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Department of Pediatrics, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3042
Retinoic acid (RA) is a potent in vitro inhibitor of cell proliferation in various malignant cell lines. The exact mechanisms of its actions, however, are not fully understood. To further elucidate the nature of this inhibition, we investigated the effects of RA in an estrogen receptor-negative human breast cancer cell line, MDA-MB-231. RA (0.01-5 µM) significantly inhibited MDA-MB-231 cell growth by 35-40% as compared with untreated controls. Similar growth inhibitory actions were observed when cells were treated with transforming growth factor ß2 (TGF-ß2), another factor with antiproliferative actions in breast cancer cells.
Both RA and TGF-ß2 increased the levels of insulin-like growth factor binding protein (IGFBP) 3 (2-3-fold) and mRNA (1.5-2-fold), whereas IGFBP-4 levels remained essentially unchanged. The direct involvement of IGFBP-3 in cell growth inhibition was further confirmed by its action on cell growth: exogenous IGFBP-3 directly and significantly inhibited MDA-MB-231 cell number by 40%. These results provided circumstantial evidence that IGFBP-3 may mediate RA and TGF-ß2 growth inhibitory actions in human breast cancer cells.
To test this hypothesis, we used an antisense IGFBP-3 oligodeoxynucleotide (ODN) which specifically inhibits IGFBP-3 expression. The antisense IGFBP-3 ODN dramatically blocked both RA- and TGF-ß2-induced increases in IGFBP-3 protein (90%) and mRNA levels (90%). This effect was not observed when RA- or TGF-ß2-exposed cells were treated with sense IGFBP-3 ODN. Moreover, antisense ODN did not significantly affect IGFBP-4 protein or mRNA levels, strongly supporting the specificity of the antisense IGFBP-3 ODN effect on IGFBP-3 mRNA.
This specific effect of antisense IGFBP-3 ODN on IGFBP-3 protein and mRNA levels was accompanied by significant attenuation of the inhibition of cell proliferation attained with RA or TGF-ß2 (approximately 40% of either RA- or TGF-ß2-induced inhibition). The control sense IGFBP-3 ODN did not reduce the growth inhibition observed with either RA or TGF-ß2.
These results indicate that IGFBP-3 is an important mediator of RA-and TGF-ß2-induced cell growth inhibition in human breast cancer cells.
1 Supported in part by Grant CA58110 from NIH (R.G.R.) and by Soros Open Society Foundation Grant 78 (Z. S. G.).
2 To whom requests for reprints should be addressed, at Department of Pediatrics, L343, School of Medicine, Oregon Health Sciences University, Portland, OR 97201-3042. Phone: (503) 394-1925; Fax: (503) 494-1933.
Received 11/ 6/95. Accepted 1/25/96.
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