Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression

Abstract

Chinese hamster ovary (CHO) cells, synchronized by selective detachment at mitosis, were treated with various concentrations of actinomycin D (AMD) or cycloheximide (CHX) either immediately, or 1, 2, or 3 hr after mitosis. Since the minimum duration of G1 phase in these cultures was 3.4 hr, the addition of RNA or protein synthesis inhibitors took place at the beginning, first third, second third, or end (G1-S boundary) of G1 phase. The kinetics of exit from G1 phase, the rate and extent of traverse of S phase, and the reaccumulation of RNA were estimated under each set of growth conditions by flow cytometry of acridine orange-stained cells. A mathematical model was constructed to describe the trajectories of the cell populations with respect to their increase in RNA and DNA content in the absence or presence of the inhibitor. The chronologic synchrony imposed on the CHO cell population began to decay within 3 hr, resulting in stochastic entrance of cells into S phase in the absence of inhibitor. Addition of AMD or CHX at 0, 1, 2, or 3 hr after mitosis, regardless of the inhibitor concentration, did not provide evidence of a critical restriction point in G1 beyond which cells were committed to enter S phase and were no longer sensitive to moderate suppression of RNA or protein synthesis. The observed kinetics of cell entrance into an traverse of S phase were consistent with an inherently heterogeneous response to serum stimulation occurring at or just after cell division.