Automated classification of mitotic catastrophe by use of the centromere fragmentation morphology.

Mitotic catastrophe is a common mode of tumor cell death. Cancer cells with a defective cell-cycle checkpoint often enter mitosis with damaged or under replicated chromosomes following genotoxic treatment. Premature condensation of the under-replicated (or damaged) chromosomes results in double-stranded DNA breaks at the centromere (centromere fragmentation). Centromere fragmentation is a morphological marker of mitotic catastrophe and is distinguished by the clustering of centromeres away from the chromosomes. We present an automated 2-step system for segmentation of cells exhibiting centromere fragmentation. The first step segments individual cells from clumps. We added two new terms, weighted local repelling term (WLRt) and weighted gradient term (WGt), in the energy functional of the traditional Chan-Vese based level set method. WLRt was used to generate a repelling force when contours of adjacent cells merged and then penalized the overlap. WGt enhances gradients between overlapping cells. The second step consists of a new algorithm, SBaN (shape-based analysis of each nucleus), which extracts features like circularity, major-axis length, minor-axis length, area, and eccentricity from each chromosome to identify cells with centromere fragmentation. The performance of SBaN algorithm for centromere fragmentation detection was statistically evaluated and the results were robust.

Thyroid hormone receptor interacting protein 13 (TRIP13) AAA-ATPase is a novel mitotic checkpoint-silencing protein.

The mitotic checkpoint (or spindle assembly checkpoint) is a fail-safe mechanism to prevent chromosome missegregation by delaying anaphase onset in the presence of defective kinetochore-microtubule attachment. The target of the checkpoint is the E3 ubiquitin ligase anaphase-promoting complex/cyclosome. Once all chromosomes are properly attached and bioriented at the metaphase plate, the checkpoint needs to be silenced. Previously, we and others have reported that TRIP13 AAA-ATPase binds to the mitotic checkpoint-silencing protein p31(comet). Here we show that endogenous TRIP13 localizes to kinetochores. TRIP13 knockdown delays metaphase-to-anaphase transition. The delay is caused by prolonged presence of the effector for the checkpoint, the mitotic checkpoint complex, and its association and inhibition of the anaphase-promoting complex/cyclosome. These results suggest that TRIP13 is a novel mitotic checkpoint-silencing protein. The ATPase activity of TRIP13 is essential for its checkpoint function, and interference with TRIP13 abolished p31(comet)-mediated mitotic checkpoint silencing. TRIP13 overexpression is a hallmark of cancer cells showing chromosomal instability, particularly in certain breast cancers with poor prognosis. We suggest that premature mitotic checkpoint silencing triggered by TRIP13 overexpression may promote cancer development.

Human mitochondrial Fis1 links to cell cycle regulators at G2/M transition.

We have previously shown that prolonged mitochondrial elongation triggers cellular senescence. Here, we report that enforced mitochondrial elongation by hFis1 depletion caused a severe defect in cell cycle progression through G2/M phase (~3-fold reduction in mitotic index; p < 0.01). Reintroduction of Myc-hFis1 to these cells induced mitochondrial fragmentation and restored the cell cycle, indicating that morphodynamic changes of mitochondria closely link to the cell cycle. In hFis1-knockdown cells, cell cycle regulators governing the G2/M phase, including cyclin A, cyclin B1, cyclin-dependent kinase1 (Cdk1), polo-like kinase1 (Plk1), aurora kinase A and Mad2, were significantly suppressed (2- to 10-fold). Notably, however, when mitochondrial fragmentation was induced by double knockdown of hFis1 and Opa1, the cells regained their ability to enter mitosis, and cell cycle regulators were rebounded. Reconstitution of the cyclin B1/Cdk1 complex, a major regulator of the G2/M transition, failed to restore mitotic entry in hFis1-depleted cells. In contrast, expression of Plk1, an upstream regulator of the cyclin B1/Cdk1 complex, or FoxM1 (forkhead box M1), a master transcriptional factor for the cell cycle regulators of G2/M phase, restored the cell cycle in these cells. Our findings suggest that mitochondrial fission molecule hFis1 ensures the proper cell division by interplay with the cell cycle machinery.

Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors.

Cell cycle checkpoint kinases serve as important therapeutic targets for various cancers. When they are inhibited by small molecules, checkpoint abrogation can induce cell death or further sensitize cancer cells to other genotoxic therapies. Particularly aberrant Cdk1 activation at the G2/M checkpoint by kinase inhibitors causing unscheduled mitotic entry and mitotic arrest was found to lead to DNA damage and cell death selectively in cancer cells. Promising drugs inhibiting kinases like Wee1 (Adavosertib), Wee1+Myt1 (PD166285), ATR (AZD6738) and Chk1 (UCN-01) have been developed, but clinical data has shown variable efficacy for them with poorly understood mechanisms of resistance. Our lab recently identified Myt1 as a predictive biomarker of acquired resistance to the Wee1 kinase inhibitor, Adavosertib. Here, we investigate the role of Myt1 overexpression in promoting resistance to inhibitors (PD166285, UCN-01 and AZD6738) of other kinases regulating cell cycle progression. We demonstrate that Myt1 confers resistance by compensating Cdk1 inhibition in the presence of these different kinase inhibitors. Myt1 overexpression leads to reduced premature mitotic entry and decreased length of mitosis eventually leading to increased survival rates in Adavosertib treated cells. Elevated Myt1 levels also conferred resistance to inhibitors of ATR or Chk1 inhibitor. Our data supports that Myt1 overexpression is a common mechanism by which cancer cells can acquire resistance to a variety of drugs entering the clinic that aim to induce mitotic catastrophe by abrogating the G2/M checkpoint.

Tripin/hSgo2 recruits MCAK to the inner centromere to correct defective kinetochore attachments.

