EGF receptors in R3230AC rat mammary carcinomas: characteristics and regulation in vitro and in vivo.

Epidermal growth factor (EGF), at 10(-11) M and 10(-10) M, stimulated [methyl-3H]thymidine incorporation into DNA and cell growth of R3230AC mammary adenocarcinomas in primary cultures, whereas at higher concentrations (10(-9) M and 10(-8) M) EGF inhibited DNA synthesis and cell growth in vitro. To determine whether these responses were receptor-mediated, 125I-EGF binding to freshly dissociated cells and primary cultures of R3230AC cells was measured and found to be time- and temperature-dependent. Specificity of EGF binding was demonstrated by 50% displacement occurring at an EGF concentration of 0.46 nM. Using 125I-EGF concentrations from 0.05 nM to 10 nM, saturable binding sites were documented; Scatchard analysis of these data produced curvilinear plots, suggesting the presence of high affinity (0.44-0.93 nM) and low affinity (1.5-4.8 nM) sites. 125I-EGF was rapidly internalized by cultured R3230AC tumor cells. By 30 min, 73% (25 degrees C) and 77% (37 degrees C) of total 125I-EGF was internalized (resistance to acid/salt extraction), whereas at 4 degrees C, cells internalized only 20% of EGF over a 3 hr incubation period. Following incubation with 125I-EGF for 2 hr at 4 degrees C, 25 degrees C, or 37 degrees C, the majority of cell-associated radioactivity eluted with intact 125I-EGF. However, when the material that dissociated from R3230AC cells after the 2 hr incubation was analyzed, 38% (25 degrees C) and 46% (37 degrees C) of the radioactivity migrated as lower molecular weight products, indicating that 125I-EGF was partially degraded intracellularly by R3230AC cells in primary culture. Pre-incubation of cells in primary culture with EGF (1-100 nM) for 30 min at 37 degrees C, led to "down-regulation" of EGF receptors; 1 nM EGF reduced the specific binding of 125I-EGF by 54% with higher concentrations (10 nM and 100 nM) reducing it further. Scatchard analysis of down-regulated cells showed a reduced number of high affinity binding sites with no change in the Kd of binding. Sialoadenectomy of rats had no effect on R3230AC tumor growth or EGF receptor levels in the tumor, liver, or uterus. Experiments to determine whether perturbations of the insulin milieu or ovariectomy would alter EGF receptors were performed. 125I-EGF binding was significantly elevated in tumors from diabetic rats (152% increase vs controls) and binding was returned to control (77% of intact rats) levels after administration of insulin to diabetic rats.(ABSTRACT TRUNCATED AT 400 WORDS)