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The Cell Cycle Explained, and How to Study it

Heather Brown
Tags: Successful Research Tips,

What is the cell cycle?

All living things consist of a diverse collection of cells, the membrane-bound fundamental unit of life. Cells function autonomously and non-autonomously within the context of their surrounding biology to enable life and carry out essential functions such as immunity, growth, and reproduction. Excluding quiescent cells, every cell, whether a unicellular organism (e.g. protozoa) or a multicellular organism (e.g. human) undergoes the evolutionarily conserved cell cycle of life. The main objective of the cell cycle is for the parent cell to yield genetically identical daughter cells (Figure 1).



Figure1. Overview of the cell cycle. Adapted from “Cell Division Cycle-Mitosis”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates.


To accomplish this, the cell cycle consists of two distinct phases: interphase and mitotic phase. Within interphase, there are additional sub-phases of G1 (Gap 1), S (Synthesis), and G2 (Gap 2). Similarly, the mitotic phase is further divided into P (Prophase), M (Metaphase), A (Anaphase), and T (Telophase). Each phase and sub-phase is characterized by specific molecular events that steer the cell through the cell cycle, described in further detail below.


Interphase

Interphase is when cell growth and DNA synthesis occur. Throughout this phase, there are cellular checkpoints to ensure nutrient availability, transcription and translation of critical cell cycle components, and accurate DNA replication for advancement through the cell cycle. These checkpoints will either inhibit entry into the next phase of the cell cycle or permit the progression of the cell cycle.
  • G1: Also called Gap 1, is the first sub-phase of interphase after the previous mitosis has completed. The cell prepares to divide again by accumulating energy and producing proteins required for DNA replication. The major checkpoint at this stage is the G1/S checkpoint that ensures the cell has made sufficient organelles and growth factors for replication and division. The cell will fail to pass this checkpoint if there is insufficient ATP or DNA damage present (Figure 2A).

  • S: In the synthesis sub-phase, the cell synthesizes a new copy of DNA. Cyclin A and Cyclin E bind cyclin-dependent kinase 2 (CDK2), which enables the phosphorylation of S-phase target proteins to up-regulate proteins required for accurate DNA synthesis, such as histones. The significant checkpoints in the S phase check for DNA structure, which is mediated via the ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related protein) protein kinases and their downstream targets checkpoint kinase 1 (CHK1) and/or CHK2. The S phase is arrested if an aberrant DNA structure is detected (Figure 2B).

  • G2: The Gap 2 sub-phase is when the cell organizes/condenses the genetic material and prepares to divide. The cell also continues to grow, accumulate nutrients, and ensure correct DNA replication/synthesis. The primary cell cycle checkpoint involved is the G2/S checkpoint which evaluates the cell size, nutrient availability, and integrity of the DNA. If the cell experiences DNA damage or insufficient volume, the cell is arrested in the G2 phase and is not a candidate for cell division (Figure 2C).


Mitotic Phase

The process of cell division occurs in the mitotic phase. The cell confronts the M checkpoint, which is the last checkpoint to assess the arrangement of the copied chromosomes to facilitate division into two daughter cells.
  • P: In prophase, the chromosomes condense, divide into two chromatids joined by the centromere, and move towards opposite poles of the cell. The centriole also divides and migrates to opposite poles of the cell (Figure 1B).

  • M: Metaphase is characterized by the chromatids lining up along the metaphase plate anchored by their respective centriole via microtubules ready to be pulled apart (Figure 1C).

  • A: The anaphase-promoting complex (APC) facilitates the cell's entry into anaphase. In anaphase, the microtubules attached to the chromatids progressively shorten, thus splitting the chromatids into two sister chromatids that will become the chromosomes of the daughter cells (Figure 1D).

  • T: Telophase is characterized by the induction of two new nuclear envelopes that form around the chromosomes to form two distinct daughter nuclei (Figure 1E). Cytokinesis then split the cytoplasm and organelles into two identical and separate daughter cells (Figure 1F).



