TERMINAL DIFFERENTIATION OF NORMAL CHICKEN ERYTHROID PROGENITORS - SHORTENING OF G(1) CORRELATES WITH LOSS OF D-CYCLIN CDK4 EXPRESSION AND ALTERED CELL-SIZE CONTROL/

Detailed knowledge is available about the molecular makeup of the cell cycle clock in dividing cells. However, comparatively little is known about cell cycle regulation during terminal differentiation. Here we describe a primary cell system in which this question can be addressed . Normal avian erythroid progenitors undergo continuous self-renewal i n suspension culture in the presence of growth factors and hormones, a llowing us to obtain large cell numbers (10(10)-10(11)). By replacing these ''self-renewal factors'' with erythropoietin and insulin, the ce lls can be induced to synchronous, terminal differentiation. During th e first 72 h, the cells undergo five cell divisions. Thereafter, they arrest in G(1) and complete their maturation into RBC without further divisions.Sixteen to 24 h after induction of differentiation, the cell cycle length decreased from about 20 to 12 h. This shortened doubling time was due to a drastic reduction of G(1) (from 12 to 5 h), while S - and G(2)-phase lengths were not affected. At the same time, the diff erentiating cells underwent an extensive and concerted switch in their gene expression pattern. During the subsequent four cell divisions, t he cell volume decreased from about 300 to less than 70 femtoliters, b ut the rate of protein synthesis normalized to cell volume remained co nstant. Interestingly, the shortening of G(1) was accompanied by a rap id down-regulation of D-type cyclins and their partner, cyclin-depende nt kinase type 4 (cdk4), while expression of S- and G(2)-M-associated cell cycle regulators (cyclin A and cdk1/cdc2) remained high until the cells arrested in G(1) 72-96 h after differentiation induction. We co nclude that concerted reprogramming of progenitor gene expression duri ng erythroid differentiation is accompanied by profoundly altered cell cycle progression involving the loss or alteration of cell size contr ol at the restriction point.