Death In Mitosis Research Articles

Antimitotic activity of DY131 and the estrogen-related receptor beta 2 (ERRβ2) splice variant in breast cancer.

Breast cancer remains a leading cause of cancer-related death in women, and triple negative breast cancer (TNBC) lacks clinically actionable therapeutic targets. Death in mitosis is a tumor suppressive mechanism that occurs in cancer cells experiencing a defective M phase. The orphan estrogen-related receptor beta (ERRβ) is a key reprogramming factor in murine embryonic and induced pluripotent stem cells. In primates, ERRβ is alternatively spliced to produce several receptor isoforms. In cellular models of glioblastoma, short form (ERRβsf) and beta2 (ERRβ2) splice variants differentially regulate cell cycle progression in response to the synthetic agonist DY131, with ERRβ2 driving arrest in G2/M.The goals of the present study are to determine the cellular function(s) of ligand-activated ERRβ splice variants in breast cancer and evaluate the potential of DY131 to serve as an antimitotic agent, particularly in TNBC. DY131 inhibits growth in a diverse panel of breast cancer cell lines, causing cell death that involves the p38 stress kinase pathway and a bimodal cell cycle arrest. ERRβ2 facilitates the block in G2/M, and DY131 delays progression from prophase to anaphase. Finally, ERRβ2 localizes to centrosomes and DY131 causes mitotic spindle defects. Targeting ERRβ2 may therefore be a promising therapeutic strategy in breast cancer.

Cell fate determination in cisplatin resistance and chemosensitization.

Understanding the determination of cell fate choices after cancer treatment will shed new light on cancer resistance. In this study, we quantitatively analyzed the individual cell fate choice in resistant UM-SCC-38 head and neck cancer cells exposed to cisplatin. Our study revealed a highly heterogeneous pattern of cell fate choices in UM-SCC-38 cells, in comparison to that of the control, non-tumorigenic keratinocyte HaCaT cells. In both UM-SCC-38 and HaCaT cell lines, the majority of cell death occurred during the immediate interphase without mitotic entry, whereas significant portions of UM-SCC-38 cells survived the treatment via either checkpoint arrest or checkpoint slippage. Interestingly, checkpoint slippage occurred predominantly in cells treated in late S and G2 phases, and cells in M-phase were hypersensitive to cisplatin. Moreover, although the cisplatin-resistant progression of mitosis exhibited no delay in general, prolonged mitosis was correlated with the induction of cell death in mitosis. The finding thus suggested a combinatorial treatment using cisplatin and an agent that blocks mitotic exit. Consistently, we showed a strong synergy between cisplatin and the proteasome inhibitor Mg132. Finally, targeting the DNA damage checkpoint using inhibitors of ATR, but not ATM, effectively sensitized UM-SCC-38 to cisplatin treatment. Surprisingly, checkpoint targeting eliminated both checkpoint arrest and checkpoint slippage, and augmented the induction of cell death in interphase without mitotic entry. Taken together, our study, by profiling cell fate determination after cisplatin treatment, reveals new insights into chemoresistance and suggests combinatorial strategies that potentially overcome cancer resistance.

Mcl-1 dynamics influence mitotic slippage and death in mitosis.

Microtubule-binding drugs such as taxol are frontline treatments for a variety of cancers but exactly how they yield patient benefit is unclear. In cell culture, inhibiting microtubule dynamics prevents spindle assembly, leading to mitotic arrest followed by either apoptosis in mitosis or slippage, whereby a cell returns to interphase without dividing. Myeloid cell leukaemia-1 (Mcl-1), a pro-survival member of the Bcl-2 family central to the intrinsic apoptosis pathway, is degraded during a prolonged mitotic arrest and may therefore act as a mitotic death timer. Consistently, we show that blocking proteasome-mediated degradation inhibits taxol-induced mitotic apoptosis in a Mcl-1-dependent manner. However, this degradation does not require the activity of either APC/C-Cdc20, FBW7 or MULE, three separate E3 ubiquitin ligases implicated in targeting Mcl-1 for degradation. This therefore challenges the notion that Mcl-1 undergoes regulated degradation during mitosis. We also show that Mcl-1 is continuously synthesized during mitosis and that blocking protein synthesis accelerates taxol induced death-in-mitosis. Modulating Mcl-1 levels also influences slippage; overexpressing Mcl-1 extends the time from mitotic entry to mitotic exit in the presence of taxol, while inhibiting Mcl-1 accelerates it. We suggest that Mcl-1 competes with Cyclin B1 for binding to components of the proteolysis machinery, thereby slowing down the slow degradation of Cyclin B1 responsible for slippage. Thus, modulating Mcl-1 dynamics influences both death-in-mitosis and slippage. However, because mitotic degradation of Mcl-1 appears not to be under the control of an E3 ligase, we suggest that the notion of network crosstalk is used with caution.

