Redox regulation of cAMP levels by ascorbate in 1,25-dihydroxy-vitamin D3-induced differentiation of HL-60

1 alpha,25-Dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] induces differentiation to monocyte-macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells ia culture. We have previously reported the stimulating effect of ascorbate on 1,25-(OH)(2)D-3-induced HL-60 cell differentiation. We show here that 1,25-(OH)(2)D-3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E-2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1,25-(OH)(2)D-3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)(2)D-3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)(2)D-3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1,25-(OH)(2)D-3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1,25-(OH)(2)D-3-induced differentiation of HL-60 cells can be explained by redox regulation of the cAMP pathway.