Open Biology ( IF 5.8 ) Pub Date : 2020-10-14 , DOI: 10.1098/rsob.200200 Eleanor Wendy Trotter 1 , Iain Michael Hagan 1
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.
中文翻译:
用 Cdk4/6 抑制剂从细胞周期停滞中释放,在人类细胞培养中产生高度同步的细胞周期进程
用于同步人类细胞系细胞周期进程的每种方法都提出了一系列独特的挑战。与通过关键细胞周期阶段短暂阻断进展的药剂进行诱导同步很受欢迎,但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,对于 DNA 复制抑制剂,会损伤 DNA。如果在研究周期中对表型的解释要保持独立于对前一个周期进展的影响,那么目标分子的产生、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 在细胞周期承诺的自然控制点(限制点)阻止细胞周期进程的能力来避免这些挑战。在先前的工作发现从 CDK4/6 抑制恢复期间生长和分裂的耦合没有变化之后,我们发现细胞周期进程高度同步。尽管我们验证了 CDK4/6 与 hTERT-RPE-1、A549、THP1 和 H1299 的诱导同步,但它在其他系中也有效,并避免了伴随胸苷阻断/释放同步的 DNA 损伤。在 72 小时停止后返回循环的能力可以实现循环外目标的诱导/操纵,而不会影响之前的循环。它在其他品系中有效,并避免了伴随胸苷阻断/释放同步的 DNA 损伤。在 72 小时停止后返回循环的能力可以实现循环外目标的诱导/操纵,而不会影响之前的循环。它在其他品系中有效,并避免了伴随胸苷阻断/释放同步的 DNA 损伤。在 72 小时停止后返回循环的能力可以实现循环外目标的诱导/操纵,而不会影响之前的循环。