Cell, Vol . 9, 3 75-381, November 1976, Copyright©1976 by MIT
Induction by Ouabain of Hemoglobin Synthesis in Cultured Friend Erythroleukemic Cells
Alan Bernstein, D . Margaret Hunt, Valerie Crichley, and Tak W . Mak The Ontario Cancer Institute, and the Department of Medical Biophysics University of Toronto 500 Sherbourne Street Toronto, Ontario M4X 1 K9, Canada
Summary Induction of erythroid differentiation in ouabainresistant murine erythroleukemia cells by ouabain is reported . Ouabain induction results in the appearance of hemoglobin-containing cells 12-24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide . The concentration of ouabain required to induce hemoglobin synthesis depends upon the K+ ion levels in the culture medium . Lowering the extracellular K+ ion concentration 2-4 fold reduced by 10-40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis . In low K+ medium (1 .8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0 .02 mM . This K+ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K+ ions in the culture medium . These results suggest that the binding of ouabain to the plasma membrane enzyme, Na/K ATPase, is required for the induction of erythroid differentiation by ouabain . A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells . The observation that the binding of ouabain to the Na/K ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K+ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells . Introduction Cultured cell lines (Friend cells), derived from the spleens of Friend virus-infected mice, can be induced in vitro to synthesize hemoglobin (Friend et al ., 1971) and to undergo a number of additional cellular changes that strikingly parallel normal erythropoiesis in vivo (Friend et al ., 1971 ; Ross, Ikawa, and Leder, 1972 ; Ostertag et al ., 1972 ; Boyer et al ., 1972 ; Reem and Friend, 1975 ; Sassa, 1976) . Induction of this differentiation process can be initiated by dimethyl sulfoxide (DMSO) (Friend et al .,
1971), as well as a wide variety of other organic solvents (Preisler and Lyman, 1975 ; Tanaka et al ., 1975 ; Preisler, Christoff, and Taylor, 1976 ; Reuben et al ., 1976) . In addition, Friend cells can be induced by the fatty acid, butyric acid (Leder and Leder, 1975), and several purines and purine analogues (Gusella and Housman, 1976) . The mechanism(s) by which these inducers initiate, and possibly maintain, hemoglobin synthesis in these cells is not known . Based on the relative solubility of different inducers in an oil/water mixture, and the observation that local anesthetics, such as procaine, inhibit the stimulation of heme synthesis in these cells, we have previously suggested that a primary event in this induction process is the interaction of the inducing compound with the plasma membrane (Bernstein et al ., 1976) . We now report that ouabain, a cardiac glycoside that binds specifically to the plasma membrane enzyme, Na+_K+_Mg++-activated ATPase (EC 3 .6 .1 .3, hereafter abbreviated Na/K ATPase), is a good inducer of wild-type, ouabain-sensitive Friend cells, and a potent inducer of erythroid differentiation in Friend cell mutants that have been selected for resistance to the cytotoxic action of ouabain . Results Isolation and Properties of Ouabain-Resistant Friend Cells Ouabain-resistant (OUAr) mutants were isolated by plating cultures, mutagenized as described in Experimental Procedures, in 1 .5 mM ouabain, a concentration of ouabain that reduced survival to
Induction by Ouabain of Hemoglobin Synthesis in Cultured Friend Erythroleukemic Cells
Alan Bernstein, D . Margaret Hunt, Valerie Crichley, and Tak W . Mak The Ontario Cancer Institute, and the Department of Medical Biophysics University of Toronto 500 Sherbourne Street Toronto, Ontario M4X 1 K9, Canada
Summary Induction of erythroid differentiation in ouabainresistant murine erythroleukemia cells by ouabain is reported . Ouabain induction results in the appearance of hemoglobin-containing cells 12-24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide . The concentration of ouabain required to induce hemoglobin synthesis depends upon the K+ ion levels in the culture medium . Lowering the extracellular K+ ion concentration 2-4 fold reduced by 10-40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis . In low K+ medium (1 .8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0 .02 mM . This K+ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K+ ions in the culture medium . These results suggest that the binding of ouabain to the plasma membrane enzyme, Na/K ATPase, is required for the induction of erythroid differentiation by ouabain . A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells . The observation that the binding of ouabain to the Na/K ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K+ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells . Introduction Cultured cell lines (Friend cells), derived from the spleens of Friend virus-infected mice, can be induced in vitro to synthesize hemoglobin (Friend et al ., 1971) and to undergo a number of additional cellular changes that strikingly parallel normal erythropoiesis in vivo (Friend et al ., 1971 ; Ross, Ikawa, and Leder, 1972 ; Ostertag et al ., 1972 ; Boyer et al ., 1972 ; Reem and Friend, 1975 ; Sassa, 1976) . Induction of this differentiation process can be initiated by dimethyl sulfoxide (DMSO) (Friend et al .,
1971), as well as a wide variety of other organic solvents (Preisler and Lyman, 1975 ; Tanaka et al ., 1975 ; Preisler, Christoff, and Taylor, 1976 ; Reuben et al ., 1976) . In addition, Friend cells can be induced by the fatty acid, butyric acid (Leder and Leder, 1975), and several purines and purine analogues (Gusella and Housman, 1976) . The mechanism(s) by which these inducers initiate, and possibly maintain, hemoglobin synthesis in these cells is not known . Based on the relative solubility of different inducers in an oil/water mixture, and the observation that local anesthetics, such as procaine, inhibit the stimulation of heme synthesis in these cells, we have previously suggested that a primary event in this induction process is the interaction of the inducing compound with the plasma membrane (Bernstein et al ., 1976) . We now report that ouabain, a cardiac glycoside that binds specifically to the plasma membrane enzyme, Na+_K+_Mg++-activated ATPase (EC 3 .6 .1 .3, hereafter abbreviated Na/K ATPase), is a good inducer of wild-type, ouabain-sensitive Friend cells, and a potent inducer of erythroid differentiation in Friend cell mutants that have been selected for resistance to the cytotoxic action of ouabain . Results Isolation and Properties of Ouabain-Resistant Friend Cells Ouabain-resistant (OUAr) mutants were isolated by plating cultures, mutagenized as described in Experimental Procedures, in 1 .5 mM ouabain, a concentration of ouabain that reduced survival to
