Densities of phosphorylated Bad (P) and total Bad (T) are given at the top of the Western blot panels, calculated as described in Section Materials and Methods. Data shown represents one of at least two impartial experiments with comparable results. of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, including those resistant to glucocorticoids, can be induced to undergo apoptosis by stimulating the cAMP signaling pathway, with enhancement by glucocorticoids, and the mechanism by which this occurs may be related to changes in Bim and Bad expression, and in all cases, to activation of Bad. model systems have suggested that it may be associated with a decrease in Ceftizoxime the expression or alteration of the glucocorticoid receptor (GR), such that the actions normally carried Mouse monoclonal to CIB1 out by the GR that lead to therapeutic benefit are muted (Moalli and Rosen, 1994; Gaynon and Carrel, 1999; Schmidt et al., 2004, 2006; Ploner et al., 2005); however, at least one study finds no correlation with GR expression or function, but finds instead a correlation with the profound attenuation of the induction of Ceftizoxime the BH3-only pro-apoptotic protein, Bim (Bachmann et al., 2005). Studies using acute lymphocytic leukemic (ALL) cells obtained from patients, as well as 15 T-ALL cell lines produced directly from patients cells without prior drug exposure in culture, also indicated that resistance could not be attributed to mutations in GR or variations in levels of its expression (Tissing et al., 2006; Bachmann et al., 2007; Beesley et al., 2009). We found that stimulation of the cAMP signaling pathway can overcome glucocorticoid resistance in chronic lymphocytic leukemia (CLL) cells, and in the ALL cell line, CCRF-CEM (Tiwari et al., 2005; Lerner and Epstein, 2006; Dong et al., 2010). The mechanism by which this synergistic effect between stimulation of the cAMP and glucocorticoid signaling pathways occurs, to induce apoptosis of glucocorticoid resistant cells, is usually, however, still not fully understood. The purpose of this study is usually to examine the mechanism(s) by which cAMP and glucocorticoid signaling synergize to induce apoptosis of leukemic and multiple myeloma cells. With respect to leukemia, it appears that the BH3-only pro-apoptotic proteins, Bim and Bad, may be very crucial regulators of apoptosis of these cells. In a DNA microarray analysis to uncover genes important in glucocorticoid-induced apoptosis of leukemic cells, Bim was identified as one of the proteins whose expression was most upregulated (Wang et al., 2003). Additionally, studies with mice made deficient for the production of Bim indicate that Bim plays a key role in mediating apoptosis of B and T lymphocytes (Hildeman et al., 2002; Mouhamad et al., 2004). And silencing of the Bim gene with RNAi inhibits glucocorticoid-induced apoptosis of leukemic cells (Abrams et al., 2004). Bim exists as three alternate spliced forms, a short form, BimS, a long form, BimL, and an extra long form, BimEL. Both the turnover and activation of BimEL have been shown to be regulated Ceftizoxime by its phosphorylation by the MAP Kinases, ERK 1/2 (Ley et al., 2004) and JNK (Putcha et al., 2003). Additionally, studies have shown that the expression of Bim at the gene level is under direct control of the Forkhead transcription factor, FOXO (FKHR; Dijkers et al., 2000). FOXO itself can be phosphorylated and inhibited by the growth promoting kinase PKB/Akt (Burgering and Medema, 2003). PKB/Akt was shown to be inhibited in lymphoma cells by stimulating the cAMP pathway with phosphodiesterase4 (PDE4) inhibitors (Smith et al., 2005), and a similar effect was also seen in mouse embryo fibroblasts (Kuiperij et al., 2005). Hence, stimulating the cAMP pathway and inhibiting PKB/Akt, would be expected to disinhibit FOXO and drive the expression of Bim. And indeed, it was shown that stimulation of the cAMP and glucocorticoid pathways in mouse S49 lymphoma and human CCRF-CEM leukemia cells resulted in a synergistic increase in the expression of Bim (Zhang and Insel,.