The cell cycle is a strictly regulated process that is responsible for appropriate cellular growth, development, and differentiation. It combines DNA replication with chromosomal segregation to ensure equal distribution of duplicated genetic material in the daughter cells.
Cell cycle phases are divided into four sequential stages that progress from quiescence (G0 phase) to proliferation (G1, S, G2, and M phases), and back to G0 phase. The deregulation of this process is considered a hallmark of tumor cell development.
Therefore, both academic and industrial drug discovery programs have strongly focused on the development of drugs targeting cell cycle phases. Using advanced flow cytometry researchers can rapidly and precisely determine different phases of the cell cycle.
Traditionally, cell cycle phases are analyzed by evaluation of a DNA histogram generated by flow cytometry. This technique is fast and reproducible and can, for a well-defined cell population, give an approximation of the number of cells in G0/G1, S, and G2/M.
However, G0 and G2 cells cannot be separated from G1 and M cells, respectively. This limitation can partly be overcome by acridine orange staining giving discrimination of G0 from G1. Furthermore, histogram analysis of tumor samples with aneuploid populations can be difficult and sometimes impossible.
In a flow cytometer cell cycle analysis based on DNA content measurement is usually analyzed on a linear scale since the differences in fluorescence are usually small. Flow cytometry software programs offer algorithms to accurately estimate the cell cycle phases.
Different proliferation- and cell cycle-related antigens have been described, allowing for the evaluation of cell kinetic parameters by detecting the expression of these antigens.
Proliferating cell nuclear antigen (PCNA) cyclin is expressed mainly during S phase and acts as a scaffold to recruit proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics.
PCNA expression has been found to correlate with tumorigenesis, mainly with the degree of malignancy, vascular infiltration, distant metastasis, and survival. This antigen has been described as a biomarker of colorectal adenocarcinoma.
Proliferating cell nuclear antigen (PCNA) cyclin is expressed mainly during S phase and acts as a scaffold to recruit proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics. PCNA expression has been found to correlate with tumorigenesis, mainly with the degree of malignancy, vascular infiltration, distant metastasis, and survival. This antigen has been described as a biomarker of colorectal adenocarcinoma.
Cyclins are a family of proteins that control the progression of cells through the cell cycle by activating cyclin-dependent kinase (CDK) enzymes. The most important cyclins involved in cell cycle processes are listed below.
|Cyclin A||Control of S phase in complex with Cdk1 or Cdk2||Cdk1 or Cdk2|
|Cyclin B||Control of G2/M phase in complex with Cdk1||Cdk1|
|Cyclin D||Control of G1 phase in complex with Cdk4 or Cdk6; Regulation of Rb/E2F transcription||Cdk4 or Cdk6|
|Cyclin E||Control of G1-S phase in complex with Cdk2; Regulation of Rb/E2F transcription||Cdk2|
|Cyclin H||Cdk-activating kinase and RNAPII transcription in complex with Cdk7||Cdk7|
Histone H3 becomes phosphorylated only during M phase of the cell cycle.
Thus, histone H3 pSer28 can be used as a specific marker for M-phase cells and combined with other proliferation tools to further segment cell cycle compartments.