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.

100-kDa polypeptides in peripheral clathrin-coated vesicles are required for receptor-mediated endocytosis.

“…This explains our observation of 100% inhibition of endocytosis in individual cells, whereas earlier experiments only observed partial inhibition of endocytosis in cell cultures (19,20). Such stochastic behavior has been observed in many other systems and could be a cell cycle effect (24,25). In our case, variable susceptibility to AP.6 injection may also be due to differential expression or use of 100-kDa polypeptide isotypes that may be present in subpopulations of peripheral adaptors (9).…”

Cell surface and nuclear changes during TNF-α-induced apoptosis in WEHI 164 murine fibrosarcoma cells

“…Lamin may be related to the shape of the nucleus, so that deformations of the nuclear membrane and changes in shape of the cell nucleus might be related to lamin degradations. Cell detachment has been described in both mitotic and apoptotic cells; however, the former retain their ability to adhere [29]. Detachment means that changes or modifications take place not only in the cytoskeleton but also at the cell membrane, with a diminution of the adhesive forces and adhesive properties of the cell to the substrate.…”

Cell cycle dependent expression and stability of the nuclear protein detected by Ki‐67 antibody in HL‐60 cells

“…The stability of this antigen was also examined in cells in which protein synthesis was inhib- ited by CHX; exposure of cells to 0.1 pg/ml of CHX suppresses the rate of protein synthesis by over 70% without affecting cell viability in short-term (up to 8 h) experiments (Traganos et al 1987). The data revealed a rapid turnover rate of the antigen in cells treated with this concentration of CHX (Figures 3 & 4).…”