Hermann L. Müller

Insulin-like growth factor binding proteins and their regulation

Insulin and the insulin-like growth factors (IGFs) share including gel chromatography, radioreceptor assays, significant structural homology, as is evident from the affinity cross-linking, Western ligand blotting (5, 6), ability of insulin to compete for occupancy of the type 1 immunoblotting and, recently, specific radioimmunoIGF receptor. The IGFs differ from insulin, however, in assays (RIAs). It has been the study of the molecular one important respect: in contrast to insulin, the IGFs biology of the IGFBPs, however, that has provided the circulate in plasma complexed to a family of binding most information concerning their structural interproteins (1-3). Although both IGF-I and IGF-I1 bind to relationship. these binding proteins (IGFBPs) with high affinity, insulin is totally incapable of competing for occupancy. These carrier proteins significantly extend the serum half-life of the IGF peptides, transport the IGFs to target cells and modulate the interaction of the IGFs with their surface membrane receptors. Furthermore, recent evidence suggests that under certain conditions, IGFBPs may be capable of acting independently, affecting cellular metabolism and replication, even in the absence of IGF peptides. The existence of IGFBPs was first inferred 20 years ago from chromatographic studies of the size distribution of IGF peptides in serum (4). It has only been in the last few years, however, that the complexity of the interactions among the IGFs, IGFBPs and IGF receptors has been fully appreciated. This has been accompanied by the realization that a full understanding of IGF action at a cellular level must take into account the modulatory role of IGFBPs. The identification and characterization of IGFBPs in various body fluids and in conditioned media from cultured cells have been facilitated by the development of a number of biochemical and assay techniques,

Insulin-like growth factor binding protein-3 concentrations and insulin-like growth factor binding protein-3 protease activity in sera of patients with malignant solid tumors or leukemia.

ABSTRACT: IGF binding proteins (IGFBP) regulate the bioavailability and bioactivity of IGF. The major IGFBP in serum is IGFBP-3. We investigated whether sera from children with malignancies show alterations in levels of IGFBP-3 as measured by Western ligand blot analysis (WLB) and RIA with αIGFBP-3gl, a specific rabbit polyclonal antibody. Furthermore, IGFBP-3 proteolysis was quantified by densitometric analysis of [125I]IGFBP-3 protease assays, and IGFBP-3 fragments were visualized by Western immunoblot with αIGFBP-3gl. We examined sera from 21 children with solid tumors, five patients with sarcoma who had reached complete remission, and 13 children with acute leukemia. Serum samples were collected at diagnosis, before initiation of therapy. Sera of 10 healthy children served as normal controls. Children with solid tumor or leukemia had significantly higher (p < 0.001) IGFBP-3 protease activity in serum than did normal controls or patients with sarcoma in complete remission. Corresponding to this finding, densitometry of WLB showed lower IGFBP-3 levels in sera of children with malignancies in comparison with normal controls. The negative correlation (p < 0.001, r =-0.80) between IGFBP-3 proteolysis, as measured by [125I]IGFBP-3 protease assay, and IGFBP-3 band density on WLB indicates that proteolysis is the probable reason for reduction of IGFBP-3 on WLB. IGFBP-3 concentrations measured by RIA were in the normal range for most patients, further indicating that differences in serum IGFBP-3 levels measured by WLB reflect protease activity. We conclude that sera from children with malignancies frequently contain protease activity that causes IGFBP-3 cleavage in a way that might affect the tissue availability of IGF. Because many tumor cells are responsive to the mitogenic actions of IGF in vitro, IGFBP-3 proteases in serum could also play a role in tumor progression and metastasis in vivo.

Synthesis and Characterization of IGF-II Analogs: Applications in the Evaluation of IGF Receptor Function and IGF-Independent Actions of IGFBPS

The elucidation of the mechanisms involved in the biological actions of the IGFs has been hampered by the complexity of the IGF system: three peptide ligands (IGF-I, IGF-II and insulin),1 three receptors (type 1 and type 2 IGF receptors and insulin receptors)2 and six distinct, but structurally related binding proteins (IGFBP-1 to IGFBP-6).3 The physiological interactions among these components of the IGF system are not completely understood, although they have all been cloned and sequenced.4

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 3 (IGFBP-3) CONCENTRATION IN CEREBROSPINAL FLUID (CSF) OF CHILDREN WITH BRAIN TUMORS OR LEUKEMIA

The major IGFBPs in CSF are IGFBP-2 and IGFBP-4. whereas IGFBP-3 is a minor component in CSF of healthy subjects. We investigated IGFBP-3 levels in CSF from patients with brain tumors, leukemia or meningitis. IGFBP-3 was measured by radioimmuno-assay with αIGFBP-3g I, a rabbit polyclonal antibody. Further, as proteolysis of IGFBP-3 is purt of the modulation of IGF activity IGFBP-3 fragmentation was quantified by densitometric analysis of [125I]IGFBP-3 protease assays. We examined CSFs of 24 children with malignant brain tumors. 18 children with leukemia and 13 children with meningitis. CSFs of 38 children, who received a lumbal puncture in order to exclude meningitis, were used to define a normalative range for IGFBP-3 concentration and IGFBP-3 protease activity in normal CSF. Elevated IGFBP-3 concentrations were found in CSF of 17 of all 24 (71%) brain tumor patients and 7 of 8 (87%) brain tumor patients who had microscopically detectable malignant cells in CSF. 13 of 14 (93%) patients with medulloblastoma or ependymoma and all 7 medulloblasioma/ependymoma patients with malignant cells in CSF had elevated IGFBP-3 concentrations in CSF. IGFBP-3 protease activity in CSF was elevated in 15 of 17 (88%) patients with histological high grade (WIIO III°/IV°) brain tumors. 5 of 6 (83%) patients with acute leukemia and microscopically detectable malignant cells in CSF at the time of diagnosis showed elevated IGFBP-3 concentrations in CSF that normalized under chemotherapy. leukemia patients without any detectable malignant cells in the CSF had normal IGFBP-3 concentrations in CSF. We conclude that in CSF of children with high grade malignant brain tumors or CNS leukemia. IGFBP-3 is elevated. We hypothesize that this phenomenon could be caused by local production of IGFBP-3 by the brain tumor tissue and secretion into CSF or by local secretion of IGFBP-3 by malignant cells that spread into CSF. This hypothesis is supported by the observation that only 2 of 13 (15%) meningitis patients had slightly elevated IGFBP-3 concentrations in CSF while high numbers of non malignant inflammatory cells were present in all cases. Further studies will analyse the origin of elevated IGFBP-3 levels in CSF of tumor patients and will show whether these findings are of diagnostic or prognostic value.