Epidermal growth factor (EGF)-nonresponsive variants of normal rat kidney cell line: Response to EGF and transforming growth factor-β☆

Abstract Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) (or TGF-α). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125 I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy- d -glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-β treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-β, whereas that of N-3 was significantly enhanced by TGF-β alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-β mRNA, but there appeared little expression of TGF-β mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-β enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.