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Effects of 8-Chloroadenosine 3',5'-Monophosphate and N⁶-Benzyl-Cyclic Adenosine 5'-Monophosphate on Cell Cycle Kinetics of HL-60 Leukemia Cells

Cellular Biochemistry Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute [S. P., G. T., Y. S. C-C.], and Center for Biologics Research and Evaluation, Division of Biochemistry and Biophysics, Food and Drug Administration [P. D. N., G. M., G. C. W.], National Institutes of Health, Bethesda, Maryland 20892

Site-selective cyclic AMP (cAMP) analogues have been shown to inhibit growth and induce differentiation in several human leukemia cell lines. However, detailed studies of the effects exerted by cAMP analogues on cell cycle kinetics have been lacking. We have examined the effects of 8-Cl-cAMP and N⁶-benzyl-cAMP on the cell cycle kinetics of the HL-60 human promyelocytic leukemia cell line. A cell cycle study was performed by univariate DNA analysis after 24–72 h of treatment with noncytotoxic concentrations of 8-Cl-cAMP and N⁶-benzyl-cAMP capable of inducing 50–60% growth inhibition in these cells. HL-60 cells treated with 5 µM 8-Cl-cAMP showed no significant change in the cell distribution in the cycle as compared to the untreated control cells, whereas the treatment with 10 µM N⁶-benzyl-cAMP transiently increased the percentage of cells in the G₀/G₁ phase after 48 h, followed by a partial recovery at 72 h. Combined treatment with low doses of 8-Cl-cAMP and N⁶-benzyl-cAMP, each of which alone produced 20% growth inhibition, exerted a growth inhibitory effect of 65% and delayed increase of the G₀/G₁ phase by 72 h. To better understand the cell cycle effects induced by 8-Cl-cAMP, flow cytometric analysis of bromodeoxyuridine incorporation was also performed. 8-Cl-cAMP treatment exhibited a slowing down of the cell cycle; thus, the delayed appearance of the G₀/G₁ cell accumulation after combined treatment could be due to this effect of 8-Cl-cAMP on the HL-60 cell cycle. At a toxic dose, 8-Cl-cAMP brought about a G₂M block, whereas N⁶-benzyl-cAMP brought about an increase of the G₀/G₁ compartment. G₂M block produced by toxic doses of 8-Cl-cAMP was not related to its adenosine metabolite since 8-Cl-adenosine did not produce any specific block in the cell cycle. Our results show, for the first time, that these site-selective cAMP analogues could affect cell cycle kinetics at different points. These data may provide the basis for combination treatments involving cAMP analogues and other agents in the treatment of human leukemia.

¹ Present address: Cattedra di Oncologia Medica, Via S. Pansini 5, 80131 Naples, Italy.

² To whom requests for reprints should be addressed, at National Cancer Institute, Building 10, Room 5B38, Bethesda, MD 20892.

Received 6/21/91. Accepted 9/24/91.