Apraiz Aintzane, Mitxelena Jone, Zubiaga Ana
Department of Cell Biology and Histology, University of the Basque Country, UPV/EHU.
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, UPV/EHU; Department of Molecular Mechanisms of Disease, University of Zurich.
J Vis Exp. 2017 Jun 6(124):55745. doi: 10.3791/55745.
The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.
细胞周期的基因表达程序是理解细胞周期依赖性过程及其在诸如癌症等疾病中所起作用的关键步骤。细胞周期调控的基因表达分析依赖于将细胞同步到特定阶段。在此，我们描述一种利用两种互补同步方案的方法，该方法常用于研究细胞周期中基因表达的周期性变化。这两种方法均基于在一个特定点瞬时阻断细胞周期。通过羟基脲（HU）处理的同步方案会导致细胞停滞在G1晚期/ S期早期，而从HU介导的停滞中释放可提供一个均匀进入S期和G2 / M期的细胞群体。通过胸腺嘧啶核苷和诺考达唑（Thy-Noc）处理的同步方案会将细胞阻断在有丝分裂早期，从Thy-Noc介导的停滞中释放可提供一个适合用于G1期和进入S期研究的同步细胞群体。应用这两种方法都需要监测细胞周期分布情况，这通常在对细胞进行碘化丙啶（PI）染色并通过流式细胞术分析DNA含量后进行。我们表明，联合使用两种同步方案是一种可靠的方法，可清晰地确定在细胞周期中差异调节的基因（即E2F1和E2F7）的转录谱，从而更好地理解它们在细胞周期过程中的作用。此外，我们表明这种方法对于研究基于药物的治疗机制（即丝裂霉素C，一种抗癌剂）很有用，因为它能够区分对基因毒性剂有反应的基因与仅受该药剂引起的细胞周期扰动影响的基因。
