Cellular differentiation

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Cellular_differentiation 

Stem cell differentiation into various tissue types.

 

Cell-count distribution featuring cellular differentiation for three types of cells (progenitor ${\displaystyle z}$, osteoblast ${\displaystyle y}$, and chondrocyte ${\displaystyle x}$) exposed to pro-osteoblast stimulus.

Cellular differentiation is the process in which a cell changes from one cell type to another. Usually, the cell changes to a more specialized type. Differentiation occurs numerous times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Some differentiation occurs in response to antigen exposure. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly controlled modifications in gene expression and are the study of epigenetics. With a few exceptions, cellular differentiation almost never involves a change in the DNA sequence itself. Although metabolic composition does get altered quite dramatically where stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. Thus, different cells can have very different physical characteristics despite having the same genome.

A specialized type of differentiation, known as terminal differentiation, is of importance in some tissues, for example vertebrate nervous system, striated muscle, epidermis and gut. During terminal differentiation, a precursor cell formerly capable of cell division, permanently leaves the cell cycle, dismantles the cell cycle machinery and often expresses a range of genes characteristic of the cell's final function (e.g. myosin and actin for a muscle cell). Differentiation may continue to occur after terminal differentiation if the capacity and functions of the cell undergo further changes.

Among dividing cells, there are multiple levels of cell potency, the cell's ability to differentiate into other cell types. A greater potency indicates a larger number of cell types that can be derived. A cell that can differentiate into all cell types, including the placental tissue, is known as totipotent. In mammals, only the zygote and subsequent blastomeres are totipotent, while in plants, many differentiated cells can become totipotent with simple laboratory techniques. A cell that can differentiate into all cell types of the adult organism is known as pluripotent. Such cells are called meristematic cells in higher plants and embryonic stem cells in animals, though some groups report the presence of adult pluripotent cells. Virally induced expression of four transcription factors Oct4, Sox2, c-Myc, and Klf4 (Yamanaka factors) is sufficient to create pluripotent (iPS) cells from adult fibroblasts. A multipotent cell is one that can differentiate into multiple different, but closely related cell types. Oligopotent cells are more restricted than multipotent, but can still differentiate into a few closely related cell types. Finally, unipotent cells can differentiate into only one cell type, but are capable of self-renewal. In cytopathology, the level of cellular differentiation is used as a measure of cancer progression. "Grade" is a marker of how differentiated a cell in a tumor is.

Mammalian cell types

See also: List of distinct cell types in the adult human body

Three basic categories of cells make up the mammalian body: germ cells, somatic cells, and stem cells. Each of the approximately 37.2 trillion (3.72x10¹³) cells in an adult human has its own copy or copies of the genome except certain cell types, such as red blood cells, that lack nuclei in their fully differentiated state. Most cells are diploid; they have two copies of each chromosome. Such cells, called somatic cells, make up most of the human body, such as skin and muscle cells. Cells differentiate to specialize for different functions.

Germ line cells are any line of cells that give rise to gametes—eggs and sperm—and thus are continuous through the generations. Stem cells, on the other hand, have the ability to divide for indefinite periods and to give rise to specialized cells. They are best described in the context of normal human development.

Development begins when a sperm fertilizes an egg and creates a single cell that has the potential to form an entire organism. In the first hours after fertilization, this cell divides into identical cells. In humans, approximately four days after fertilization and after several cycles of cell division, these cells begin to specialize, forming a hollow sphere of cells, called a blastocyst. The blastocyst has an outer layer of cells, and inside this hollow sphere, there is a cluster of cells called the inner cell mass. The cells of the inner cell mass go on to form virtually all of the tissues of the human body. Although the cells of the inner cell mass can form virtually every type of cell found in the human body, they cannot form an organism. These cells are referred to as pluripotent.

Pluripotent stem cells undergo further specialization into multipotent progenitor cells that then give rise to functional cells. Examples of stem and progenitor cells include:

• Radial glial cells (embryonic neural stem cells) that give rise to excitatory neurons in the fetal brain through the process of neurogenesis.
• Hematopoietic stem cells (adult stem cells) from the bone marrow that give rise to red blood cells, white blood cells, and platelets
• Mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells
• Epithelial stem cells (progenitor cells) that give rise to the various types of skin cells
• Muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue.

A pathway that is guided by the cell adhesion molecules consisting of four amino acids, arginine, glycine, asparagine, and serine, is created as the cellular blastomere differentiates from the single-layered blastula to the three primary layers of germ cells in mammals, namely the ectoderm, mesoderm and endoderm (listed from most distal (exterior) to proximal (interior)). The ectoderm ends up forming the skin and the nervous system, the mesoderm forms the bones and muscular tissue, and the endoderm forms the internal organ tissues.

Dedifferentiation

Micrograph of a liposarcoma with some dedifferentiation, that is not identifiable as a liposarcoma, (left edge of image) and a differentiated component (with lipoblasts and increased vascularity (right of image)). Fully differentiated (morphologically benign) adipose tissue (center of the image) has few blood vessels. H&E stain.

Dedifferentiation, or integration, is a cellular process often seen in more basal life forms such as worms and amphibians in which a partially or terminally differentiated cell reverts to an earlier developmental stage, usually as part of a regenerative process. Dedifferentiation also occurs in plants. Cells in cell culture can lose properties they originally had, such as protein expression, or change shape. This process is also termed dedifferentiation.

Some believe dedifferentiation is an aberration of the normal development cycle that results in cancer, whereas others believe it to be a natural part of the immune response lost by humans at some point as a result of evolution.

A small molecule dubbed reversine, a purine analog, has been discovered that has proven to induce dedifferentiation in myotubes. These dedifferentiated cells could then redifferentiate into osteoblasts and adipocytes.

Diagram exposing several methods used to revert adult somatic cells to totipotency or pluripotency.

Mechanisms

See also: Embryonic differentiation waves

Mechanisms of cellular differentiation.

