Differential sensitivity to 5-bromodeoxyuridine during the S phase of synchronized myogenic cells.
Differential sensitivity to 5-bromodeoxyuridine during the S phase of synchronized myogenic cells.
复制标题
同步化生肌细胞 S 期对 5-溴脱氧尿苷的敏感性不同。
DOI：
10.1016/0012-1606(76)90165-2
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发表时间：
1976
影响因子：
2.7
通讯作者：
Richard Bischoff
中科院分区：
文献类型：
--
作者：
John Lough;Richard Bischoff
同步的肌原性细胞培养物已被用于证明在S期的片段期间对BUdR的差异敏感性。通过两种方法实现细胞的同步。首先，在含有DNA合成抑制剂FUdR的培养基中启动细胞。在体外19小时后用TdR逆转FUdR阻断后，使同步化的细胞用BUdR复制它们的DNA，持续对应于早期和晚期S的时期。在与FUdR同步期间标记的细胞的百分比的测定表明，约90%的细胞的循环群体在细胞周期的G1S界面处积累，并且在与TdR的阻断逆转之后的S期的持续时间没有改变。由于BUdR在同步点之后立即脉冲至这些培养物，因此获得高度的同步。在第二种同步方法中，在用秋水仙胺进行的中期阻滞中收集在BUdR脉冲期间处于G2、晚期S或早期S的细胞群，并选择性地从培养物中去除。用有丝分裂选择方法，同步化点发生在BUdR脉冲后几小时。在两种方法中，使细胞恢复肌生成，并在体外约50小时后对融合核的百分比进行评分。在这两种同步方法中，BUdR掺入早期复制DNA导致成肌细胞融合显著下降，而掺入晚期复制DNA则没有影响。这一结果不能归因于S. 4小时S期进一步分馏成1小时的周期表明，BUdR敏感的目标是在DNA合成的第二个小时复制。
Synchronized myogenic cell cultures have been used to demonstrate differential sensitivity to BUdR during segments of the S period. Synchronization of the cells was achieved by two methods. First, cells were initiated in medium containing FUdR, an inhibitor of DNA synthesis. Following FUdR blockade reversal with TdR after 19 hr in vitro, the synchronized cells were allowed to replicate their DNA with BUdR for periods corresponding to early and late S. Determinations of percentage labeled cells during synchronization with FUdR indicate that about 90% of the cycling population of cells accumulates at the G1 S interface of the cell-cycle and that the duration of the S period following blockade reversal with TdR is not altered. Since BUdR is pulsed to these cultures immediately after the point of synchronization, a high degree of synchrony is obtained. In the second method of synchrony, cohorts of cells which had been in G2, late S, or early S during a BUdR pulse were collected in metaphase arrest with Colcemid and selectively removed from the cultures. With the mitotic selection method the point of synchronization occurred several hours after the BUdR pulse. In both methods the cells were allowed to resume myogenesis and scored for percentage fused nuclei after approx 50 hr in vitro. With both methods of synchrony, BUdR incorporation into early replicating DNA results in a striking decline in myoblast fusion, whereas incorporation into late replicating DNA is without effect. The results cannot be attributed to a disproportionate uptake of nucleotide during early S. Further fractionation of the 4-hr S phase into 1-hr periods indicates that the BUdR sensitive target is replicated during the second hr of DNA synthesis.