growth factor binding protein (IGFBP) proteolysis: occurrence, identification, role and regulation. Growth Horm IGF Res, 1999. 9(2): p. 85-95. 96	 ?	 ?130. Maile, L.A., et al., Active and inhibitory components of the insulin-like growth factor binding protein-3 protease system in adult serum, interstitial, and synovial fluid. Endocrinology, 1998. 139(12): p. 4772-81. 131. Nam, T.J., W.H. Busby, Jr., and D.R. Clemmons, Characterization and determination of the relative abundance of two types of insulin-like growth factor binding protein-5 proteases that are secreted by human fibroblasts. Endocrinology, 1996. 137(12): p. 5530-6. 132. Thrailkill, K.M., et al., Characterization of insulin-like growth factor-binding protein 5-degrading proteases produced throughout murine osteoblast differentiation. Endocrinology, 1995. 136(8): p. 3527-33. 133. Busby, W.H., Jr., et al., The complement component C1s is the protease that accounts for cleavage of insulin-like growth factor-binding protein-5 in fibroblast medium. J Biol Chem, 2000. 275(48): p. 37638-44. 134. Sim, R.B. and A. Laich, Serine proteases of the complement system. Biochem Soc Trans, 2000. 28(5): p. 545-50. 135. Cohen, P., et al., Prostate-specific antigen (PSA) is an insulin-like growth factor binding protein-3 protease found in seminal plasma. J Clin Endocrinol Metab, 1992. 75(4): p. 1046-53. 136. Braulke, T., et al., Proteolysis of IGFBPs by cathepsin D in vitro and in cathepsin D-deficient mice. Prog Growth Factor Res, 1995. 6(2-4): p. 265-71. 137. Conover, C.A. and D.D. De Leon, Acid-activated insulin-like growth factor-binding protein-3 proteolysis in normal and transformed cells. Role of cathepsin D. J Biol Chem, 1994. 269(10): p. 7076-80. 97	 ?	 ?138. Braulke, T., et al., Alteration in pancreatic immunoreactivity of insulin-like growth factor (IGF)-binding protein (IGFBP)-6 and in intracellular degradation of IGFBP-3 in fibroblasts of IGF-II receptor/IGF-II-deficient mice. Horm Metab Res, 1999. 31(2-3): p. 235-41. 139. Zwad, O., et al., Decreased intracellular degradation of insulin-like growth factor binding protein-3 in cathepsin L-deficient fibroblasts. FEBS Lett, 2002. 510(3): p. 211-5. 140. Olivo-Marston, S.E., et al., Genetic reduction of circulating insulin-like growth factor-1 inhibits azoxymethane-induced colon tumorigenesis in mice. Mol Carcinog, 2009. 48(12): p. 1071-6. 141. Wu, Y., et al., Reduced circulating insulin-like growth factor I levels delay the onset of chemically and genetically induced mammary tumors. Cancer Res, 2003. 63(15): p. 4384-8. 142. Wu, Y., et al., Circulating insulin-like growth factor-I levels regulate colon cancer growth and metastasis. Cancer Res, 2002. 62(4): p. 1030-5. 143. Dunn, S.E., et al., Dietary restriction reduces insulin-like growth factor I levels, which modulates apoptosis, cell proliferation, and tumor progression in p53-deficient mice. Cancer Res, 1997. 57(21): p. 4667-72. 144. Carboni, J.M., et al., Tumor development by transgenic expression of a constitutively active insulin-like growth factor I receptor. Cancer Res, 2005. 65(9): p. 3781-7. 98	 ?	 ?145. Lopez, T. and D. Hanahan, Elevated levels of IGF-1 receptor convey invasive and metastatic capability in a mouse model of pancreatic islet tumorigenesis. Cancer Cell, 2002. 1(4): p. 339-53. 146. Kim, K.W., et al., Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma. Cancer Res, 1998. 58(2): p. 348-51. 147. Moromisato, D.Y., et al., Effect of hypoxia on lung, heart, and liver insulin-like growth factor-I gene and receptor expression in the newborn rat. Crit Care Med, 1996. 24(6): p. 919-24. 148. Feldser, D., et al., Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2. Cancer Res, 1999. 59(16): p. 3915-8. 149. Zelzer, E., et al., Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT. EMBO J, 1998. 17(17): p. 5085-94. 150. Grulich-Henn, J., et al., Transport of insulin-like growth factor-I across endothelial cell monolayers and its binding to the subendothelial matrix. Exp Clin Endocrinol Diabetes, 2002. 110(2): p. 67-73. 