In 1858 the pathologist Rudolph Virchow coined the cell doctrine which states that "When a cell arises, there must have been a previous cell, just as animals can only arise from animals and plants from plants." This doctrine is founded on the understanding that whether one is examining a single-celled organism or an animal as complex as man, the product is a result of repeated rounds of cell growth and division. Most eukaryotic cells will proceed through an ordered series of events (described in the section below) in which the cell duplicates its contents and then divides into two cells. This cycle of duplication and division is called the cell cycle. In order to maintain the fidelity of the developing organism this process of cell division in multicellular organisms must be highly ordered and tightly regulated. The loss of control (as discussed in the sections below) will lead to abnormal development and is the cause of cancer.
The eukaryotic cell cycle is composed of four steps, or phases, as depicted in the Figure below.
Of the four phases depicted in the Figure, the two critical steps are DNA replication, which occurs during S-phase, and the physical process of cell division which occurs during M-phase (for mitosis). If we start at the beginning of the process, a cell undergoes a period where all of the necessary machinery for the process of DNA replication is synthesized. This process occurs during what is referred to as a gap between S-phase and M-phase and is termed G1. Following DNA replication, the cell pauses in another gap phase termed G2 where all the machinery necessary for cell division is synthesized. M-phase is composed of two discreet steps: mitosis, which constitutes the pairing and separation of the duplicated chromosomes, and cytokinesis which is the physical process whereby the cell splits into two daughter cells. Not all cells continue to divide during the life-span of an organism. Many cells undergo what is referred to as terminal differentiation and become quiescent and no longer divide. Cells in this phase of their life-cycle are said to reside in another gap phase called G0. Under certain conditions, such as that resulting from an external signal stimulating cell growth, cells can exit the quiescent state and re-enter the cell cycle.back to the top
G1: The first gap in the normal cell cycle is called G1 and is the period when the necessary proteins for DNA replication are synthesized. However, this phase of the cell cycle is not only characterized by synthesis of replication machinery. During this period the cell must monitor both the internal and external environments to ensure that all the preparations for DNA synthesis have been completed and that overall conditions for cell division are favorable. As discussed below, there is a major check-point in a normal cell cycle that is critical for ensuring that all is well for the cell to enter S-phase.
S-phase: The duplication of the cellular content of DNA occurs during S-phase, so-called because this is the phase when DNA is synthesized. This phase of the cell cycle is the longest taking 10–12 hours of a typical 24hr eukaryotic cell cycle.
G2: During the second gap phase of the cell cycle the cell undertakes the synthesis of the proteins required to assemble the machinery required for separation of the duplicated chromosomes (the process called mitosis) and ultimately division of the parental cell into two daughter cells (the process termed cytokinesis). Like the G1 phase, the G2 phase is also a stage when the internal and external environments are monitored to ensure that faithful replication of the DNA has occurred and that conditions are favorable for cytokinesis. In addition, as for the G1 phase there is a major check-point at the end of the G2 phase that controls the entry into M-phase.
M-phase: During M-phase there is an ordered series of events that leads to the alignment and separation of the duplicated chromosomes (called sister chromatids) This process is divided into distinct steps that were originally identified and characterized through light microscopic observations of dividing cells. The steps of mitosis are termed prophase, prometaphase, metaphase, anaphase and telophase. Although cytokinesis is the process by which the parental cell is physically separated into two new daughter cells, it actually begins during anaphase. The processes that occur during M-phase require much less time than those of S-phase, generally lasting only 1–2hrs.
During prophase the duplicated chromosomes condense while outside the nucleus the mitotic spindle assembles between the two centrosomes. The centrosome is an organelle that serves as the main microtubule organizing center that is involved in the attachment of microtubules to the sister chromatids.
During prometaphase the nuclear membrane breaks apart and the chromosomes can attach to spindle microtubules and begin active movement.
During metaphase the chromosomes are aligned at the equator of the spindle midway between the spindle poles. The sister chromatids are attached to opposite poles of the spindle.
During anaphase the sister chromatids synchronously separate to form the two sets of daughter chromosomes. Each sister chromatid is slowly pulled towards the spindle pole it faces.
