EXPRESSION OF CYCLINS-B AND CYCLINS-E IN INDIVIDUAL MOLT-4 CELLS AND IN STIMULATED HUMAN-LYMPHOCYTES DURING THEIR PROGRESSION THROUGH THE CELL-CYCLE
- JP GONG
- F TRAGANOS
- Z DARZYNKIEWICZ
Published online on: Wednesday, December 1, 1993
Cyclins B (B1) and E, integral components of the cell cycle drive machinery consisting of p34cdc2 and p33cdk2 cyclin-dependent kinases and their regulatory kinases and phosphatases, were detected in human leukemic MOLT-4 cells and in mitogen-stimulated normal peripheral blood lymphocytes immunocytochemically, using commercially available antibodies. Flow cytometric bivariate analysis of DNA content and cyclin B or E made it possible to correlate expression of these proteins in individual cells with their position in the cycle, without the necessity for cell synchronization. In both cell systems, cyclin B was expressed almost exclusively in G2 and M cells: cells in G1 and throughout most of S phase, were negative. Cells arrested in G2 by gamma radiation or the DNA topoisomerase II inhibitor m-AMSA for up to 4 h, had very high levels of cyclin B. Cells arrested in metaphase by vinblastine also strongly expressed cyclin B, although to a lesser degree than cells arrested in G2. Expression of cyclin E was maximal in late G1 and in early S, while its expression progressively decreased during the remainder of S phase. Two compartments of the G1 phase, G1A and G1B representing cells that were cyclin E negative and positive, respectively, were discriminated. The kinetics of cell exit from G1A, under conditions of stathmokinesis induced by vinblastine, showed a stochastic component; the half-time of MOLT-4 cell residence in G1A was 5.6 h. Nonstimulated (G0) lymphocytes did not express cyclin E; their stimulation by phytohemagglutinin led to the appearance of a subpopulation of cyclin E positive cells as early as 18 h after addition of the mitogen. Maximal expression of cyclin E occurred in G1 lymphocytes just prior to cell entrance to S, and in early S phase cells. Thus, expression of cyclin E can be used as a marker of lymphocyte stimulation (G0 to G1 transition). The combined use of cyclin B and E antibodies can identify late G1, S and G2+M cells, and thus it may be applied to measure the fraction of cycling cells in cell populations, e.g. the tumor growth fraction.