An Agent or Agents Produced by Virus-transformed Cells Cause Unregulated Ruffling in Untransformed Cells

K N R K cells (a normal rat kidney [NRK] cell line transformed by Kirsten murine sarcoma vires) in sparse culture exhibit a highly ruffled morphology, but the cause of this ruffling is unknown. In this study, we have demonstrated that the continuous, excess ruffling on K N R K cells is caused by one or more soluble agents secreted by the K N R K cells themselves. To do this study, an assay for ruffling responses in live cell cultures was defined, and its reproducibility was demonstrated. This assay permitted observation of the kinetics of ruffling responses (percentage of cells ruffled as a function of time after stimulation). This method was used to compare the kinetics of ruffling induced by insulin, epidermal growth factor, fibroblast growth factor, glucose, and K N R K cell conditioned medium (CM). Ruffling was elicited on N R K cells by each of the polypeptide mitogens and nutrients, but, in each case, this ruffling subsided spontaneously within an hour. CM from K N R K cells also caused ruffling movements on untransformed N R K cells, but this ruffling continued for at least 20 h. This response was largely blocked by premixing the K N R K cell CM with rabbit IgG against rat transforming growth factor, type a, (TGF-a). K N R K cells made quiescent (ruffle free) by a pH shift (from 7.4 to 8.4) responded to insulin, glucose, and K N R K cell CM with kinetics similar to those observed for each of these factors in N R K cells. The unusual feature for the ruffle-inducing agent(s) produced by K N R K cells was that this activity was not subject, in either N R K or K N R K cells, to the cellular off-regulation that limits the responses to insulin or glucose. Thus, the continuous ruffling of K N R K cells is caused by their own unregulated ruffleinducing agent or agents, which appear to include TGF-a. This work also demonstrates that kinetic analysis of cellular responses to exogenous factors can provide new insights into the regulatory mechanisms involved in the normal limitation of these responses. UFFLING on the free cell margins of sparsely cultured ceils can be elicited by various hormone-like growth factors including epidermal growth factor (EGF) 1 (7, 8, 14, 16), nerve growth factor (8), platelet-derived growth factor (28), and even fresh serum-containing medium. With each factor, ruffling appears to subside spontaneously in < 1 h at 37"C, indicating cellular off-regulating mechanisms. Kinetic analysis of these responses is limited by most methods used to observe ruffling. Scanning electron microscopy provides excellent detail in the ruffles themselves but does not permit repeated observations on the same set of cells over an experimental time course and is too cumbersome to confirm repeatedly kinetic observations under numerous experimental conditions. An alternative technique, time-lapse cinematography, permits elegant visualization of ruffling activities over time on the same cells, but the number of ceils that can be observed at the required high magnification is too limited to permit extensive experimentation. The most useful approach to the analysis of ruffling kinetics is to observe living cells directly with the aid of inverted phase microscopy. Goshima ~ Abbreviations used in this paper: EGF, epidermal growth factor; KNRK, Kirsten murine sarcoma virus-transformed NRK; NKR, normal rat kidney; TGF, transforming growth factor; TRA, tumor ruffling agent. et al. (13) used this direct method to observe the ruffling induced by insulin treatment of KB cells. By repeatedly counting the percentage of ruffled cells in the same specimen over a period of time after the addition of insulin, these authors demonstrated the transient kinetics of insulin-induced ruffling at several concentrations and at several temperatures. They also observed that glucose caused a small ruffling response, but the duration of that response was not reported. Increased and continuous ruffling is a frequently observed correlate of malignant transformation (1, 19, 21). Neither the molecular mechanism underlying this alteration nor the significance of it is known. In particular, it is not known how or if the ruffling of transformed cells relates to the transient mitogen-induced ruffling of normal cells. In the present study, we compared ruffling responses in a normal rat kidney cell line (NRK) and a line of Kirsten murine sarcoma virustransformed cells (KNRK cells). Our objective was to determine the cause of the apparently unregulated (not transient) ruffling observed in the transformed line. Several polypeptide mitogens (insulin, EGF, and fibroblast growth factor) and low molecular weight nutrients (glucose and vitamins) were tested for ruffle-inducing capacity. The following factors were studied extensively: Insulin was chosen as a representative poly© The Rockefeller University Press, 0021-9525/86/04/1224/06 $1.00 The Journal of CeU Biology, Volume 102, April 1986 1224-1229 1224 on A uust 7, 2017 jcb.rress.org D ow nladed fom peptide mitogen because several other early insulin effects, including increased hexose (9, 12, 17) and amino acid transport (t 3), have been described. Glucose was chosen because it is a simple, chargeless nutrient for which membrane transport systems have been extensively studied, transport is augmented after insulin stimulation or as a component of the transformed phenotype (10, 15, 32), and ruffle-inducing capacity has been reported (l 3). In addition, we tested conditioned buffer from both transformed and untransformed cells. We considered and tested four independent hypotheses which together appear to exhaust the potential alternative explanations for this phenomenon: (a) Glucose (or nutrient)-induced ruffling is not subject to off-regulation as is the ruffling induced by peptide mitogens, and KNRK cells have an enhanced glucose ruffling response. (b) KNRK cells no longer require any stimulation to ruffle. (c) K N R K cells have lost the cellular off-regulatory control of responses to exogenous factors. (d) KNRK cells produce their own ruffle-inducing factor or factors that are not off-regulated. It was important to be able to observe, at brief intervals, the individual effects of a series of treatments and to monitor both the onset and the cessation of ruffling within a specific specimen. To do that, we used the direct observation method of Goshima et al. (13), first testing to ensure that our observation methods gave valid and reproducible results. Materials and Methods NRK ( 11 ) and KNRK (25) cells were cultured in sparse monolayers ( 10% fetal bovine serum, Waymouth's medium MB752/1, 37*C, 5% COz atmosphere). On day 2 after passage, complete medium was replaced with nutrient deficient Waymouth's medium, made without glucose, amino acids, and vitamins (i.e., a buffer with the same salt composition as Waymouth's medium BM752/1). Complete Waymouth's medium with or without fetal bovine serum was used in indicated experiments. After 2--4 h, cells were examined by inverted phase microscopy using a 25X objective lens for total magnification of-250. Ruffling was quantified by rapidly scanning visually several areas within each culture dish, counting numbers of cells with and without marginal ruffles, and recording the percentage of cells ruffled. Approximately 100 cells were counted for each data point. A cell was considered ruffled if more than ~2-3% (by visual estimate) of the free margin was ruffling. Ruffling was scored just before and at brief intervals after addition of the ruffling agent. For each type of agent, specificity of ruffling was tested by the addition of an equal volume of blank carrier with the same mixing techniques. This ensured that mechanical factors or slight changes in pH or salt concentration had not caused the observed ruffling. Insulin was from Sigma Chemical Co., St. Louis, MO (bovine pancreas crystalline, 1-5500), epidermal growth factor and fibroblast growth factor were from Collaborative Research Inc., Lexington, MA); vitamins were a mixture of the vitamins in Waymouth's medium MB752/1 as described by Gibeo (Grand Island, NY). Antiserum to TGF-a was a gift from Dr. Daniel R. Twardzik (Oncogen, Seattle. WA) (18).