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Summary of the Cell Cycle and the regulatory components discussed on this page.

Regulation of Cell Division

Introduction

Dysregulation of Cell Division Leading to Death

The cell cycle is broken down into 4 main phases: G1 phase, S phase, G2 phase and M phase. The ultimate goal is cell division. M phase is where the chromosomes segregate and the cell physically divides into two daughter cells [1].

Cell division (M-phase) at first glance appears to be a simple and common process. However, as each step is broken down it becomes evident how many processes there are and how many things could go wrong, illustrating that cell division is anything but simple. During cell division there are various steps that need tight regulation, for instance the entry into M-phase, the spindle checkpoint, the reorganization of the actin cytoskeleton, distribution of organelles and fragmentation of the nuclear envelope [1].

The regulation of cell division is essential and even the breakdown of one seemingly small component of this regulation could be catastrophic [2]. Due to the immense nature of the regulation of cell division, for the purpose of this page, only a couple of key regulatory checkpoints and their components will be discussed.

Mitogens will be addressed as they play a role in the commencement of G1/S phase. Although mitogens act before M-phase, they are an extremely important regulatory component of cell division [3].

The first checkpoint addressed will be the entry checkpoint to mitosis, where the regulation ensures that DNA replication is complete and cell division commences as a result of the activation of the Mitosis Promoting Factor (MPF)[4].

The second checkpoint being addressed is the Spindle formation checkpoint, where the regulation ensures that all chromosomes are attached to the appropriate kinetochores before activation of the Anaphase Promoting Complex (APC) and hence the separation of chromosomes in anaphase [5].

The complexity of the regulation of cell division means that there is a plethora of information to be gained in research. Single components of complex pathways can have a tremendous effect on whether cell division occurs or not. Dysregulation can result in the breakdown of cell division in a mitotic catastrophe, halting of cell division or irregular cell division [6].

History

File:Metaphasecolchicine.jpeg
In Reider and Pallazo's 1992 paper, a set of stills from a time lapse showing the effect of the drug colchicine to keep cells at metaphase and study the Spindle Checkpoints.
Date Discovery
1950 The discovery of growth factors were established. Nerve growth factors were one of the first growth-regulating signal substances that was discovered by Rita Levi Montalcinigrowth. [7]
1951 Discovery of a distinct S-phase was achieved by Howard and Pelc.[8]
1960 S-phase cell cycle detection and measurement began with the incorporation of radioactive nucleosides to detect DNA synthesis. [9]
1970 The dependence of S-phase on M-phase initially came from cell fusion experiments who were performed by Potu Rao and Robert Johnson.[10]
1970 Zirkle was one of the first to realise the existance of the spindle checkpoint in the transition from metaphase to anaphase by holding back single chromosomes from reaching the metaphase plate. He observed that anaphase was postponed until the chromosomes all arrived. [11]
1971 Maturation Promoting Factor (MPF) was described as a cytoplasmic activity appearing during meiosis in amphibian oocytes that could cause maturation when injected into resting oocyte.[12]
1980 The first mitogen-activated protein kinase that is called ERK1 (MAPK3) in mammals was discovered. [13]
1983 Timothy Hunt discovered cyclins by studying the fertilisation of sea urchin eggs.[14].
1988 The relationship between Cyclin B and cdc2 was discovered by Maller, Newport & Beach. They concluded that cdc2 protein is a component of Maturation-Promoting Factor (MPF). [15]
1992 Reider and Palazzo further researched the “checkpoint mechanism” and discovered a checkpoint that paused the cell division in the middle of metaphase to anaphase transition if these mitotic checkpoints were not reached. [16]
1999 The concept of "sister chromatid separation" during mitosis was discovered.[17]

Mitogens and Cell Division

Mitogens

A mitogen is an extracellular substance, such as a growth factor, that stimulates cell proliferation. The rate of cell proliferation in unicellular organisms is highly dependant on the availability of nutrients in its environment. However, the cells in multicellular organisms only divide when new cells are necessary. Therefore nutrients alone is not sufficient for the process of cell proliferation [18]. In order to aid this process, cells receive stimulatory extracellular signals via specific mitogens of other cells. One such mitogen is Platelet-derived growth factor (PDGF). Mitogens effect cell division by overcoming the intracellular braking mechanisms that block progression through the cell cycle, therefore increasing the rate of cell division.

