The time-course of cyclic AMP signaling is critical for leukemia U-937 cell differentiation,☆☆

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Abstract

The regulation of the cAMP signaling is intimately involved in several cellular processes, including cell differentiation. Here, we provide strong evidence supporting that the time-course of cAMP signal is critical for leukemia U-937 cell differentiation. Three stimulating-cAMP agents were used to analyze the correlation between cAMP time-course and cell differentiation. All three agents denoted similar cAMP maximal responses in dose–response experiments. The kinetic of desensitization showed differential characteristics, while H2 receptor desensitized homologously without affecting PGE2 or forskolin effect, PGE2 response showed mixed desensitization characterized by a homologous initial phase followed by a heterologous phase. Regarding forskolin, long-term stimuli attenuated PGE2 and H2 agonist response without affecting adenylyl cyclase activity. In the absence of phosphodiesterase inhibitors, the three agents induced similar maximal cAMP levels after 5 min, but only that induced by the H2 agonist returned to basal levels. Consistent with this observation, H2 agonist was not able to induce U-937 cell maturation in contrast to PGE2 and forskolin, supporting the importance of time-course signaling in the determination of cell behavior.

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Materials and methods

Materials. Cell culture medium, isobutylmethylxanthine (IBMX), cAMP, dbcAMP, forskolin, prostaglandin E2 (PGE2), Fura 2 acetoxymethyl ester (Fura 2-AM), bovine serum albumin (BSA), and rhC5a were obtained from Sigma Chemical Company (St. Louis, MO). Fetal calf serum was purchased from Natocor (Argentina). Amthamine and 6[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane-carboxamide (HTMT dimaleate) were from Tocris Cookson (Ballwin, MO). H-7 was obtained from Calbiochem (La Jolla,

Cyclic AMP elevation in response to different agents in U-937 cells

Cyclic AMP levels were induced in U-937 cells by amthamine and PGE2, two agonists that stimulate G protein-coupled receptors (GPCR), histamine H2 receptor, and PGE2 receptors, respectively. Forskolin, a direct adenylyl cyclase-activating agent, was also used to increase cAMP levels. All agents elevated cAMP levels in a concentration-dependent manner, with EC50 values of 465 ± 95 nM (n=5), 39 ± 6 nM (n=3), and 40 ± 7 μM (n=3) for amthamine, PGE2, and forskolin, respectively. These agents evoked similar

Discussion

The formation of the different blood cells is essential for the development of a normal individual. New blood cells belonging to different cell lineages are formed from stem cells not only during embryogenesis but also during the adult lifetime. Abnormalities in the normal developmental program of blood cell formation result in various types of hematological diseases, including leukemia which involves an uncoupling of the regulatory mechanisms that normally integrate cell proliferation with

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    This study was supported by grants from the Universidad Buenos Aires (Grant B022) and Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (PICT 9805-03514).

    ☆☆

    Abbreviations: APL, acute promyelocytic leukemia; cAMP, cyclic adenosine monophosphate; dbcAMP, dibutyryl cAMP; PDE, phosphodiesterase; GRK, G protein-coupled receptor kinase; GPCR, G protein-coupled receptor; IBMX, isobutylmethylxanthine; PGE2, prostaglandin E2; Fura 2-AM, Fura 2 acetoxymethyl ester; BSA, bovine serum albumin; rhC5a, recombinant human C5a; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.

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