Dimethylsulfoxide (DMSO) converts almost all of the undifferentiated murine erythroleukemia cells (MEL or Friend cells, clone 745A) in a culture to differentiated cells that contain high levels of hemoglobin and that stop growing after a limited number of cell divisions. Contrary to other reports-that amiloride strongly inhibits DMSO-induced differentiation in MEL cells-in this laboratory, inhibition by amiloride, tested with DMSO over a range of concentrations in two kinds of media and at various cell densities, was found to be only weak or absent. Similarly, amiloride did not inhibit induction by N,N'hexamethylene bis-acetarnide (HMBA). As expectzd from previous findings with other cell systems, amiloride inhibited protein synthesis and cell multiplication.
Since the original report that DMSO can induce hemoglobin production and terminal differentiation in MEL cells (Friend et al., 19711, many other inducers have been found, including the potent agent HMBA and its congeners (Fibach et al., 1977). A number of agents that antagonize the action of inducers have also been described (Marks et al., 1982;Abrahm and Rovera, 1981). Reports that amiloride, an inhibitor of Nat transport in epithelial tissues (Benos, 19821, prevents the inducer effect of DMSO, were particularly i n t r i y n g , and led to a proposal that Na+ entry and Na+/Ca + exchange are involved in MEL differentiation (Levenson et al., 1980;1982; 1983;Smith et al., 1982).
Because of current interest in the possible role of ions in cell growth and differentiation, in the present study, the effect of amiloride on MEL cells was reexamined.
MATERIALS AND METHODS
Cell cultures
MEL cells (clone 745A) originated in the laboratory of Dr Charlotte Friend and were thawed from frozen stocks at intervals. Cells were grown in media obtained from GIBCO (Grand Island, NY): either MEM alpha medium without nucleosides and with 15% Reheis or Hyclone fetal bovine serum (FBS) or Dulbecco's MEM (DMEM) with 10% FBS. Both media contained penicillin-streptomycin-neomycin (50-50-100 pg/ml) antibiotics (GIBCO) in COZ-air adjusted to produce pH 7.2-7.4 in the medium (slightly different levels of COZ were needed for each medium) at 37Β°C in 6 cm Nunc plastic dishes. Growth in DMEM was about 25% faster than in MEM alpha medium. For all experiments, cells were taken from exponentially growing cultures. Subclones of clone 745A were obtained from suspensions in medium containing 1.3% Methocel.