Progression from G1 to S phase

The G1 phase is the first phase of the cell cycle. Cells must be activated in order to progress into the S phase. Mitogens stimulate this process by triggering the cells to enter S phase in the G1 phase at which point the cells begin to divide. Entry into the S phase of the cell cycle is regulated by the G1 cyclins (cyclin D) by initiating expression of G1/S and S cyclins. This is done by removing inhibitory proteins from G1/S cyclin-CDK (cyclin dependant kinase) complexes [19]. Throughout the G1 phase of the cell cycle there are several mechanisms that act to suppress Cdk activity therefore inhibiting entry in to S phase. Mitogens act in a way to stimulate Cdk activity in order to allow S phase to begin. This occurs by mitogens binding to cell-surface receptors to initiate a complex array of intracellular signals that penetrate deep into the cytoplasm and nucleus [18]. The activation of G1-Cdk and G1/S-Cdk complexes by mitogens will overcome inhibitory barriers that usually block the cell cycle from progressing in to the S phase.

The impact of mitogens on the cell cycle. A. Cell cycle progression is dependent on CYCLIN-CDK kinase activity. B. Mitogen starvation results in cell cycle arrest in G1 through degradation or suppression of CYCLIN D and CKI-mediated inhibition of CYCLIN E-CDK2

Mitogens are responsible for signalling cells to proliferate and therefore activate Cdk in order to initiate the S phase. Therefore in their absence the G1 phase is maintained and the cell cycle will arrest. A specialised non-dividing state, G0, can be achieved if cells partly disassemble from their cell- cycle control system [18]. Many cells in our body exist in G0 phase therefore their cell-cycle control system is completely dismantled and the expression of specific Cdk and cyclin encoding genes being permanently turned off. Therefore resulting in the arrest of cell division.The presence of retinoblastoma proteins (pRB) prevents expression of the G1/S cyclins, therefore restricting entry into S phase[20]. Mitogens are responsible for the initiation of phosphorylation of pRB proteins and dissociate them from E2F transcription factors[20]. The complexes responsible for the phosphorylation of the pRB proteins are cyclinD-CDK4 and cyclinD-CDK6. Mitogens stimulate cell division by increasing the expression of cyclin D in the G1 phase.

In order to increase the expression of cyclin D, mitogens act via a “classic tyrosine kinase pathway” [19]. When bound to mitogens, their receptors become active and therefore phosphorylate one another on the cytoplasmic domains. A guanine nucleotide exchange factor for Ras recognises these phosphorylated domains resulting in an increase in the amount of Ras-GTP. Activation of the small GTPase Ras is one of the early steps of mitogen activation. The activation of Ras leads to mitogen activated kinase pathways (MAP) being activated with the final kinase phosphorylating transcription factors. This induces the expression of early response genes such as FOS and MYC. Separate complexes are formed by these proteins, which causes activation of G1 cyclin (cyclin D) expression [19].


Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF) is one of many mitogens that are involved in extracellular regulation of cell division. It has been suggested that PDGF is a fundamental component involved in scar formation by functioning as a wound hormone. It is thought to do so by increasing the number of fibroblast population in the wound tissue [18]. It also effects the types and total amount of matrix components that are being synthesised in early wound healing thereby effecting scar formation. The results of research done by Savage et al. (1987) confirmed that the mitogenic effects of PDGF have been shown to have more of an effect on normal skin fibroblasts than scar-derived fibroblasts.

PDGF has a crucial role during the development process, however limited evidence is available for normal physiological function in adults[21]. However, it has been shown that increased activity of PDGF is linked with some diseases. In mammals, four genes have been identified that encode for four different PDGF chains (PDGF-A, PDGF-B, PDGF-C, PDGF-D)[22]. The heterodimer, PDGF-AB, has been distinguished in human platelets and has been shown to have different signalling properties to homodimers. However, research shows that homodimers are more prevalent than heterodimers and there is no overlap better PDGF-A and PDGF-B homodimers, therefore the function of PDGF-AB remains unclear. The core domain found in mammalian PDGFs are crucial for receptor binding and activation, which is necessary for progression into the S phase of the cell cycle[21].

