Monocytically differentiating HL60 cells proliferate rapidly before they mature

1α,25-Dihydroxyvitamin D₃ (D₃) 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 D₃ addition, there is also evidence that D₃ 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, D₃ was added at various stages of the cell cycle, the progeny were counted, and the proportions of mature monocytes were determined. Initially, the D₃-treated cells proliferated at an accelerated rate, and they matured only later. If cells encountered D₃ early in G1 they divided two to four times before maturing, and if they encountered D₃ 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 D₃, 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 D₃. Instead, an encounter with D₃ 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 D₃-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 D₃ addition, but they also show that the process of D₃-induced monocytic differentiation is much more complex than has previously been realized.