During telophase the two daughter chromosomes arrive at the spindle poles and decondense. A new nuclear envelope forms around each set of chromosomes which forms the two new nuclei. This process marks the end of mitosis and sets the stage for cytokinesis.back to the top
It should seem obvious that the processes that drive a cell through the cell cycle must be highly regulated so as to ensure that the resultant daughter cells are viable and each contains the complement of DNA found in the original parental cell. There are many "parts" to the systems that control the transit through a eukaryotic cell cycle. These "parts" include mechanisms to control the timing of events so that each individual process is turned on and off at the appropriate time, mechanisms to initiate each event in the correct order and to also ensure that each event is triggered only once per cell cycle, controls to ensure events occur in a linear, irreversible direction, redundancy, or back-ups to ensure the cycle functions properly even in the context of some malfunctioning parts, and systems that are adaptable so that cell cycle events can be modified in the context of different cell types and/or environmental conditions.
Many of the most important discoveries about the mechanisms that control events of the cell cycle were elucidated using yeasts which are single cell eukaryotes. By analysis of various mutants that inactivated genes encoding essential components of cell cycle control systems in yeast many important control genes were identified. These genes were identified as cell division cycle genes or cdc genes. Thus, many cell cycle control genes in mammalian cells are also called cdc genes. Much of the control of the progression through the phases of a cell cycle are exerted at checkpoints. There are many such checkpoints but the two most critical are those that occur near the end of G1 prior to S-phase entry and those near the end of G2 prior to mitosis.
As indicated above, there is the need for cell cycle control mechanisms to exert their influences at specific times during each transit through a cell cycle. The heart of this timing control is the responsibility of a family of protein kinases that are called cyclin-dependent kinases, CDKs. The kinase activity of these enzymes rises and falls as the cell progresses through a cell cycle. Different CDKs operate at different points in the cell cycle. As would be expected, the oscillating changes in the activity of CDKs leads to oscillating changes in the phosphorylation of various intracellular proteins. These phosphorylations alter the activity of the modified proteins which then effect changes in events of the cell cycle. The cyclical activity of each CDK is controlled by a complex series of proteins, the most important of which are the cyclins, hence the name of the enzymes as cyclin-dependent kinases. The CDKs are absolutely dependent upon their interaction with the cyclins for activity. Unless they are tightly bound CDKs have no kinase activity. The cyclins were originally idenitified because they undergo a cycle of synthesis and degradation at specific points in each cell cycle. Thus, whereas the levels of the various CDKs remain fairly constant throughout the cell cycle, their activities changes in concert with the fluctuations of the cyclins.
Four different classes of cyclins have been defined on the basis of the stage of the cell cycle in which they bind and activate CDKs. These four classes are G1-cyclins, G1/S-cyclins, S-cyclins, and M-cyclins. The cyclin nomenclature and associated CDK in mammalian cells are listed in the following Table.
|Cyclin-CDK Complex||Cyclin||CDK Partner|
|G1-CDK||cyclin D*||CDK4, CDK6|
*There are three D cyclins in mammals: D1, D2, and D3
**CDK1 is the same as CDC2 in fission yeast and CDC28 in budding yeast
The G1-cyclins are not found in all eukaryotic cells but in those where they are synthesized they promote passage through a restriction point in late G1 called Start. The G1/S-cyclins bind to their cognate CDKs at the end of G1 and it is this interaction that is required to commit the cell to the process of DNA replication in S-phase. The S-cyclins bind to their cognate CDKs during S-phase and it is this interaction that is required for the initiation of DNA synthesis. The M-cyclins bind to their cognate CDKs and in so doing promote the events of mitosis.
Although CDKs are inactive unless bound to a cyclin, there is more to the activation process than just the interaction of the two parts of the complex. When cyclins bind to CDKs they alter the conformation of the CDK resulting in exposure of a domain that is the site of phosphorylation by another kinase called CDK-activating kinase (CAK). Following phosphorylation the cyclin-CDK complex is fully active.
