In previous articles, I have discussed the exquisite design and irreducible complexity of the mitotic cell division apparatus. These previous articles are linked below:
Here, I want to draw attention to a review article by Levine and Holland, published in 2018, on "the impact of mitotic errors on cell proliferation and tumorigenesis."Noting that "mitosis is a delicate event that must be executed with high fidelity to ensure genomic stability," the paper underscores the critical nature of various aspects of the eukaryotic cell cycle machinery.
Spindle Assembly Checkpoint (SAC) Defects
I have previously written (here and here) about the spindle assembly checkpoint, which ensures that chromosome segregation only occurs after a proper association between kinetochores and microtubules has been achieved. But let me give a brief refresher. The anaphase promoting complex or cyclosome (APC/C), an E3 ubiquitin ligase, together with its coactivator Cdc20, drives the progression from metaphase to anaphase by adding a chain of ubiquitin peptides to the protein securin, as well as cyclin B, targeting them for destruction by the proteasome. Securin inhibits an enzyme called separase. When securin is degraded by the proteasome, it liberates separase to cleave the cohesin ring that tethers the sister chromatids together. This facilitates the sister chromatid separation, thereby driving entry into anaphase of the cell cycle. Destruction of cyclin B also inactivates cyclin-dependent kinase 1 (Cdk1), which is necessary for cell division to be completed. The spindle assembly checkpoint ensures that the APC/C only carries out its ubiquitylation if, and only if, a proper association between the kinetochores and microtubules has been accomplished. The mitotic checkpoint complex (consisting of BubR1, Mad2, Bub3, and Cdc20) bind to the APC/C, inhibiting it. Once it has been determined that the connections between the kinetochores and microtubules are secure (i.e., upon satisfaction of the spindle assembly checkpoint), these proteins release APC/C to drive anaphase entry.
Levine and Holland note that "In mammals, inactivation of the SAC leads to dramatic chromosome segregation errors; thus, the SAC is essential for organismal development and the viability of most mammalian cells." Indeed, "mutations that weaken the SAC can result in precocious anaphase onset before complete kinetochore attachment, which dramatically increases the probability of chromosome missegregation." They further observe,
Mouse models have shown that attenuating the SAC promotes aneuploidy and genome instability in vivo. Moreover, mutations in the SAC proteins TRIP13 and BUBR1 cause mosaic variegated aneuploidy (MVA), a rare disorder characterized by high levels of aneuploidy and an increased incidence of tumorigenesis. [Internal citations omitted.]
The TRIP13 protein, referenced above, is an AAA+ ATPase that, in cooperation with p31, catalyzes the conversion of the closed (active) form of Mad2, which binds Cdc20 to inhibit the activity of the APC/C, back to its open (inactive) form, thereby disassembling the MCC and facilitating activation of the APC/C.
Cohesion Defects
As noted above, the two identical sister chromatids of each mitotic chromosome are tethered together by a protein called cohesin, which is cut by the enzyme separase to facilitate segregation during anaphase. Levine and Holland comment,
Consistently, recent work has suggested that the deterioration of cohesion with advanced maternal age is a leading cause of meiotic defects and age-related aneuploidy in female oocytes. Genes involved in sister chromatid cohesion have also been found to be mutated in colorectal cancers and a wide range of myeloid neoplasms. [Internal citations omitted.]
Consistent with this, experimental work has shown that, when a mouse is deficient in the SA1 subunit of cohesin, it results in embryonic lethality. This indicates that cohesin plays a crucial role in development. Mouse embryonic fibroblasts that lacked SA1 showed a reduced proliferation and an increased level of aneuploidy, in consequence of chromosome segregation defects.
Another study explored the roles of meiotic cohesin subunits Rec8 and Rad21L by creating double-knockout mice. The result, again, was severe meiotic defects, including the failure to assemble axial elements of the synaptonemal complex.
Merotelic Attachments
Merotelic attachments occur when a single kinetochore is attached to microtubules from both spindle poles. Unlike other attachment errors, this sort of incorrect attachment does not trigger the spindle assembly checkpoint. Aurora B kinase plays a crucial role in correcting merotelic attachments by phosphorylating kinetochore substrates, thereby destabilizing them and resulting in the detachment of incorrect microtubule-kinetochore interactions. Indeed, inhibition of aurora B kinase results in an increased frequency of merotelic kinetochores.
Though Levine and Holland observe that a majority of merotelic attachments correctly segregate during anaphase, "a proportion of chromosomes with these attachments are delayed in their segregation and end up lagging in the middle of the spindle." These lagging chromosomes can lead to aneuploidy or be sequestered into micronuclei, where they are prone to DNA damage and chromosomal rearrangements. Levine and Holland note that "Merotelic attachments are thus likely to be a major source of genetic instability in human tumors."
