Induced cell cycle arrest
[1] It is an artificial activation of naturally occurring cell cycle checkpoints, induced by exogenous stimuli controlled by an experimenter.
While in G1 the cell synthesizes messenger RNA (mRNA) and proteins in preparation for subsequent steps of interphase leading to mitosis.
This natural process can be mimicked in a lab through the overexpression of p27Kip1, which results in induced cell cycle arrest in G1 phase.
When grown in a media without some essential amino acids, such as methionine, some cells arrest in early G1 phase.
Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.
It is a reversible inhibitor of eukaryotic nuclear DNA replication that blocks progression past the S phase.
Its mechanism is the inhibition of DNA polymerase A and D. A structural study found that this is thought to occur through binding the alpha active site of the polymerase and "rotating the template guanine," which prevents deoxycytidine triphosphate (dCTP) from binding.
[24] This was demonstrated in cancer cell lines and downregulates expression of B-cell lymphoma-extra large (Bcl-XL), an anti-apoptotic protein that prevents the release of mitochondrial contents like cytochrome c. G2 phase is the final part of interphase and directly precedes mitosis.
Destruction of a cell's endogenous cyclin messenger RNA can arrest frog egg extracts in interphase and prevent them from entering mitosis.
It is composed of four phases - prophase, metaphase, anaphase, and telophase - and involves the condensation of the chromosomes in the nucleus, the dissolution of the nuclear envelope, and the separation of sister chromatids by spindle fibers.
As mitosis concludes, the spindle fibers disappear and the nuclear membrane reforms around each of the two sets of chromosomes.
[28] Cells treated with nocodazole arrest with a G2 or M phase DNA content, which can be verified with flow cytometry.
[31][32] It acts through a specific binding site on the microtubule polymer, and as such does not require GTP or other cofactors to induce tubulin polymerization.
Flow cytometry is a technique of measuring physical and chemical characteristics of a population of cells using lasers and fluorophore dyes covalently linked to protein markers.
Staining with DNA dyes propidium iodide or 4',6'-diamidino-2-phenylindole (DAPI) allows delineation or sorting of cells between G1, S, or G2/M phases.
The secondary antibody is then visualized through staining or immunofluorescence, allowing indirect detection of the original target protein.
[38] FUCCI is a system that takes advantage of cell cycle phase-specific expression of proteins and their degradation by the ubiquitin-proteasome pathway.