Each specialized cell type in an organism expresses a subset of all the genes that constitute the genome of that species. Each cell type is defined by its particular pattern of regulated gene expression. Cell differentiation is thus a transition of a cell from one cell type to another and it involves a switch from one pattern of gene expression to another. Cellular differentiation during development can be understood as the result of a gene regulatory network. A regulatory gene and its cis-regulatory modules are nodes in a gene regulatory network; they receive input and create output elsewhere in the network. The systems biology approach to developmental biology emphasizes the importance of investigating how developmental mechanisms interact to produce predictable patterns (morphogenesis). However, an alternative view has been proposed recently. Based on stochastic gene expression, cellular differentiation is the result of a Darwinian selective process occurring among cells. In this frame, protein and gene networks are the result of cellular processes and not their cause.

An overview of major signal transduction pathways.

While evolutionarily conserved molecular processes are involved in the cellular mechanisms underlying these switches, in animal species these are very different from the well-characterized gene regulatory mechanisms of bacteria, and even from those of the animals' closest unicellular relatives. Specifically, cell differentiation in animals is highly dependent on biomolecular condensates of regulatory proteins and enhancer DNA sequences.

Cellular differentiation is often controlled by cell signaling. Many of the signal molecules that convey information from cell to cell during the control of cellular differentiation are called growth factors. Although the details of specific signal transduction pathways vary, these pathways often share the following general steps. A ligand produced by one cell binds to a receptor in the extracellular region of another cell, inducing a conformational change in the receptor. The shape of the cytoplasmic domain of the receptor changes, and the receptor acquires enzymatic activity. The receptor then catalyzes reactions that phosphorylate other proteins, activating them. A cascade of phosphorylation reactions eventually activates a dormant transcription factor or cytoskeletal protein, thus contributing to the differentiation process in the target cell. Cells and tissues can vary in competence, their ability to respond to external signals.

Signal induction refers to cascades of signaling events, during which a cell or tissue signals to another cell or tissue to influence its developmental fate. Yamamoto and Jeffery investigated the role of the lens in eye formation in cave- and surface-dwelling fish, a striking example of induction. Through reciprocal transplants, Yamamoto and Jeffery found that the lens vesicle of surface fish can induce other parts of the eye to develop in cave- and surface-dwelling fish, while the lens vesicle of the cave-dwelling fish cannot.

Other important mechanisms fall under the category of asymmetric cell divisions, divisions that give rise to daughter cells with distinct developmental fates. Asymmetric cell divisions can occur because of asymmetrically expressed maternal cytoplasmic determinants or because of signaling. In the former mechanism, distinct daughter cells are created during cytokinesis because of an uneven distribution of regulatory molecules in the parent cell; the distinct cytoplasm that each daughter cell inherits results in a distinct pattern of differentiation for each daughter cell. A well-studied example of pattern formation by asymmetric divisions is body axis patterning in Drosophila. RNA molecules are an important type of intracellular differentiation control signal. The molecular and genetic basis of asymmetric cell divisions has also been studied in green algae of the genus Volvox, a model system for studying how unicellular organisms can evolve into multicellular organisms. In Volvox carteri, the 16 cells in the anterior hemisphere of a 32-cell embryo divide asymmetrically, each producing one large and one small daughter cell. The size of the cell at the end of all cell divisions determines whether it becomes a specialized germ or somatic cell.

Epigenetic control

Main article: Epigenetics in stem cell differentiation

Since each cell, regardless of cell type, possesses the same genome, determination of cell type must occur at the level of gene expression. While the regulation of gene expression can occur through cis- and trans-regulatory elements including a gene's promoter and enhancers, the problem arises as to how this expression pattern is maintained over numerous generations of cell division. As it turns out, epigenetic processes play a crucial role in regulating the decision to adopt a stem, progenitor, or mature cell fate. This section will focus primarily on mammalian stem cells.

In systems biology and mathematical modeling of gene regulatory networks, cell-fate determination is predicted to exhibit certain dynamics, such as attractor-convergence (the attractor can be an equilibrium point, limit cycle or strange attractor) or oscillatory.

Importance of epigenetic control

The first question that can be asked is the extent and complexity of the role of epigenetic processes in the determination of cell fate. A clear answer to this question can be seen in the 2011 paper by Lister R, et al.  on aberrant epigenomic programming in human induced pluripotent stem cells. As induced pluripotent stem cells (iPSCs) are thought to mimic embryonic stem cells in their pluripotent properties, few epigenetic differences should exist between them. To test this prediction, the authors conducted whole-genome profiling of DNA methylation patterns in several human embryonic stem cell (ESC), iPSC, and progenitor cell lines.

Female adipose cells, lung fibroblasts, and foreskin fibroblasts were reprogrammed into induced pluripotent state with the OCT4, SOX2, KLF4, and MYC genes. Patterns of DNA methylation in ESCs, iPSCs, somatic cells were compared. Lister R, et al. observed significant resemblance in methylation levels between embryonic and induced pluripotent cells. Around 80% of CG dinucleotides in ESCs and iPSCs were methylated, the same was true of only 60% of CG dinucleotides in somatic cells. In addition, somatic cells possessed minimal levels of cytosine methylation in non-CG dinucleotides, while induced pluripotent cells possessed similar levels of methylation as embryonic stem cells, between 0.5 and 1.5%. Thus, consistent with their respective transcriptional activities, DNA methylation patterns, at least on the genomic level, are similar between ESCs and iPSCs.

However, upon examining methylation patterns more closely, the authors discovered 1175 regions of differential CG dinucleotide methylation between at least one ES or iPS cell line. By comparing these regions of differential methylation with regions of cytosine methylation in the original somatic cells, 44-49% of differentially methylated regions reflected methylation patterns of the respective progenitor somatic cells, while 51-56% of these regions were dissimilar to both the progenitor and embryonic cell lines. In vitro-induced differentiation of iPSC lines saw transmission of 88% and 46% of hyper and hypo-methylated differentially methylated regions, respectively.

Two conclusions are readily apparent from this study. First, epigenetic processes are heavily involved in cell fate determination, as seen from the similar levels of cytosine methylation between induced pluripotent and embryonic stem cells, consistent with their respective patterns of transcription. Second, the mechanisms of reprogramming (and by extension, differentiation) are very complex and cannot be easily duplicated, as seen by the significant number of differentially methylated regions between ES and iPS cell lines. Now that these two points have been established, we can examine some of the epigenetic mechanisms that are thought to regulate cellular differentiation.

