Mitogenic and Antiapoptotic Effects of Insulin-like Growth Factor Binding Protein-6 in the Human Osteoblastic Osteosarcoma Cell Line Saos-2/B-10 Christoph Schmid, Claudia Keller, Martina Gosteli-Peter, and Ju ¨ rgen Zapf Division of Endocrinology and Diabetes, Department of Internal Medicine, University Hospital, Ra ¨ mistrasse 100, CH-8091 Zu ¨ rich, Switzerland Received August 27, 1999 Insulin-like growth factor (IGF) I is a potent mitogen for human osteosarcoma cells such as the Saos-2/B-10 cell line. IGF binding proteins (IGFBPs) prevent stim- ulation of DNA synthesis by IGFs. In contrast to re- combinant human (rh) IGFBP-2, -3, -4, and -5, 10 –100 nM rhIGFBP-6 stimulated [ 3 H]thymidine incorpora- tion into DNA and multiplication of Saos-2/B-10 cells. Upon withdrawal of serum, 30 nM IGFBP-6 also de- creased apoptosis (within 4 h) and increased protein content and sodium-dependent phosphate uptake (within 24 h), but less potently than IGF I. 125 I-labeled rhIGFBP-6 did not bind to the cells, and cold IGFBP-6 did not affect 125 I-labeled IGF I binding. Production of IGF I, IGF II, and IGFBP-6 by the cells or significant degradation of rhIGFBP-6 could not be detected within 24 h of incubation. Thus, among the rhIGFBPs tested, rhIGFBP-6 is unique in stimulating osteosar- coma cell growth. Furthermore, it has an antiapop- totic effect. © 1999 Academic Press Six different high affinity insulin-like growth fac- tor binding proteins (IGFBP-1 to IGFBP-6) of mo- lecular mass 22–32 kDa have been identified and cloned (1). IGFBP-6 was isolated from cerebrospinal fluid (2) and also from the medium of fibroblasts (3). IGFBP-6 binds IGF II with a 100-fold higher affinity than IGF I (4) and is abundant in adult human serum; serum concentrations are in the range of 5–10 nM (1, 5). IGF I is a potent mitogen for Saos-2/B-10 cells (6); IGF II is 2- to 3-fold less potent. While testing the specificity of the biological activity of rhIGFBPs, we found that IGFBP-6 but not IGFBP-5 stimulated DNA synthesis in these cells (7). Since stimulatory effects of an IGFBP in a cell line which expresses little IGF I and IGF II were unexpected, we undertook the present study. MATERIALS AND METHODS Cell culture. Saos-2/B-10 cells (kindly provided by Drs. S. B. and G. A. Rodan) (8) were grown and passaged in medium supplemented with fetal calf serum (FCS, 10%) as described (6, 9). Cells were plated in Falcon multiwell tissue culture dishes at a density of 2  10 5 per 9.6-cm 2 surface area (35 mm diameter), kept for 3 days in 5% FCS- containing growth medium, then rinsed with serum-free medium, and the medium was replaced by serum-free Ham’s F12 medium containing gentamycin (50 g/ml), glutamine (2 mmol/liter) and charcoal-treated bovine serum albumin (BSA) at 1 g/liter. Recombi- nant human (rh) IGF I and rhIGF II were from former Ciba–Geigy AG, Basel. RhIGFBP-2 was a gift from Dr. J. Shuster, Chiron (Em- eryville, CA), rhIGFBP-3 from Dr. A. Sommer, Celtrix (Santa Clara, CA). RhIGFBP-4 and rhIGFBP-6 were expressed in yeast and puri- fied as described (4). Aliquots of test agents were added directly to the cultures. [ 3 H]Thymidine incorporation into DNA. Confluent cultures were incubated in 1 ml serum-free F12 medium containing 1 g/liter BSA and test agents for 18 h, pulsed with [ 3 H]thymidine (Amersham, 85 Ci/mmol; 1 Ci per dish) and further incubated for 3 h at 37°C. DNA was precipitated and incorporated radioactivity was measured (6, 9). Photometric enzyme immunoassay for determination of cytoplas- mic histone-associated DNA fragments. An ELISA kit (Cell Death Detection ELISA PLUS from Boehringer-Mannheim) was used to mea- sure cytosolic oligonucleosome-bound DNA. 42,000 cells were seeded in 24 multiwell (2-cm 2 surface area) dishes and grown for 3 days in FCS-containing medium, rinsed with serum-free medium and incu- bated for 4 h in 200 l serum-free Ham’s F12 medium containing 1 g/l BSA and test agents. The ELISA monitors enrichment of oligo- nucleosomes in the cells by monoclonal antibodies directed against DNA and histones. To avoid potential effects caused by differences in cell adherence and recovery, both the cell supernatant and the cell layer lysate were analyzed. The media were combined with 2  400 l phosphate-buffered saline (PBS), pH 7.3, which was used for washing the cell layers (to harvest detached and loosely adhering cells). After centrifugation for 10 min at room temperature at 200g, 200 l of lysis buffer was added to the pellet, and a 20-l aliquot was used for the assay. The cell layer on the dishes was lysed directly into 400 l lysis buffer, centrifuged for 10 min at 200g at room temper- ature, and 20 l was used for analysis as described by the supplier. Phosphate transport studies. Cells were grown on 9.6-cm 2 surface area dishes for 3 days in 5% FCS-containing medium, rinsed with serum-free medium and exposed to test medium. 32 PO 4 uptake stud- ies were performed in buffer containing 140 mM NaCl or choline Biochemical and Biophysical Research Communications 263, 786 –789 (1999) Article ID bbrc.1999.1451, available online at http://www.idealibrary.com on 786 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.