History of Platelet-Derived Growth Factor

File:How Platelets work in the body.jpg
Platelets act in the body by producing clots at the site of tissue damage to prevent excessive bleeding. PDGF is a growth-promoted "activity" that is released from activated platelets

PDGF was first isolated while the cellular and molecular mechanisms that underly the lesions of atherosclerosis were being investigated[23]. It was observed that cultured fibroblasts do not proliferate when given plasma but in fact do so when they are given serum. Plasma and serum are prepared using very different methods. In order to prepare plasma, cells from blood are removed without allowing clotting to occur whereas serum is prepared by removing the remaining cell-free liquid from the blood after the blood has clotted. Platelets are miniature cells that are found in blood that are imperative to the process of blood clotting at sites of tissue damage. They are necessary in order to stop excessive bleeding and also release other factors that stimulate healing[24].

When the process of blood clotting occurs it triggers the involved platelets in the blood clot to release the contents of their secretory vesicles. “The superior ability of serum to support cell proliferation suggested that platelets contain one or more mitogens”[24]. Further research showed that extracts of platelets were able to substitute for serum in order to stimulate fibroblast proliferation, therefore supporting this hypothesis. It was shown that the critical factor in the platelet extracts that allowed this to occur was a protein that was later purified and confirmed as PDGF. As mentioned earlier, it has also been shown that PDGF in the body has a particularly crucial role in increasing the rate of cell division of fibroblasts during wound healing [18]. However, it is not isolated to stimulation of fibroblast cell division. PDGF is also involved in the stimulation of proliferation of other cells such as smooth muscle cells, and neuroglial cells.

Entry into M-phase

During different stages of the cell cycle, there are certain events that take place such as DNA replication, division of cells (M-phase) and the spindle checkpoint [1]. These events need to be operated with one another so that they occur in the appropriate order. For example a cell cannot divide unless its genome has already been replicated. Therefore to assure these types of processes occur correctly, cell cycle presents specific checkpoints such as G1 (Restriction) checkpoint, G2 checkpoint and the spindle check point [25]. Entry to M-phase requires the activation of G2 checkpoint. Nevertheless before the role of G2 phase is established, it is important to describe briefly the M-phase.

What is M-phase?

M-phase also known as mitotic phase, is a period of the eukaryotic cell cycle during which the nucleus and the cytoplasm divide. M-phase involves a series of dramatic procedures that begin with nuclear division, consists of chromosome segregation and ends with cytokinesis. In mammalian cell this occurs in less than an hour, therefore require less time [25].

The Role of G2-Phase

G2 is a checkpoint between S-phase and Mitosis. It is an extra gap phase that is inserted in most cell cycles to allow more time for growth and repair. It is relatively passive part of the cell cycle during interphase. G2 checkpoint assures replicated DNA is distributed to daughter cells without any damage or mutation. It achieves this with certain control mechanisms. For instance if DNA replication is not completed, G2 checkpoint senses this point, then generates a signal that leads to cell cycle arrest. Functioning of G2 checkpoint therefore prevents initiation of M-phase, so cells remain in G2 until the genome has been completely replicated. Also in response to DNA damage, the G2 checkpoint will arrest damaged cells, delaying entry into mitosis until the damage has been repaired [25].

Once replicated DNA is safe, then regulation ensures the cell is ready to enter the M-phase. This occurs at the end of G2 phase, hence G2 involves activation of key regulators such as Cyclins, Cyclin-Dependent Kinases (CDKs) and Mitosis-Promoting Factor.[24]

Cyclin-Dependent kinases (CDKs)

CDKs enzymes are present throughout the cell cycle, but they are inactive at the absence of cyclins. In other words cyclins activate CDKs and need to bind to them in order to reach a maximum at a certain point, particularly when moving from one cell phase to another[24]. CDKs are constitutively expressed in cells whereas cyclins are synthesized and degraded at specific stages of the cell cycle[24]. Animal cells contain at least nine CDKs, however only four of them (CDK 1, 2, 4 & 6) are directly involved in cell cycle control. Cyclin B/CDK1 complex is one of the main key regulators that is involved in transition of G2 checkpoint to M-phase.

