Multidrug resistance proteins (MRPs) and cAMP efflux: new potential targets for leukemia differentiation therapy

COPSEL SABRINA; GARCIA CORINA; SHAYO CARINA; DAVIO CARLOS

Fundacion Pablo Cassara, Buenos Aires

Simposio; International Symposium on Drug Transport and Metabolism; 2009

Over the last decades the concept of differentiation therapy, whereby immature cells are stimulated to develop into their mature phenotype, aroused considerable interest. Many efforts join to evaluate new differentiation agents for the treatment of leukemia where early hematopoietic progenitors are thought to exhibit maturation arrest. The best evidence of this therapy success has been the treatment of acute promyelocytic leukemia (APL) with the all-trans retinoic acid or with arsenic trioxide. In addition, agents that increase cyclic AMP (cAMP)-mediated signaling, as cyclic nucleotide phosphodiesterases (PDEs) inhibitors, augment the ability of these therapies to induce differentiation in APL blast cells1. cAMP, one of the most common second messengers, plays an important role in response to hormonal signals for cell proliferation, differentiation, and apoptosis in hematopoietic development. Several mechanisms are involved in the regulation of cAMP levels including its degradation by PDEs and the modulation of G protein-coupled receptor (GPCR) mediated production. The human promonocytic leukemic U937 cell line is able to differentiate into monocyte loosing their malignant capacity. Thus, these cells are an appropriated model to study hematopoietic cell differentiation mechanisms. Previous reports from our laboratory established an important correlation between duration and intensity of cAMP signaling induced by different agents, and evoked cellular responses such as proliferation and differentiation in U937 cells2,3,4.   As cAMP levels are critical for leukemic cell differentiation and the capability of MRP4, MRP5 and MRP8 to transport cAMP out of the cells5, the aim of the present work was to characterize cAMP efflux mechanism in U937 cell line and to investigate the role of MRPs on the differentiation process. Time course cAMP accumulation studies using agents that increased cAMP levels such as amthamine (H2 receptor agonist), isoproterenol (beta-adrenergic receptor agonist), or forskolin (an adenylyl cyclase activator), showed that there is cAMP efflux and it is independent of the stimuli used. Furthermore, we observed that this process is dependent on both, time post-stimuli and cAMP intracellular concentration. After confirming MRP4, MRP5 and MRP8 expression in U937 cells, we investigated their relevance in cAMP efflux process using MRPs´ specific inhibitors (probenecid, verapamil and MK-571). Thus, MRPs´ inhibition significantly increased cAMP intracellular levels and inhibited cell proliferation by 90%. This increase in cAMP levels induced the expression of CD11b, CD88 and augmented the chemotactic response to complement 5a (C5a), all markers of monocytic differentiation.