Cell cycle acts as a relay station, connecting growth signalling networks with the initiation of DNA synthesis, and is therefore a potentially attractive therapeutic target. Assessing cell cycle distribution and proliferation is essential to study cell growth, differentiation, senescence and apoptosis. This helps to evaluate the underlying mechanisms, therapeutic efficacies of anticancer drugs and can be indicative of wider effects upon the cells, often used in the broader context or in conjunction with other target engagement assays.
Proliferating cells sequentially undergo transition through G1 – S – G2 and M phases, and under certain circumstances enter G0, as resting or quiescent state. During DNA damage, the progression of cell cycle is interrupted at certain checkpoints leading to failed chromosomal alignment and mitotic arrest or delays. These can be characterized at individual stages to exactly pinpoint disruption of the normal processes. o2h scientists can implement flow cytometry-based analysis to profile mitotic pathways and evaluate your compounds for cancer therapies. Whole-cell staining with propidium iodide (PI) and antibodies towards specific proteins or surface markers helps to concurrently analyse cellular characteristics within distinct cell populations G0/G1, S, and G2/M. The software helps to quantitatively determine phase distribution along with characteristics like ploidy, apoptosis, senescence, cell type etc.
Case Study
The cell cycle can be divided into 5 main stages: G₀ phase where cells are resting and not undergoing any division, G₁ phase where cells are preparing to undergo DNA replication, S phase where cells are actively replicating their DNA, G₂ phase where cells have completed DNA replication and M phase where cells are undergoing mitosis to split into 2 daughter cells. At o2h, we have developed flow cytometry-based assays which allow the cell cycle phase of cells to be discovered and any alterations to these that result from treatment detected.
Figure 1: Cell cycle stages of HeLa cells in exponential growth phase in culture determined by staining of DNA content.
These stages can also be manipulated in culture to trap cells in one part of the cell cycle or to enrich for certain populations. Figure 2 shows the effects of a double thymidine block on the progression of cells through the cell cycle. As can be seen, this traps the majority of cells into the G₀/G₁ phase before releasing the cells allows them to progress through the cell cycle.
Figure 2: The effects of a thymidine block on cell cycle progression in HeLa cells. Left panel shows cells which have undergone a double thymidine block, the majority of cells are trapped in the early G₀/G₁ phases of the cycle. The middle panel shows the cells 2 hours after the release of the block where the majority of cells are in S phase. The right panel shows the cell cycle stage 8 hours after release where most of the cells have progressed into the G₂/M phase of the cell cycle.
To gain additional insight into the stages of the cell cycle, it is possible to combine DNA content staining with other markers which can separate cell cycle stages, such as Phospho-Histone H3 to distinguish the G₂ and M phases. As can be seen in figure 3, a relatively small proportion of cells are in M phase actively undergoing mitosis, and this can be enriched by using nocodazole, an agent that traps cells in the mitotic phase. Being able to detect cells in the M phased can be used to investigate the rate of cell division which is linked to a range of therapeutic opportunities.
Figure 3: Separation of the G₂ and M phases of the cell cycle by phosphor-Histone H3 and PI staining. HeLa cells growing in culture treated with vehicle (right 2 panels) or nocodazole (left 2 panels) and co-stained for DNA content and phosphor-histone H3 (Ser10). This shows that nocodazole traps cells in the M phase and that pH3 can be used to separate the G₂ and M phases.
At o2h discovery, we are experts in cellular and biochemical assays giving us the ability to combine the types of cell cycle and DNA ploidy analysis shown here with other assays including cell death and phenotypic screens, allowing us to provide you with excellent layers of depth of analysis with a high throughput in a bespoke manner to fit your needs.
To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.