Mechanisms of epigenetic regulation

Pioneer factors (Oct4, Sox2, Nanog)

Three transcription factors, OCT4, SOX2, and NANOG – the first two of which are used in induced pluripotent stem cell (iPSC) reprogramming, along with Klf4 and c-Myc – are highly expressed in undifferentiated embryonic stem cells and are necessary for the maintenance of their pluripotency. It is thought that they achieve this through alterations in chromatin structure, such as histone modification and DNA methylation, to restrict or permit the transcription of target genes. While highly expressed, their levels require a precise balance to maintain pluripotency, perturbation of which will promote differentiation towards different lineages based on how the gene expression levels change. Differential regulation of Oct-4 and SOX2 levels have been shown to precede germ layer fate selection. Increased levels of Oct4 and decreased levels of Sox2 promote a mesendodermal fate, with Oct4 actively suppressing genes associated with a neural ectodermal fate. Similarly, Increased levels of Sox2 and decreased levels of Oct4 promote differentiation towards a neural ectodermal fate, with Sox2 inhibiting differentiation towards a mesendodermal fate. Regardless of the lineage cells differentiate down, suppression of NANOG has been identified as a necessary prerequisite for differentiation.

Polycomb repressive complex (PRC2)

In the realm of gene silencing, Polycomb repressive complex 2, one of two classes of the Polycomb group (PcG) family of proteins, catalyzes the di- and tri-methylation of histone H3 lysine 27 (H3K27me2/me3). By binding to the H3K27me2/3-tagged nucleosome, PRC1 (also a complex of PcG family proteins) catalyzes the mono-ubiquitinylation of histone H2A at lysine 119 (H2AK119Ub1), blocking RNA polymerase II activity and resulting in transcriptional suppression. PcG knockout ES cells do not differentiate efficiently into the three germ layers, and deletion of the PRC1 and PRC2 genes leads to increased expression of lineage-affiliated genes and unscheduled differentiation. Presumably, PcG complexes are responsible for transcriptionally repressing differentiation and development-promoting genes.

Trithorax group proteins (TrxG)

Alternately, upon receiving differentiation signals, PcG proteins are recruited to promoters of pluripotency transcription factors. PcG-deficient ES cells can begin differentiation but cannot maintain the differentiated phenotype. Simultaneously, differentiation and development-promoting genes are activated by Trithorax group (TrxG) chromatin regulators and lose their repression. TrxG proteins are recruited at regions of high transcriptional activity, where they catalyze the trimethylation of histone H3 lysine 4 (H3K4me3) and promote gene activation through histone acetylation. PcG and TrxG complexes engage in direct competition and are thought to be functionally antagonistic, creating at differentiation and development-promoting loci what is termed a "bivalent domain" and rendering these genes sensitive to rapid induction or repression.

DNA methylation

Regulation of gene expression is further achieved through DNA methylation, in which the DNA methyltransferase-mediated methylation of cytosine residues in CpG dinucleotides maintains heritable repression by controlling DNA accessibility. The majority of CpG sites in embryonic stem cells are unmethylated and appear to be associated with H3K4me3-carrying nucleosomes. Upon differentiation, a small number of genes, including OCT4 and NANOG, are methylated and their promoters repressed to prevent their further expression. Consistently, DNA methylation-deficient embryonic stem cells rapidly enter apoptosis upon in vitro differentiation.

Nucleosome positioning

While the DNA sequence of most cells of an organism is the same, the binding patterns of transcription factors and the corresponding gene expression patterns are different. To a large extent, differences in transcription factor binding are determined by the chromatin accessibility of their binding sites through histone modification and/or pioneer factors. In particular, it is important to know whether a nucleosome is covering a given genomic binding site or not. This can be determined using a chromatin immunoprecipitation (ChIP) assay.

Histone acetylation and methylation

DNA-nucleosome interactions are characterized by two states: either tightly bound by nucleosomes and transcriptionally inactive, called heterochromatin, or loosely bound and usually, but not always, transcriptionally active, called euchromatin. The epigenetic processes of histone methylation and acetylation, and their inverses demethylation and deacetylation primarily account for these changes. The effects of acetylation and deacetylation are more predictable. An acetyl group is either added to or removed from the positively charged Lysine residues in histones by enzymes called histone acetyltransferases or histone deacteylases, respectively. The acetyl group prevents Lysine's association with the negatively charged DNA backbone. Methylation is not as straightforward, as neither methylation nor demethylation consistently correlate with either gene activation or repression. However, certain methylations have been repeatedly shown to either activate or repress genes. The trimethylation of lysine 4 on histone 3 (H3K4Me3) is associated with gene activation, whereas trimethylation of lysine 27 on histone 3 represses genes.

In stem cells

During differentiation, stem cells change their gene expression profiles. Recent studies have implicated a role for nucleosome positioning and histone modifications during this process. There are two components of this process: turning off the expression of embryonic stem cell (ESC) genes, and the activation of cell fate genes. Lysine specific demethylase 1 (KDM1A) is thought to prevent the use of enhancer regions of pluripotency genes, thereby inhibiting their transcription. It interacts with Mi-2/NuRD complex (nucleosome remodelling and histone deacetylase) complex, giving an instance where methylation and acetylation are not discrete and mutually exclusive, but intertwined processes.

Role of signaling in epigenetic control

A final question to ask concerns the role of cell signaling in influencing the epigenetic processes governing differentiation. Such a role should exist, as it would be reasonable to think that extrinsic signaling can lead to epigenetic remodeling, just as it can lead to changes in gene expression through the activation or repression of different transcription factors. Little direct data is available concerning the specific signals that influence the epigenome, and the majority of current knowledge about the subject consists of speculations on plausible candidate regulators of epigenetic remodeling. We will first discuss several major candidates thought to be involved in the induction and maintenance of both embryonic stem cells and their differentiated progeny, and then turn to one example of specific signaling pathways in which more direct evidence exists for its role in epigenetic change.

The first major candidate is Wnt signaling pathway. The Wnt pathway is involved in all stages of differentiation, and the ligand Wnt3a can substitute for the overexpression of c-Myc in the generation of induced pluripotent stem cells. On the other hand, disruption of β-catenin, a component of the Wnt signaling pathway, leads to decreased proliferation of neural progenitors.

