Examining and explaining the mechanisms of centrosomal aberrations

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The centrosome is a little non-membranous organelle (1-2 mm in diameter) normally localized at the periphery of nucleus. Its primary function is to nucleate (anchor) microtubules, meaning it has a key role in the establishment of the interphase cytoplasmic microtubule network and bipolar mitotic spindles hence often denoted as a major microtubule organizing center (MTOC). The centrosome in animal cells consists of a pair of centrioles, which are joined by fibers connecting their proximal ends, and a number of different proteins surrounding the centriole pair, which are referred to as pericentriolar material (PCM) as a whole. The centrioles in the pair structurally differ from each other; one having appendages at the distal ends this is known as the mother centriole and the other without appendages which is the daughter centriole. These appendages appear to be essential for anchoring microtubules. The daughter centriole acquires the appendages in late G2 phase of the cell cycle. centrosome.jpg

During interphase, centrosomes organize the cytoplasmic microtubule network, which is involved in vesicle transport, proper distribution of small organelles, and establishment of cellular shape and polarity. During mitosis, centrosomes become the core structures of spindle poles and direct the formation of mitotic spindles. On account of each daughter cell receiving only one centrosome at the time of cytokinesis, the centrosome, like DNA, must duplicate once prior to the next mitosis. Thus, at any given time in the cell cycle, cells have either are either unduplicated or have two duplicated centrosomes. Because centrosome and DNA are the only two organelles subject to semi conservative duplication once every single cell cycle, animal cells are furnished with structures that coordinate these two events, to make certain these two organelles to duplicate only once. By late G2, two mature centrosomes are generated and move to the opposite ends of the cell. These then become spindle poles that direct the formation of bipolar mitotic spindles.

Functions of centrosomes include 1) formation of the network of microtubules (spindle fibers) that participate in making the cytoskeleton 2) signaling that it is okay to continue to cytokinesis. Abolition of both centrosomes with a laser beam inhibits cytokinesis even with the condition that mitosis has been completed normally, 3) signaling that it is okay for the daughter cells to start another round of the cell cycle characteristically to duplicate their chromosomes in the subsequent S phase. Abolition of one centrosome with a laser beam still warrants cytokinesis but the daughter cells does not advance to a new S phase. 4) The vertebrate centrosome is a very organized organelle that among other functions serves as the cell microtubule organizing center a.k.a MTOC. For this function, many proteins engage in the nucleation (gamma-tubulin, pericentrin, polo kinases, aurora kinases), anchoring (dynactin, centriolin) and release microtubules from the centrosome.

The bipolarity of the mitotic spindles is crucial for the accurate segregation of duplicated chromosomes; failure to form proper bipolar mitotic spindles, due to numeral as well as functional abnormalities of centrosomes, results in chromosome segregation errors. The consequential addition or subtraction of chromosomes greatly accelerates tumour progression, because addition or subtraction of even a single chromosome can simultaneously introduce multiple genetic alterations required for the acquisition of malignant phenotypes (for example, loss of tumoursuppressor genes or acquisition of oncogenes). Indeed, almost all solid tumours are highly aneuploid, and chromosome gain or loss in most cases can be attributed to Indeed, chromosome segregation errors due to mitotic defects. Among several potential mechanisms for chromosome instability in cancer cells, much attention has recently been given to numeral and functional abnormality of centrosomes because of its prevalence in almost all types of solid tumours and certain cases of leukaemia and lymphoma, and a strong association between centrosome abnormality and a high degree of aneuploidy in cancers.

The presence of two centrosomes at mitosis is crucial for the formation of bipolar mitotic spindles. Thus, numerical integrity of centrosomes is carefully controlled, and retraction of this control results in centrosome amplification, which leads to the formation of aberrant mitotic spindles with multiple (>2) spindle poles. Cells with amplified centrosomes also form 'pseudo-bipolar' spindles. Amplified centrosomes manage to position on a bipolar axis by unknown mechanisms, and form mitotic spindles, which structurally resemble 'true' bipolar spindles organized by two centrosomes. It is also conceivable that some multipolar spindles can transform to pseudo-bipolar spindles during mitosis. Cells with pseudo-bipolar spindles seem to undergo normal cytokinesis lacking any chromosome segregation errors. However, even these pseudo-bipolar spindles often encounter a risk of chromosome destabilization: one or a few amplified centrosomes fault to line up on the bipolar axis, yet they are actively complete, nucleating microtubules which capture chromosomes. Aneuploid daughter cells can be generated, and this depends on which daughter cell acquires the chromosomes.

In most cases, tripolar spindles can be subject to cytokinesis, and some daughter cells are functional yet suffer severe aneuploidy (panel a). A cell with mitotic spindles with >3 spindle poles fail to undergo cytokinesis (panel b), and becomes either a bi-nucleated or large mono-nucleated cell. Depending on the status of p53, this cell can either becomes arrested or continues cell cycling to become a multi-nucleated cell. Some cells with amplified centrosomes form 'pseudo-bipolar'spindles (panel c). When this happens, a normal mitotic process takes place. Another mitotic aberration that is associated with numeral abnormality of centrosomes is the formation of monopolar spindles, which occurs when centrosomes fail to duplicate. A cell with monopolar spindles cannot undergo cytokinesis, and often becomes large and mononucleated. Like the mitotic spindles with >3 poles, the cells with monopolar spindles will exit mitosis without cytokinesis. Depending on the p53 status, these cells will either undergo cell-cycle arrest and/or cell death or continue further cell cycling (panel d).