Phosphorylation of XIAP by CDK1-cyclin-B1 controls mitotic cell death.

ABSTRACTRegulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that X-linked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1–cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1–cyclin-B1.

AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest.

Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. However, how cells survive during prolonged mitotic arrest is not well understood. We show here that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells.

Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.

Abstract 693: AXL tyrosine kinase inhibition selectively sensitizes mesenchymal cancer cells to antimitotic agents

Abstract Molecularly-targeted drug therapies have revolutionized cancer treatment, however, resistance remains a major limitation to their overall efficacy. Epithelial to mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine kinase inhibitors (TKIs), independent of mutational resistance mechanisms. AXL is a receptor tyrosine kinase (RTK) associated with EMT that has been implicated in drug resistance, and has emerged as a candidate therapeutic target. Across 643 human cancer cell lines that were analyzed, we found that elevated AXL was strongly associated with a mesenchymal phenotype, particularly in triple negative breast cancer and non-small cell lung cancer. Here we experimentally established a model system to induced EMT by exposure to TGF-β and we observed that the resulting TGF-β-induced mesenchymal cells exhibit substantially increased AXL expression, become relatively drug-resistant to their appropriate TKI and cross-resistant to a variety of anti-cancer agents. We tested the ability of AXL inhibition to restore erlotinib sensitivity, in both a TGF-β-induced resistance model and in two different models of acquired resistance to erlotinib. Here, our observations do not support the previously reported role for AXL inhibition in overcoming acquired resistance to TKIs in the EMT context. However, in an unbiased screen of small molecule inhibitors of cancer-relevant processes, we discovered that AXL inhibition was specifically synergistic with anti-mitotic agents in killing cancer cells that had undergone EMT and demonstrated associated TKI resistance. Inhibition of AXL in combination with anti-mitotic agents enhances death in mitosis by promoting cells to enter mitosis through the down-regulation of CDK1, resulting in G2M cell cycle arrest. In summary, our findings highlight the utility of combination drug screening to reveal novel potentially useful anti-cancer drug combinations to improve clinical responses. While we did not identify a functional role for AXL in the context of acquired resistance to TKI therapies, we did observe that AXL inhibition may be an effective therapy in the context of mesenchymal tumors, specifically when used in combination with anti-mitotic agents. These findings reveal a novel therapeutic strategy for tumors displaying mesenchymal features that otherwise render them treatment-refractory. Citation Format: Catherine Wilson, Thinh Pham, Xiaofen Ye, Eva Lin, Sara Chan, Erin McNamara, Richard M. Neve, Lisa Belmont, Hartmut Koeppen, Robert L. Yauch, Avi Ashkenazi, Jeff Settleman. AXL tyrosine kinase inhibition selectively sensitizes mesenchymal cancer cells to antimitotic agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 693. doi:10.1158/1538-7445.AM2014-693

Identification of a mitotic death signature in cancer cell lines

This study examined the molecular mechanism of action of anti-mitotic drugs. The hypothesis was tested that death in mitosis occurs through sustained mitotic arrest with robust Cdk1 signaling causing complete phosphorylation of Mcl-1 and Bcl-xL, and conversely, that mitotic slippage is associated with incomplete phosphorylation of Mcl-1/Bcl-xL. The results, obtained from studying six different cancer cell lines, strongly support the hypothesis and identify for the first time a unique molecular signature for mitotic death. The findings represent an important advance in understanding anti-mitotic drug action and provide insight into cancer cell susceptibility to such drugs which has important clinical implications.

A Rationale to Enhance the Response to Antimitotic Therapy in Acute Myeloid Leukemia