Figure 2. Cell cycle checkpoints. The major checkpoints are G1/S checkpoint (A), synthesis checkpoint (B), G2/M checkpoint (C), and M/G1 checkpoint (D). Adapted from “Cell Cycle Checkpoints”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates.



How is the cell cycle regulated?

The cell cycle is spatially and temporally regulated through complex genetic networks. This intricate network of pathways regulates cell cycle checkpoints, DNA repair mechanisms, genetic recombination, and programmed cell death. Dysfunction at any step throughout this cascade can cause abnormal cell division/proliferation. Dysfunction of the cell cycle underlies many human pathological conditions, such as cancer; thus, a crucial step to understanding these conditions is to reveal the mechanisms underlying alterations in cell cycle progression that result in dysfunction.
Schematic showing the different cyclins that are expressed in each sub-phase of the cell cycle

Figure 3. Schematic showing the different cyclins that are expressed in each sub-phase of the cell cycle. Adapted from “Cyclins - Cell Cycle Regulators”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates

As described above, the cells face several cell cycle checkpoints as they advance through the cell cycle. Cyclins play a significant role in regulating these checkpoints because they bind to cyclin-dependent kinases (CDKs), which phosphorylate target proteins to activate them. Cyclins and CDKs are evolutionarily conserved and are present in many species. Specific cyclins and CDKs are expressed in each phase of the cell cycle, resulting in the activation of proteins required for the progression of that particular phase of the cell cycle (Figure 3).

Another essential protein that regulates the cell cycle is p53 (Proto-oncogene 53). p53 works on multiple levels to ensure that cells do not pass on their damaged DNA through cell division. p53 stops the cell cycle at the G1/S checkpoint by triggering the production of CDK inhibitor (CKI) proteins. The CKI proteins bind to cyclin-CDK complexes and block their activity so that the cell can accomplish DNA repair via the activation of DNA repair enzymes. If DNA damage is irreparable, p53 initiates apoptosis (programmed cell death) to avoid passing on damaged DNA. Apoptosis is a critical component of the cell cycle to rid the organism of potentially hazardous cells so they do not continue to proliferate.

Senescent cells are characterized by G1 growth arrest involving the repression of genes that drive cell cycle progression and the up-regulation of cell cycle inhibitors. While cellular senescence increases with age and can contribute to many age-related diseases, cellular senescence can also be a protective mechanism to avoid the malignant transformation of damaged cells. Typically, mature cells do not undergo cell division (such as mature somatic cells). However, cells can come out of and enter the cell cycle in the context of injury and repair. For example, if exposed to a mitogen, a cell in senescence will re-enter the cell cycle.


What types of diseases are a result of a disrupted cell cycle?

Many human diseases are rooted in cell cycle dysfunction. For example, cancer, kidney disease, and Alzheimer's disease have cell cycle dysfunction etiology. Specifically, neuroblastoma is one of the most common solid tumors diagnosed in children and is characterized by its uncontrolled proliferation in neural tissue. Holzer et al. showed that neuroblastoma's progression is partly due to the up-regulation of the proto-oncogene N-MYC and can be successfully treated with the drug Nifurtimox. They used the NUCLEAR-ID® Green cell cycle kit with flow cytometry analysis to reveal that neuroblastoma cells treated with Nifurtimox are arrested in the G2/M phase since the drug reduces the expression of N-MYC in the cells. This study revealed a potential treatment for neuroblastoma.

Further, atherosclerotic renal artery stenosis (kidney disease) is partly driven by cellular senescence, resulting in the pathological decline of the regenerative capacity of mesenchymal stem cells (MSCs) and scarring/deterioration of renal function. Hermann et al. used the Cellular senescence activity assay to show that hypoxia pre-conditioning increases regenerative capacity, thus avoiding cellular senescence in MSCs in the kidney that may serve as a potential treatment for nephropathies.

