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

Abstract

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 ,uM 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 IpM) treatment resulted in 20% growth inhibition by day 3. When 8-Cl-cAMP (10 pM) and uM) were both added, growth was almost completely arrested. The growth inhibition was accompanied by megakaryocytic differentiation in K-562 cells. The untreated control cells expressed little or no detectable levels of glycoprotein nwb-rn surface antigen complex. 8-Cl-cAMP (30 ,uM) treatment for 3 days substantially increased the antigen expression, while N6-benzyl-cAMP caused little or no change in the antigen expression. When cells were treated with 8-Cl-cAMP in combination with N6-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 I protein kinase regulatory subunits (RI. and Rllp), the catalytic subunit, and c-myc during 8-Cl-cAMP treatment. The steady-state level of RUp, cAMP receptor mRNA sharply increased within 1 hr of treatment and remained elevated for 3 days, while that of the RIU 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.A major defect ofcancer cells is their inability to differentiate normally (1-4). This defect may be partly reversible, and study of such models may help to elucidate the mechanism responsible for differentiation. The K-562 human leukemic cell line, which carries a Philadelphia chromosome and was established from a patient with chronic myelogenous leukemia in blast crisis (5), is such a model. This cell line is considered to consist of a multipotent hematopoietic stem cell, since it responds to inducers of erythroid differentiation (6-8), megakaryocytic differentiation (9, 10), and granulocytic differentiation (11,12). The cellular mechanisms responsible for these processes, however, are not known.cAMP, an intracellular regulatory agent, has long been considered to have a role in the control of cell proliferation and differentiation in a variety of cell types (13-16). cAMP exerts its effect in eukaryotes by binding to the regulatory subunits of either type I or type II cAMP-dependent protein kinase (17, 18). Differential expression ofthese protein kinase isozymes has been observed d...

Dual Anticancer Activity of 8-Cl-cAMP: Inhibition of Cell Proliferation and Induction of Apoptotic Cell Death

“…8-Cl-cAMP-treated cancer cells showed a decrease of RI␣ and type I PKA, an increase of RII␤ and type II PKA, and inhibition of cell growth (11)(12)(13). Especially, ras-transformed mouse fibroblast (DT) cells showed morphological differentiation and human leukemic cells showed differentiated characteristics upon the treatment with 8-Cl-cAMP (6,14). We also found that 8-Cl-cAMP induces apoptotic cell death in human cancer cells including SH-SY5Y human neu-roblastoma cells and HL60 human leukemic cells (unpublished results).…”

“…Suppression of type I PKA using antisense oligonucleotide against RI␣ mRNA successfully induced growth inhibition of cancer cells in vitro and in vivo (3,4). A group of site-selective cAMP analogues inhibited cell growth in a variety of cancer cells and induced differentiation in leukemic cells (5,6). In addition, introduction of RII␤ subunit of PKA, which resulted in the increase of type II PKA and the downregulation of type I PKA, suppressed growth and transformed phenotype of transformed or cancer cells (7)(8)(9).…”

Site‐selective 8‐C1‐cAMP which causes growth inhibition and differentiation increases DNA (CRE)‐binding activity in cancer cells

“…The cell lines used in this study were cancer cells, such as LS-174T (human colon carcinoma) 15,111, K-562 (chronic myelogenous human leukemia) [12,13], PC12 (rodent pheochromocytoma), and TMK-1 and MKN-1 (8-Cl-cAMPresponsive and -unresponsive human gastric cancers, respectively) [14], that were demonstrated previously to be responsive to treatment with site-selective CAMP analogs. The cells were grown under standard conditions and treated for l-3 days with 8Cl-CAMP at indicated concentrations as previously described [5,12].…”

“…Site-selective CAMP analogs also have induced differentiation in several leukemic cell lines [12]. The molecular events underlying these changes have not been elucidated but are suggested to involve specific factors that play a regulatory role in transcription [13].…”

“…The molecular events underlying these changes have not been elucidated but are suggested to involve specific factors that play a regulatory role in transcription [13]. In this study, we examined whether or not the regulatory role of site-selective CAMP analogs on the growth and differentiation of cancer cells would involve modulation of gene transcription.…”