Coregulation of NDC80 Complex Subunits Determines the Fidelity of the Spindle-Assembly Checkpoint and Mitosis.

NDC80 complex (NDC80C) is composed of four subunits (SPC24, SPC25, NDC80, and NUF2) and is vital for kinetochore-microtubule (KT-MT) attachment during mitosis. Paradoxically, NDC80C also functions in the activation of the spindle-assembly checkpoint (SAC). This raises an interesting question regarding how mitosis is regulated when NDC80C levels are compromised. Using a degron-mediated depletion system, we found that acute silencing of SPC24 triggered a transient mitotic arrest followed by mitotic slippage. SPC24-deficient cells were unable to sustain SAC activation despite the loss of KT-MT interaction. Intriguingly, our results revealed that other subunits of the NDC80C were co-downregulated with SPC24 at a posttranslational level. Silencing any individual subunit of NDC80C likewise reduced the expression of the entire complex. We found that the SPC24-SPC25 and NDC80-NUF2 subcomplexes could be individually stabilized using ectopically expressed subunits. The synergism of SPC24 downregulation with drugs that promote either mitotic arrest or mitotic slippage further underscored the dual roles of NDC80C in KT-MT interaction and SAC maintenance. The tight coordinated regulation of NDC80C subunits suggests that targeting individual subunits could disrupt mitotic progression and provide new avenues for therapeutic intervention. IMPLICATIONS: These results highlight the tight coordinated regulation of NDC80C subunits and their potential as targets for antimitotic therapies.

NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosomes in hepatocellular carcinoma.

BACKGROUND: Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The success of cell entry determines the effect of sEV on recipient cells. Here, we aimed to delineate the mechanisms underlying the uptake of sEV in HCC. METHODS: Macropinocytosis was examined by the ability of cells to internalize dextran and sEV. Macropinocytosis was analyzed in Na(+)/H(+) exchanger 7 (NHE7)-knockdown and -overexpressing cells. The properties of cells were studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. The expression of NHE7 in patients' liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7. RESULTS: The data revealed that macropinocytosis controlled the internalization of sEVs and their oncogenic effect on recipient cells. It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and non-metastatic HCC cells. Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) limited the entry of sEVs and compromised cell aggressiveness. Mechanistically, we delineated that high level of NHE7, a sodium-hydrogen exchanger, alkalized intracellular pH and acidized endosomal pH, leading to the maturation of macropinosomes. Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis. Clinically, NHE7 expression was upregulated and linked to dismal prognosis of HCC. CONCLUSIONS: This study advances the understanding that NHE7 enhances sEV uptake by macropinocytosis to promote the malignant properties of HCC cells. Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.

Cyclin A-CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry.

Cyclin A and CDC25A are both activators of cyclin-dependent kinases (CDKs): cyclin A acts as an activating subunit of CDKs and CDC25A a phosphatase of the inhibitory phosphorylation sites of the CDKs. In this study, we uncovered an inverse relationship between the two CDK activators. As cyclin A is an essential gene, we generated a conditional silencing cell line using a combination of CRISPR-Cas9 and degron-tagged cyclin A. Destruction of cyclin A promoted an acute accumulation of CDC25A. The increase of CDC25A after cyclin A depletion occurred throughout the cell cycle and was independent on cell cycle delay caused by cyclin A deficiency. Moreover, we determined that the inverse relationship with cyclin A was specific for CDC25A and not for other CDC25 family members or kinases that regulate the same sites in CDKs. Unexpectedly, the upregulation of CDC25A was mainly caused by an increase in transcriptional activity instead of a change in the stability of the protein. Reversing the accumulation of CDC25A severely delayed G2-M in cyclin A-depleted cells. Taken together, these data provide evidence of a compensatory mechanism involving CDC25A that ensures timely mitotic entry at different levels of cyclin A.

Midline 1 interacting protein 1 promotes cancer metastasis through FOS-like 1-mediated matrix metalloproteinase 9 signaling in HCC.