151. Lee, O.H., et al., Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models. Br J Cancer, 2000. 82(2): p. 385-91. 152. Shigematsu, S., et al., IGF-1 regulates migration and angiogenesis of human endothelial cells. Endocr J, 1999. 46 Suppl: p. S59-62. 153. Mira, E., et al., Insulin-like growth factor I-triggered cell migration and invasion are mediated by matrix metalloproteinase-9. Endocrinology, 1999. 140(4): p. 1657-64. 99	 ?	 ?154. Zhang, D., et al., Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals. J Biol Chem, 2004. 279(19): p. 19683-90. 155. Chan, J.M., et al., Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science, 1998. 279(5350): p. 563-6. 156. Hankinson, S.E., et al., Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet, 1998. 351(9113): p. 1393-6. 157. Yu, H., et al., Plasma levels of insulin-like growth factor-I and lung cancer risk: a case-control analysis. J Natl Cancer Inst, 1999. 91(2): p. 151-6. 158. Ma, J., et al., Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J Natl Cancer Inst, 1999. 91(7): p. 620-5. 159. Shevah, O. and Z. Laron, Patients with congenital deficiency of IGF-I seem protected from the development of malignancies: a preliminary report. Growth Horm IGF Res, 2007. 17(1): p. 54-7. 160. Chan, J.M., et al., Insulin-like growth factor-I (IGF-I) and IGF binding protein-3 as predictors of advanced-stage prostate cancer. J Natl Cancer Inst, 2002. 94(14): p. 1099-106. 161. Brunner, J.E., et al., Colon cancer and polyps in acromegaly: increased risk associated with family history of colon cancer. Clin Endocrinol (Oxf), 1990. 32(1): p. 65-71. 162. Pines, A., et al., Gastrointestinal tumors in acromegalic patients. Am J Gastroenterol, 1985. 80(4): p. 266-9. 100	 ?	 ?163. Ron, E., et al., Acromegaly and gastrointestinal cancer. Cancer, 1991. 68(8): p. 1673-7. 164. Renehan, A.G. and B.M. Brennan, Acromegaly, growth hormone and cancer risk. Best Pract Res Clin Endocrinol Metab, 2008. 22(4): p. 639-57. 165. Baris, D., et al., Acromegaly and cancer risk: a cohort study in Sweden and Denmark. Cancer Causes Control, 2002. 13(5): p. 395-400. 166. Kauppinen-Makelin, R., et al., Increased cancer incidence in acromegaly--a nationwide survey. Clin Endocrinol (Oxf), 2010. 72(2): p. 278-9. 167. Orme, S.M., et al., Mortality and cancer incidence in acromegaly: a retrospective cohort study. United Kingdom Acromegaly Study Group. J Clin Endocrinol Metab, 1998. 83(8): p. 2730-4. 168. Watanabe, S., et al., Leukemia and other malignancies among GH users. J Pediatr Endocrinol, 1993. 6(1): p. 99-108. 169. Banerjee, I. and P.E. Clayton, Growth hormone treatment and cancer risk. Endocrinol Metab Clin North Am, 2007. 36(1): p. 247-63. 170. Bell, J., et al., Long-term safety of recombinant human growth hormone in children. J Clin Endocrinol Metab, 2010. 95(1): p. 167-77. 171. Ho, K.K., Consensus guidelines for the diagnosis and treatment of adults with GH deficiency II: a statement of the GH Research Society in association with the European Society for Pediatric Endocrinology, Lawson Wilkins Society, European Society of Endocrinology, Japan Endocrine Society, and Endocrine Society of Australia. Eur J Endocrinol, 2007. 157(6): p. 695-700. 101	 ?	 ?172. Midyett, L.K., et al., Recombinant insulin-like growth factor (IGF)-I treatment in short children with low IGF-I levels: first-year results from a randomized clinical trial. J Clin Endocrinol Metab, 2010. 95(2): p. 611-9. 173. Chernausek, S.D., et al., Long-term treatment with recombinant insulin-like growth factor (IGF)-I in children with severe IGF-I deficiency due to growth hormone insensitivity. J Clin Endocrinol Metab, 2007. 92(3): p. 902-10. 174. Prostate cancer statistics. American Cancer Society and Canadian Cancer Society. 175. Pollak, M., Insulin-like growth factors and prostate cancer. Epidemiol Rev, 2001. 23(1): p. 59-66. 176. Djavan, B., et al., Insulin-like growth factors and prostate cancer. World J Urol, 2001. 19(4): p. 225-33. 177. Wolk, A., et al., Insulin-like growth factor 1 and prostate cancer risk: a population-based, case-control study. J Natl Cancer Inst, 1998. 