Academic literature on the topic 'Mitotic slippage'

Biochemical studies suggest that caspase activity is required for a functional mitotic checkpoint (MC) and mitotic slippage. To test this directly, we followed nontransformed human telomerase immortalized human retinal pigment epithelia (RPE-1) cells through mitosis after inhibiting or depleting selected caspases. We found that inhibiting caspases individually, in combination, or in toto did not affect the duration or fidelity of mitosis in otherwise untreated cells. When satisfaction of the MC was prevented with 500 nM nocodazole or 2.5 μM dimethylenastron (an Eg5 inhibitor), 92–100% of RPE-1 cells slipped from mitosis in the presence of pan-caspase inhibitors or after simultaneously depleting caspase-3 and -9, and they did so with the same kinetics (∼21–22 h) as after treatment with nocodazole or Eg5 inhibitors alone. Surprisingly, inhibiting or depleting caspase-9 alone doubled the number of nocodazole-treated, but not Eg5-inhibited, cells that died in mitosis. In addition, inhibiting or depleting caspase-9 and -3 together accelerated the rate of slippage ∼40% (to ∼13–15 h). Finally, nocodazole-treated cells that recently slipped through mitosis in the presence or absence of pan-caspase inhibitors contained numerous BubR1 foci in their nuclei. From these data, we conclude that caspase activity is not required for a functional MC or for mitotic slippage.

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.

Antimicrotubule agents are commonly utilised as front-line therapies against several malignancies, either by themselves or as combination therapies. Cell-based studies have pinpointed the anti-proliferative basis of action to be a consequence of perturbation of microtubule dynamics leading to sustained activation of the spindle assembly checkpoint, prolonged mitotic arrest and mitotic cell death. However, depending on the biological context and cell type, cells may take an alternative route besides mitotic cell death via a process known as mitotic slippage. Here, mitotically arrested cells ‘slip’ to the next interphase without undergoing proper chromosome segregation and cytokinesis. These post-slippage cells in turn have two main cell fates, either cell death or a G1 arrest ensuing in senescence. In this review, we take a look at the factors determining mitotic cell death vs mitotic slippage, post-slippage cell fates and accompanying features, and their consequences for antimicrotubule drug treatment outcomes.

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.

The spindle assembly checkpoint arrests mitotic cells by preventing degradation of cyclin B1 by the anaphase-promoting complex/cyclosome, but some cells evade this checkpoint and slip out of mitosis. Balachandran et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601083) show that the E3 ligase CRL2ZYG11 degrades cyclin B1, allowing mitotic slippage.

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is known to target the degradation of cyclin B1, which is crucial for mitotic progression in animal cells. In this study, we show that the ubiquitin ligase CRL2ZYG-11 redundantly targets the degradation of cyclin B1 in Caenorhabditis elegans and human cells. In C. elegans, both CRL2ZYG-11 and APC/C are required for proper progression through meiotic divisions. In human cells, inactivation of CRL2ZYG11A/B has minimal effects on mitotic progression when APC/C is active. However, when APC/C is inactivated or cyclin B1 is overexpressed, CRL2ZYG11A/B-mediated degradation of cyclin B1 is required for normal progression through metaphase. Mitotic cells arrested by the spindle assembly checkpoint, which inactivates APC/C, often exit mitosis in a process termed “mitotic slippage,” which generates tetraploid cells and limits the effectiveness of antimitotic chemotherapy drugs. We show that ZYG11A/B subunit knockdown, or broad cullin–RING ubiquitin ligase inactivation with the small molecule MLN4924, inhibits mitotic slippage in human cells, suggesting the potential for antimitotic combination therapy.

The response of leukaemia cells to therapeutic agents includes cell cycle arrest and apoptosis. The former response is useful in retarding disease progression, but induction of the latter is essential for disease eradication. Cell death is often related to toxicity so reducing drug-concentration or sensitising target cells to apoptosis is desirable. The relationship between the cell cycle and cell death has been at the centre of recent investigation focusing on mechanisms of cell death that are not driven directly by apoptotic responses. These mechanisms included mitotic catastrophe and mitotic slippage. The K562 myeloid leukaemia cell line exhibits a combination of p53 negativity and carries the Bcr-Abl t (9:22) Philadelphia chromosome. Bcr-Abl is a powerful anti-apoptotic translocation and is the hallmark of chronic myeloid leukaemia (CML). The absence of p53-mediated apoptosis and the anti-apoptotic effects of Bcr-abl delays drug-induced cell death, leaving a window of opportunity to investigate the effects of different agents on leukaemia cells. My investigations show that when DNA-targeting agents are used against myeloid leukaemia cells, G2 cell cycle arrest and apoptosis do not occur together i.e. cell cycle arrest precludes cell death; cells may escape G2 arrest as a result of mitotic slippage. In contrast, when anti-mitotic agents are used, it is necessary to induce mitotic arrest to subsequently induce apoptosis; thus lower concentrations are more effective in inducing apoptosis than higher drug concentrations. Evidence is provided suggesting reduced concentrations of both genotoxic agents and anti-mitotic agents may share a common pathway in inducing cell death that is related to events at mitosis and I suggest that this pathway has potential for exploitation by new agents currently in clinical trials, such as UCN-01, Purvanol, Roscovitine and agents that target the passenger proteins, in reducing the concentration of more conventional agents required to kill the Bcr-Abl positive leukaemias.

Faithful chromosome segregation during mitosis is fundamental for cell viability and genome stability. For a correct division, all kinetochores must be attached to the mitotic spindle and cohesion must be timely removed. Anaphase is triggered by the Anaphase Promoting Complex bound to its regulatory subunit Cdc20 (APC-Cdc20) that polyubiquitylates securin (Pds1 in budding yeast), whose role is to maintain inactive the protease separase (Esp1 in budding yeast) until anaphase onset. Once active, separase cleaves cohesin, thus triggering sister chromatid separation. Separase also promotes cyclinB proteolysis and mitotic exit due to its involvement in the Cdc14-early anaphase release (FEAR) pathway that promotes a partial activation of the Cdc14 phophatase, which is in turn key for CDK inactivation and mitotic exit. Cdc14 is maintained inactive throughout most of the cell cycle bound to its inhibitor Net1/Cfi1 and trapped in the nucleolus. At the beginning of anaphase Cdc14 is released from the nucleolus into the nucleus by the FEAR pathway; subsequently, Cdc14 is released also in the cytoplasm by the MEN (Mitotic Exit Network) pathway. In this way Cdc14 is fully active and can trigger mitotic exit by cyclinB-CDK inactivation. The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism conserved in all eukaryotic organisms that ensures the correct segregation of the genetic material. In fact, it inhibits the metaphase to anaphase transition until all kinetochores are properly attached to the mitotic spindle by inactivating the APC-Cdc20 complex, thus providing the time for error correction. Cells do not arrest indefinitely upon SAC activation. After a variable period of time cells escape from the metaphase arrest also in the presence of a damaged mitotic spindle or faulty kinetochore attachments to spindle microtubules. This process is referred to as adaptation or mitotic slippage and is often involved in the resistance to chemotherapeutic compounds that target the mitotic spindle. In spite of its importance, the adaptation process is still little known. Within this context, the goals of my Ph.D. were: (1) to characterize the molecular mechanisms underlying SAC adaptation and (2) to search for factors involved in this process. For these purposes we used the yeast Saccharomyces cerevisiae as a model organism. (1) We characterized the adaptation process in either the presence or the absence of mitotic spindle perturbations. We depolymerized spindles by using two different drugs that alter microtubule dynamics, i.e. nocodazole and benomyl, whereas we induced SAC hyperactivation without spindle damage by overproducing Mad2 (GAL1-MAD2 cells), one of the key proteins for SAC signal generation and maintenance. We observed that in all the conditions cells are able to adapt, but with different kinetics. In particular, cells adapt faster in benomyl, while in nocodazole and with high levels of Mad2 cells need more time to slip out of mitosis. The few data available about SAC adaptation in higher eukaryotes indicate that SAC adaptation is accompanied by chromatid separation, a decrease in mitotic CDK activity and mitotic exit. Indeed, like in mammalian cells, yeast securin and cyclinB are degraded and sister chromatids are separated during adaptation. In addition, cyclinB stabilization, as well as Cdc20 and Cdc5 (polo kinase) inactivation, markedly delay adaptation, while the only yeast CKI (Sic1) is not involved in this process. Finally, when yeast cells adapt the SAC is likely to be turned off, as shown by the disassembly of the Mad1/Bub3 checkpoint complex. (2) To search for factors involved in SAC adaptation, we performed a genetic screen using GAL1-MAD2 cells. In particular, we screened for mutants that would remain arrested for prolonged times in mitosis upon MAD2 overexpression. We identified Rsc2, a non-essential component of the RSC chromatin remodelling complex, as a regulator of SAC adaptation in yeast. We demonstrated that RSCRsc2 is involved in fine tuning mitotic exit during the unperturbed cell cycle. Its activity becomes particularly important in conditions that would activate the SAC, as it contributes to cyclinB degradation. In the absence of Rsc2 Net1 phosphorylation and the early anaphase release of Cdc14 from the nucleolus are impaired, whereas expression of a dominant allele of CDC14 that loosens Net1 inhibition (CDC14TAB6-1) is sufficient to restore mitotic exit in conditions where Rsc2 becomes essential for this process. We further demonstrated that the ATPase activity of RSC is required for mitotic exit regulation, suggesting that its chromatin-remodelling activity is involved in this process. By studying possible genetic interactions between the RSC2 deletion and FEAR or MEN mutations, we found that RSC2 deletion confers synthetic lethality or sickness to MEN but not to FEAR mutants. Altogether, our data suggest that RSCRsc2 is a novel component of the FEAR pathway. Finally, we demonstrated that Rsc2 interacts in vivo and in vitro with the polo kinase Cdc5, which controls mitotic exit at different levels. Since RSC binds to acetylated histone tails, it is possible that histone transacetylases are also involved in SAC adaptation. We tested if the SAGA (Spt-Ada-Gcn5 Acetyltransferase) complex is involved in SAC adaptation by deleting ADA2 or GCN5 in yeast. Indeed, SAGA seems involved in adaptation, although the contribution of Ada2 and Gcn5 in the process differs depending on the conditions used to activate the SAC. Finally, since we found that upon treatment with benomyl (a microtubule destabilizer) cells adapt dividing nuclei, we wondered if SAC adaptation could be linked to the presence of cytoplasmic microtubules that are still partially detectable in these conditions. We therefore asked whether motor proteins and microtubule regulators are involved in mitotic slippage. Indeed, we found that in the absence of Kip2 and Bik1, which specifically bind to cytoplasmic microtubules, cells divide nuclei and exit mitosis slower than wild type cells, demonstrating that cytoplasmic microtubules and associated proteins could accelerate SAC adaptation. In conclusion, SAC adaptation is a very complex process whose timing probably depends on the interplay between different mechanisms. An important aim for a complete comprehension of this process, as well as for the development of new and more efficient cancer therapies, will be to identify novel factors implicated in adaptation and clarify how their function might be linked to one another.

Background. Temozolomide (TMZ) is a methylating drug that is commonly used in the treatment of glioma. Although many features are still unclear, its general mechanism of action is well described. TMZ induces O6-methylguanine (O6MeG) lesions in DNA, which, in the absence of repair by O6-methylguanine methyltransferase (MGMT), mispair with thymine and start a futile cycle of repair-resynthesis events. The resultant DNA double-strand breaks (DSBs) activate the components of G2 checkpoint, and cells with a 4N DNA content accumulate and remain arrested at the G2/M boundary for several days. Cell death subsequently occurs by senescence, necrosis, or mitotic catastrophe, while apoptosis has been ruled out in many studies. Moreover, the effect of multiple TMZ treatments on G2 arrest and apoptosis induction is not clear. Repair of methylating drug-induced DNA lesions requires monoubiquitination of PCNA and FANCD2. Loss of either protein or inhibition of their monoubiquitination increases drug toxicity. USP1 is a hydrolase that removes monoubiquitin from PCNA and FANCD2, and can potentially play a role in TMZ mechanism of action. Materials and methods. U87, U251 (TMZ-sensitive, low MGMT), and GBM8 (TMZ-resistant, high MGMT) cell lines were used for experiments. The treatment was scheduled with 100μM TMZ for 3 hours for 1, 2, or 3 consecutive days. Cell cycle progression was studied with both FACS-based analysis and a novel time-lapse microscopic real-time analysis using FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator), and apoptosis was measured with FACS-based Annexin V-PI analysis. To address the possible role of USP1 in TMZ action, we examined expression of USP1 at the mRNA levels in expression microarray databases derived from primary GBM. We also used siRNA targeting USP1 to modulate USP1 expression, and studied the effect of USP1 downregulation on TMZ-induced G2 arrest, cell death, and clonogenicity. Results. Compared to single treatment, multiple TMZ treatments cause a significant reduction of clonogenicity in TMZ-sensitive cells and induce a significant increase of apoptosis, particularly in a late stage. However, multiple treatments don’t have any major effect on cell cycle profile. Time-lapse microscopic analysis with FUCCI system showed that TMZ-sensitive glioma cells arrest at the G2 checkpoint for less than 48 hours and, in the presence of an activated G2 checkpoint, they replicate their DNA without cellular division, re-enter the cell cycle at the next G1 phase, and repeat the cycle, ultimately giving rise to polyploid cells. siRNA-mediated suppression of USP1 had no effect on cell cycle progression or the extent of temozolomide-induced G2 arrest. However, while USP1 knockdown alone had minimal effect on cell death, it increased temozolomide-induced loss of clonagenicity both in TMZ-sensitive and TMZ-resistant cells. Further examination of the mechanism of cell death suggested that while control cells, control cells exposed to TMZ, or USP1-suppressed cells rarely underwent apoptotic cell death, temozolomide-treated cells in which USP1 levels were suppressed underwent high rates of apoptosis. Conclusions. The present studies show that TMZ can induce apoptosis in TMZ-sensitive glioma cells, which is visible after 3 days but significant after 7 days. Multiple TMZ treatments don’t affect cell cycle profile, but significantly increase apoptosis. Moreover, time-lapse studies suggest a novel mechanism of action for TMZ, alternative to the one commonly accepted. These results have significant implications for the development of TMZ resistance. Furthermore, rather than sensitizing cells to DNA damaging agents, USP1 appears to suppress latent apoptotic pathways and to protect cells from temozolomide-induced apoptosis. These results identify a new function for USP1 and suggest that suppression of USP1 and/or USP1 controlled pathway may be a means to enhance the cytotoxic potential of temozolomide and to sensitize TMZ-resistant GBM cells
Introduzione. La temozolomide (TMZ) è un farmaco alchilante frequentemente utilizzato nella chemioterapia dei gliomi. Nonostante molti aspetti siano ancora enigmatici, il suo meccanismo di azione generale è ben noto. La TMZ induce metilazione della guanina nel DNA (O6MeG) che, in assenza di riparazione ad opera di O6-methylguanine methyltransferase (MGMT), si appaia con una timina innescando un ciclo futile di riparazione e risintesi. Ne risultano rotture del DNA a doppio filamento (DSBs) che attivano i componenti del checkpoint in G2, e le cellule con DNA 4N si accumulano e arrestano in G2 per parecchi giorni. Le cellule muoiono poi per senescenza, necrosi, o catastrofe mitotica, mentre l’apoptosi è stata a lungo negata. Inoltre non è chiaro l’effetto di somministrazioni multiple di TMZ sull’arresto in G2/M e sull’induzione di apoptosi. La riparazione delle lesioni al DNA causate dai farmaci alchilanti richiede la monoubiquitinazione di PCNA e FANCD2; la perdita di una delle due proteine o l’inibizione della loro monoubiquitinazione potenzia la tossicità indotta dagli agenti metilanti. USP1 è una idrolasi in grado di rimuovere la monoubiquitina da PCNA e FANCD2, e per questo può essere un regolatore della risposta alla TMZ. Materiali e metodi. Sono state utilizzate le linee cellulari U87, U251 (TMZ-sensibili, bassi livelli di MGMT) e GBM8 (TMZ-resistenti, alti livelli di MGMT). Il protocollo di trattamento prevede 1, 2 o 3 dosi di TMZ 100μM per 3 ore. La progressione nel ciclo cellulare è stata studiata sia con FACS sia con una nuova tecnica di microscopia time-lapse in tempo reale (FUCCI, Fluorescent Ubiquitination-based Cell Cycle Indicator), mentre l’apoptosi è stata verificata al citofluorimetro con il metodo dell’annessina V-Propidio Ioduro. Per verificare il possibile ruolo di USP1 nell’azione della TMZ, dopo aver esaminato su databases di mRNA microarray l’espressione di USP1 nei glioblastomi, le cellule sono state transfettate con RNA a interferenza contro USP1 o di controllo. È quindi stato studiato l’effetto della soppressione dei livelli di USP1 sull’arresto in G2/M, la morte cellulare e la clonogenicità indotte dalla TMZ. Risultati. Trattamenti multipli con TMZ riducono la clonogenicità delle cellule di glioma sensibili al farmaco in maniera significativamente superiore rispetto al trattamento singolo, non modificano l’entità dell’arresto in G2, mentre inducono un significativo aumento dell’apoptosi in particolare in fase tardiva. L’analisi in time-lapse con il sistema FUCCI ha mostrato che le cellule sensibili alla TMZ subiscono un arresto in G2 inferiore alle 48 ore. Inoltre, in presenza di attivazione del checkpoint in G2, replicano il DNA ma non si dividono, rientrando nel ciclo cellulare in G1 e dando origine a cellule poliploidi. La soppressione dei livelli di USP1 da sola ha effetti minimi sulla progressione del ciclo cellulare e sulla morte cellulare sia nelle cellule sensibili che in quelle resistenti alla TMZ. Allo stesso modo, la soppressione dei livelli di USP1 non altera l’entità dell’arresto in G2/M indotto dalla TMZ. Tuttavia il knockdown di USP1 sorprendentemente incrementa la perdita di clonogenicità indotta dalla TMZ sia nelle cellule sensibili che in quelle resistenti. A differenza delle cellule di controllo in cui USP1 è stato soppresso, o di quelle con normale espressione di USP1 e trattate con TMZ, le cellule USP1-knockdown trattate con TMZ subiscono un’alta percentuale di morte per apoptosi. Conclusioni. I risultati dei nostri studi hanno mostrato che il trattamento delle cellule di glioma con TMZ può indurre apoptosi, e che questa è evidenziabile già dopo 3 giorni, sebbene diventi significativa solo tardivamente. Trattamenti multipli non modificano l’entità dell’arresto in G2, ma aumentano significativamente l’apoptosi. Inoltre gli studi di time-lapse permettono di proporre un nuovo meccanismo di azione per la TMZ, diverso da quello finora comunemente accettato, con significative implicazioni sullo sviluppo della resistenza al farmaco. La deubiquitinasi USP1, piuttosto che impedire l’attivazione di PCNA e FANCD2 e inibire in questo modo la riparazione del danno al DNA indotto dagli agenti metilanti, come indirettamente suggerito da studi precedenti, sembra invece sopprimere vie apoptotiche latenti e proteggere le cellule dall’apoptosi indotta dalla TMZ. La soppressione di USP1 o delle vie controllate da USP1 può rappresentare un modo per incrementare il potenziale citotossico della TMZ e per sensibilizzare GBM prima resistenti

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Hereditary spastic paraplegia type 54 is a rare autosomal recessive neurological gait disorder characterized by paraplegia, muscle spasticity, and intellectual disability. This length-dependent distal axonopathy is caused by mutations in the DDHD2 gene, which encodes the intracellular phospholipase A1 DDHD2. Little is known about the molecular function of the DDHD2 protein, especially in the context of HSP54. Thus, there is a need to further investigate its molecular functions and investigate the impact of DDHD2 deficiency in disease-relevant cells. Here, lipidomic profiling of dermal fibroblasts derived from three unrelated patients has revealed 19 glycerophosphoethanolamine species at differential levels in patients relative to unaffected controls. However, patient cells appear to have an unaffected Golgi apparatus morphology and lipid droplet formation, despite DDHD2’s proposed roles in these processes. To study the gene function in neuronal cells, I transdifferentiated the fibroblasts into induced neuronal precursor cells and found all the patient cells arrested in the G0/G1 phase of upon conversion. Given that these cell lines are unsustainable, I generated a stable knockdown cell line in the highly proliferative HEK293A to study the molecular biology of DDHD2. The knockdown cells had a reduced growth, were delayed in the G2/M phase of the cell cycle, and became multinucleated. I then treated the cells with antineoplastic compounds paclitaxel and nocodazole and found more knockdown cells in G0/G1 than controls, suggesting the possible occurrence of mitotic slippage. Lastly, I report a novel subcellular localization for DDHD2 at the microtubule organization center.