CELL CYCLE-DEPENDENT GENE-EXPRESSION IN V-POINT-ARRESTED BALB/C-3T3 CELLS

Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G, at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G, and enter S phase. As data presented here show that mRNA synthesis is needed for 2-3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid-G1 arrest protocol to analyze gene expression in late G1. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G1, genes normally expressed in late G, were expressed only after release from the V point. The expression of late G1 genes in cells released from the V point was temporally similar, in respect to G1 location, as was seen in stimulation of quiescent G0 cells. As this protocol effectively divides gene expression into early (pre-V point) and late (post-V point) categories, it should be useful in studies of growth factor-modulated events that regulate traverse of late G, and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G1, is required for BALB/c-3T3 fibroblasts to traverse late G1 and initiate DNA synthesis.