Each approach used to synchronize cell cycle progression of human cell lines presents a unique set of challenges. Induction synchrony with agents that transiently block progression through key cell cycle stages are popular, but change stoichiometries of cell cycle regulators, invoke compensatory changes in growth rate and, for DNA replication inhibitors, damage DNA. The production, replacement or manipulation of a target molecule must be exceptionally rapid if the interpretation of phenotypes in the cycle under study is to remain independent of impacts upon progression through the preceding cycle. We show how these challenges are avoided by exploiting the ability of the Cdk4/6 inhibitors, palbociclib, ribociclib and abemaciclib to arrest cell cycle progression at the natural control point for cell cycle commitment: the restriction point. After previous work found no change in the coupling of growth and division during recovery from CDK4/6 inhibition, we find high degrees of synchrony in cell cycle progression. Although we validate CDK4/6 induction synchronization with hTERT-RPE-1, A549, THP1 and H1299, it is effective in other lines and avoids the DNA damage that accompanies synchronization by thymidine block/release. Competence to return to cycle after 72 h arrest enables out of cycle target induction/manipulation, without impacting upon preceding cycles.

每种用于同步人类细胞系细胞周期进程的方法都面临着一系列独特的挑战。使用短暂阻断细胞周期关键阶段进展的药物进行同步诱导很受欢迎，但会改变细胞周期调节剂的化学计量，引起生长速率的补偿性变化，并且对于 DNA 复制抑制剂来说，会损伤 DNA。如果对所研究的周期中表型的解释要保持独立于对前一个周期进展的影响，则目标分子的产生、替换或操作必须异常迅速。我们展示了如何通过利用 Cdk4/6 抑制剂 palbociclib、ribociclib 和 abemaciclib 在细胞周期定型的自然控制点（限制点）处阻止细胞周期进展的能力来避免这些挑战。之前的工作发现从 CDK4/6 抑制恢复期间生长和分裂的耦合没有变化，我们发现细胞周期进程具有高度同步性。尽管我们验证了 CDK4/6 诱导同步与 hTERT-RPE-1、A549、THP1 和 H1299，但它在其他细胞系中也有效，并且避免了胸苷阻断/释放同步伴随的 DNA 损伤。 72 小时逮捕后返回循环的能力可以实现循环外目标诱导/操纵，而不影响之前的循环。