Maturation and Differentiation of B16 Melanoma Cells Induced by Theophylline Treatment2

Abstract

Recent studies suggested that 3′5′-cyclic AMP (cAMP) may be involved in the regulation of cell proliferation and differentiation. Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, elevated intracellular cAMP. A melanotic clone of the 816 melanoma was treated with theophylline and studied in vitro and in vivo. With 12 hours after 1.0 mM theophylline was added to growing cultures, the number of cells incorporating tritiated thymidine (³H-TDR) and the rate of uptake of ³H-TDR into DNA were significantly reduced. After 7 days, the number of cells in the control cultures increased twenty-four times, whereas theophylline-treated cells increased only sixfold. Compared to the controls, the theophylline-treated cells contained ten times the melanin and an elevated cAMP content. Stimulation of melanogenesis and inhibition of proliferation increased progressively with the duration of exposure to theophylline. After 5 days of culture with theophylline, cells were assayed for plating efficiency in theophylline-free medium. Although the number of colony-forming cells was unaffected by previous exposure to theophylline, the colonies were composed of fewer cells, which indicated that reduced proliferative capacity persisted after theophylline removal. Theophylline-treated cells inoculated into syngeneic hosts were less tumorigenic than untreated cells. However, theophylline treatment of hosts bearing 816 tumors failed to reduce the tumor growth rate, and theophylline did not potentiate the growth inhibition resulting from treatment with the synthetic polyribonucleotide, polyinosinic-polycytidylic acid.