Olson J E, Winston J T, Whitlock J A, Pledger W J
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
J Cell Physiol. 1993 Feb;154(2):333-42. doi: 10.1002/jcp.1041540217.
Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G1 at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G1 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 G1 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 G1 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.
在缺乏氨基酸的培养基中被密度抑制的BALB/c - 3T3细胞，在被刺激增殖时会在G1期中期停滞于一个称为V点的位置。从V点停滞状态释放的细胞需要6小时才能穿过G1期晚期并进入S期。由于此处呈现的数据表明，细胞从V点释放后2 - 3小时需要mRNA合成，在此之后抑制mRNA合成并不妨碍进入S期，我们利用这种G1期中期停滞方案来分析G1期晚期的基因表达。我们发现，尽管在缺乏氨基酸的培养基中刺激细胞并不抑制在G1期早期表达的基因的诱导，但通常在G1期晚期表达的基因仅在从V点释放后才表达。从V点释放的细胞中G1期晚期基因的表达，就G1期位置而言，在时间上与静止的G0细胞受到刺激时的情况相似。由于该方案有效地将基因表达分为早期（V点之前）和晚期（V点之后）两类，它在研究调节G1期晚期进程和DNA合成起始的生长因子调节事件中应该会很有用。此外，我们使用c - myb反义寡核苷酸来表明，在G1期晚期出现的c - myb表达是BALB/c - 3T3成纤维细胞穿过G1期晚期并启动DNA合成所必需的。