90(12): p. 911-5. 178. Krueckl, S.L., et al., Increased insulin-like growth factor I receptor expression and signaling are components of androgen-independent progression in a lineage-derived prostate cancer progression model. Cancer Res, 2004. 64(23): p. 8620-9. 179. Pollak, M., W. Beamer, and J.C. Zhang, Insulin-like growth factors and prostate cancer. Cancer Metastasis Rev, 1998. 17(4): p. 383-90. 180. Nickerson, T., et al., In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of 102	 ?	 ?insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR). Cancer Res, 2001. 61(16): p. 6276-80. 181. Huynh, H., R.M. Seyam, and G.B. Brock, Reduction of ventral prostate weight by finasteride is associated with suppression of insulin-like growth factor I (IGF-I) and IGF-I receptor genes and with an increase in IGF binding protein 3. Cancer Res, 1998. 58(2): p. 215-8. 182. Nickerson, T., et al., Castration-induced apoptosis of androgen-dependent shionogi carcinoma is associated with increased expression of genes encoding insulin-like growth factor-binding proteins. Cancer Res, 1999. 59(14): p. 3392-5. 183. Thomas, L.N., et al., Insulin-like growth factor binding protein 5 is associated with involution of the ventral prostate in castrated and finasteride-treated rats. Prostate, 1998. 35(4): p. 273-8. 184. Nickerson, T. and M. Pollak, Bicalutamide (Casodex)-induced prostate regression involves increased expression of genes encoding insulin-like growth factor binding proteins. Urology, 1999. 54(6): p. 1120-5. 185. Miyakoshi, N., et al., Evidence that IGF-binding protein-5 functions as a growth factor. J Clin Invest, 2001. 107(1): p. 73-81. 186. Jones, J.I., et al., Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I. J Cell Biol, 1993. 121(3): p. 679-87. 187. Bautista, C.M., D.J. Baylink, and S. Mohan, Isolation of a novel insulin-like growth factor (IGF) binding protein from human bone: a potential candidate for fixing IGF-II in human bone. Biochem Biophys Res Commun, 1991. 176(2): p. 756-63. 103	 ?	 ?188. Mohan, S. and D.J. Baylink, Insulin-like growth factor system components and the coupling of bone formation to resorption. Horm Res, 1996. 45 Suppl 1: p. 59-62. 189. Xu, C., et al., Regulation of global gene expression in the bone marrow microenvironment by androgen: androgen ablation increases insulin-like growth factor binding protein-5 expression. Prostate, 2007. 67(15): p. 1621-9. 190. Bubendorf, L., et al., Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol, 2000. 31(5): p. 578-83. 191. Olmos, D., et al., Targeting the Insulin-Like Growth Factor 1 Receptor in Ewing's Sarcoma: Reality and Expectations. Sarcoma, 2011. 2011: p. 402508. 192. Weroha, S.J. and P. Haluska, IGF-1 receptor inhibitors in clinical trials--early lessons. J Mammary Gland Biol Neoplasia, 2008. 13(4): p. 471-83. 193. Furukawa, J., et al., Antisense oligonucleotide targeting of insulin-like growth factor-1 receptor (IGF-1R) in prostate cancer. Prostate, 2010. 70(2): p. 206-18. 194. Gao, J., et al., Dual IGF-I/II-neutralizing antibody MEDI-573 potently inhibits IGF signaling and tumor growth. Cancer Res, 2011. 71(3): p. 1029-40. 195. Menefee M, e.a. MEDI-573, a dual IGF-1/-2 neutralizing antibody, blocks IGF-1R and IR-A signaling and maintains glucose homeostasis in a Phase 1 study for advanced solid tumors. in 22nd EORTCNCI-AACR Symposium. 2010. Philadelphia (PA). 196. Grimberg, A., P53 and IGFBP-3: apoptosis and cancer protection. Mol Genet Metab, 2000. 70(2): p. 85-98. 104	 ?	 ?197. Grimberg, A. and P. Cohen, Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis. J Cell Physiol, 2000. 183(1): p. 1-9. 198. Yamada, P.M. and K.W. Lee, Perspectives in mammalian IGFBP-3 biology: local vs. systemic action. Am J Physiol Cell Physiol, 2009. 296(5): p. C954-76. 199. Hwa, V., Y. Oh, and R.G. Rosenfeld, The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr Rev, 1999. 20(6): p. 761-87. 200. Andress, D.L., et al., Carboxy-truncated insulin-like growth factor binding protein-5 stimulates mitogenesis in osteoblast-like cells. Biochem Biophys Res Commun, 1993. 