Growth factors comprise the second major set of candidates of epigenetic regulators of cellular differentiation. These morphogens are crucial for development, and include bone morphogenetic proteins, transforming growth factors (TGFs), and fibroblast growth factors (FGFs). TGFs and FGFs have been shown to sustain expression of OCT4, SOX2, and NANOG by downstream signaling to Smad proteins. Depletion of growth factors promotes the differentiation of ESCs, while genes with bivalent chromatin can become either more restrictive or permissive in their transcription.

Several other signaling pathways are also considered to be primary candidates. Cytokine leukemia inhibitory factors are associated with the maintenance of mouse ESCs in an undifferentiated state. This is achieved through its activation of the Jak-STAT3 pathway, which has been shown to be necessary and sufficient towards maintaining mouse ESC pluripotency. Retinoic acid can induce differentiation of human and mouse ESCs, and Notch signaling is involved in the proliferation and self-renewal of stem cells. Finally, Sonic hedgehog, in addition to its role as a morphogen, promotes embryonic stem cell differentiation and the self-renewal of somatic stem cells.

The problem, of course, is that the candidacy of these signaling pathways was inferred primarily on the basis of their role in development and cellular differentiation. While epigenetic regulation is necessary for driving cellular differentiation, they are certainly not sufficient for this process. Direct modulation of gene expression through modification of transcription factors plays a key role that must be distinguished from heritable epigenetic changes that can persist even in the absence of the original environmental signals. Only a few examples of signaling pathways leading to epigenetic changes that alter cell fate currently exist, and we will focus on one of them.

Expression of Shh (Sonic hedgehog) upregulates the production of BMI1, a component of the PcG complex that recognizes H3K27me3. This occurs in a Gli-dependent manner, as Gli1 and Gli2 are downstream effectors of the Hedgehog signaling pathway. In culture, Bmi1 mediates the Hedgehog pathway's ability to promote human mammary stem cell self-renewal. In both humans and mice, researchers showed Bmi1 to be highly expressed in proliferating immature cerebellar granule cell precursors. When Bmi1 was knocked out in mice, impaired cerebellar development resulted, leading to significant reductions in postnatal brain mass along with abnormalities in motor control and behavior.^[42] A separate study showed a significant decrease in neural stem cell proliferation along with increased astrocyte proliferation in Bmi null mice.

An alternative model of cellular differentiation during embryogenesis is that positional information is based on mechanical signalling by the cytoskeleton using Embryonic differentiation waves. The mechanical signal is then epigenetically transduced via signal transduction systems (of which specific molecules such as Wnt are part) to result in differential gene expression.

In summary, the role of signaling in the epigenetic control of cell fate in mammals is largely unknown, but distinct examples exist that indicate the likely existence of further such mechanisms.

Effect of matrix elasticity

In order to fulfill the purpose of regenerating a variety of tissues, adult stems are known to migrate from their niches, adhere to new extracellular matrices (ECM) and differentiate. The ductility of these microenvironments are unique to different tissue types. The ECM surrounding brain, muscle and bone tissues range from soft to stiff. The transduction of the stem cells into these cells types is not directed solely by chemokine cues and cell to cell signaling. The elasticity of the microenvironment can also affect the differentiation of mesenchymal stem cells (MSCs which originate in bone marrow.) When MSCs are placed on substrates of the same stiffness as brain, muscle and bone ECM, the MSCs take on properties of those respective cell types. Matrix sensing requires the cell to pull against the matrix at focal adhesions, which triggers a cellular mechano-transducer to generate a signal to be informed what force is needed to deform the matrix. To determine the key players in matrix-elasticity-driven lineage specification in MSCs, different matrix microenvironments were mimicked. From these experiments, it was concluded that focal adhesions of the MSCs were the cellular mechano-transducer sensing the differences of the matrix elasticity. The non-muscle myosin IIa-c isoforms generates the forces in the cell that lead to signaling of early commitment markers. Nonmuscle myosin IIa generates the least force increasing to non-muscle myosin IIc. There are also factors in the cell that inhibit non-muscle myosin II, such as blebbistatin. This makes the cell effectively blind to the surrounding matrix. Researchers have obtained some success in inducing stem cell-like properties in HEK 239 cells by providing a soft matrix without the use of diffusing factors. The stem-cell properties appear to be linked to tension in the cells' actin network. One identified mechanism for matrix-induced differentiation is tension-induced proteins, which remodel chromatin in response to mechanical stretch. The RhoA pathway is also implicated in this process.

Evolutionary history

See also: Bangiomorpha

A billion-years-old, likely holozoan, protist, Bicellum brasieri with two types of cells, shows that the evolution of differentiated multicellularity, possibly but not necessarily of animal lineages, occurred at least 1 billion years ago and possibly mainly in freshwater lakes rather than the ocean.

Cell cycle

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Cell_cycle 

Life cycle of the cell

 

Onion (Allium) cells in different phases of the cell cycle. Growth in an 'organism' is carefully controlled by regulating the cell cycle.

 

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Cell cycle in Deinococcus radiodurans

The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subsequently the partitioning of its cytoplasm and other components into two daughter cells in a process called cell division.

In cells with nuclei (eukaryotes), (i.e., animal, plant, fungal, and protist cells), the cell cycle is divided into two main stages: interphase and the mitotic (M) phase (including mitosis and cytokinesis). During interphase, the cell grows, accumulating nutrients needed for mitosis, and replicates its DNA and some of its organelles. During the mitotic phase, the replicated chromosomes, organelles, and cytoplasm separate into two new daughter cells. To ensure the proper replication of cellular components and division, there are control mechanisms known as cell cycle checkpoints after each of the key steps of the cycle that determine if the cell can progress to the next phase.

In cells without nuclei (prokaryotes), (i.e., bacteria and archaea), the cell cycle is divided into the B, C, and D periods. The B period extends from the end of cell division to the beginning of DNA replication. DNA replication occurs during the C period. The D period refers to the stage between the end of DNA replication and the splitting of the bacterial cell into two daughter cells.

The cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed. After cell division, each of the daughter cells begin the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of the cell division.

