Induced cell cycle arrest is the use of a chemical or genetic manipulation to artificially halt progression through the cell cycle. Cellular processes like genome duplication and cell division stop. [1] It can be temporary or permanent. [1] It is an artificial activation of naturally occurring cell cycle checkpoints, induced by exogenous stimuli controlled by an experimenter.
In an academic research context, cell cycle arrest is typically performed in model organisms and cell extracts, such as Saccharomyces cerevisiae (yeast) or Xenopus oocytes (frog eggs). [2] [3] Frog egg cell extracts have been used extensively in cell cycle research because they are relatively large, reaching a diameter of 1mm, and so contain large amounts of protein, making protein levels more easily measurable. [4]
There are a variety of reasons a researcher may want to temporarily or permanently prevent progress through the cell cycle.
In some experiments, a researcher may want to control and synchronize the time when a group of cells progress to the next phase of the cell cycle. [5] The cells can be induced to arrest as they arrive (at different time points) at a certain phase, so that when the arrest is lifted (for instance, rescuing cell cycle progression by introducing another chemical) all the cells resume cell cycle progression at the same time. In addition to this method acting as a scientific control for when the cells resume the cell cycle, this can be used to investigate necessity and sufficiency.
Another reason synchrony is important is the control for amount of DNA content, which varies at different parts of the cell cycle based on whether DNA replication has occurred since the last round of completed mitosis and cytokinesis. [6]
Furthermore, synchronization of large numbers of cells into the same phase allows for the collection of large enough groups of cells in the same cycle for the use in other assays, such as western blot and RNA sequencing. [7]
Researchers may be investigating mechanisms of DNA damage repair. Given that some of the mechanisms below of inducing cell cycle arrest involve damaging the DNA, this allows investigation into how the cell responds to damage of its genetic material. [8]
Genetic engineering of cells with specific gene knockouts can also result in cells that arrest at different phases of the cell cycle. Examples include:
G1 phase is the first of the four phases of the cell cycle, and is part of interphase. While in G1 the cell synthesizes messenger RNA (mRNA) and proteins in preparation for subsequent steps of interphase leading to mitosis. In human somatic cells, the cell cycle lasts about 18 hours, and the G1 phase makes up about 1/3 of that time. [13] On the other hand, in frog, sea urchin, and fruit fly embryos, the G1 phase is extremely brief and instead is a slight gap between cytokinesis and S phase. [13]
α-factor is a pheromone secreted by Saccharomyces cerevisiae that arrests the yeast cells in G1 phase. It does so by inhibiting the enzyme adenylate cyclase. [2] The enzyme catalyzes the conversion of adenosine triphosphate (ATP) to 3',5'-cyclic AMP (cAMP) and pyrophosphate. [14]
Contact inhibition is a method of arresting cells when neighboring cells come into contact with each other. It results in a single layer of arrested cells of arrested cells, and is a process that is notably missing in cancer cells. The suspected mechanism is dependent on p27Kip1, a cyclin-dependent kinase inhibitor. [15] p27Kip1 protein levels are elevated in arresting cells. This natural process can be mimicked in a lab through the overexpression of p27Kip1, which results in induced cell cycle arrest in G1 phase. [16]
Mimosine is a plant amino acid that has been shown to reversibly inhibit progression beyond G1 phase in some human cells, including lymphoblastoid cells. [5] Its proposed mechanism of action is an iron/zinc chelator that depletes iron within the cell. This induces double-strand breaks in the DNA, inhibiting DNA replication. This may involve blocking the action of an iron-dependent ribonucleotide reductase. It may also inhibit transcription of serine hydroxymethyltransferase, which has zinc dependence. [17]
In cell culture, serum is the growth medium in which the cells are grown and contains vital nutrients. The use of serum deprivation - partially or completely removing the serum and its nutrients - has been shown to arrest and synchronize cell cycle progression in G0 phase, for example in neonatal mammalian astrocytes [18] and human foreskin fibroblasts. [19]
Amino acid starvation is a similar approach. When grown in a media without some essential amino acids, such as methionine, some cells arrest in early G1 phase. [5]
S phase follows G1 phase via the G1/S transition and precedes G2 phase in interphase and is the part of the cell cycle in which DNA is replicated. 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. Pre-replication complexes assembled before S phase are converted into active replication forks. [20] Driving this conversion is Cdc7 and S-phase cyclin-dependent kinases, which are both upregulated after the G1/S transition. [20]