BACKGROUND AND AIMS: Understanding the mechanisms of HCC progression and metastasis is crucial to improve early diagnosis and treatment. This study aimed to identify key molecular targets involved in HCC metastasis. APPROACH AND RESULTS: Using whole-transcriptome sequencing of patients' HCCs, we identified and validated midline 1 interacting protein 1 (MID1IP1) as one of the most significantly upregulated genes in metastatic HCCs, suggesting its potential role in HCC metastasis. Clinicopathological correlation demonstrated that MID1IP1 upregulation significantly correlated with more aggressive tumor phenotypes and poorer patient overall survival rates. Functionally, overexpression of MID1IP1 significantly promoted the migratory and invasive abilities and enhanced the sphere-forming ability and expression of cancer stemness-related genes of HCC cells, whereas its stable knockdown abrogated these effects. Perturbation of MID1IP1 led to significant tumor shrinkage and reduced pulmonary metastases in an orthotopic liver injection mouse model and reduced pulmonary metastases in a tail-vein injection model in vivo . Mechanistically, SP1 transcriptional factor was found to be an upstream driver of MID1IP1 transcription. Furthermore, transcriptomic sequencing on MID1IP1-overexpressing HCC cells identified FOS-like 1 (FRA1) as a critical downstream mediator of MID1IP1. MID1IP1 upregulated FRA1 to subsequently promote its transcriptional activity and extracellular matrix degradation activity of matrix metalloproteinase MMP9, while knockdown of FRA1 effectively abolished the MID1IP1-induced migratory and invasive abilities. CONCLUSIONS: Our study identified MID1IP1 as a regulator in promoting FRA1-mediated-MMP9 signaling and demonstrated its role in HCC metastasis. Targeting MID1IP1-mediated FRA1 pathway may serve as a potential therapeutic strategy against HCC progression.

S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos.

OBJECTIVE: Growing evidence indicates that tumour cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) are critical mediators of tumourigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown. DESIGN: The functions and mechanisms of S100A10 and EV-S100A10 in HCC progression were investigated in vitro and in vivo. Neutralising antibody (NA) to S100A10 was used to evaluate the significance of EV-S100A10. RESULTS: Functionally, S100A10 promoted HCC initiation, self-renewal, chemoresistance and metastasis in vitro and in vivo. Of significance, we found that S100A10 was secreted by HCC cells into EVs both in vitro and in the plasma of patients with HCC. S100A10-enriched EVs enhanced the stemness and metastatic ability of HCC cells, upregulated epidermal growth factor receptor (EGFR), AKT and ERK signalling, and promoted epithelial-mesenchymal transition. EV-S100A10 also functioned as a chemoattractant in HCC cell motility. Of significance, S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αⅤ. Importantly, blockage of EV-S100A10 with S100A10-NA significantly abrogated these enhancing effects. CONCLUSION: Altogether, our results uncovered that S100A10 promotes HCC progression significantly via its transfer in EVs and regulating the protein cargoes of EVs. EV-S100A10 may be a potential therapeutic target and biomarker for HCC progression.

Effect of Antiviral Treatment on Hepatitis B Virus Integration and Hepatocyte Clonal Expansion.

BACKGROUND: This study investigated the effect of nucleos(t)ide analogue (NUC) treatment on hepatitis B virus (HBV) DNA integration and hepatocyte clonal expansion, both of which are implicated in hepatocellular carcinoma (HCC) in chronic hepatitis B. METHODS: Twenty-eight patients receiving NUCs (11 lamivudine, 7 telbivudine, 10 entecavir) were included. All had liver biopsies at baseline and year 1, and 7 had a third biopsy at year 10. HBV DNA integration and hepatocyte clone size were assessed by inverse polymerase chain reaction. RESULTS: All patients had detectable HBV integration at baseline, with a median integration frequency of 1.01 × 109 per liver and hepatocyte clone size of 2.41 × 105. Neither integration frequency nor hepatocyte clone size correlated with age and HBV virologic parameters. After 1 year of treatment, HBV integration was still detectable in all patients, with a median of 5.74 × 108 integration per liver (0.22 log reduction; P = .008) and hepatocyte clone size of 1.22 × 105 (0.40 log reduction; P = .002). HBV integration remained detectable at year 10 of treatment, with a median integration frequency of 4.84 × 107 integration per liver (0.93 log reduction from baseline) and hepatocyte clone size of 2.55 × 104 (1.02 log reduction from baseline). From baseline through year 1 to year 10, there was a decreasing trend in both integration frequency and hepatocyte clone size (P = .066 and.018, respectively). CONCLUSIONS: NUCs reduced both HBV DNA integration and hepatocyte clonal expansion, suggesting another alternative pathway besides direct viral suppression to reduce HCC risk. Our findings supported the notion for a long-term NUC treatment to prevent HCC.

Quantitative differences between cyclin-dependent kinases underlie the unique functions of CDK1 in human cells.