195(1): p. 25-30. 201. Binoux, M., Insulin-like growth factor binding proteins (IGFBPs): physiological and clinical implications. J Pediatr Endocrinol Metab, 1996. 9 Suppl 3: p. 285-8. 202. Kalus, W., et al., Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions. EMBO J, 1998. 17(22): p. 6558-72. 203. Imai, Y., et al., Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions. J Biol Chem, 2000. 275(24): p. 18188-94. 204. Allan, G.J., et al., Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action. Endocrinology, 2006. 147(1): p. 338-49. 105	 ?	 ?205. Arai, T., et al., Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein 5 alters heparin binding and its change in affinity for IGF-I response to heparin. J Biol Chem, 1996. 271(11): p. 6099-106. 206. Song, H., et al., The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain. J Mol Endocrinol, 2001. 26(3): p. 229-39. 207. Parker, A., et al., Identification of the extracellular matrix binding sites for insulin-like growth factor-binding protein 5. J Biol Chem, 1996. 271(23): p. 13523-9. 208. Nam, T.J., et al., Thrombospondin and osteopontin bind to insulin-like growth factor (IGF)-binding protein-5 leading to an alteration in IGF-I-stimulated cell growth. Endocrinology, 2000. 141(3): p. 1100-6. 209. Nam, T.J., W. Busby, Jr., and D.R. Clemmons, Insulin-like growth factor binding protein-5 binds to plasminogen activator inhibitor-I. Endocrinology, 1997. 138(7): p. 2972-8. 210. Golemis, E.A., K.D. Tew, and D. Dadke, Protein interaction-targeted drug discovery: evaluating critical issues. Biotechniques, 2002. 32(3): p. 636-8, 640, 642 passim. 211. Phizicky, E.M. and S. Fields, Protein-protein interactions: methods for detection and analysis. Microbiol Rev, 1995. 59(1): p. 94-123. 212. Fang, B., et al., Label-free electrochemical detection of DNA using ferrocene-containing cationic polythiophene and PNA probes on nanogold modified electrodes. Biosens Bioelectron, 2008. 23(7): p. 1175-9. 106	 ?	 ?213. Rich, R.L. and D.G. Myszka, Higher-throughput, label-free, real-time molecular interaction analysis. Anal Biochem, 2007. 361(1): p. 1-6. 214. Cross, G.H., et al., A new quantitative optical biosensor for protein characterisation. Biosens Bioelectron, 2003. 19(4): p. 383-90. 215. Wartchow, C.A., et al., Biosensor-based small molecule fragment screening with biolayer interferometry. J Comput Aided Mol Des, 2011. 25(7): p. 669-76. 216. Francis, G.L., et al., Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency. J Mol Endocrinol, 1992. 8(3): p. 213-23. 217. King, R., et al., Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3, following their expression in Escherichia coli as fusion proteins. J Mol Endocrinol, 1992. 8(1): p. 29-41. 218. Green, N.M., Avidin. Adv Protein Chem, 1975. 29: p. 85-133. 219. Sheikh, M.S., et al., Identification of the insulin-like growth factor binding proteins 5 and 6 (IGFBP-5 and 6) in human breast cancer cells. Biochem Biophys Res Commun, 1992. 183(3): p. 1003-10. 220. Adams, S., et al., Pharmacokinetics and bioavailability of rhIGF-I/IGFBP-3 in the rat and monkey. Prog Growth Factor Res, 1995. 6(2-4): p. 347-56. 221. Rajaram, S., D.J. Baylink, and S. Mohan, Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr Rev, 1997. 18(6): p. 801-31. 107	 ?	 ?222. Miyakoshi, N., et al., Effects of recombinant insulin-like growth factor-binding protein-4 on bone formation parameters in mice. Endocrinology, 1999. 140(12): p. 5719-28. 223. Beattie, J., et al., Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study. Mol Cell Biochem, 2008. 307(1-2): p. 221-36. 224. Abdiche, Y., et al., Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal Biochem, 2008. 377(2): p. 209-17. 225. Loddick, S.A., et al., Displacement of insulin-like growth factors from their binding proteins as a potential treatment for stroke. Proc Natl Acad Sci U S A, 1998. 95(4): p. 1894-8.             108	 ?	 ?Appendix Figure 3.1 and 3.2   

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0074271/manifest

Comment

Related Items