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for centromere cohesion (Kitajima et al., 2006). In this study, we show that hSgo2 exhibits a dynamic distribution pattern, and that its localization depends on the BUB1 and Aurora B kinases. hSgo2 is concentrated at the inner centromere of unattached kinetochores, but extends toward the kinetochores that are under tension. This localization pattern is reminiscent of MCAK, which is a microtubule depolymerase that is believed to be a key component of the error correction mechanism at kinetochores. Indeed, we found that hSgo2 is essential for MCAK to localize to the centromere. Delocalization of MCAK accounts for why cells depleted of hSgo2 exhibit kinetochore attachment defects that go uncorrected, despite a transient delay in the onset of anaphase. Consequently, these cells exhibit a high frequency of lagging chromosomes when they enter anaphase. We confirmed that hSgo2 is associated with PP2A, and we propose that it contributes to the spatial regulation of MCAK activity within inner centromere and kinetochore.

hZwint-1 bridges the inner and outer kinetochore: identification of the kinetochore localization domain and the hZw10-interaction domain.

Accurate chromosome segregation in mitosis is required to maintain genetic stability. hZwint-1 [human Zw10 (Zeste white 10)-interacting protein 1] is a kinetochore protein known to interact with the kinetochore checkpoint protein hZw10. hZw10, along with its partners Rod (Roughdeal) and hZwilch, form a complex which recruits dynein-dynactin and Mad1-Mad2 complexes to the kinetochore and are essential components of the mitotic checkpoint. hZwint-1 localizes to the kinetochore in prophase, before hZw10 localization, and remains at the kinetochore until anaphase, after hZw10 has dissociated. This difference in localization timing may reflect a role for hZwint-1 as a structural kinetochore protein. In addition to hZw10, we have found that hZwint-1 interacts with components of the conserved Ndc80 and Mis12 complexes in yeast two-hybrid and GST (glutathione transferase) pull-down assays. Furthermore, hZwint-1 was found to have stable FRAP (fluorescence recovery after photobleaching) dynamics similar to hHec1, hSpc24 and hMis12. As such, we proposed that hZwint-1 is a structural protein, part of the inner kinetochore scaffold and recruits hZw10 to the kinetochore. To test this, we performed mutagenesis-based domain mapping to determine which regions of hZwint-1 are necessary for kinetochore localization and which are required for interaction with hZw10. hZwint-1 localizes to the kinetochore through the N-terminal region and interacts with hZw10 through the C-terminal coiled-coil domain. The two domains are at opposite ends of the protein as expected for a protein that bridges the inner and outer kinetochore.

Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.

Cancer cells typically heavily rely on the G2/M checkpoint to survive endogenous and exogenous DNA damage, such as genotoxic stress due to genome instability or radiation and chemotherapy. The key regulator of the G2/M checkpoint, the cyclin-dependent kinase 1 (CDK1), is tightly controlled, including by its phosphorylation state. This posttranslational modification, which is determined by the opposing activities of the phosphatase cdc25 and the kinase Wee1, allows for a more rapid response to cellular stress than via the synthesis or degradation of modulatory interacting proteins, such as p21 or cyclin B. Reducing Wee1 activity results in ectopic activation of CDK1 activity and drives premature entry into mitosis with unrepaired or under-replicated DNA and causing mitotic catastrophe. Here, we review efforts to use small molecule inhibitors of Wee1 for therapeutic purposes, including strategies to combine Wee1 inhibition with genotoxic agents, such as radiation therapy or drugs inducing replication stress, or inhibitors of pathways that show synthetic lethality with Wee1. Furthermore, it become increasingly clear that Wee1 inhibition can also modulate therapeutic immune responses. We will discuss the mechanisms underlying combination treatments identifying both cell intrinsic and systemic anti-tumor activities.

Cell Synchronization Techniques for Studying Mitosis.

Cell synchronization allows the examination of cell cycle progression. Nocodazole and other microtubule poisons have been used extensively to interfere with microtubule function and arrest cells in mitosis. Since microtubules are important for many cellular functions, alternative cell cycle synchronization techniques independent of microtubule inhibition are also used for synchronizing cells in mitosis. Here we describe using nocodazole, STLC, and combining thymidine block with MG132 to synchronize cells in mitosis. These inhibitors are reversible and mitotic cells can be released into the G1 phase synchronously. These techniques can be applied to both Western blot and timelapse imaging to study mitotic progression.

Mutations of the DNA repair gene PNKP in a patient with microcephaly, seizures, and developmental delay (MCSZ) presenting with a high-grade brain tumor.

Polynucleotide Kinase-Phosphatase (PNKP) is a bifunctional enzyme that possesses both DNA 3'-phosphatase and DNA 5'-kinase activities, which are required for processing termini of single- and double-strand breaks generated by reactive oxygen species (ROS), ionizing radiation and topoisomerase I poisons. Even though PNKP is central to DNA repair, there have been no reports linking PNKP mutations in a Microcephaly, Seizures, and Developmental Delay (MSCZ) patient to cancer. Here, we characterized the biochemical significance of 2 germ-line point mutations in the PNKP gene of a 3-year old male with MSCZ who presented with a high-grade brain tumor (glioblastoma multiforme) within the cerebellum. Functional and biochemical studies demonstrated these PNKP mutations significantly diminished DNA kinase/phosphatase activities, altered its cellular distribution, caused defective repair of DNA single/double stranded breaks, and were associated with a higher propensity for oncogenic transformation. Our findings indicate that specific PNKP mutations may contribute to tumor initiation within susceptible cells in the CNS by limiting DNA damage repair and increasing rates of spontaneous mutations resulting in pediatric glioma associated driver mutations such as ATRX and TP53.