Kinetochore-Microtubule Stability
It is also essential to proper chromosome segregation that improper kinetochore-microtubule attachments are corrected. This involves the detachment of microtubules from incorrectly attached kinetochores in order to facilitate proper biorientation (i.e., attachment to opposite poles). Cancerous cells often exhibit hyperstable kinetochore-microtubule attachments, which allow incorrect attachments to persist. Levine and Holland note that "overexpression of Mad2 or loss of STAG2 has been shown to result in hyperstabilized K-MT attachments," though "the molecular defects that cause an increase in K-MT attachment stability in most cells with [chromosomal instability] remain unclear."
Centrosome Amplification
Centrosome amplification refers to a situation where the presence of extra centrosomes contributes to chromosomal instability and aneuploidy. This can lead to the formation of multipolar mitotic spindles, resulting in segregation of the chromosomes into more than two daughter cells. There are, therefore, mechanisms in place to prevent a second round of centrosome duplication. During mitosis, the two centrioles within a centrosome are "engaged" (tethered to one another) and are unable to duplicate again until they are disengaged. This process is regulated by Polo-like kinase (Plk) and Separase.
Cancer cells with extra centrosomes survive by a mechanism known as centrosome clustering, whereby they group these additional centrosomes into functional spindle poles during mitotic division. This enables the cell to proceed with bipolar division rather than generating a lethal multipolar spindle which would result in massive missegregation of chromosomes. A minus-end-directed kinesin-14 motor protein, called HSET/KIFC1, plays an important role in centrosome clustering (though it is dispensable in healthy cells). Levine and Holland note that "Live-cell imaging has revealed that the progeny of multipolar divisions are frequently inviable, since daughter cells are unlikely to inherit a full complement of chromosomes." Moreover,
A recent study revealed that centrosome clustering in epithelial cells was inhibited by E-Cadherin, which increases cortical contractility and suppresses centrosome movement. Loss of E-Cadherin is frequently observed in breast cancer cells with high levels of centrosome amplification, suggesting that cancer cells can select for genetic changes that enable efficient centrosome clustering. [Internal citations omitted.]
Though cancerous cells can form a pseudobipolar spindle by centrosome clustering in order to promote normal segregation, this significantly increases the risk of merotelic attachments, resulting in chromosome missegregation and lagging chromosomes.
Timing of Centrosome Separation
Incorrect timing of separation of the centrosomes can also contribute to the missegregation of chromosomes. Following centrosome duplication, they remain connected by a protein bridge, which has to dissolve before the commencement of mitosis, to facilitate proper bipolar spindle formation.
Levine and Holland observe that "Both delaying and accelerating centrosome separation elevate the frequency of chromosome misattachments to the mitotic spindle, leading to chromosome segregation errors." USP44 is a centrosome-localized deubiquitinase, which is crucial for timely separation of the centrosomes. Indeed, "USP44 knockout mice are pronze to aneuploidization and spontaneous tumor formation." A motor protein, called EG5/KIF11, is responsible for promoting centrosome separation. When it is overexpressed, the consequence is chromosome missegregation and tumor development. Moreover, a tumor suppressor protein, PTEN, helps load EG5 onto centrosomes. Mutations affecting PTEN's ability to promote EG5 loading onto centrosomes also result in aneuploidy.
Tetraploidy
Tetraploidy refers to a genomically unstable condition in which cells have twice the normal set of chromosomes. Tetraploidy can arise through multiple mechanisms, including cytokinesis failure, cell fusion, or endoreduplication (a round of DNA replication without cell division). The authors note,
Tetraploidy generally occurs through one of three main mechanisms. The first is cytokinesis failure, where daughter cells fail to separate following cell division (Fig. 1E). Second, tetraploidization can occur as a result of cell fusion, which can occur spontaneously or as a result of viral infection. Finally, tetraploid cells can arise by endoreduplication, in which two rounds of DNA replication occur without an intervening cell division. Of these three main pathways, cytokinesis failure or mitotic failure is likely to be the major mechanism contributing to the production of tetraploid cells in premalignant lesions. [Internal citations omitted.]
The authors also note that this can occur by "retention of chromatin in the middle of the spindle, which can induce furrow regression." Moreover, "Cytokinesis failure has also been reported to occur following entosis, where viable cell that are internalized by neighboring cells block furrow ingression."
Tetraploid cells possess an abnormally high number of chromosomes, predisposing them to chromosomal instability via multipolar spindle formation.
Irreducible Complexity
This review article furthers the argument that I have developed elsewhere that the eukaryotic cell division cycle is elegantly engineered and irreducibly complex. There are so many things that need to go right for a successful cell division, and it seems to be quite unlikely that such a system could arise by numerous, successive, slight modifications while retaining viability at every stage. A superior explanation for such a tightly controlled and exquisitely designed system is that it arose by virtue of an intelligent cause.