Induction of megakaryocytic differentiation and modulation of protein kinase gene expression by site-selective cAMP analogs in K-562 human leukemic cells

Two classes (site 1- and site 2-selective) of cAMP analogs, which either alone or in combination demonstrate a preference for binding to type II rather than type I cAMP-dependent protein kinase isozyme, potently inhibit growth in a spectrum of human cancer cell lines in culture. Treatment of K-562 human leukemic cells for 3 days with 30 and 10 μM 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP) (site 1-selective) resulted in 60% and 20% growth inhibition, respectively (with over 90% viability). N-Benzyl-cAMP (site 2-selective) (30 μM) treatment resulted in 20% growth inhibition by day 3. When 8-Cl-cAMP (10 μM) and N-benzyl-cAMP (30 μM) were both added, growth was almost completely arrested. The growth inhibition was accompanied by megakarocytic differentiation in K-562 cells. The untreated control cells expressed little or no detectable levels of glycoprotein II(b)-III(a) surface antigen complex. 8-Cl-cAMP (30 μM) treatment for 3 days substantially increased the antigen expression, while N-benzyl-cAMP caused little or no change in the antigen expression. When cells were treated with 8-Cl-cAMP in combination with N-benzyl-cAMP, antigen expression was synergistically enhanced, and cells demonstrated megakaryocyte morphology. By Northern blotting, we examined the mRNA levels of the type I and type II protein kinase regulatory subunits (RI(α) and RII(β)), the catalytic subunit, and c-myc during 8-Cl-cAMP treatment. The steady-state level of RII(β) cAMP receptor mRNA sharply increased within 1 hr of treatment and remained elevated for 3 days, while that of the RI(α) receptor markedly decreased to below control level within 6 hr and remained low during treatment. However, 8-Cl-cAMP did not affect the mRNA level of the catalytic subunit. 8-Cl-cAMP treatment also brought about a rapid decrease in c-myc mRNA. Thus, differential regulation of cAMP receptor genes is an early event in cAMP-induced differentiation and growth control of K-562 leukemia cells.