Discrimination of G2 and mitotic cells by flow cytometry based on different expression of cyclins A and B1.
Cyclins, the regulatory subunits of their respective cyclin-dependent kinases, are the key components of the cell-cycle progression machinery. Some cyclins are expressed discontinuously during the cell cycle, their synthesis and degradation being strictly scheduled. The presence of these cyclins in the cell, therefore, provides landmarks of the cell cycle, in addition to DNA replication and mitosis. Cyclin A is expressed in late S and G2 phase and degraded during mitosis just prior to metaphase. Degradation of another "mitotic" cyclin, cyclin B1, occurs later, at the transition from metaphase to anaphase. Based on the difference in time of degradation of cyclin A versus cyclin B1 it was possible, in the present study, to discriminate between G2 and mitotic (postprophase) MOLT-4 leukemic cells, by multiparameter (cellular DNA content versus cyclin expression) flow cytometry. The cells arrested in metaphase by Vinblastine were cyclin A negative and had an elevated level of cyclin B1. The cells arrested in G2 by the DNA topoisomerase II inhibitor m-AMSA had a very high level of cyclin B1 expression and unchanged expression of cyclin A. During stathmokinesis induced by Vinblastine the percentage of mitotic cells estimated by analysis of cellular DNA content and cyclin A expression was identical to that estimated by the alternative method based on in situ DNA denaturation followed by staining with acridine orange. Thus, differences in expression of cyclins A and B1 make it possible to discriminate cells that have the same DNA content but reside in different phases of the cycle, such as DNA diploid cells in G2 versus tetraploid G1 cells or mitotic versus G2 cells.
Gong
J
J
Cancer Research Institute, New York Medical College, Valhalla 10523, USA.
Traganos
F
F
Darzynkiewicz
Z
Z
D002453:Cell Cycle / Q000502:physiology*; D002463:Cell Membrane Permeability; D016213:Cyclins / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D016195:G2 Phase / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D008938:Mitosis / Q000502:physiology; D014407:Tumor Cells, Cultured
D016213:Cyclins; D004273:DNA, Neoplasm