Co-induction of Mad1 and c-Myc in activated normal B lymphocytes

The purpose of the present study was to examine the expression of the Myc network proteins c-Myc, Mad1 and Max in normal cells under different growth and differentiation conditions. A dominant view has been that Mad1 as a c-Myc antagonist plays a role in growth inhibition linked to differentiation. Of particular interest to us was therefore to study the regulation of Mad1 in cells undergoing differentiation in the absence of growth cessation. To do so we utilized normal B lymphocytes isolated from peripheral blood. The cells were induced to concomitant proliferation and differentiation by stimulation with a combination of anti-IgM antibodies (anti-mu) and the phorbol ester TPA. Thus, by 72 h of stimulation the percentage of plasmablasts increased from 3 to 17%, and the percentage of lymphocytes decreased from 89 to 27%. The most intriguing observation we made using this cell system was a pronounced coinduction of Mad1 and c-Myc. The levels of c-Myc and Mad1 mRNAs and proteins increased within 3 h of anti-mu stimulation, and the levels were further enhanced by TPA. Furthermore, the expressions of both c-Myc and Mad1 were reduced by forskolin, which also inhibited the anti-mu + TPA driven growth and differentiation of the B lymphocytes. The level of Max remained virtually unchanged. Taken together, our results indicate that a high level of Mad1 in normal human B cells is linked to differentiation and not to growth inhibition. Furthermore, the results demonstrate that Mad1 and c-Myc are not necessarily expressed in a reciprocal manner, which underlines an independent role of Mad1 unrelated to its function as a c-Myc antagonist.