Expression of cyclins in cell cycle.jpg

Cyclins

Cyclins are from the family of proteins that are involved in regulation of cell division. They control the progression of cells through the cell cycle by activating cyclin dependent kinases enzymes. Timothy Hunt discovered cyclins by studying the fertilization of sea urchin eggs. In the experiment, while the level of proteins were measured in newly fertilized eggs, it was found that one protein had shortly disappeared at the end of cell division and then gradually appeared again as eggs began the next round of division. He named this protein as “cyclin” and concluded that this protein was driving the cell cycle. Furthermore, Hunt and other scientists deducted that making and destroying cyclins were essential for cell division[14]. There are many different types of cyclins (Cyclin: A, B, C, D, E, F, G & H), but cyclin A, B, D and E are particularly important in cell cycle. Cyclin-B is made in G2 phase and M-phases of the cell cycle. It combines with CDK1 to form the M-phase Promoting Factor (MPF). The cyclin-B/CDK1 complex has shown to encourage many of the preliminary events in mitosis such as chromatin condensation, lamin disassembly, nuclear envelope vesiculation and interaction of centromeres with microtubules emanating from the spindle [25]. The activity of this enzyme therefore seems to control a rate limiting step in the cell cycle. In the absence of cyclin-B, CDK1 has no protein kinase activity but when they both combine, three phosphorylation occurs (CDK1 being phosphorylated on threonine 161, 12 and tyrosine 15). This phosphorylation produces a primed but inactive complex, so at the G2 to M-phase transition, a phosphate known as Cdc25C removes the inhibitory phosphates, generating active kinase which drives the cell into mitosis[24].


Activity of Cyclins with CDKs.gif

Metaphase to Anaphase Transition

Anaphase Promoting Complex

A 3D model of human anaphase promoting complex obtained by cryo-negative staining electron microscopy (EM) and angular reconstitution. Human APC/C is composed of two large domains, known as 'platform' and 'arc lamp'.Elsevier. b A 3D model of budding yeast APC obtained by cryo-EM and angular reconstitution.


The main function of APC is to transition the cell from metaphase to anaphase by degrading proteins that inhibit anaphase.


Cell division is caused by degradation of regulatory proteins and the pathway used in the process is ubiquitin-dependent. A poly-ubiquitin chain is attached to a protein substrate by ubiquitin-ligase and it is “tagged” for the degradation by the 26S proteasome. APC is one of these ubiquitin ligases. [26] APC triggers the transition of metaphase to anaphase by “tagging” proteins to be degraded (usually securin and S and M cyclins). [27] Degraded securin releases separase, which triggers the separation of the sister chromatids together in the cleavage of cohesin, which normally keeps the sister chromatids together. [28]

Figure A shows the role of APC in the degradation of Securing to trigger the separation of the sister chromatids.[29]


Activation

Activator subunits, cdc20 and cdh 1, drives the APC. [30] The cell division cycle 20 homolog (CDC20) and and fizzy/cell division cycle related 1 protein (cdh1) mediate the substrate recognition. CDC20 activated APC during prophase-metaphase and chd1 activated APC in mitotic exit and G1. APC activity is regulated by phosphorylation of APC - governed by three different kinases. These kinases are: cyclin-dependent kinase 1 (CDK1), Cyclin B, polio-like kinase (PLK1) and cAMP-dependent protein kinase (PKA). The phosphorylation process regulates CDC20 ‘s binding to APC. Phosphorylation of CDC20 by CDK1 is required for CDC20 activation of APC and for the regulation of the spindle checkpoint. [31]


Activated APC (either APC/hCDK1 or APC/CDC20) complexes ubiquitinate different substrates depending on the phase of cell division. Anaphase is the beginning of sister chromatid separation. The degradation of securin causes the cleavage of the cohesion complex, allowing the separation. [32] Cyclin A and Cyclin B also cause separation of the sister chromatids and Cyclin B proteolysis inhibits CDK1.