Phases

The eukaryotic cell cycle consists of four distinct phases: G₁ phase, S phase (synthesis), G₂ phase (collectively known as interphase) and M phase (mitosis and cytokinesis). M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's nucleus divides, and cytokinesis, in which the cell's cytoplasm divides forming two daughter cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G₀ phase.

Schematic of the cell cycle. Outer ring: I = Interphase, M = Mitosis; inner ring: M = Mitosis, G₁ = Gap 1, G₂ = Gap 2, S = Synthesis; not in ring: G₀ = Gap 0/Resting

State	Phase	Abbreviation	Description
Resting	Gap 0	G0	A phase where the cell has left the cycle and has stopped dividing.
Interphase	Gap 1	G1	Cells increase in size in Gap 1. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis.
	Synthesis	S	DNA replication occurs during this phase.
	Gap 2	G2	During the gap between DNA synthesis and mitosis, the cell will continue to grow. The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis) phase and divide.
Cell division	Mitosis	M	Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells. A checkpoint in the middle of mitosis (Metaphase Checkpoint) ensures that the cell is ready to complete cell division.

After cell division, each of the daughter cells begin the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of cell division.

G₀ phase (quiescence)

Plant cell cycle

 

Animal cell cycle

 

Main article: G0 phase

G₀ is a resting phase where the cell has left the cycle and has stopped dividing. The cell cycle starts with this phase. Non-proliferative (non-dividing) cells in multicellular eukaryotes generally enter the quiescent G₀ state from G₁ and may remain quiescent for long periods of time, possibly indefinitely (as is often the case for neurons). This is very common for cells that are fully differentiated. Some cells enter the G₀ phase semi-permanently and are considered post-mitotic, e.g., some liver, kidney, and stomach cells. Many cells do not enter G₀ and continue to divide throughout an organism's life, e.g., epithelial cells.

The word "post-mitotic" is sometimes used to refer to both quiescent and senescent cells. Cellular senescence occurs in response to DNA damage and external stress and usually constitutes an arrest in G₁. Cellular senescence may make a cell's progeny nonviable; it is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis.

Interphase

Main article: Interphase

Interphase represent the phase between two successive M phases. Interphase is a series of changes that takes place in a newly formed cell and its nucleus before it becomes capable of division again. It is also called preparatory phase or intermitosis. Typically interphase lasts for at least 91% of the total time required for the cell cycle.

Interphase proceeds in three stages, G₁, S, and G₂, followed by the cycle of mitosis and cytokinesis. The cell's nuclear DNA contents are duplicated during S phase.

G₁ phase (First growth phase or Post mitotic gap phase)

Main article: G1 phase

The first phase within interphase, from the end of the previous M phase until the beginning of DNA synthesis, is called G₁ (G indicating gap). It is also called the growth phase. During this phase, the biosynthetic activities of the cell, which are considerably slowed down during M phase, resume at a high rate. The duration of G₁ is highly variable, even among different cells of the same species. In this phase, the cell increases its supply of proteins, increases the number of organelles (such as mitochondria, ribosomes), and grows in size. In G₁ phase, a cell has three options.

• To continue cell cycle and enter S phase
• Stop cell cycle and enter G₀ phase for undergoing differentiation.
• Become arrested in G₁ phase hence it may enter G₀ phase or re-enter cell cycle.

The deciding point is called check point (Restriction point). This check point is called the restriction point or START and is regulated by G₁/S cyclins, which cause transition from G₁ to S phase. Passage through the G₁ check point commits the cell to division.

S phase (DNA replication)

Main article: S phase

The ensuing S phase starts when DNA synthesis commences; when it is complete, all of the chromosomes have been replicated, i.e., each chromosome consists of two sister chromatids. Thus, during this phase, the amount of DNA in the cell has doubled, though the ploidy and number of chromosomes are unchanged. Rates of RNA transcription and protein synthesis are very low during this phase. An exception to this is histone production, most of which occurs during the S phase.

G₂ phase (growth)

Main article: G2 phase

G₂ phase occurs after DNA replication and is a period of protein synthesis and rapid cell growth to prepare the cell for mitosis. During this phase microtubules begin to reorganize to form a spindle (preprophase). Before proceeding to mitotic phase, cells must be checked at the G₂ checkpoint for any DNA damage within the chromosomes. The G₂ checkpoint is mainly regulated by the tumor protein p53. If the DNA is damaged, p53 will either repair the DNA or trigger the apoptosis of the cell. If p53 is dysfunctional or mutated, cells with damaged DNA may continue through the cell cycle, leading to the development of cancer.

Mitotic phase (chromosome separation)

Main article: Mitosis

The relatively brief M phase consists of nuclear division (karyokinesis). It is a relatively short period of the cell cycle. M phase is complex and highly regulated. The sequence of events is divided into phases, corresponding to the completion of one set of activities and the start of the next. These phases are sequentially known as:

• prophase
• prometaphase
• metaphase
• anaphase
• telophase

Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets in two nuclei. During the process of mitosis the pairs of chromosomes condense and attach to microtubules that pull the sister chromatids to opposite sides of the cell.

Mitosis occurs exclusively in eukaryotic cells, but occurs in different ways in different species. For example, animal cells undergo an "open" mitosis, where the nuclear envelope breaks down before the chromosomes separate, while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis, where chromosomes divide within an intact cell nucleus.

Cytokinesis phase (separation of all cell components)

Main article: Cytokinesis

Mitosis is immediately followed by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the division of the mother cell into two daughter cells, genetically identical to each other and to their parent cell. This accounts for approximately 10% of the cell cycle.

Because cytokinesis usually occurs in conjunction with mitosis, "mitosis" is often used interchangeably with "M phase". However, there are many cells where mitosis and cytokinesis occur separately, forming single cells with multiple nuclei in a process called endoreplication. This occurs most notably among the fungi and slime molds, but is found in various groups. Even in animals, cytokinesis and mitosis may occur independently, for instance during certain stages of fruit fly embryonic development. Errors in mitosis can result in cell death through apoptosis or cause mutations that may lead to cancer.