In addition to control of CDK kinase activity by cyclin binding and CAK phosphorylation, control is exerted to inhibit CDK activity through interaction with inhibitory proteins as well as by inhibitory phosphorylation events. Thus, there is extremely tight control on the overall activity of each CDK. One of the inhibitory kinases that phosphorylates CDKs is called Wee1. The inhibitory phosphorylations are removed through the action of a phosphatase called CDC25. The action of these two regulatory enzymes on CDK activity is most important at the level of the M-CDK activity at the onset of mitosis. Proteins that bind to and inhibit cyclin-CDK complexes are called CDK inhibitory proteins (CKI, for cyclin-kinase inhibitor). Mammalian cells express two classes of CKI. These are called CIPs for CDK inhibitory proteins and INK4 for inhibitors of kinase 4. The CIPs bind and inhibit CDK1, CDK2, CDK4, and CDK6 complexes, whereas the INK4s bind and inhibit only the CDK4 and CDK6 complexes. There are at least three CIP proteins in mammalian cells and these are identified as p21Cip1/WAF1 (gene symbol=CDKN1A), p27KIP1 (gene symbol=CDKN1B), and p57KIP2 (gene symbol=CDKN1C). The expression of each of these CIPs is controlled by specific events that may have occurred during cell cycle transit. For example p21Cip1 expression is induced in response to DNA damage. This induction is under the control of the action of the tumor suppressor protein p53 (see below). There are at least four INK4 proteins that are each identified by their molecular weights: p15INK4B, p16INK4A, and p18INK4C (these were the first 3 characterized) as well as p19INK4. The p16INK4A protein is also a tumor suppressor since loss of its function leads to cancer. All the INK4 proteins contain 4 tandem repeats of a sequence of amino acids that were first identified in ankyrin and are thus referred to as ankyrin repeats.
As indicated above, many cells reside in a resting or quiescent state but can be stimulated by external signals to re-enter the cell cycle. These external growth promoting signals are the result of growth factors binding to their receptors. Most growth factors induce the expression of genes that are referred to as early and delayed-response genes. The activation of early response genes occurs in response to growth factor receptor-mediated signal transduction resulting in phosphorylation and activation of transcription factor proteins that are already present in the cell. Many of the induced early response genes are themselves transcription factors that in turn activate the expression of delayed-response genes. In the context of the cell cycle, these delayed-response genes encode proteins of the G1-CDK complexes.
One such early response gene is the proto-oncogene MYC. With respect to the cell cycle some of the genes turned on by activation of MYC are cyclin D, proteins of the ubiquitin ligase complex called SCF (Skp1/cullin/F-box protein) and the members of the E2F transcription factor family. There are six members of the E2F family: E2F1 through E2F6). The synthesis of cyclin D will result in the activation of G1-CDK complexes. The synthesis of components of SCF leads to the degradation of p27KIP1 which normally inhibits G1-CDK complexes. The synthesis of E2F family members results in increased synthesis of proteins involved in DNA synthesis as well as the synthesis of the S-phase cyclins A and E and CDK2. Regulation of E2F activity by the tumor suppressor pRB will be discussed below.
The cyclical degradation of the cyclins is effected through the action of several different ubiquitin ligase complexes. The action of ubiquitin ligases in protein turn-over is discussed in more detail in the Protein Modifications page. There are two important ubiquitin ligase complexes that control the turn-over of cyclins and other cell cycle regulating proteins. One is the SCF complex which functions to control the transit from G1 to S-phase and the other is called anaphase promoting complex (APC) which controls the levels of the M-phase cyclins as well as other regulators of mitosis.
