Activation of the cAMP signaling pathway increases apoptosis in human beta-precursor cells and is associated with downregulation of Mcl-1 expression

During B- and T-cell ontogeny, extensive apoptosis occurs at distinct stage s of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10(+) B-precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) i ncreases apoptosis in the CD10+ cells in a dose-dependent manner (19%-94% w ith 0-1,000 mu M forskolin after 48 hours incubation, IC50 = 150 mu M). Hig h levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8-chlorophenylthio-cAMP (8-CPT-cAMP). Specific involvement of cAMP -dependent protein kinase (PKA) was demonstrated by the ability of a cAMP a ntagonist, Rp-isomer of 8-bromoadenosine- 3',. 5'-monophosphorothioate (Rp- 8-Br-cAMPS), to reverse the apoptosis Increasing effect of the complementar y cAMP agonist, Sp-8-Br-cAMPS. Furthermore, we investigated the expression of Bcl-2 family proteins. We found that treatment of the cells with forskol in or 8-CPT-cAMP for 48 hours resulted in a fourfold decline in the express ion of Mcl-1 (n = 6, P = 0.002) compared to control cells. The expression o f Bcl-2, Bcl-x(L), or Bar was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in re sponse to 8-CPT-cAMP (1.3-fold, n = 6, P = 0.045) compared to B-precursor c ells, and a smaller decrease in Mcl-1 levels (1.5-fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulatio n of apoptosis in B-progenitor and mature B cells and suggest that cAMP-inc reased apoptosis could be mediated, at least in part, by a decrease in Mcl- 1 levels. (C) 1999 Wiley-Liss, Inc.