Regulation of eukaryotic cell cycle

Levels of the three major cyclin types oscillate during the cell cycle (top), providing the basis for oscillations in the cyclin–Cdk complexes that drive cell-cycle events (bottom). In general, Cdk levels are constant and in large excess over cyclin levels; thus, cyclin–Cdk complexes form in parallel with cyclin levels. The enzymatic activities of cyclin–Cdk complexes also tend to rise and fall in parallel with cyclin levels, although in some cases Cdk inhibitor proteins or phosphorylation introduce a delay between the formation and activation of cyclin–Cdk complexes. Formation of active G1/S–Cdk complexes commits the cell to a new division cycle at the Start checkpoint in late G1. G1/S–Cdks then activate the S–Cdk complexes that initiate DNA replication at the beginning of S phase. M–Cdk activation occurs after the completion of S phase, resulting in progression through the G2/M checkpoint and assembly of the mitotic spindle. APC activation then triggers sister-chromatid separation at the metaphase-to-anaphase transition. APC activity also causes the destruction of S and M cyclins and thus the inactivation of Cdks, which promotes the completion of mitosis and cytokinesis. APC activity is maintained in G1 until G1/S–Cdk activity rises again and commits the cell to the next cycle. This scheme serves only as a general guide and does not apply to all cell types.

Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. The molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.

Role of cyclins and CDKs

Nobel Laureate Paul Nurse

Nobel Laureate Tim Hunt

Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine a cell's progress through the cell cycle. Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine for their discovery of these central molecules. Many of the genes encoding cyclins and CDKs are conserved among all eukaryotes, but in general, more complex organisms have more elaborate cell cycle control systems that incorporate more individual components. Many of the relevant genes were first identified by studying yeast, especially Saccharomyces cerevisiae; genetic nomenclature in yeast dubs many of these genes cdc (for "cell division cycle") followed by an identifying number, e.g. cdc25 or cdc20.

Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine the downstream proteins targeted. CDKs are constitutively expressed in cells whereas cyclins are synthesised at specific stages of the cell cycle, in response to various molecular signals.

General mechanism of cyclin-CDK interaction

Upon receiving a pro-mitotic extracellular signal, G₁ cyclin-CDK complexes become active to prepare the cell for S phase, promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication. The G₁ cyclin-CDK complexes also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is targeted for proteolytic degradation by the proteasome. However, results from a recent study of E2F transcriptional dynamics at the single-cell level argue that the role of G1 cyclin-CDK activities, in particular cyclin D-CDK4/6, is to tune the timing rather than the commitment of cell cycle entry.

Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G₁ phase on DNA replication origins. The phosphorylation serves two purposes: to activate each already-assembled pre-replication complex, and to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. The reason for prevention of gaps in replication is fairly clear, because daughter cells that are missing all or part of crucial genes will die. However, for reasons related to gene copy number effects, possession of extra copies of certain genes is also deleterious to the daughter cells.

Mitotic cyclin-CDK complexes, which are synthesized but inactivated during S and G₂ phases, promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly. A critical complex activated during this process is a ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes degradation of structural proteins associated with the chromosomal kinetochore. APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed.

Specific action of cyclin-CDK complexes

Cyclin D is the first cyclin produced in the cells that enter the cell cycle, in response to extracellular signals (e.g. growth factors). Cyclin D levels stay low in resting cells that are not proliferating. Additionally, CDK4/6 and CDK2 are also inactive because CDK4/6 are bound by INK4 family members (e.g., p16), limiting kinase activity. Meanwhile, CDK2 complexes are inhibited by the CIP/KIP proteins such as p21 and p27, When it is time for a cell to enter the cell cycle, which is triggered by a mitogenic stimuli, levels of cyclin D increase. In response to this trigger, cyclin D binds to existing CDK4/6, forming the active cyclin D-CDK4/6 complex. Cyclin D-CDK4/6 complexes in turn mono-phosphorylates the retinoblastoma susceptibility protein (Rb) to pRb. The un-phosphorylated Rb tumour suppressor functions in inducing cell cycle exit and maintaining G0 arrest (senescence).

In the last few decades, a model has been widely accepted whereby pRB proteins are inactivated by cyclin D-Cdk4/6-mediated phosphorylation. Rb has 14+ potential phosphorylation sites. Cyclin D-Cdk 4/6 progressively phosphorylates Rb to hyperphosphorylated state, which triggers dissociation of pRB–E2F complexes, thereby inducing G1/S cell cycle gene expression and progression into S phase.

However, scientific observations from a recent study show that Rb is present in three types of isoforms: (1) un-phosphorylated Rb in G0 state; (2) mono-phosphorylated Rb, also referred to as "hypo-phosphorylated' or 'partially' phosphorylated Rb in early G1 state; and (3) inactive hyper-phosphorylated Rb in late G1 state. In early G1 cells, mono-phosphorylated Rb exits as 14 different isoforms, one of each has distinct E2F binding affinity. Rb has been found to associate with hundreds of different proteins and the idea that different mono-phosphorylated Rb isoforms have different protein partners was very appealing. A recent report confirmed that mono-phosphorylation controls Rb's association with other proteins and generates functional distinct forms of Rb. All different mono-phosphorylated Rb isoforms inhibit E2F transcriptional program and are able to arrest cells in G1-phase. Importantly, different mono-phosphorylated forms of RB have distinct transcriptional outputs that are extended beyond E2F regulation.

In general, the binding of pRb to E2F inhibits the E2F target gene expression of certain G1/S and S transition genes including E-type cyclins. The partial phosphorylation of RB de-represses the Rb-mediated suppression of E2F target gene expression, begins the expression of cyclin E. The molecular mechanism that causes the cell switched to cyclin E activation is currently not known, but as cyclin E levels rise, the active cyclin E-CDK2 complex is formed, bringing Rb to be inactivated by hyper-phosphorylation. Hyperphosphorylated Rb is completely dissociated from E2F, enabling further expression of a wide range of E2F target genes are required for driving cells to proceed into S phase. Recently, it has been identified that cyclin D-Cdk4/6 binds to a C-terminal alpha-helix region of Rb that is only distinguishable to cyclin D rather than other cyclins, cyclin E, A and B. This observation based on the structural analysis of Rb phosphorylation supports that Rb is phosphorylated in a different level through multiple Cyclin-Cdk complexes. This also makes feasible the current model of a simultaneous switch-like inactivation of all mono-phosphorylated Rb isoforms through one type of Rb hyper-phosphorylation mechanism. In addition, mutational analysis of the cyclin D- Cdk 4/6 specific Rb C-terminal helix shows that disruptions of cyclin D-Cdk 4/6 binding to Rb prevents Rb phosphorylation, arrests cells in G1, and bolsters Rb's functions in tumor suppressor. This cyclin-Cdk driven cell cycle transitional mechanism governs a cell committed to the cell cycle that allows cell proliferation. A cancerous cell growth often accompanies with deregulation of Cyclin D-Cdk 4/6 activity.