One important function of APC is to control the initiation of sister chromatid separation which begins at the metaphase-anaphase transition. The attachment of the sister chromatids to the opposite poles of the mitotic spindles occurs early during mitosis. The ability of the sister chromatids to be pulled apart is initially inhibited because they are bound together by a protein complex termed cohesin complex. The cohesin complex is deposited along the chromosomes as they are duplicated during S-phase. Anaphase can only begin with the disruption of the cohesin complex. The breakdown of the cohesin complex is brought about as a consequence of the activation of the ubiquitin ligase activity of the APC. APC targets a protein called securin. Securin functions to inhibit the protease called separase and the action of separase is to degrade the proteins of the cohesin complex, thus allowing sister chromatid separation.back to the top
Tumor suppressors are so called because cancer ensues as a result of a loss of their normal function, i.e. these proteins suppress the ability of cancer to develop. It would seem obvious, therefore, that one import function of tumor suppressors would be control of the progression of a cell through a round of the cell cycle. If cells are able to synthesize damaged DNA before it is repaired or to divide when the DNA is damaged then the resulting daughter cells can pass on the resultant DNA damage to their progeny. The result can be catastrophic resulting in cancer. For this reason, the two most important check points in the eukaryotic cell cycle are the G1-S transition and the entry into mitosis. The former prevents DNA replication prior to repair of damaged DNA and the latter prevents damage that may have occurred to the DNA during replication to propagated into daughter cells during mitosis. Following the isolation and characterization of two tumor suppressor genes in particular it was found that they function to control the ability of cells to progress through these two important checkpoints. The protein encoded by the retinoblastoma susceptibility gene (pRB) and the p53 protein are both tumor suppressors. The function of pRB is to act as a brake preventing cells from exiting G1 and that of p53 is to inhibit progression from S-phase to M-phase.
The best understood effect of G1-CDK activity is that exerted on transcription factors of the E2F family, hereafter referred to simply as E2F. In the context of the cell cycle regulation, E2F activates the expression of cyclin A, cyclin E and CDK2. These proteins are components of the S-CDK complexes necessary for progression through S-phase. The activity of E2F is itself controlled via interaction with pRB. When pRB binds E2F it can no longer function as a transcription factor as it is sequestered in the cytosol. Interaction of pRB and E2Fcorrelates to the state of phosphorylation of pRB and the affinity between the two proteins is highest when pRB is hypophosphorylated. Phosphorylation of pRB is maximal at the start of S phase and lowest after mitosis and entry into G1. Stimulation of quiescent cells with mitogen induces phosphorylation of pRB, while in contrast, differentiation induces hypophosphorylation of pRB. One of the most significant substrates for phosphorylation by the G1 cyclin-CDK complexes is pRB. When pRB is phosphorylated by G1 cyclin-CDK complexes it releases E2F allowing E2F to transcriptionally activate its target genes. When E2F activates the expression of S-CDK complex proteins these complexes also target pRB for phosphorylation, thus maintaining the cell in a pro cell cycle progression state.
Regulation of E2F activity by pRB. During early G1 the transcription factor E2F is inhibited by interactoin with pRB in the cytosol. Activation of the G1 cyclin-CDK complex (cyclin D-CDK4/6) results in the phosphorylation of pRB which then releases E2F. Free from pRB, E2F migrates to the nucleus where it activates the transcription of several genes including the cyclin E gene and the E2F gene itself. The autoregulation of E2F allows for high level activity of this critical cell cycle regulatory factor. In addition, the activation of cyclin E expression results in formation of active cyclin E-CDK2 complexes which keep pRB phosphorylated ensuring transit through to S-phase of the cell cycle.
One major function of the p53 protein, which is active as a homotetrameric transcription factor, is to serve as a component of the checkpoint that controls whether cells enter as well as progress through S-phase. The action of p53 is induced in response to DNA damage. Under normal circumstances p53 levels remain very low due to its interaction with a member of the ubiquitin ligase family called MDM2. MDM2 is so named since it was isolated as an amplified gene in the tumorigenic mouse cell line 3T3DM. In response to DNA damage, e.g. as a result of uv-irradiation or γ-irradiation, cells activate several kinases including checkpoint kinase 2 (CHK2) and ataxia telangiectasia mutated (ATM). One target of these kinases is p53. ATM also phosphorylates MDM2. When p53 is phosphorylated it is released from MDM2 and can carry out its transcriptional activation functions. One target of p53 is the cyclin inhibitor p21Cip1 gene. Activation of p21Cip1 leads to increased inhibition of the cyclin D1-CDK4 and cyclin E-CDK2 complexes thereby halting progression through the cell cycle either prior to S-phase entry or during S-phase. As a consequence of p53-induced synthesis of p21 expression, there is a convergence between the roles of p53 and pRB (as outlined above) in regulation of cyclin-CDK complexes. In either case the aim is to allow the cell to repair its damaged DNA prior to replication or mitosis.
