ABSTRACT
Insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate IGF action at cellular level through inhibition or, alternatively, potentiation, where their limited proteolysis is a contributory mechanism. Under basal conditions, neuroblastoma cells secrete IGFs (essentially IGF-II), IGFBPs (IGFBP-4 and predominantly IGFBP-2 that is partially proteolysed), and proteases, including tissue-type plasminogen (PLG) activator, whose activity is inhibited by PLG activator inhibitor-1. Neuroblastoma cells were used to investigate the influence of the plasmin system, transforming growth factor-beta retinoic acid on cell growth and the IGF system. In cells treated with 5 micrograms/ml PLG, proliferation was stimulated, an effect that was inhibited in the presence of either alpha IR-3 (which blocks the type 1 IGF receptor) or anti-IGF-II antibodies. There was a parallel increase in IGFBP-2 proteolysis, which resulted in a 5-fold loss of affinity for IGF-II. In the presence of 1 ng/ml transforming growth factor-beta, PLG-induced mitogenesis and IGFBP-2 proteolysis were reduced, and Northern blot analysis revealed increased PLG activator inhibitor-1 mRNA. Conversely, with 2 microM retinoic acid, the mitogenic effect of PLG, IGFBP-2 proteolysis, and tissue-type PLG activator mRNAs were increased. Therefore, IGF-II mediates autocrine proliferation in neuroblastoma cells under the control of IGFBPs secreted by the cells, its bioavailability being enhanced as a result of plasmin-induced IGFBP-2 proteolysis.
Subject(s)
Cell Division , Insulin-Like Growth Factor Binding Protein 2/metabolism , Neuroblastoma/pathology , Transforming Growth Factor beta/pharmacology , Tretinoin/pharmacology , Blotting, Northern , Fibrinolysin/metabolism , Humans , Immunoblotting , Plasminogen Activator Inhibitor 1/genetics , RNA, Messenger/metabolism , Tissue Plasminogen Activator/pharmacology , Tumor Cells, CulturedABSTRACT
Insulin-like growth factors (IGF-I and -II) play an active role in cell proliferation. In biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBPs), at least 6 species of which have been identified to date, but with poorly defined functions. One of these IGFBPs, IGFBP-2, is secreted by most cell lines and appears to be involved in cell proliferation. A human epidermoid carcinoma cell line, KB 3.1, which produces IGFBP-1 and -3 and small amounts of IGFBP-4, but no IGFBP-2, was stably transfected with an expression vector comprising IGFBP-2 complementary DNA (cDNA), whose expression was placed under the control of the constitutive and ubiquitous cytomegalovirus promoter. After an s.c. injection of these IGFBP-2-expressing KB 3.1 cells into nude mice, tumours developed more quickly than in controls, they were 3 to 4 times larger and grew about 3 times as fast. Concomitant with IGFBP-2 expression in these tumours, were a decrease in IGFBP-1 expression and an increase in IGFBP-3 proteolysis, both of which increase the bioavailability of the IGF-II produced by the cells. The increased IGFBP-3 proteolysis most probably resulted from amplified expression of tissue-type plasminogen activator (t-PA) and depression of its inhibitor (PAI-I) observed in IGFBP-2-expressing xenografts. Our findings suggest that IGFBP-2 plays a role in this model of experimental tumorigenesis via a mechanism that remains unclear, but appears to involve increased protease activity and IGF-II bioavailability.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Insulin-Like Growth Factor Binding Protein 1/metabolism , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor Binding Protein 3/metabolism , Animals , Blotting, Northern , Blotting, Western , Carcinogenicity Tests , DNA Probes , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Nude , Tumor Cells, Cultured/metabolismABSTRACT
BACKGROUND: The insulin-like growth factors (IGFs) are involved in the growth and differentiation of neuroblastoma cells. In all biological fluids, they are non-covalently bound to high-affinity binding proteins (IGFBP-1 to -6) which modulate their bioavailability. We previously showed that IGFBP-6 expression is linked to the arrest of growth in neuroblastoma cells, whereas IGFBP-2 is associated with proliferation. PROCEDURE: To study the role of IGFBP-6 in cell growth, we stably IGR-N-91 neuroblastoma cells with a plasmid containing sequences coding for IGFBP-6 under the control of the cytomegalovirus (CMV) promoter. RESULTS: The incidence and size of tumors generated by injecting IGFBP-6-expressing cells into nude mice were reduced by factors of 2 and 5, respectively, as compared with those generated by injection by control cells. Northern blot analyses if xenografts revealed weaker expression of IGF-II, type 2 IGF receptor and IGFBP-2 mRNAs in IGFBP-6-expressing cthan in control xenografts. IGFBP-6 may therefore reduce the expression of IGF-II (which induces tumour development) at a transcriptional level. Conversely, containing IGFBP-2 cDNA under the control of CMV promoter grew three to four times as fast as normal control xenografts. Northern blot analyses revealed weaker expression of intact IGFBP-3 and IGFBP-1 in IGFBP-2-expressing than in control xenografts. CONCLUSIONS: IGFBP-1 and intact IGFBP-3 expression both enhance IGF bioavailability which promotes tumour growth. Although the mechanisms of action of IGFBP-2 and IGFBP-6 remain to be elucidated, an inverse relationship appears to exist between the two binding proteins, IGFBP-2 being involved in proliferation and IGFBP-6 in its arrest.