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Characterization of Insulin-Like Growth Factor Binding Proteins from Human Breast Cancer Cells

Department of Pediatrics, Division of Pediatric Endocrinology, Stanford University Medical Center Stanford, California 94305

Address requests for reprints to: Dr. Daisy D. De Leon, S-322 Pediatric Endocrinology, Stanford University Medical Center, Stanford, California 94305-5119.

Abstract

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (M_r = 150 k) and a small GH-independent BP (M_r = 28–42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent M_r = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent M_r = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative. These data demonstrate that the IGF-BPs secreted by breast cancer cells are heterogeneous and that the major secreted forms are structurally and immunologically distinct from the small IGF-BP from amniotic fluid and HEP G2. Understanding the role of these proteins is of great interest in view of the potential effects of the IGFs in breast cancer and the possible modulation of IGF actions by these IGF-BPs.

FOOTNOTES

This work was supported in part by NIH Grants DK-28229, DK-36054, CA-42106 (to R.G.R.), Biomedical Support Grant RR-05353 from the American Cancer Society (to D.M.W.), and Grant DK-24085 (to R.L.H.).

^* Recipient of Research Career Development Award DK-01275 from the NIH.

Received for publication September 7, 1988. Revision received December 21, 1988. Accepted for publication December 21, 1988.

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