īesides these markers, the in vivo reporters of cell cycle phases can be used. Therefore, the Ki-67 protein is degraded continuously during the G1 and G0 phases and thus is a graded rather than a binary marker, both for cell cycle progression and time since entry into quiescence. It depends on the time that an individual cell has spent in the G0 phase. The level of Ki-67 protein during G0 and G1 phases in individual cells is highly heterogeneous. On the other hand, the depletion of the Cdh1 protein, which is an activator of the Anaphase Promoting Complex, stabilizes the Ki-67 protein. Recent studies on the Ki-67 protein have indicated that this protein undergoes proteasome-mediated degradation during the G1 phase and upon cell cycle exit. Initial studies on the Ki-67 protein indicated that the Ki-67 protein is present during every phase of the cell cycle in asynchronously cycling cells and absent in non-dividing cells. Another option for G0 phase identification is Ki-67 antibody staining. It relies on the assumption that cells in the G0 phase have a lower level of RNA compared to the G1-phase cells, and therefore it allows for the distinguishing of G0 cells. The determination of G0 cells can be achieved by the application of pyronin Y for staining of RNA. Thus, mathematical approaches were developed for the estimation of fractions of cells in the G1/G0 phases, S phase, and G2/M phases. As the cells with the same DNA content can differently bind fluorescent dyes to their DNA, DNA content does not allow easy discrimination between the G1/G0, S, and G2/M phases. As PI also stains RNA, the protocols based on PI also include the step with the treatment of cells with RNase. For the staining of the living cells, Hoechst 33342 is commonly used. The most frequently used protocols for DNA staining of fixed cells are based on the use of fluorescent, stoichiometrically DNA binding dyes such as propidium iodide (PI, Figure 3), 4,6-diamidino-2-phenylindole (DAPI), Hoechst 33258, or Hoechst 33342. The histogram of DNA content thus enables the estimation of the number of cells in G1/G0, S, and G2/M phases. During determination of the amount of DNA, DNA is stained by fluorescent dyes, and the fluorescent signal is analyzed. The cells in G1 and G0 have half the content of DNA as compared to G2 and M cells. The approach that is likely the most frequent is based on the analysis of DNA content. Various approaches are used to identify the distinct cell cycle phases. The G2/M checkpoint prevents cells with damaged DNA from entering mitosis and allows for DNA repair. Another important checkpoint is the G1/S checkpoint that controls the DNA integrity and stops DNA synthesis when cells suffer, for example, from extracellular stresses. When cells pass through the restriction point, no growth factors are necessary for transition to the S phase. In the early G1 phase, until cells reach the restriction point, the growth factors are required for their transition through the G1 phase leading to the S phase. During the cell cycle, several important control points (checkpoints) are present ( Figure 2). In human cells, there are supposed to be around 20 various CDKs and 29 various cyclins. The activation of cyclins/CDKs is induced by mitogenic signals and inhibited by the activation of the cell cycle checkpoints in response to DNA damage. The formation of cyclin/CDKs complexes controls the cell cycle progression by the phosphorylation of the targets, such as tumor suppressor protein retinoblastoma (Rb). An important role in these steps is played by cyclin-dependent kinases (CDKs Figure 2) regulated by their interactions with cyclins and CDK inhibitors (CKIs for a review see, e.g., ). The progression of cells through the cell cycle is controlled by highly orchestrated steps reacting to intracellular and extracellular signals. They are the G1 (gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis) phases ( Figure 2). The cell cycle is composed of four distinct phases.
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