There are several types of functional defect of the centrosome that interfere with the formation of bipolar spindles. Two duplicated centrosomes are physically connected, and stay close to each other until late G2 when they separate and migrate to opposite ends. Failure of the duplicated centrosomes to separate results in the formation of aberrant mitotic spindles with a single spindle pole (monopolar spindles), as seen in cells exposed to Eg5 inhibitors. The ability of the centrosome to properly nucleate and/or anchor microtubules is essential for the formation of bipolar mitotic spindles, which is acquired after duplication by recruiting many critical pericentriolar material components (eg. PLK1, LATS2 etc for centrosome maturation). The failure to undergo an appropriate centrosome maturation process results in failure to form proper mitotic spindles as seen in cells inhibited for polo-like kinase 1. During mitosis, centrosomes are subjected to the strong pulling forces exerted by the microtubules attached to chromosomes, and thus they need to structurally fortify themselves before mitosis (proteins involved are, for example, PLK1). The failure to do so results in centrosome fragmentation; centrosomes are physically ripped apart by the forces exerted by microtubules. The resulting acentriolar centrosomal fragments still retain the ability to nucleate microtubules, and act as extra centrosomes. The centrioles are tightly paired throughout the cell cycle except during initiation of centrosome duplication. The abrogation of the mechanism underlying the centriole pairing leads to uncontrolled splitting of centrioles, which results in the generation of extra centrosomes that contain only one centriole, which retain the ability to nucleate microtubules.

Mitotic aberrations associated with numeral and functional abnormalities of centrosomes such as generation of misaligned or fragmented chromosomes, failure of spindle attachment abnormal spindle tension at kinetochores trigger spindle checkpoint, leading to mitotic catastrophe and cell death. Cells with amplified centrosomes frequently undergo cell death owing to prolonged activation of the spindle checkpoint function. When exposed to DNA synthesis inhibitors, for example hydroxyurea (HU), which depletes dNTPs, cells become captured at the transition from G1 to S phase and also in S-phase. In these cells, centrosomes continue to reduplicate in the absence of DNA synthesis if p53 is either lost or mutated. In the presence of functionally flawless p53, p53 is maintained in response to stress associated with prolonged arrest, leading to upregulation of p21, which in turn perpetually prevents CDK2, hence blocking centrosome duplication. On the other hand, in the non appearance of p53, CDK2 activity is unchecked and turned on, which triggers centrosome reduplication, resulting in centrosome amplification.

There are four possible mechanisms that can result in the generation of amplified centrosomes; multiple rounds of centrosome duplication in single cell cycle, failed cytokinesis, resulting in genome doubling and number of centrosomes, untimely split of paired centrioles to form individual centrosomes, De novo formation of MTOCs without centrioles that function as centrosomes as shown below.

Many proteins that are frequently mutated in cancers take part in the maintenance of centrosome duplication and also the numerical integrity of centrosomes. Those proteins belong to one of three functional groups: cell-cycle regulation, DNA-damage response and/or repair, nucleocytoplasmic transport. Mutational activation or inactivation of all these proteins leads to supernumerary centrosomes, which in turn increases the frequency of mitotic defects and chromosome segregation errors, and thus promotes tumor progression. In general, the positive regulators of centrosome duplication are oncogenic, and the negative regulators are tumor suppressors. Cyclin E/Cdk 2, a known activator of S-phase entry, has a major role in the start of centrosome duplication. The late-G1-specific activation of Cyclin E/Cdk 2 organizes the beginning of centrosome and DNA duplication. Unduplicated centrosomes quickly respond to active CDK2-cyclin E and start duplication. On the other hand, newly duplicated centrosomes do not reduplicate in the existence of active CDK2-cyclin E immediately. The analysis of cells that are halted by exposure to DNA-synthesis inhibitors demonstrates that duplicated centrosomes continually reduplicate in the absence DNA synthesis, however, they do so only after a reasonable length of time (about 20 hours), suggesting that duplicated centrosomes need to regain duplication competency before reinitiating duplication. Several CDK2-cyclin E targets have been identified, including nucleophosmin (NPM) that participates in various cellular events, including DNA duplication and nucleocytoplasmic transport. Within the unduplicated centrosome, NPM localizes between the paired centrioles, probably functioning in centriole pairing.On phosphorylation by CDK2-cyclin E, most NPM dissociates from centrosomes, leading to splitting of the paired centrioles, which is an initial event of centrosome duplication. After phosphorylation by CDK2-cyclin E, NPM acquires an increased binding affinity for ROCK2 and can strongly activate ROCK2 at centrosomes which rapidly drives centrosome duplication.

A strong correlation between the occurrence of centrosome amplification and the degree of aneuploidy has been observed. Interestingly, loss or inactivating mutation of certain tumor suppressor proteins, most notably p53, results in centrosome amplification. p53 has been implicated in the control of centrosome duplication and numeral homeostasis of centrosomes. This is because centrosome amplification and consequential mitotic aberrations are frequent in embryonic fibroblasts as well as various tissues of p53-deficient mouse. These observations imply that destabilization of chromosomes due to centrosome amplification contributes to the cancer susceptibility phenotype associated with loss or mutational inactivation of p53. The mechanism for centrosome amplification is associated with loss of p53. Centrosomes undergo multiple rounds of duplication in rodent cells exposed to DNA synthesis inhibitors such as aphidicolin (Aph) or hydroxyurea (HU). However this occurs only when p53 is either mutated or lost; in the presence of wild-type p53, centrosome re-duplication is also blocked by exposure to DNA synthesis inhibitors. p53 is up-regulated upon prolonged exposure to Aph or HU, which in turn transactivates p21. The increased levels of p21 block the initiation of centrosome duplication via continuous inhibition of CDK2/cyclin E. In contrast, in cells lacking p53, the cellular stress imposed by DNA synthesis inhibitors does not up-regulate p21, hence allowing activation of CDK2/cyclin E, which triggers centrosome re-duplication.

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