Conditioned medium (CM) from a human mammary carcinoma cell line, MCF-7, and ten individual clones derived from these cells was examined for the presence of transforming growth factors (TGFs). Concentrated CM from all of the MCF-7 cell lines was found to stimulate the anchorage-independent growth of normal rat kidney cells in soft agar and to inhibit the binding of epidermal growth factor (EGF) to mouse NIH/3T3 fibroblasts and to A431 human epidermoid carcinoma cell membranes. The soft agar stimulating activity was heat stable but sensitive to treatment with dithiothreitol. EGF receptors were measured on the MCF-7 cell lines to determine whether the amount of TGFs associated with the CM from the various cell lines was correlated with the level of EGF receptors being expressed on these cells. Moreover, the intrinsic cloning efficiency of these lines in soft agar was measured to ascertain if any correlation might exist between the level of TGFs associated with these cells and the abil...

At least one member of the TGF-β family, TGF-β1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-β family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-β1 and TGF-β2 on the anchorage-independent growth of the estrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-β1 and TGF-β2 caused a 75–90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (< 100) MCF7 cells. T-47D cells responded to TGF-β only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (> 500) MCF7 cells was not inhibited by TGF-β1 or TGF-β2. TGF-β-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-β-related polypeptide. TGF-β1 and TGF-β2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-β effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-β. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-β is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-β effects in vivo.

Stromal-epithelial interactions modulate growth and development in normal and neoplastic mammary gland. The release of IGF binding proteins (IGFBPs) by the stromal compartment of the mammary gland may play a modulating role in the IGF-mediated proliferation of mammary epithelium. Therefore, the IGFBP-expression pattern of the canine mammary tumor cell line U335 (CMT-U335), which has a mesenchymal phenotype, was determined. In addition, the effects of IGFs and all trans retinoic acid (RA) on DNA synthesis, and IGFBP secretion and distribution were examined. The IGFBPs secreted by CMT-U335 were characterized as IGFBP-2, -4, -5, and -6. Moreover, CMT-U335 appeared to be a suitable mammary mesenchymal cell line for study of the regulatory factors of IGFBP expression and the mechanism(s) involved. IGFs and RA enhanced IGFBP concentrations in cell-conditioned medium with IGF-I and RA having an additive effect. The IGF-I-stimulated DNA synthesis, however, was inhibited by RA. The difference between IGF-I and RA was an enhanced IGFBP-5 binding to the extracellular matrix (ECM) by RA, whereas IGF-I reduced binding to the ECM. Because high doses of insulin had no significant effects on IGFBP concentrations in the medium, it is concluded that IGF-I-induced changes in IGFBP concentrations are not mediated by type-IIGF receptors and may be the consequence of IGFBP redistribution.