One of the most intriguing features of cell-cycle control is that, although there are multiple cyclin-dependent kinases (CDKs) in higher eukaryotes, a single CDK is responsible for both G1-S and G2-M in yeasts. By leveraging a rapid conditional silencing system in human cell lines, we confirm that CDK1 assumes the role of G1-S CDK in the absence of CDK2. Unexpectedly, CDK1 deficiency does not prevent mitotic entry. Nonetheless, inadequate phosphorylation of mitotic substrates by noncanonical cyclin B-CDK2 complexes does not allow progression beyond metaphase and underscores deleterious late mitotic events, including the uncoupling of anaphase A and B and cytokinesis. Elevation of CDK2 to a level similar to CDK1 overcomes the mitotic defects caused by CDK1 deficiency, indicating that the relatively low concentration of CDK2 accounts for the defective anaphase. Collectively, these results reveal that the difference between G2-M and G1-S CDKs in human cells is essentially quantitative.

The Conditional Knockout Analogous System: CRISPR-Mediated Knockout Together with Inducible Degron and Transcription-Controlled Expression.

The revolutionary CRISPR technology opens a new era of cell biology in mammalian cells. The InDel mutation is induced by CRISPR and results in the frameshift mutation of the gene. Owing to the nature of CRISPR induced knockout, the conditional knockout using CRISPR technology is not common. With the recent development of the small molecule-inducible degron system, an analogous system to the classical genetic conditional knockout has become feasible. By integrating CRISPR-knockout, the tetracycline-controlled transcriptional and auxin-induced degradation post-translational control of protein expression, a method imitating the conditional knockout is developed. We herein describe the detailed protocol for the generation of a conditional protein inactivation in human cancer cells. The system is especially useful to study essential gene function in aneuploidy cancer cells where gain in copy number is common.

Aurora kinases and DNA damage response.

It is well established that Aurora kinases perform critical functions during mitosis. It has become increasingly clear that the Aurora kinases also perform a myriad of non-mitotic functions including DNA damage response. The available evidence indicates that inhibition Aurora kinase A (AURKA) may contribute to the G2 DNA damage checkpoint through AURKA's functions in PLK1 and CDC25B activation. Both AURKA and Aurora kinase B (AURKB) are also essential in mitotic DNA damage response that guard against DNA damage-induced chromosome segregation errors, including the control of abscission checkpoint and prevention of micronuclei formation. Dysregulation of Aurora kinases can trigger DNA damage in mitosis that is sensed in the subsequent G1 by a p53-dependent postmitotic checkpoint. Aurora kinases are themselves linked to the G1 DNA damage checkpoint through p53 and p73 pathways. Finally, several lines of evidence provide a connection between Aurora kinases and DNA repair and apoptotic pathways. Although more studies are required to provide a comprehensive picture of how cells respond to DNA damage, these findings indicate that both AURKA and AURKB are inextricably linked to pathways guarding against DNA damage. They also provide a rationale to support more detailed studies on the synergism between small-molecule inhibitors against Aurora kinases and DNA-damaging agents in cancer therapies.

Mitotic slippage is determined by p31comet and the weakening of the spindle-assembly checkpoint.

Mitotic slippage involves cells exiting mitosis without proper chromosome segregation. Although degradation of cyclin B1 during prolonged mitotic arrest is believed to trigger mitotic slippage, its upstream regulation remains obscure. Whether mitotic slippage is caused by APC/CCDC20 activity that is able to escape spindle-assembly checkpoint (SAC)-mediated inhibition, or is actively promoted by a change in SAC activity remains an outstanding issue. We found that a major culprit for mitotic slippage involves reduction of MAD2 at the kinetochores, resulting in a progressive weakening of SAC during mitotic arrest. A further level of control of the timing of mitotic slippage is through p31comet-mediated suppression of MAD2 activation. The loss of kinetochore MAD2 was dependent on APC/CCDC20, indicating a feedback control of APC/C to SAC during prolonged mitotic arrest. The gradual weakening of SAC during mitotic arrest enables APC/CCDC20 to degrade cyclin B1, cumulating in the cell exiting mitosis by mitotic slippage.

Synergism between ATM and PARP1 Inhibition Involves DNA Damage and Abrogating the G2 DNA Damage Checkpoint.

