Immunodetection and modulation of cellular growth with antibodies against native transforming growth factor-beta 1
Keski-Oja, J.; Lyons, R.M.; Moses, H.L.
Cancer Research 47(24 Pt 1): 6451-6458
1987
ISSN/ISBN: 0008-5472 PMID: 2890433 Document Number: 290351
In an attempt to identify and quantitate latent and active forms of transforming growth factor .beta. (TGF.beta.) without the use of cell cultures and to test for autocrine stimulation by TGF.beta., rabbit antibodies were raised against native human and porcine platelet-derived TGF.beta. A radioimmunoassay for TGF.beta. was developed using radioiodinated TGF.beta., anti-TGF.beta. antibodies, and protein A. Inhibition in the radioimmunoassay was achieved with nanogram quantities of TGF.beta., comparable to the sensitivity of radioreceptor assays. Analyses of the TGF.beta. levels of conditioned medium from cultured cells indicated that the latent form(s) of TGF.beta. is not detectable in the radioimmunoassay established using antibodies raised against native TGF.beta. Immunoprecipitation analysis of radiolabeled conditioned medium revealed a specific Mr 25,000 band only after acidification. A Mr 62,000 protein was observed with and without prior acidification of the medium but could not be competed with unlabeled TGF.beta. in the immunoprecipitation indicating antigenic unrelatedness. The anti-TGF.beta. IgG inhibited the binding of [125I]TGF.beta. to the cell surface receptors in a radioreceptor assay. TGF.beta. inhibition of A549 cell growth was reversed by the antibodies, which also neutralized the growth inhibitory effects of TGF.beta. on AKR-2B cells in a monolayer [3H]thymidine incorporation assay as demonstrated by prevention of TGF.beta. inhibition of insulin and epidermal growth factor-stimulated DNA synthesis. The antibodies also effectively inhibited spontaneous soft agar growth of AKR-MCA fibroblasts, providing evidence for autocrine secretion of TGF.beta. as a mechanism of their anchorage-independent growth.