CORRELATION BETWEEN DNA-REPLICATION AND EXPRESSION OF CYCLIN-A AND CYCLIN-B1 IN INDIVIDUAL MOLT-4 CELLS

Cyclins A and B1 activate cyclin-dependent kinases CDK2 and CDC2, whic h regulate cell progression through S and G(2). Expression of these cy clins is generally measured in populations of synchronized cells, by i mmunoblotting. Such studies neither provide information regarding inte rcellular variability in cyclin expression nor yield precise data on a time relationship between initiation and termination of DNA replicati on in relation to cyclin expression. Furthermore, cell synchronization by DNA polymerase inhibitors or excess of thymidine induces cell grow th imbalance and alters expression of cyclins, thereby introducing an experimental bias. Using a novel flow cytometric method of detection o f incorporated bromodeoxyuridine (BrdUrd) in the present study, we hav e been able to correlate expression of immunocytochemically discerned cyclins A and B1 with incorporation of BrdUrd and the cell cycle posit ion of individual MOLT-4 cells. On the basis of differences in amount of incorporated BrdUrd and DNA content, the following cohorts of cells in narrow windows of the cell cycle mere identified: (a) cells initia ting and (b) terminating DNA replication during a 1-h pulse of BrdUrd; (c) cells replicating DNA throughout the duration of BrdUrd pulse; (d ) G(1) cells; and (e) G(2) cells that remained in G(2) for at least 1 h after exiting S phase. These populations were characterized with res pect to expression of cyclins A and B1. Expression of cyclin A was an early event of S phase, and 84% of cells entering S phase during 1 h o f exposure to BrdUrd mere already cyclin A positive. More than 95% of S-phase cells, as well as the cells exiting S during BrdUrd pulse, wer e also cyclin A positive. The maximal rate of accumulation of cyclin A was seen during the first hour of progression through S phase. In con trast, the maximal accumulation rate of cyclin B1 showed cells during the first hour of progression through G(2). A strong correlation betwe en expression of cyclin A and the rate of DNA replication, estimated b y the degree of BrdUrd incorporation (r = 0.99), was observed.