Last, central nervous system disorders (CNS) can result from dysregulation of the intrinsic cellular programs that mediate the progression of the cell cycle and cell death. For example, cell cycle checkpoint proteins are essential players in the pathogenesis of Alzheimer's disease. Specifically, Helal et al. used the cell cycle checkpoint inhibitor, BML-259, to show that chemical inhibition of CDK5 resulted in preventing tau hyper-phosphorylation and neurofibrillary tangle formation, providing an additional target/candidatein drug development to treat Alzheimer's.


How can one study the cell cycle? What are the Pro Tips?



Figure 4. Live cells treated with different drugs show inhibition of cell cycle progression at different phases. Testing by flow cytometry using NUCLEAR-ID® Red cell cycle kit (GFP-CERTIFIED®)
Studying the cell cycle in healthy and disease contexts helps advance research and lead to discoveries that can translate into treatments and cures for diseases of the cell cycle. Recent advancements in technology allow researchers to study the cell cycle in detail. For example, Enzo's NUCLEAR-ID® Red cell cycle kit (GFP-CERTIFIED®) (ENZ-51008) is convenient for studying cell cycle progression by various applications such as flow cytometry without any pre-treatment with RNase. The data in Figure 4 show drug treatments arresting the cell cycle at different phases in a cancer cell line. The data were generated using this kit by flow cytometry. Pro tips are described below to maximize success when studying the cell cycle.

Pro Tip: Always ensure you use reliable and robust controls in your cell cycle experiments. For example, Reveromycin A (ALX-380-216) is a potent G1-cell cycle inhibitor, or use the controls included with your Enzo kit. The controls will reveal if your methods are sufficient (i.e., your experiment worked), and they will give you a vantage point to compare your experimental groups and assess differences.

Pro Tip: Make sure your cells are healthy for your experiments. For example, if you are screening compounds that arrest the cell cycle as a cancer treatment, make sure that your cells begin healthily and in the log phase of growth so that your results are not confounded with the fact that your cells were already dying or sick. Reagents such as cell viability assays can help to test for healthy cells. This may require some optimization to define appropriate parameters and select the right media, etc.


Continuing research on the cell cycle is essential to provide insight into the etiology of many diseases and provide a foundation for developing treatments in the future. Enzo's solutions to study the cell cycle are cited in hundreds of peer-reviewed publications, and are trusted by the research and medical community. Do you need help studying the cell cycle? Contact our application scientists today!

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References:

  1. Zhivotovsky, B. and Orrenius, S. (2010), Cell cycle and cell death in disease: past, present and future. Journal of Internal Medicine, 268: 395-409. https://doi.org/10.1111/j.1365-2796.2010.02282.x
  2. Bartee, L., Shriner, W., & Creech, C. (n.d.). Control of the Cell Cycle. In Principles of Biology. essay, Open Oregon Educational Resources.
  3. Cobb, L., 2021. The Cell Cycle Analysis. [online] Labome.com. Available at: https://www.labome.com/method/The-Cell-Cycle-Analysis.html.
  4. Cabanillas Stanchi, K. M., Bruchelt, G., Handgretinger, R., & Holzer, U. (2015). Nifurtimox reduces N-Myc expression and aerobic glycolysis in neuroblastoma. Cancer biology & therapy, 16(9), 1353–1363. https://doi.org/10.1080/15384047.2015.1070987
  5. Diabetic Kidney Disease Alters the Transcriptome and Function of Human Adipose-Derived Mesenchymal Stromal Cells but Maintains Immunomodulatory and Paracrine Activities Important for Renal Repair | Diabetes.
  6. Helal, C. J., Sanner, M. A., Cooper, C. B., Gant, T., Adam, M., Lucas, J. C., Kang, Z., Kupchinsky, S., Ahlijanian, M. K., Tate, B., Menniti, F. S., Kelly, K., & Peterson, M. (2004, September 29). Discovery and SAR of 2-aminothiazole inhibitors of cyclin-dependent kinase 5/P25 as a potential treatment for alzheimer's disease. Bioorganic & Medicinal Chemistry Letters. Retrieved November 19, 2021, from https://www.sciencedirect.com/science/article/pii/S0960894X04011060?via%3Dihub.

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