Use of flow microfluorometry in detailed analysis of effects of chemical agents on cell cycle progression.
Use of flow microfluorometry in detailed analysis of effects of chemical agents on cell cycle progression.
复制标题
使用流式显微荧光测定法详细分析化学试剂对细胞周期进展的影响。
作者：
R. Tobey;H. Crissman
建立了一种测定化疗药物对体外培养的同步化CHO中国仓鼠细胞周期进程的影响的技术。直肠结合放射自显影和细胞计数技术与DNA分布模式的分析，通过洛斯阿拉莫斯流动显微荧光计。通过在异亮氨酸缺乏培养基中生长，使CHO细胞在G1停滞状态下蓄积;然后将细胞重悬于含有异亮氨酸和供试药物的新鲜完全培养基中，以在药物存在下重新启动循环遍历。10小时后，对培养物的DNA含量的群体分布进行流式显微荧光分析，得到G1、S和G2或M的细胞分数。对于具有可逆效应的药物，将细胞重悬于含胸苷-3H的新鲜无药物培养基中后获得确证性细胞周期数据;并定期取出等分试样，以测定穿过能力，即使在缓慢或非进展的群体中也是如此。四种药物对细胞周期进程有不同的影响进行了检查，即，羟基脲，阿糖胞苷，博来霉素，喜树碱。羟基脲（10-3 m）和阿糖胞苷（5 µg/ml）均可显著降低细胞进入S期的速率，导致细胞在G1-S边界蓄积。这两种药物都不能完全阻止细胞启动DNA合成。博莱霉素（100 µg/ml）允许基因组复制的起始和完成以接近正常的速率发生，但细胞在G2期积累，大多数细胞失去了进入有丝分裂的能力。喜树碱（1 µg/ml）降低了周期进展的总体速率，并允许少数细胞复制完整的DNA补体。这些体外研究结果的化疗方案设计的关系进行了讨论。
A technique has been developed for determining effects of chemotherapeutic agents on cell cycle progression in synchronized line CHO Chinese hamster cells grown in vitro . The proctocol combines autoradiographic and cell-enumeration techniques with analysis of DNA distribution patterns by means of the Los Alamos flow microfluorometer. CHO cells were accumulated in a state of G1 arrest by growth in isoleucine-deficient medium; then the cells were resuspended in fresh, complete medium containing isoleucine plus a test agent to reinitiate cycle traverse in the presence of the drug. Ten hr later, flow microfluorometric analysis of the population distribution of DNA contents for the cultures yielded the fractions of cells in G1, S, and G2 or M. For agents with reversible effects, confirmatory cell cycle data were obtained after resuspension of the cells in fresh, drug-free medium containing thymidine-3H; and aliquots were removed at intervals for determination of traverse capacity, even in slowly or nonprogressing populations. Four agents with differing effects on cell cycle progression were examined, namely, hydroxyurea, cytosine arabinoside, bleomycin, and camptothecin. Both hydroxyurea (10-3 m) and cytosine arabinoside (5 µg/ml) grossly decreased the rate of progression of cells into S phase, resulting in an accumulation of cells at the G1-S boundary. Neither agent completely prevented cells from initiating DNA synthesis. Bleomycin (100 µg/ml) allowed initiation and completion of genome replication to occur at a nearly normal rate, but cells accumulated in G2 and most cells lost the capacity to enter mitosis. Camptothecin (1 µg/ml) reduced the overall rate of cycle progression and allowed a few cells to replicate a complete complement of DNA. The relationship of these in vitro findings to the chemotherapy regimen design is discussed.