Aphidicolin is an antibiotic isolated from the fungus Cephalosporum aphidicola. 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. [21] This S phase block induces apoptosis in HeLa cells. [5]
Hydroxyurea (HU) is a small molecule drug that inhibits the enzyme ribonucleotide reductase (RNR), preventing the catalysis of converting deoxyribonucleotides (DNTs) to ribonucleotides. It is hypothesized that there is tyrosyl free radical within RNR that is disabled by HU. [6] [22] The free radicals are necessary for the reduction of the DNTs and are scavenged by HU instead. [23] HU has been shown to arrest cells in both S phase (healthy cells) and immediately before cytokinesis (mutant cells). [22]
23-(2,3-dichlorophenoxy)propyl aminoethanol (2,3-DCPE) is a small-molecule that induces S phase arrest. [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. It will only be entered in regular cells if the DNA replication in S phase is completed successfully. It is a period of rapid cell growth and protein synthesis during which the cell prepares itself for mitosis.
Cyclins are proteins that control progression through the cell cycle by activating cyclin-dependent kinases. Destruction of a cell's endogenous cyclin messenger RNA can arrest frog egg extracts in interphase and prevent them from entering mitosis. [3] Introduction of exogenous cyclin mRNA is also sufficient to rescue cell cycle progression. [3] One method of this destruction is through the use of antisense oligonucleotides, pieces of RNA that bind to the cyclin mRNA and prevent the mRNA from being translated into cyclin protein. [25] This can actually be used to destroy phase-specific cyclins beyond just G2 - for instance, destruction of cyclin D1 mRNA by antisense oligonucleotides prevents progression from G1 phase to S phase. [26]
Mitosis is the final part of the cell cycle and follows interphase. 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. After successful mitosis, the cell physically splits into two identical daughter cells in a process called cytokinesis, and this concludes a full round of the cell cycle. Each of these new cells could then potentially re-enter G1 phase and begin the cell cycle again. [27]
Nocodazole is a chemical agent that interferes with the polymerization of microtubules. [28] Cells treated with nocodazole arrest with a G2 or M phase DNA content, which can be verified with flow cytometry. From microscopy it has been determined they do enter mitosis but they cannot form the spindles necessary for metaphase because the microtubules cannot polymerize. [29] Research into the mechanism has hinted at it potentially preventing tubulin from forming its alpha/beta heterodimer. [30]
Taxol works in the opposite way of nocodazole, instead stabilizing the microtubule polymer and preventing it from disassembly. It also causes M phase arrest, as the spindle that is supposed to pull apart sister chromatids is unable to disassemble. [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. [33]
Temperature has been shown to regulate HeLa cell cycle progression. Mitosis was found to be the most temperature-sensitive part of the cell cycle. [34] Pre-cytokinesis mitotic arrest was visible through accumulation of cells in mitosis in below-normal temperatures between 24 and 31 °C (75.2-87.8 °F). [34]
There are several methods that can be used to verify that cells have been arrested in the proper phase.
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. [35] The stronger the signal, the more of a particular protein is present. 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. [36]
Immunoblotting is the detection of specific proteins in a tissue sample or extract. Primary antibodies recognize and bind the protein in question, and secondary antibodies are added that recognize the primary antibodies. The secondary antibody is then visualized through staining or immunofluorescence, allowing indirect detection of the original target protein.
Immunoblotting can be performed to detect the presence of cyclins, proteins that regulate the cell cycle. [37] Different classes of cyclins are up- and down-regulated at different parts of the cell cycle. Measurement of the cyclins from an extract of an arrested cell can determine what phase the cell is in. For example, a peak of cyclin E protein would indicate the G1/S transition, a cyclin A peak would indicate late G2 phase, and a cyclin B peak would indicate mitosis. [38]
FUCCI is a system that takes advantage of cell cycle phase-specific expression of proteins and their degradation by the ubiquitin-proteasome pathway. Two fluorescent probes - Cdt1 and Geminin conjugated to fluorescent proteins - allow for real-time visualization of the cell cycle phase a cell is in. [39]
The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA and some of its organelles, and subsequently the partitioning of its cytoplasm, chromosomes and other components into two daughter cells in a process called cell division.
