Monocytically differentiating HL60 cells proliferate rapidly before they mature

1alpha,25-Dihydroxyvitamin D(3) (D(3)) provokes growth arrest and monocytic differentiation in myeloid cells. Although it is usually assumed that the cellular events leading to growth arrest start within one cell cycle of D(3) addition, there is also evidence that D(3) provokes the expression of proliferation-related genes and accelerates cell division. Herein we clarify the relationship between proliferation and maturation in differentiating HL60 cells. Cells were cultured singly, D(3) was added at various stages of the cell cycle, the progeny were counted, and the proportions of mature monocytes were determined. Initially, the D(3)-treated cells proliferated at an accelerated rate, and they matured only later. If cells encountered D(3) early in G1 they divided two to four times before maturing, and if they encountered D(3) later in the cell cycle they underwent an extra division. Indomethacin slows HL60 cell multiplication by prolonging G1, and when these slower-growing cells were exposed to D(3), they matured after the usual period but underwent one division less than indomethacin-free cells. Contrary to common assumptions, we conclude that promyeloid cells do not initiate growth arrest or monocytic maturation immediately after exposure to D(3). Instead, an encounter with D(3) early in G1 sets in train a complex differentiation program. This consists of 2-3 days of rapid proliferation-probably employing cell cycles with a shortened G1 phase-that is followed by growth arrest and maturation. As a result, a single D(3)-treated promyeloid cell gives rise to 10 or more mature monocytes. These observations not only explain why "differentiating" cells express proliferation-related characteristics soon after D(3) addition, but they also show that the process of D(3)-induced monocytic differentiation is much more complex than has previously been realized.