The hyperphosphorylated Rb dissociates from the E2F/DP1/Rb complex (which was bound to the E2F responsive genes, effectively "blocking" them from transcription), activating E2F. Activation of E2F results in transcription of various genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc. Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from G₁ to S phase (G₁/S, which initiates the G₂/M transition). Cyclin B-cdk1 complex activation causes breakdown of nuclear envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit mitosis. A quantitative study of E2F transcriptional dynamics at the single-cell level by using engineered fluorescent reporter cells provided a quantitative framework for understanding the control logic of cell cycle entry, challenging the canonical textbook model. Genes that regulate the amplitude of E2F accumulation, such as Myc, determine the commitment in cell cycle and S phase entry. G1 cyclin-CDK activities are not the driver of cell cycle entry. Instead, they primarily tune the timing of E2F increase, thereby modulating the pace of cell cycle progression.

Inhibitors

Endogenous

Overview of signal transduction pathways involved in apoptosis, also known as "programmed cell death"

Two families of genes, the cip/kip (CDK interacting protein/Kinase inhibitory protein) family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) family, prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.

The cip/kip family includes the genes p21, p27 and p57. They halt the cell cycle in G₁ phase by binding to and inactivating cyclin-CDK complexes. p21 is activated by p53 (which, in turn, is triggered by DNA damage e.g. due to radiation). p27 is activated by Transforming Growth Factor β (TGF β), a growth inhibitor.

The INK4a/ARF family includes p16^INK4a, which binds to CDK4 and arrests the cell cycle in G₁ phase, and p14^ARF which prevents p53 degradation.

Synthetic

Synthetic inhibitors of Cdc25 could also be useful for the arrest of cell cycle and therefore be useful as antineoplastic and anticancer agents.

Many human cancers possess the hyper-activated Cdk 4/6 activities. Given the observations of cyclin D-Cdk 4/6 functions, inhibition of Cdk 4/6 should result in preventing a malignant tumor from proliferating. Consequently, scientists have tried to invent the synthetic Cdk4/6 inhibitor as Cdk4/6 has been characterized to be a therapeutic target for anti-tumor effectiveness. Three Cdk4/6 inhibitors - palbociclib, ribociclib, and abemaciclib - currently received FDA approval for clinical use to treat advanced-stage or metastatic, hormone-receptor-positive (HR-positive, HR+), HER2-negative (HER2-) breast cancer. For example, palbociclib is an orally active CDK4/6 inhibitor which has demonstrated improved outcomes for ER-positive/HER2-negative advanced breast cancer. The main side effect is neutropenia which can be managed by dose reduction.

Cdk4/6 targeted therapy will only treat cancer types where Rb is expressed. Cancer cells with loss of Rb have primary resistance to Cdk4/6 inhibitors.

Transcriptional regulatory network

Current evidence suggests that a semi-autonomous transcriptional network acts in concert with the CDK-cyclin machinery to regulate the cell cycle. Several gene expression studies in Saccharomyces cerevisiae have identified 800–1200 genes that change expression over the course of the cell cycle. They are transcribed at high levels at specific points in the cell cycle, and remain at lower levels throughout the rest of the cycle. While the set of identified genes differs between studies due to the computational methods and criteria used to identify them, each study indicates that a large portion of yeast genes are temporally regulated.

Many periodically expressed genes are driven by transcription factors that are also periodically expressed. One screen of single-gene knockouts identified 48 transcription factors (about 20% of all non-essential transcription factors) that show cell cycle progression defects. Genome-wide studies using high throughput technologies have identified the transcription factors that bind to the promoters of yeast genes, and correlating these findings with temporal expression patterns have allowed the identification of transcription factors that drive phase-specific gene expression. The expression profiles of these transcription factors are driven by the transcription factors that peak in the prior phase, and computational models have shown that a CDK-autonomous network of these transcription factors is sufficient to produce steady-state oscillations in gene expression).

Experimental evidence also suggests that gene expression can oscillate with the period seen in dividing wild-type cells independently of the CDK machinery. Orlando et al. used microarrays to measure the expression of a set of 1,271 genes that they identified as periodic in both wild type cells and cells lacking all S-phase and mitotic cyclins (clb1,2,3,4,5,6). Of the 1,271 genes assayed, 882 continued to be expressed in the cyclin-deficient cells at the same time as in the wild type cells, despite the fact that the cyclin-deficient cells arrest at the border between G₁ and S phase. However, 833 of the genes assayed changed behavior between the wild type and mutant cells, indicating that these genes are likely directly or indirectly regulated by the CDK-cyclin machinery. Some genes that continued to be expressed on time in the mutant cells were also expressed at different levels in the mutant and wild type cells. These findings suggest that while the transcriptional network may oscillate independently of the CDK-cyclin oscillator, they are coupled in a manner that requires both to ensure the proper timing of cell cycle events. Other work indicates that phosphorylation, a post-translational modification, of cell cycle transcription factors by Cdk1 may alter the localization or activity of the transcription factors in order to tightly control timing of target genes.

While oscillatory transcription plays a key role in the progression of the yeast cell cycle, the CDK-cyclin machinery operates independently in the early embryonic cell cycle. Before the midblastula transition, zygotic transcription does not occur and all needed proteins, such as the B-type cyclins, are translated from maternally loaded mRNA.

DNA replication and DNA replication origin activity

Analyses of synchronized cultures of Saccharomyces cerevisiae under conditions that prevent DNA replication initiation without delaying cell cycle progression showed that origin licensing decreases the expression of genes with origins near their 3' ends, revealing that downstream origins can regulate the expression of upstream genes. This confirms previous predictions from mathematical modeling of a global causal coordination between DNA replication origin activity and mRNA expression, and shows that mathematical modeling of DNA microarray data can be used to correctly predict previously unknown biological modes of regulation.

