Abstract
The murine pre-B-lymphocyte cell line 70Z /3 may be induced to differentiate to a surface immunoglobulin-positive phenotype by the polyclonal B-cell mitogen, lipopolysaccharide. This is accomplished via activation of an amiloride-sensitive Na+-uptake system ( Rosoff , P. M., and Cantley , L. C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7547-7550). Here we show that the active tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) also induces surface IgM expression in 70Z /3 cells. TPA also appears to work by activating a plasma membrane Na+/H+ exchange system. A significant increase in cellular Na+ was detected within 10 min after TPA addition and by 2 h an 80% increase was observed. Amiloride blocked both the induction of surface IgM and the increase in cellular Na+. Further evidence that both TPA and lipopolysaccharide activate Na+/H+ exchange was provided by measurements of intracellular pH with carboxyfluorescein. Both TPA and lipopolysaccharide caused a 0.15 unit increase in cytoplasmic pH within 5 min after addition to the cells at 37 degrees C. The pH change required high extracellular Na+ and was inhibited by amiloride. These data suggest a mechanism by which phorbol esters affect cellular growth and differentiation.
Highlights
The murine pre-B-lymphocyte cell line 70Z/3 may lipid-dependent proteinkinase [7]
We show that the activetumor promoter 12-0-tetradecanoylphorbol 13-acetate (TPA) alsoinduces surface IgM expression in 70Z/3 cells.TPA appears to work by activating a plasma membrane Na+/H+exchange system
Phorbol Esters In.duce 70213 Cell Differentiation-When 70Z/3 cells were exposed to low concentrations of the potent tumor promoter TPA for 24 h and tested for the presence of surface IgM, 74% ofthe cells differentiated to the surfacIgeM positive phenotype (Table I)
Summary
LPS,TPA, 4a-phorbol 12,13-didecanoate, and amiloride were used a t 10 pg/ml, 50 nM, 50 nM, and 66 pM, respectively, and were present for 24h. Surface IgM positive cells were determined at the end of the 24-h incubation period by immunofluorescencemicroscopy as described under "Materials and Methods." At least 500 cells were examined in each experiment. Cell viability was greater than 90% as judged by exclusion of trypan blue in all experiments.
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