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Abstract

Vimentin expression throughout the cell cycle has been analyzed at the single−cell level in asynchronously growing MPC−11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12−O−tetradecanoylphorbol−13−acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short−term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid− and late S phase. In parallel, the G1−phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin−positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1−phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1−phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA−induced vimentin synthesis is regulated in a cell cycle−dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase