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Cancer Biology & Therapy Dec 2023Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of...
Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)-treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials.
Topics: Humans; Animals; Mice; Cyclin-Dependent Kinase 2; Centrosome; Anaphase; Mitosis; Aneuploidy; Neoplasms
PubMed: 38031910
DOI: 10.1080/15384047.2023.2279241 -
Nature Communications Apr 2024Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/C) prevents cell-cycle entry by targeting...
Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/C) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/C activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/C mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/C activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.
Topics: Proteomics; Cell Cycle; Anaphase-Promoting Complex-Cyclosome; Cell Cycle Proteins; Phosphorylation; Protein Stability; NIMA-Interacting Peptidylprolyl Isomerase; Mitosis
PubMed: 38622115
DOI: 10.1038/s41467-024-47427-w -
Stem Cell Research & Therapy Dec 2023Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in...
BACKGROUND
Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.
METHODS
After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.
RESULTS
Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.
CONCLUSION
This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.
Topics: Humans; Stem Cells; Limbal Stem Cells; Limbus Corneae; Epithelium, Corneal; Spindle Apparatus; Endotoxins
PubMed: 38093301
DOI: 10.1186/s13287-023-03586-z -
BioRxiv : the Preprint Server For... Aug 2023In many species, early embryonic mitoses proceed at a very rapid pace, but how this pace is achieved is not understood. Here we show that in the early embryo, cyclin B3...
In many species, early embryonic mitoses proceed at a very rapid pace, but how this pace is achieved is not understood. Here we show that in the early embryo, cyclin B3 is the dominant driver of rapid embryonic mitoses. Metazoans typically have three cyclin B isoforms that associate with and activate Cdk1 kinase to orchestrate mitotic events: the related cyclins B1 and B2 and the more divergent cyclin B3. We show that whereas embryos expressing cyclins B1 and B2 support slow mitosis (NEBD to Anaphase ~ 600s), the presence of cyclin B3 dominantly drives the ~3-fold faster mitosis observed in wildtype embryos. CYB-1/2-driven mitosis is longer than CYB-3-driven mitosis primarily because the progression of mitotic events itself is slower, rather than delayed anaphase onset due to activation of the spindle checkpoint or inhibitory phosphorylation of the anaphase activator CDC-20. Addition of cyclin B1 to cyclin B3-only mitosis introduces an ~60s delay between the completion of chromosome alignment and anaphase onset, which likely ensures segregation fidelity; this delay is mediated by inhibitory phosphorylation on CDC-20. Thus, the dominance of cyclin B3 in driving mitotic events, coupled to introduction of a short cyclin B1-dependent delay in anaphase onset, sets the rapid pace and ensures fidelity of mitoses in the early embryo.
PubMed: 37609212
DOI: 10.1101/2023.08.11.553011 -
Life Science Alliance Dec 2023SiR-DNA/SiR-Hoechst is a far-red fluorescent DNA probe that is routinely used for live-cell imaging of cell nuclei in interphase and chromosomes during mitosis. Despite...
SiR-DNA/SiR-Hoechst is a far-red fluorescent DNA probe that is routinely used for live-cell imaging of cell nuclei in interphase and chromosomes during mitosis. Despite being reported to induce DNA damage, SiR-DNA has been used in more than 300 research articles, covering topics like mitosis, chromatin biology, cancer research, cytoskeletal research, and DNA damage response. Here, we used live-cell imaging to perform a comprehensive analysis of the effects of SiR-DNA on mitosis of four human cell lines (RPE-1, DLD-1, HeLa, and U2OS). We report a dose-, time-, and light-dependent effect of SiR-DNA on chromosome segregation. We found that, upon the exposure to light during imaging, nanomolar concentrations of SiR-DNA induce non-centromeric chromosome entanglement that severely impairs sister chromatid segregation and spindle elongation during anaphase. This causes DNA damage that is passed forward to the following cell cycle, thereby having a detrimental effect on genome integrity. Our findings highlight the drawbacks in using SiR-DNA for investigation of late mitotic events and DNA damage-related topics and urge the use of alternative labeling strategies to study these processes.
Topics: Humans; Anaphase; DNA; Chromatin; Chromosome Segregation; DNA Damage; HeLa Cells
PubMed: 37726128
DOI: 10.26508/lsa.202302260 -
BioRxiv : the Preprint Server For... Nov 2023Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase APC/C...
Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear if APC/C maintains all types of arrest. Here by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological CDK4/6 inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves cyclin-dependent kinases acting in an atypical order to inactivate RB-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.
PubMed: 37986787
DOI: 10.1101/2023.11.09.566394 -
BioRxiv : the Preprint Server For... Jan 2024Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the...
Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the interface between chromosomes and spindle microtubules and as such are under tight control by complex regulatory circuitry. As part of the chromosomal passenger complex (CPC), the Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint, a feedback control system that delays the onset of anaphase by inhibiting the anaphase-promoting complex/cyclosome. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, whose kinetochores are composed of a unique set of structural and regulatory proteins. Kinetoplastids do not have a canonical spindle checkpoint and it remains unclear how their kinetochores are regulated to ensure the fidelity and timing of chromosome segregation. Here, we show in , the kinetoplastid parasite that causes African sleeping sickness, that inhibition of Aurora B using an analogue-sensitive approach arrests cells in metaphase, with a reduction in properly bi-oriented kinetochores. Aurora B phosphorylates several kinetochore proteins , including the N-terminal region of the divergent Bub1-like protein KKT14. Depletion of KKT14 partially overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 protein results in a prominent delay in the metaphase-to-anaphase transition. Finally, we demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes massive chromosome mis-segregation in anaphase. Our results indicate that the CPC and KKT14 are involved in an unconventional pathway controlling mitotic exit and error-free chromosome segregation in trypanosomes.
PubMed: 38293145
DOI: 10.1101/2024.01.20.576407 -
Life Science Alliance Nov 2023In metazoans, Polo-like kinase (PLK1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation, spindle assembly and progression...
In metazoans, Polo-like kinase (PLK1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation, spindle assembly and progression through mitosis. Here we show that a mutation in the mitochondria-localized protein SPD-3 affects mitotic events by inducing elevated levels of PLK-1 in early embryos. SPD-3 mutant embryos contain abnormally positioned mitotic chromosomes, show a delay in anaphase onset and asymmetrically disassemble the nuclear lamina. We found that more PLK-1 accumulated on centrosomes, nuclear envelope, nucleoplasm, and chromatin before NEBD, suggesting that PLK-1 overexpression is responsible for some of the observed mitotic phenotypes. In agreement with this, the chromosome positioning defects of the mutant could be rescued by reducing PLK-1 levels. Our data suggests that the mitochondrial SPD-3 protein affects chromosome positioning and nuclear envelope integrity by up-regulating the endogenous levels of PLK-1 during early embryogenesis in This finding suggests a novel link between mitochondria and nuclear envelope dynamics and chromosome positioning by increasing the amount of a key mitotic regulator, PLK-1, providing a novel link between mitochondria and mitosis.
Topics: Animals; Mitochondrial Proteins; Caenorhabditis elegans; Cell Cycle; Mitosis; Cell Nucleus
PubMed: 37684042
DOI: 10.26508/lsa.202302011 -
Cancers Apr 2024The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro...
The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.
PubMed: 38730707
DOI: 10.3390/cancers16091755 -
BMC Biology Mar 2024Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses....
BACKGROUND
Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress.
RESULTS
We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20 N-terminus through a canonical MAPK docking site. Most important, the Cdc20 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit.
CONCLUSIONS
Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.
Topics: Schizosaccharomyces; Anaphase-Promoting Complex-Cyclosome; Schizosaccharomyces pombe Proteins; Cdc20 Proteins; Cell Cycle Proteins; Mitosis; Spindle Apparatus
PubMed: 38523261
DOI: 10.1186/s12915-024-01865-6