Cell division is the process by which a parent cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukaryotes, there are two distinct types of cell division: a vegetative division (mitosis), producing daughter cells genetically identical to the parent cell, and a cell division that produces haploid gametes for sexual reproduction (meiosis), reducing the number of chromosomes from two of each type in the diploid parent cell to one of each type in the daughter cells. Mitosis is a part of the cell cycle, in which, replicated chromosomes are separated into two new nuclei. Cell division gives rise to genetically identical cells in which the total number of chromosomes is maintained. In general, mitosis is preceded by the S stage of interphase and is followed by telophase and cytokinesis; which divides the cytoplasm, organelles, and cell membrane of one cell into two new cells containing roughly equal shares of these cellular components. The different stages of mitosis all together define the M phase of an animal cell cycle—the division of the mother cell into two genetically identical daughter cells. To ensure proper progression through the cell cycle, DNA damage is detected and repaired at various checkpoints throughout the cycle. These checkpoints can halt progression through the cell cycle by inhibiting certain cyclin-CDK complexes. Meiosis undergoes two divisions resulting in four haploid daughter cells. Homologous chromosomes are separated in the first division of meiosis, such that each daughter cell has one copy of each chromosome. These chromosomes have already been replicated and have two sister chromatids which are then separated during the second division of meiosis. Both of these cell division cycles are used in the process of sexual reproduction at some point in their life cycle. Both are believed to be present in the last eukaryotic common ancestor.
Interphase is the active portion of the cell cycle that includes the G1, S, and G2 phases, where the cell grows, replicates its DNA, and prepares for mitosis, respectively. Interphase was formerly called the "resting phase," but the cell in interphase is not simply dormant. Calling it so would be misleading since a cell in interphase is very busy synthesizing proteins, transcribing DNA into RNA, engulfing extracellular material, and processing signals, to name just a few activities. The cell is quiescent only in G0. Interphase is the phase of the cell cycle in which a typical cell spends most of its life. Interphase is the "daily living" or metabolic phase of the cell, in which the cell obtains nutrients and metabolizes them, grows, replicates its DNA in preparation for mitosis, and conducts other "normal" cell functions.
The G1 phase, gap 1 phase, or growth 1 phase, is the first of four phases of the cell cycle that takes place in eukaryotic cell division. In this part of interphase, the cell synthesizes mRNA and proteins in preparation for subsequent steps leading to mitosis. G1 phase ends when the cell moves into the S phase of interphase. Around 30 to 40 percent of cell cycle time is spent in the G1 phase.
Anaphase-promoting complex is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cullin (Apc2) and RING (Apc11) subunit much like SCF. Other parts of the APC/C have unknown functions but are highly conserved.
Cell growth refers to an increase in the total mass of a cell, including both cytoplasmic, nuclear and organelle volume. Cell growth occurs when the overall rate of cellular biosynthesis is greater than the overall rate of cellular degradation.
Cyclins are proteins that control the progression of a cell through the cell cycle by activating cyclin-dependent kinases (CDK).
S phase (Synthesis phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 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.
G2 phase, Gap 2 phase, or Growth 2 phase, is the third subphase of interphase in the cell cycle directly preceding mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated. G2 phase ends with the onset of prophase, the first phase of mitosis in which the cell’s chromatin condenses into chromosomes.
Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: the G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein.
The G1/S transition is a stage in the cell cycle at the boundary between the G1 phase, in which the cell grows, and the S phase, during which DNA is replicated. It is governed by cell cycle checkpoints to ensure cell cycle integrity and the subsequent S phase can pause in response to improperly or partially replicated DNA. During this transition the cell makes decisions to become quiescent, differentiate, make DNA repairs, or proliferate based on environmental cues and molecular signaling inputs. The G1/S transition occurs late in G1 and the absence or improper application of this highly regulated checkpoint can lead to cellular transformation and disease states such as cancer.
Cyclin A is a member of the cyclin family, a group of proteins that function in regulating progression through the cell cycle. The stages that a cell passes through that culminate in its division and replication are collectively known as the cell cycle Since the successful division and replication of a cell is essential for its survival, the cell cycle is tightly regulated by several components to ensure the efficient and error-free progression through the cell cycle. One such regulatory component is cyclin A which plays a role in the regulation of two different cell cycle stages.
A cyclin-dependent kinase inhibitor protein(also known as CKIs, CDIs, or CDKIs) is a protein that inhibits the enzyme cyclin-dependent kinase (CDK) and Cyclin activity by stopping the cell cycle if there are unfavorable conditions, therefore, acting as tumor suppressors. Cell cycle progression is stopped by Cyclin-dependent kinase inhibitor protein at the G1 phase. CKIs are vital proteins within the control system that point out whether the processes of DNA synthesis, mitosis, and cytokines control one another. When a malfunction hinders the successful completion of DNA synthesis in the G1 phase, it triggers a signal that delays or halts the progression to the S phase. Cyclin-dependent kinase inhibitor proteins are essential in the regulation of the cell cycle. If cell mutations surpass the cell cycle checkpoints during cell cycle regulation, it can result in various types of cancer.
Cell synchronization is a process by which cells in a culture at different stages of the cell cycle are brought to the same phase. Cell synchrony is a vital process in the study of cells progressing through the cell cycle as it allows population-wide data to be collected rather than relying solely on single-cell experiments. The types of synchronization are broadly categorized into two groups; physical fractionization and chemical blockade.
Cyclin-dependent kinase 1 also known as CDK1 or cell division cycle protein 2 homolog is a highly conserved protein that functions as a serine/threonine protein kinase, and is a key player in cell cycle regulation. It has been highly studied in the budding yeast S. cerevisiae, and the fission yeast S. pombe, where it is encoded by genes cdc28 and cdc2, respectively. With its cyclin partners, Cdk1 forms complexes that phosphorylate a variety of target substrates ; phosphorylation of these proteins leads to cell cycle progression.
Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in the fission yeast Schizosaccharomyces pombe. Wee1 has a molecular mass of 96 kDa and is a key regulator of cell cycle progression. It influences cell size by inhibiting the entry into mitosis, through inhibiting Cdk1. Wee1 has homologues in many other organisms, including mammals.
A series of biochemical switches control transitions between and within the various phases of the cell cycle. The cell cycle is a series of complex, ordered, sequential events that control how a single cell divides into two cells, and involves several different phases. The phases include the G1 and G2 phases, DNA replication or S phase, and the actual process of cell division, mitosis or M phase. During the M phase, the chromosomes separate and cytokinesis occurs.
Cdc14 and Cdc14 are a gene and its protein product respectively. Cdc14 is found in most of the eukaryotes. Cdc14 was defined by Hartwell in his famous screen for loci that control the cell cycle of Saccharomyces cerevisiae. Cdc14 was later shown to encode a protein phosphatase. Cdc14 is dual-specificity, which means it has serine/threonine and tyrosine-directed activity. A preference for serines next to proline is reported. Many early studies, especially in the budding yeast Saccharomyces cerevisiae, demonstrated that the protein plays a key role in regulating late mitotic processes. However, more recent work in a range of systems suggests that its cellular function is more complex.
In cell biology, eukaryotes possess a regulatory system that ensures that DNA replication occurs only once per cell cycle.
The G2-M DNA damage checkpoint is an important cell cycle checkpoint in eukaryotic organisms that ensures that cells don't initiate mitosis until damaged or incompletely replicated DNA is sufficiently repaired. Cells with a defective G2-M checkpoint will undergo apoptosis or death after cell division if they enter the M phase before repairing their DNA. The defining biochemical feature of this checkpoint is the activation of M-phase cyclin-CDK complexes, which phosphorylate proteins that promote spindle assembly and bring the cell to metaphase.
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