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Immunodetection and Modulation of Cellular Growth with Antibodies against Native Transforming Growth Factor-ß¹

Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

In an attempt to identify and quantitate latent and active forms of transforming growth factor ß (TGFß) without the use of cell cultures and to test for autocrine stimulation by TGFß, rabbit antibodies were raised against native human and porcine platelet-derived TGFß. A radio-immunoassay for TGFß was developed using radioiodinated TGFß, anti-TGFß antibodies, and protein A. Inhibition in the radioimmunoassay was achieved with nanogram quantities of TGFß, comparable to the sensitivity of radioreceptor assays. Analyses of the TGFß levels of conditioned medium from cultured cells indicated that the latent form(s) of TGFß is not detectable in the radioimmunoassay established using antibodies raised against native TGFß. Immunoprecipitation analysis of radiolabeled conditioned medium revealed a specific M_r 25,000 band only after acidification. A M_r 62,000 protein was observed with and without prior acidification of the medium but could not be competed with unlabeled TGFß in the immunoprecipitation indicating antigenic unrelatedness. The anti-TGFß IgG inhibited the binding of [¹²⁵I]TGFß to the cell surface receptors in a radioreceptor assay. TGFß inhibition of A549 cell growth was reversed by the antibodies, which also neutralized the growth inhibitory effects of TGFß on AKR-2B cells in a monolayer [³H]thymidine incorporation assay as demonstrated by prevention of TGFß 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 TGFß as a mechanism of their anchorage-independent growth.

¹ Supported by Grant CA 42572 awarded by the National Cancer Institute, Department of Health and Human Services, and by a contract from R & D Systems, Minneapolis, MN.

² To whom requests for reprints should be addressed.

Received 4/ 6/87. Revised 8/28/87. Accepted 9/ 8/87.