Through the G1 phase, APC degrades factors that regulate spindle-pole separation and spindle disassembly, including kinesin-like DNA-binding protein (KID). The degradation of KID is mediated by APC/CDH1 and APC/CDC20 complexes. CDC20 is a main activator through G1 phase.[33] Addtionally, APC degrades Aurora-A kinase (localised in the spindles) in G1 [34] as well as CDC25A in mitotic exit and G1 through APC/CDH1. [35]

Spindle Checkpoint

When the spindle-checkpoint is activated, it inhibits the activation of APC, thus keeping the cell from transitioning between metaphase and anaphase. The spindle-checkpoint is only inactivated when all kinetochores of sister chromatids are attached to microtubules stemming from opposite chromatids. Even a single unconnected kinetochore can activate the spindle checkpoint [36].

The Spindle Assembly Checkpoint (SAC) functions to make sure the kinetochores and spindle microtubules are properly connected. CDC20 ans SAC proteins are concentrated at the kinetochores during prometaphase until every kinetochore is attached. [37]

The Spindle Checkpoint blocks anaphase by inhibiting the APC. The checkpoint complex also activates a protein, BUB1, through CENP-E (a centromete protein) to block anaphase. [38]

Mitotic Arrest Deficient

Mitotic Arrest Deficient (MAD1) is a protein that functions in the Spindle Assembly Checkpoint. MAD1 uses the anaphase inhibitor MAD2, sending MAD2 to unattached kinetochores. MAD1 is phosphorylated during the formation of the mitotic checkpoint complex and it inhibits APC. [39]

MAD1 is localised in the unattached kinetochores and is essential to the function of the SAC. [40] It is regulated by kinases MPS1 and BUB1 to send it to the unattached kinetochored. Spindle checkpoint inhibitor p31 inhibits MAD1 if the kinetochore is attached to the spindle properly. [41]


Figure 2. MAD2B and CLTA co-localize at the mitotic spindle

Disease

Disease Description
Chronic Myeloid Leukaemia (CML)
The Philadelphia chromosome
A somatic translocation of chromosomes 9 and 22 called the "Philadelphia chromosome" results in an BCR-ABL chimeric gene that acts as an oncogene and impacts various signalling pathways. As a result, patients with CML can exhibit enlargement of the spleen due to the accumulation of neoplastic leukocytes and splenic infarctions due to hyperviscosity of the blood [42].
Ovarian Clear Cell Carcinoma
Emi1 and it's upregulation in Ovarian clear cell carcinoma
A study found that over expresseion of Early Mitosis Inhibitor 1 (Emi1) was present in 82% of clear cell carcinomas. Clear cell carcinomas are more aggressive than other forms, and it is thought that the upregulation of Emi1 results in the upregualtion of different cyclins and disregulation of APC [43].
Retinoblastoma: Paediatric cancer of the eye The G1 checkpoint is an important barrier to proliferation under growth inhibitory conditions. It is frequently mutated in human tumors by various perturbations in the retinoblastoma tumor suppressor (Rb) pathway (Malumbres and Barbacid 2001). Retinoblastoma is initiated by the loss of function of the RB1 gene[44].
Epithelial Cancer
PDGF deregulation and its implications for several diseases
Paracrine PDGF signalling can trigger stromal recruitment and may be involved in epithelial–mesenchymal transition, thereby affecting tumor growth, angiogenesis, invasion, and metastasis[45]

Current Research

Polo-like Kinases and their effect on different phases of cell division

Due to the impact that CDK's have on the regulation of cell division, some current research is exploring the importance of associated protein kinases such as Polo-like kinases (Plks) [46]. Barr et al (2004) reviewed the current research of the impact of Plks on cell division.

The correct levels of Plks is essential for normal cell division. The deregulation or upregulation of Plks can result in activation of checkpoints such as the mitotic entry or spindle checkpoints. Plk1 is thought to have an impact in the feedback loops responsible for the activation of the maturation promoting factor (MPF), although the exact molecular mechanism is unclear.

