Immunodetection and modulation of cellular growth with antibodies against native transforming growth factor-beta 1

Abstract

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.