PARP inhibitors have emerged as effective chemotherapeutic agents for BRCA1/BRCA2-deficient cancers. Another DNA damage response protein, ATM, is also increasingly being recognized as a target for synthetic lethality with PARP inhibitors. As ATM functions in both cell cycle arrest and DNA repair after DNA damage, how cells respond to inhibition of ATM and PARP1 is yet to be defined precisely. We found that loss of ATM function, either in an ATM-deficient background or after treatment with ATM inhibitors (KU-60019 or AZD0156), results in spontaneous DNA damage and an increase in PARylation. When PARP1 is also deleted or inhibited with inhibitors (olaparib or veliparib), the massive increase in DNA damage activates the G2 DNA damage checkpoint kinase cascade involving ATR, CHK1/2, and WEE1. Our data indicated that the role of ATM in DNA repair is critical for the synergism with PARP inhibitors. Bypass of the G2 DNA damage checkpoint in the absence of ATM functions occurs only after a delay. The relative insensitivity of PARP1-deficient cells to PARP inhibitors suggested that other PARP isoforms played a relatively minor role in comparison with PARP1 in synergism with ATMi. As deletion of PARP1 also increased sensitivity to ATM inhibitors, trapping of PARP1 on DNA may not be the only mechanism involved in the synergism between PARP1 and ATM inhibition. Collectively, these studies provide a mechanistic foundation for therapies targeting ATM and PARP1.

Conditional gene inactivation by combining tetracycline-mediated transcriptional repression and auxin-inducible degron-mediated degradation.

Characterizing the functions of essential cell cycle control genes requires tight and rapid inducible gene inactivation. Drawbacks of current conditional depletion approaches include slow responses and incomplete depletion. We demonstrated that by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) system together, AID-tagged proteins can be downregulated more efficiently than the individual technology alone. When used in conjunction with CRISPR-Cas9-mediated disruption of the endogenous locus, this system facilitates the analysis of essential genes by allowing rapid and tight conditional depletion, as we have demonstrated using several cell cycle-regulatory genes including cyclin A, CDK2, and TRIP13. The vectors constructed in this study allow expression of AID-fusion proteins under the control of tetracycline-controlled promoters and should be useful in studies requiring rapid and tight suppression of gene expression in mammalian cells.

Pharmacological inactivation of CHK1 and WEE1 induces mitotic catastrophe in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer. As radiotherapy is the primary treatment for NPC, this offers a rationale to investigate if uncoupling the DNA damage responses can sensitize this cancer type. The G2 DNA damage checkpoint is controlled by a cascade of protein kinases: ATM/ATR, which phosphorylates CHK1/CHK2, which in turn phosphorylates WEE1. A number of small molecule inhibitors have been developed against these kinases as potential therapeutic agents. Here we demonstrated that compare to that in immortalized nasopharyngeal epithelial cells, ATR, CHK1, and WEE1 were overexpressed in NPC cell lines. Inhibitors of these kinases were unable to promote extensive mitotic catastrophe in ionizing radiation-treated NPC cells, indicating that they are not very effective radiosensitizer for this cancer. In the absence of prior irradiation, however, mitotic catastrophe could be induced with inhibitors against CHK1 (AZD7762) or WEE1 (MK-1775). NPC cells were more sensitive to WEE1 inactivation than nasopharyngeal epithelial cells. Targeting CHK1 and WEE1 together induced more extensive mitotic catastrophe than the individual components alone. Taken together, our results show that NPC cells depend on CHK1 and WEE1 activity for growth and that inhibitors of these kinases may serve as potential therapeutics for NPC.

Co-inhibition of polo-like kinase 1 and Aurora kinases promotes mitotic catastrophe.

Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells.

Pharmacological targeting the ATR-CHK1-WEE1 axis involves balancing cell growth stimulation and apoptosis.

The ATR-CHK1-WEE1 kinase cascade's functions in the DNA damage checkpoints are well established. Moreover, its roles in the unperturbed cell cycle are also increasingly being recognized. In this connection, a number of small-molecule inhibitors of ATR, CHK1, and WEE1 are being evaluated in clinical trials. Understanding precisely how cells respond to different concentrations of inhibitors is therefore of paramount importance and has broad clinical implications. Here we present evidence that in the absence of DNA damage, pharmacological inactivation of ATR was less effective in inducing mitotic catastrophe than inhibition of WEE1 and CHK1. Small-molecule inhibitors of CHK1 (AZD7762) or WEE1 (MK-1775) induced mitotic catastrophe, as characterized by dephosphorylation of CDK1(Tyr15), phosphorylation of histone H39(Ser10), and apoptosis. Unexpectedly, partial inhibition of WEE1 and CHK1 had the opposite effect of accelerating the cell cycle without inducing apoptosis, thereby increasing the overall cell proliferation. This was also corroborated by the finding that cell proliferation was enhanced by kinase-inactive versions of WEE1. We demonstrated that these potential limitations of the inhibitors could be overcome by targeting more than one components of the ATR-CHK1-WEE1 simultaneously. These observations reveal insights into the complex responses to pharmacological inactivation of the ATR-CHK1-WEE1 axis.

Synergism between inhibitors of Aurora A and KIF11 overcomes KIF15-dependent drug resistance.

