A completely serum-free assay method has been used to compare the mitogenic activities of polypeptide growth factors and estrogens with MCF-7 and T47D human breast cancer cells in culture. The lines were maintained in a viable, slowly dividing condition in Ham's F12 and Dulbecco's modified Eagle's medium (1:1) supplemented with sodium bicarbonate (2.2 g/liter), 15 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, human transferrin (10 µg/ml), and bovine serum albumin (200 µg/ml) (designated Tf/BSA). This medium allowed the assay of mitogenic activities as measured by multiple rounds of cell division and permitted comparisons of the biological potencies of growth factors within functional families as well as of dissimilar mitogens. Insulin-like growth factor I (IGF-I) was the most potent mitogen studied, showing ED50 values of 160 pg/ml and 1.7 ng/ml with the MCF-7 and T47D cells, respectively. Insulin-like growth factor II and insulin were less active, with ED50 values of 0.55 and 1.2 ng/ml with MCF-7 cells and 4.3 and 10 ng/ml with the T47D cell line, respectively. Mitogens sharing epidermal growth factor-like functional properties had ED50 values from 35 pg/ml to 2.5 ng/ml, while transforming growth factor type β and platelet-derived growth factor had no detectable stimulatory effects. Basic fibroblast growth factor had ED50 values of 0.42 ng/ml and 3.7 ng/ml for the MCF-7 and T47D cells, respectively, while acidic fibroblast growth factor was nearly inactive. In phenol red-free Tf/BSA, 17β-estradiol caused a 60% increase in MCF-7 cell numbers over controls in 8 days while having no effect on growth of the T47D cell line. From MCF-7 conditioned Tf/BSA medium, IGF-I was identified by biological activity, by radioimmunoassay (≈2 pg/ml) and by estimation of molecular weight (8,000) under dissociating conditions. The concentration of IGF-I was not affected by 17β-estradiol treatment. The data indicate that induction of acid stable, low molecular weight autocrine growth factors involved more regulation than defined by estrogens alone. The minimal effects of 17β-estradiol in Tf/BSA opened several possibilities including the putative roles of other serum-borne hromones, growth factors and regulators in autocrine growth factor induction.
This work was supported by National Cancer Institute Grants CA-38024 and CA-26617, American Cancer Society Grant number BC-255 and Grant number 2225 from The Council for Tobacco Research, USA, Inc.