Plks also can inhibit the APC by phosphorylation and although the exact mechanisms are also unknown, it is known that Plk1 is associated with kinetochores.

Inhibition of APC by Emi1.


Current research done by Reimann et al. (2001) presents the discovery of a new early mitotic inhibitor called Emi1[47]. Emi1 inhibits the Anaphase Promoting Complex (APC) by binding to Cdc20. Cdc20 is a component which is essential for the activation of APC[48]. APC is an important regulatory complex in cell division as it triggers the transition between metaphase and anaphase, but there has been some unknown components surrounding the mechanism of its activation.

The discovery of this inhibitor answers some of the questions about how APC is regulated and activated but there are questions left unanswered. Firstly, a question that may be asked with regards to destruction of the protein is what happens if the protein isn't destroyed after its interaction with Cdc20? The presence of nondestructable isoforms of the protein results in somatic cells not being able to commence through mitosis. The protein, after removal, is destroyed by proteolysis, mediated by the attachment of ubiquitin. Further mechanisms surrounding Emi1's destruction are currently being pursued by researchers Reimann et al.

Another question that presents itself is are there any other inhibitory mechanisms or proteins interacting with Emi1?


Research done by Mocciaro et al. (2012) looks at particular mechanisms of regulation of the two E3 enzymes Skp1–cullin1–F-box complex (SCF) and the anaphase promoting complex (APC) with regards to ubiquitylation[49]. The research of Reimann et al. suggests that if certain components such as Emi1 are not destroyed by ubiquitin-dependent mechanisms, then the cell cannot progress through cell division[47]. This reinforces the importance of ubiquitin-dependant destruction of inhibitory proteins and hence the importance of the enzyme cascade leading to ubiquitylation.

Mocciaro et al. discuss new signals and specific targeting of cell division regulators and groups together ubiquitylation enzymes that work in collaboration. Despite giving an overview of the current understanding of the regulation of SCF and APC, it was made clear that there is still a plethora of information to be uncovered in the topic in terms of cross-talk and mechanisms.

Future Research

Cytoskeletal rearrangement during different phases of cell division

As research on the topic of cell division regulation continues to progress, many questions seem to arise. The following are suggestions of what could be considered in possible future research.

  • Sensor mechanisms that check DNA replication and activate M-Cdk [1]
  • How M-Cdk initiates the morphological changes that occur at the start of cell division, ie actin cytoskeleton rearrangement [50]
  • What triggers APC? It’s known that a Cdc20-APC complex forms and that M-Cdk activity is required, but still unclear what kinases phosphorylate the complex [36].
  • Why do cells with a defective spindle-attachment checkpoint still undergo anaphase with normal timing? Useful accessory or essential component.
  • Can checkpoints in cell division be targeted in chemotherapy therapies as cancer tends to find a way to get through the normal regulation of the checkpoints [1]?
  • How is the actin cytoskeleton regulated in cell division [50]?


The Actin Cytoskeleton and Tropomyosin

Impact of tropomyosin in the regulation of the actin cytoskeleton in cell division is a field for future research. We know that different isoforms can impact cellular activity but don't know a lot about it's impact on cell division.

Tropomyosin is a coiled-coil actin binding protein that resides in the main groove of actin filaments. It that has over 40 different isoforms that occur from 4 genes. Tropomyosin regulates the formation of different actin filament populations as discussed in the Microfilaments lecture [51]. It helps to create different actin populations by regulating how other actin-associated proteins bind to actin ie ADF/cofilin, myosin II [52].

Actin cytoskeleton rearrangement occurs upon entry into cell division as well as the exit, cytokinesis. It has been shown that cytokinesis can be affected by specific isoforms of tropomyosins, even the overexpression of a single isoform is enough to cause cytokinesis failure [53]. So the following questions could be asked:

  • How does tropomyosin regulate cell division? Does it affect the rearrangement of the cytoskeleton upon entry into M-phase?
  • What affect do the different isoforms have on cell division?
  • As the Tm5NM1 isoform is upregulated in cancer, does it have an impact on the replicative potential typical of cancer cells [54]?

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2013 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7

Dr Mark Hill 2013, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G