Human osteosarcoma (U-2 OS) cells express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors and synthesize IGF-II: autocrine growth stimulation by IGF-II via the IGF-I receptor.
Recently, insulin-like growth factor-I and -II (IGF-I and -II) have been implicated in the growth promotion of tumors in vivo and tumor cells in vitro. We have studied the human osteosarcoma cell line U-2 OS in order 1) to gain more insight into the growth promoting actions of the IGFs and 2) to establish an in vitro tissue culture model of IGF action in human tumor cells. Specific binding of 125I-IGF-I and 125I-IGF-II to IGF-I receptors and IGF-II/mannose-6-phosphate (M6P) receptors on U-2 OS cells was demonstrated in competitive binding experiments and in affinity crosslinking experiments. Western blotting of cell extracts confirmed the expression of the IGF-II/M6P receptor. In addition, in Northern blotting experiments using total RNA from U-2 OS cells IGF-I receptor RNA of 11 kb and IGF-II/M6P receptor RNA of approximately 9 kb were detected. Solution hybridization experiments confirmed the presence of IGF-I receptor and IGF-II/M6P receptor RNA. In a subset of experiments DNA synthesis was measured as 3H-thymidine uptake into cellular DNA of U-2 OS cells. Normal rat serum stimulated DNA synthesis maximally. IGF-I-deficient serum from hypophysectomized rats as well as IGF-I or IGF-II without serum were approximately twofold and tenfold, respectively, less potent than serum in stimulating 3H-thymidine uptake. The concentrations of IGF-I and IGF-II needed for half maximal stimulation of DNA synthesis corresponded well with the respective concentrations required for half maximal inhibition of 125I-IGF-I binding to U-2 OS cells. The anti-IGF-I receptor antibody alpha IR3 blocked the IGF-I and IGF-II stimulated increase of 3H-thymidine uptake. In addition, basal DNA synthesis was partially inhibited by the anti-IGF-I receptor antibody. These data suggest that U-2 OS cells synthesize and secrete IGF-like peptides. Northern blotting experiments confirmed that U-2 OS cells express an IGF-II RNA species of 5.3 kb but no IGF-I transcripts. In a series of RNase protection assays, protected RNA fragments were detected with an IGF-II riboprobe. Also, cell-conditioned medium from U-2 OS cells contained 1-2 ng/ml IGF-II immunoreactivity as measured in an IGF-binding protein blocked IGF-II radioimmunoassay.


CONCLUSIONS
1) U-2 OS cells express IGF-I and IGF-II/M6P receptors. 2) U-2 OS tumor cells respond to the addition of exogenous IGF-I and IGF-II with an increase of DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
Raile
K
K
Department of Pediatric Endocrinology, Children's Hospital, University of Munich, Germany.
Höflich
A
A
Kessler
U
U
Yang
Y
Y
Pfuender
M
M
Blum
W F
WF
Kolb
H
H
Schwarz
H P
HP
Kiess
W
W
D000818:Animals; D001667:Binding, Competitive; D001769:Blood; D015152:Blotting, Northern; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D017077:Culture Media, Conditioned; D015342:DNA Probes; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000096:biosynthesis; D015870:Gene Expression; D005910:Glioma; D006801:Humans; D007016:Hypophysectomy; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology*; D008099:Liver / Q000502:physiology; D012516:Osteosarcoma / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis* / Q000187:drug effects / Q000302:isolation & purification; D017527:Receptor, IGF Type 2 / Q000096:biosynthesis* / Q000302:isolation & purification; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D015342:DNA Probes; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017527:Receptor, IGF Type 2; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; D013936:Thymidine