Owen C. Blair

Incorporation of 3H-leucine, 3H-lysine, and 3H-tryptophan during the cell cycle of Chinese hamster ovary cells: a flow cytometric analysis.

Exponentially growing Chinese hamster ovary cells were pulse labeled with 3H-leucine, 3H-lysine, or 3H-tryptophan, fixed, and stained by either the acriflavine-Feulgen procedure or with fluorescein isothiocyanate (FITC). Protein content as determined by FITC fluorescence was representative of protein content determined biochemically by the method of Lowry. Utilizing a fluorescence-activated cell sorter, 3H-labeled cells were sorted according to their DNA or protein content and the incorporation of 3H-leucine, 3H-lysine, or 3H-tryptophan determined by liquid scintillation counting. The rates of 3H-leucine, 3H-lysine, and 3H-tryptophan incorporation increased with respect to increasing DNA content (G1, mid-S, G2+M). The rate of 3H-lysine incorporation increased continuously with increasing protein content, whereas the rates of 3H-leucine and 3H-tryptophan incorporation were constant initially with an increase in incorporation near mid-cycle followed by a slight decrease. Matrix algebra modeling of the increase in protein content suggests that 3H-lysine incorporation is consistent with a sigmoidal increase in protein content, however, 3H-leucine and 3H-tryptophan incorporation do not follow either the exponential, linear, or sigmoidal models. Matrix algebra simulation of the FITC protein distribution indicates that while the rate of protein accumulation is not linear, the exponential and sigmoidal models fit the experimental data equally well.

Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin

Tiazofurin is an effective inducer of the maturation of HL-60 promyelocytic leukemia cells, as monitored by increased phagocytic ability and the capacity to reduce nitroblue tetrazolium (NBT). The antimetabolite acts as a potent inhibitor of IMP dehydrogenase, which results in a profound depression in the cellular levels of guanine nucleotides. Flow cytometric analysis of DNA histograms indicated that the commitment of HL-60 cells to differentiate when exposed to tiazofurin was preceded by a transient delay in the G1 phase of the cell cycle. HL-60 leukemia cells enriched in the various phases of the cell cycle by centrifugal elutriation were utilized to further characterize the relationship between the phase of the cell cycle and the commitment to enter a pathway of differentiation. Fractions composed mainly of G1 cells demonstrated an increased capacity to mature when exposed to tiazofurin, whereas fractions containing cells from the S and G2 + M phases of the cell cycle had a lower ability to enter a differentiation pathway. The findings suggest that the commitment of HL-60 cells to mature when exposed to tiazofurin is mediated during the G1 phase of the cell cycle.