Department of Biology, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen, Denmark.
Department of Biology, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen, Denmark; Department of Biochemistry and Molecular Biology, Faculty of Medicine, Complutense University, Madrid, Spain.
Cell Calcium. 2020 Jun;88:102207. doi: 10.1016/j.ceca.2020.102207. Epub 2020 Apr 30.
Calmodulin (CaM) is the principle mediator of the Ca signal in all eukaryotic cells. A huge variety of basic cellular processes including cell cycle control, proliferation, secretion and motility, among many others are governed by CaM, which regulates activities of myriads of target proteins. Mammalian CaM is encoded by three genes localized on different chromosomes all producing an identical protein. In this study, we have generated HeLa human cancer cells conditionally expressing CaM in a genetic background with all three genes inactivated by CRISPR/Cas9. We demonstrate that downregulation of ectopically expressed CaM is achieved after 120 h, when cells are arrested in the M phase of the cell cycle. We show for the first time that CaM downregulation in human cancer cells is followed by a multinucleated senescent state as indicated by expression of β-galactosidase as well as cell morphology typical for senescent cells. Our newly generated genetic system may be useful for the analysis of other CaM regulated processes in eukaryotic cells in the absence of endogenous CaM genes.
钙调蛋白（CaM）是所有真核细胞中钙信号的主要介质。包括细胞周期控制、增殖、分泌和运动在内的大量基本细胞过程都受 CaM 调控，它调节着无数靶蛋白的活性。哺乳动物 CaM 由三个定位于不同染色体上的基因编码，所有基因都通过 CRISPR/Cas9 失活。在这项研究中，我们在遗传背景中生成了条件表达 CaM 的 HeLa 人癌细胞，该背景中所有三个基因均被 CRISPR/Cas9 失活。我们证明，在用药物诱导细胞周期 M 期阻滞 120 小时后，异位表达的 CaM 被下调。我们首次表明，CaM 在人癌细胞中的下调会导致多倍体衰老状态，这表现为β-半乳糖苷酶的表达以及衰老细胞特有的细胞形态。我们新生成的遗传系统可能对分析真核细胞中其他受 CaM 调控的过程有用，而无需内源性 CaM 基因。