Checkpoints

Main article: Cell cycle checkpoint

Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage. The cell cannot proceed to the next phase until checkpoint requirements have been met. Checkpoints typically consist of a network of regulatory proteins that monitor and dictate the progression of the cell through the different stages of the cell cycle.

It is estimated that in normal human cells about 1% of single-strand DNA damages are converted to about 50 endogenous DNA double-strand breaks per cell per cell cycle. Although such double-strand breaks are usually repaired with high fidelity, errors in their repair are considered to contribute significantly to the rate of cancer in humans.

There are several checkpoints to ensure that damaged or incomplete DNA is not passed on to daughter cells. Three main checkpoints exist: the G₁/S checkpoint, the G₂/M checkpoint and the metaphase (mitotic) checkpoint. Another checkpoint is the Go checkpoint, in which the cells are checked for maturity. If the cells fail to pass this checkpoint by not being ready yet, they will be discarded from dividing.

G₁/S transition is a rate-limiting step in the cell cycle and is also known as restriction point. This is where the cell checks whether it has enough raw materials to fully replicate its DNA (nucleotide bases, DNA synthase, chromatin, etc.). An unhealthy or malnourished cell will get stuck at this checkpoint.

The G₂/M checkpoint is where the cell ensures that it has enough cytoplasm and phospholipids for two daughter cells. But sometimes more importantly, it checks to see if it is the right time to replicate. There are some situations where many cells need to all replicate simultaneously (for example, a growing embryo should have a symmetric cell distribution until it reaches the mid-blastula transition). This is done by controlling the G₂/M checkpoint.

The metaphase checkpoint is a fairly minor checkpoint, in that once a cell is in metaphase, it has committed to undergoing mitosis. However that's not to say it isn't important. In this checkpoint, the cell checks to ensure that the spindle has formed and that all of the chromosomes are aligned at the spindle equator before anaphase begins.

While these are the three "main" checkpoints, not all cells have to pass through each of these checkpoints in this order to replicate. Many types of cancer are caused by mutations that allow the cells to speed through the various checkpoints or even skip them altogether. Going from S to M to S phase almost consecutively. Because these cells have lost their checkpoints, any DNA mutations that may have occurred are disregarded and passed on to the daughter cells. This is one reason why cancer cells have a tendency to exponentially accrue mutations. Aside from cancer cells, many fully differentiated cell types no longer replicate so they leave the cell cycle and stay in G₀ until their death. Thus removing the need for cellular checkpoints. An alternative model of the cell cycle response to DNA damage has also been proposed, known as the postreplication checkpoint.

Checkpoint regulation plays an important role in an organism's development. In sexual reproduction, when egg fertilization occurs, when the sperm binds to the egg, it releases signalling factors that notify the egg that it has been fertilized. Among other things, this induces the now fertilized oocyte to return from its previously dormant, G₀, state back into the cell cycle and on to mitotic replication and division.

p53 plays an important role in triggering the control mechanisms at both G₁/S and G₂/M checkpoints. In addition to p53, checkpoint regulators are being heavily researched for their roles in cancer growth and proliferation.

Fluorescence imaging of the cell cycle

Fluorescent proteins visualize the cell cycle progression. IFP2.0-hGem(1/110) fluorescence is shown in green and highlights the S/G₂/M phases. smURFP-hCdtI(30/120) fluorescence is shown in red and highlights the G₀/G₁ phases.

Pioneering work by Atsushi Miyawaki and coworkers developed the fluorescent ubiquitination-based cell cycle indicator (FUCCI), which enables fluorescence imaging of the cell cycle. Originally, a green fluorescent protein, mAG, was fused to hGem(1/110) and an orange fluorescent protein (mKO₂) was fused to hCdt1(30/120). Note, these fusions are fragments that contain a nuclear localization signal and ubiquitination sites for degradation, but are not functional proteins. The green fluorescent protein is made during the S, G₂, or M phase and degraded during the G₀ or G₁ phase, while the orange fluorescent protein is made during the G₀ or G₁ phase and destroyed during the S, G₂, or M phase. A far-red and near-infrared FUCCI was developed using a cyanobacteria-derived fluorescent protein (smURFP) and a bacteriophytochrome-derived fluorescent protein (movie found at this link).

Role in tumor formation

A disregulation of the cell cycle components may lead to tumor formation. As mentioned above, when some genes like the cell cycle inhibitors, RB, p53 etc. mutate, they may cause the cell to multiply uncontrollably, forming a tumor. Although the duration of cell cycle in tumor cells is equal to or longer than that of normal cell cycle, the proportion of cells that are in active cell division (versus quiescent cells in G₀ phase) in tumors is much higher than that in normal tissue. Thus there is a net increase in cell number as the number of cells that die by apoptosis or senescence remains the same.

The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation. This fact is made use of in cancer treatment; by a process known as debulking, a significant mass of the tumor is removed which pushes a significant number of the remaining tumor cells from G₀ to G₁ phase (due to increased availability of nutrients, oxygen, growth factors etc.). Radiation or chemotherapy following the debulking procedure kills these cells which have newly entered the cell cycle.

The fastest cycling mammalian cells in culture, crypt cells in the intestinal epithelium, have a cycle time as short as 9 to 10 hours. Stem cells in resting mouse skin may have a cycle time of more than 200 hours. Most of this difference is due to the varying length of G₁, the most variable phase of the cycle. M and S do not vary much.

In general, cells are most radiosensitive in late M and G₂ phases and most resistant in late S phase.

For cells with a longer cell cycle time and a significantly long G₁ phase, there is a second peak of resistance late in G₁.

The pattern of resistance and sensitivity correlates with the level of sulfhydryl compounds in the cell. Sulfhydryls are natural substances that protect cells from radiation damage and tend to be at their highest levels in S and at their lowest near mitosis.

Homologous recombination (HR) is an accurate process for repairing DNA double-strand breaks. HR is nearly absent in G1 phase, is most active in S phase, and declines in G₂/M. Non-homologous end joining, a less accurate and more mutagenic process for repairing double strand breaks, is active throughout the cell cycle.