Activation of p53 by cellular stress and DNA damage. Normal regulation of the level of p53 protein is carried out by various ubiquitin ligases (e.g. MDM2) that ubiquitinate p53 resulting in its degradation in the proteosome. In response to DNA damage, or cellular stress, several kinases become activated that hyperphosphorylate p53 allowing the protein to be released from MDM2. Phospho-p53 enters the nucleus and activates the transcription of many target genes, with the CDK inhibitory protein (CIP) gene, p21CIP being shown. Increased levels of p21CIP results in inhibition of several cyclin-CDK complexes causing cell cycle arrest.
Even given the limited discussion of the functions of pRB and p53 it is still easy to understand how loss of either protein function can lead to aberrant cell cycle progression and the potential for the development of cancer.back to the top
The process of cell division must occur in a highly ordered and accurate manner so as to ensure that each daughter cell receives an identical copy of the parental cells genome. Obviously the first step in this process is the accurate replication of the genome as described in the DNA Metabolism page. The processes described above relate to the regulation of the progressive steps taken as a cell progresses to cytokinesis. This section will discuss the biochemical processes undertaken to effect accurate separation of the duplicated DNA (the sister chromatids) and cytokinesis.
Following duplication of the chromosomes the sister chromatids are held together through multisubunit protein complexes referred to as cohesins (described briefly above). These complexes are found all along the length of each chromatid as the DNA is replicated. Following DNA replication the chromosomes condense and this is the role of proteins called condensins. Chromosome condensation is the first easily identifiable sign that a cell is about to enter M-phase. Cohesins and condensins are structurally related and act in concert to prepare the chromosomes for mitosis.
The task of separating the sister chromatids such that each daughter cell receives one copy of each chromosome is carried out by the mitotic spindle which is composed of microtubules and several proteins that interact with them. An additional cytoskeletal structure is required for the actual separation of the cell into two new cells. This structure is referred to as the contractile ring. The process of mitosis occurs through a highly ordered series of five steps as outlined above. The actual separation of the parental cell into two daughter cells can be considered the sixth step in mitosis. Whereas, prophase, prometaphase, metaphase, anaphase, and telophase occur in a strictly controlled sequential fashion, cytokinesis begins in anaphase and continues until the cell divides.
During interphase the microtubule machinery is in a constant state of dynamic instability. Individual microtubules are growing or shrinking at any given moment. During prophase the activation of M-CDK complexes initiates a change in the microtubule structures to one where there are a large number of shorter microtubules surrounding each centrosome. The centrosomes are cytoplasmic nucleation sites for the mitotic spindles. M-CDK complexes initiate these changes via the phosphorylation of microtubule motor proteins and microtubule-associated proteins (MAPs).
During prometaphase the nuclear envelope abruptly breaks down as a consequence of M-CDK complexes phosphorylating the nuclear lamina. The dissolution of the nuclear membrane allows the microtubules to access the mitotic spindles. When microtubules attach to the mitotic spindle they become stabilized. The microtubules eventually become attached at the kinetochore which is a complex protein structure that assembles onto the highly condensed DNA at the centromere. The chromosomes are pulled back and forth by the microtubules eventually becoming aligned equidistant from the two spindle poles. The alignment of the chromosomes forms the metaphase plate. The chromosomes oscillate about the metaphase plate awaiting the signal that will induce the sister chromatids to separate. This phase of mitosis is referred to as the spindle-attachment checkpoint and it ensures that cells do not enter anaphase until all of the chromosomes are attached to both poles of the mitotic spindles. As described above, sister chromatids begin to separate with the activation of the APC. As each sister chromatid is pulled towards one of the poles of the mitotic spindle the kinetochore microtubules depolymerize.
By the end of anaphase, the daughter chromosomes have separated to opposite ends of the cells and have begun to decondense which signals the onset of telophase. Telophase is denoted by the reassembly of the nuclear envelope around each group of daughter chromosomes. During this process the lamins that make of the nuclear lamina are dephosphorylated allowing them to re-associate with the nuclear envelope. Following the formation of the new nuclear envelopes, the chromosomes decondense into their interphase state and transcriptional activity begins anew. The cell is now ready for the final process, complete separation into two daughter cells.back to the top