The mitotic kinesin KIF11 (also called Eg5) plays critical roles in spindle functions. Although a number of small-molecule inhibitors of KIF11 are currently in clinical development, drug-resistance could be developed through compensation by another kinesin called KIF15. Using a newly developed infrared-based cell system, we discovered that the effectiveness of one of the latest generations of KIF11 inhibitor (SB743921) could be enhanced with several inhibitors of Aurora A kinase. Evidence including live-cell imaging and isobologram analysis indicated that targeting KIF11 and Aurora A together promoted monoastral spindle formation and mitotic catastrophe synergistically, supporting a model of parallel pathways of centrosome regulation by Aurora A and KIF11. We also developed a KIF15-dependent SB743921-resistance cell model. Significantly, the drug-resistance could also be overcome with Aurora A inhibitors. These results provide a molecular basis for increasing the effectiveness of Aurora A and KIF11 inhibitors and tackling problems of drug resistance.

Tetraploidization increases sensitivity to Aurora B kinase inhibition.

Aurora kinases are overexpressed in many cancers and are targets for anticancer drugs. The yeast homolog of Aurora B kinase, IPL1, was found to be a ploidy-specific lethality gene. Given that polyploidization is a common feature of many cancers, we hypothesized polyploidization also sensitizes mammalian cells to inhibition of Aurora kinases. Using two models of apparent diploid vs. tetraploid cell lines (one based on the hepatocellular carcinoma cell line Hep3B and another on untransformed mouse fibroblasts), we found that tetraploid cells were more sensitive to Aurora B inhibition than their diploid counterparts. Apoptosis could be induced in tetraploid cells by two different Aurora B inhibitors. Furthermore, tetraploid cells were sensitive to Aurora B inhibition but were not affected by Aurora A inhibition. Interestingly, the underlying mechanism was due to mitotic slippage and the subsequent excessive genome reduplication. In support of this, abolition of cytokinesis with dihydrocytochalasin B resulted in similar effects on tetraploid cells as Aurora B inhibition. These results indicate that inhibition of Aurora B or cytokinesis can promote apoptosis effectively in polyploid cancer cells.

Depletion of p31comet protein promotes sensitivity to antimitotic drugs.

Antimitotic spindle poisons are among the most important chemotherapeutic agents available. However, precocious mitotic exit by mitotic slippage limits the cytotoxicity of spindle poisons. The MAD2-binding protein p31(comet) is implicated in silencing the spindle assembly checkpoint after all kinetochores are attached to spindles. In this study, we report that the levels of p31(comet) and MAD2 in different cell lines are closely linked with susceptibility to mitotic slippage. Down-regulation of p31(comet) increased the sensitivity of multiple cancer cell lines to spindle poisons, including nocodazole, vincristine, and Taxol. In the absence of p31(comet), lower concentrations of spindle poisons were required to induce mitotic block. The delay in checkpoint silencing was induced by an accumulation of mitotic checkpoint complexes. The increase in the duration of mitotic block after p31(comet) depletion resulted in a dramatic increase in mitotic cell death upon challenge with spindle poisons. Significantly, cells that are normally prone to mitotic slippage and resistant to spindle disruption-mediated mitotic death were also sensitized after p31(comet) depletion. These results highlight the importance of p31(comet) in checkpoint silencing and its potential as a target for antimitotic therapies.

How protein kinases co-ordinate mitosis in animal cells.

Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

Determinants of mitotic catastrophe on abrogation of the G2 DNA damage checkpoint by UCN-01.

Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G(2) DNA damage checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the checkpoint and induce unscheduled mitosis in irradiated cells. Precisely, how cells behave following checkpoint abrogation remains to be defined. In this study, we tracked the fates of individual cells after checkpoint abrogation, focusing in particular on whether they undergo mitotic catastrophe. Surprisingly, while a subset of UCN-01-treated cells were immediately eliminated during the first mitosis after checkpoint abrogation, about half remained viable and progressed into G(1). Both the delay of mitotic entry and the level of mitotic catastrophe were dependent on the dose of radiation. Although the level of mitotic catastrophe was specific for different cell lines, it could be promoted by extending the mitosis. In supporting this idea, weakening of the spindle-assembly checkpoint, by either depleting MAD2 or overexpressing the MAD2-binding protein p31(comet), suppressed mitotic catastrophe. Conversely, delaying of mitotic exit by depleting either p31(comet) or CDC20 tipped the balance toward mitotic catastrophe. These results underscore the interplay between the level of DNA damage and the effectiveness of the spindle-assembly checkpoint in determining whether checkpoint-abrogated cells are eliminated during mitosis.