NIH 3T3 CELLS TRANSFECTED WITH A YEAST H-ATPASE HAVE ALTERED SENSITIVITY TO INSULIN, INSULIN GROWTH-FACTOR-I, AND PLATELET-DERIVED GROWTH FACTOR-AA()

The role of intracellular pH (pH(in)) in the regulation of cell growth in both normal and transformed cells is a topic of considerable contr oversy. In an effort to study this relationship NIH 3T3 cells were sta bly transfected with the gene for the yeast H+-ATPase, constitutively elevating their pH(in). The resulting cell line, RN1a, has a transform ed phenotype: The cells are serum independent for growth, clone in sof t agar, and form tumors in nude mice, In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum-independent growth, using defined med ia to investigate the effects of specific growth factors on the transf ected and parental NIH 3T3 cells. While both cell lines show similar g rowth increases in response to platelet-derived growth factor (PDGF)-B B and epidermal growth factor (ECF), they respond differently to insul in, insulin-like growth factor-I (ICF-I) and PDGF-AA. RN1a cells exhib it increased growth at nanomolar concentrations of insulin but the par ental cells had only a relatively minor response to insulin at 10 mu M . Both cell lines showed some response to IGF-I in the nanomolar range but the response of RN1a cells was much larger. Differences in insuli n and IGF-I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF-AA. RN1a cells are re latively insensitive to stimulation by PDGF-AA and express fewer PDGF alpha receptors as shown by Northern blots and receptor-binding studie s. We propose a unifying hypothesis in which the H+-ATPase activates a downstream element in the PDGF-AA signal transduction pathway that co mplements insulin and IGF-I signals, while leading to downregulation o f the PDGF alpha receptor. (C) 1994 Wiley-Liss, Inc.