Evidence for cell cycle phase-specific initiation of a program of HL-60 cell myeloid differentiation mediated by inducer uptake.

The question of whether the initial regulatory event, which directs an uncommitted precursor cell toward terminal differentiation, is cell cycle phase specific was examined using the human promyelocytic leukemia cell line, HL-60. While the HL-60 system does not reflect all of the features of normal hematopoiesis, it does provide a relatively well-defined in vitro experimental system which can be useful for examining aspects of the differentiation process. HL-60 cells were induced to undergo myeloid differentiation by retinoic acid. The subsequent differentiation kinetics of HL-60 populations initially enriched in different cell cycle phases was measured. This was compared to the cellular uptake of retinoic acid as a function of cell cycle position. If the initial differentiation-regulating event were cell cycle phase independent, then the kinetics of differentiation would be independent of the cell cycle status of the initial population. Flow cytometric cell sorting, based on cellular narrow angle and orthogonal light scatter intensity spectra, was used to select G1-enriched and S + G2 + M-enriched cell populations without pharmacological perturbation. These two populations were each induced to undergo myeloid differentiation with 10(-6) M beta-all-trans-retinoic acid. The kinetics of G1/0 arrest associated with terminal cell differentiation, as well as phenotypic differentiation, assayed by development of oxidative metabolism, was measured for both populations. The kinetics of differentiation differed for the two populations, indicating that the initial differentiation-regulating event was cell cycle phase specific. For both of the initial cell populations, significant phenotypic differentiation followed approximately 24 hr after enrichment in the relative number of S-phase cells. When exponentially proliferating HL-60 cells were exposed to a 1-hr pulse of 10(-5) M [3H]retinoic acid and then flow cytometrically sorted by DNA content, cells in late S + G2 + M had an approximately 10-fold higher uptake than cells in G1 or early S. The results indicate that cellular regulation of myeloid differentiation first becomes responsive to the inducer, retinoic acid, in S phase when uptake is enhanced.