AbstractAbstract 1332Acute myeloid leukemia (AML) is known to respond only moderately to antimitotic therapy while acute lymphoblastic leukemias can be efficiently targeted using spindle-disrupting agents. The underlying molecular cause for this clinical phenomenon is unknown. Recent evidence suggests that response to antimitotic therapy substantially depends on the stability of the critical mitotic regulator cyclin B. The ability to keep cyclin B expression levels stable during a mitotic block is associated with a good response leading to cell death in mitosis.At the metaphase to anaphase transition of an unperturbed cell division, cyclin B is targeted for degradation by the anaphase-promoting complex/cyclosome (APC/C) to trigger chromosome separation. The spindle assembly checkpoint (SAC) is a surveillance mechanism to ensure that APC/C-mediated ubiquitylation is restricted to cells that show proper attachment of all chromosomes to a functional mitotic spindle. In case of spindle disruption or unattached chromosomes, the spindle checkpoint stays active which leads to interference with APC/C-dependent proteolysis of cyclin B blocking cells in prometaphase until every chromosome is attached to the mitotic spindle.We recently developed a cell line-based reporter system which allows monitoring of cyclin B degradation under various conditions (Schnerch et al. Cell Cycle 2012). Here, we identified a pattern of slow degradation of cyclin B which continues through a mitotic block in case of chromosomal misalignment in unperturbed cell cycles. Remarkably, we also found prolonged slow degradation to trigger aberrant exit from mitosis in such cells giving rise to tetraploid cells. Therefore, a reduction in slow degradation appears as a promising rationale to foster a mitotic arrest and enhance cell death in mitosis during antimitotic therapy by preventing such mitotic slippage. We exposed our reporter cells to low concentrations of proteasome inhibitor during a spindle poison induced mitotic block to assess whether proteasome inhibition is capable of modulating slow degradation. Importantly, very low doses of proteasome inhibitor were sufficient to reduced the extent of cyclin B slow degradation during the mitotic block. Moreover, we demonstrate that low doses of proteasome inhibitor render the AML cell line Kasumi-1 responsive to low, non-disruptive concentrations of spindle poison (nocodazole and vincristine) leading to remarkable increases in the G2M-fraction. To the best of our knowledge there is no evidence so far that low doses of proteasome inhibitor exert antimitotic effects by interference with protein degradation during mitosis. Importantly, concentration of bortezomib of 1–2ng/ml (such as found in the serum of patients for up to 72h following administration of 1.3mg/m2 bortezomib subcutaneously) were found to exert synergistic effects with antimitotic therapy. Increases in the percentage of G2M cells by 38% were observed in Kasumi-1 cells for the combination of vincristine and bortezomib. Based on these findings, we currently apply our system to probe combinations of proteasome inhibitor with modern tailored therapies that exert their antimitotic effects by activation of the SAC, such as inhibitors of the motor protein Eg5 or of the mitotic kinases Polo-like kinase 1 (Plk1) or Aurora A and B.Using our cell line-based reporter system, we provide evidence in the in vitro setting that modulating slow degradation during antimitotic therapy by proteasome inhibition is a promising rationale to enhance the efficacy of antimitotic drugs. Drug concentrations used are based on published pharmacokinetics in humans and suggest feasibility of the drug combination in vivo. Our approach of targeted drug combinations may provide highly efficient treatment alternatives for patients that are not eligible for induction treatment. Disclosures:No relevant conflicts of interest to declare.

Abstract 14: Role of Cdk1 and Bcl-2 proteins in mitotic cell death

Abstract Tumor cells exhibit variable responses to microtubule inhibitors (MTIs) and an understanding of the molecular basis of this variability and the determinants of resistance and sensitivity to these agents is critical for appropriate patient selection while minimizing associated toxicity. Our studies have focused on elucidation of the mechanism of action of MTIs including vinca alkaloids and taxanes. Using vinblastine-treated KB-3 cells, a HeLa subline, as a model system, we have shown previously that death in mitosis is associated with high levels of phosphorylation of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL and Mcl-1) catalyzed by cyclin dependent kinase-1 (Cdk1)/cyclin B. In this study, we sought to determine whether these results reflected a more general trend, and conversely, whether escape from mitotic arrest (mitotic slippage) was associated with defective Cdk1 activation and/or reduced levels of phosphorylation of anti-apoptotic Bcl-2 proteins. For this purpose, we used DLD-1 and HT-29 colon carcinoma cells lines that stably express GFP-tagged histone H2B which enable cell fate to be studied by examination of nuclear morphology by fluorescent microscopy and time lapse imaging. In confirmation of previous findings, DLD-1 cells on treatment with paclitaxel exhibited extensive mitotic slippage after mitotic arrest and then either survived or died in subsequent interphase. In contrast, the vast majority of HT29 cells upon paclitaxel treatment died in mitosis. To determine if a relationship existed between these different fate profiles and the extent/duration of Cdk1-mediated Bcl-2 protein phosphorylation, cells were synchronized by double thymidine block, treated with paclitaxel, and extracts made at 4-h intervals over a 48-h time-course for analysis by immunoblotting. In DLD-1 cells, both Mcl-1 and Bcl-xL were transiently and partially phosphorylated and PARP cleavage was incomplete, consistent with the majority of the cells surviving through mitotic arrest. In contrast, in HT-29 cells, paclitaxel induced robust phosphorylation and subsequent degradation of Mcl-1 as well as extensive Bcl-xL phosphorylation. In addition, PARP was completely cleaved consistent with widespread mitotic cell death. Interestingly, PARP cleavage was observed to occur in HT-29 cells only under conditions where Bcl-xL was phosphorylated and Mcl-1 was degraded, indicating that mitotic death requires simultaneous functional elimination of both these anti-apoptotic Bcl-2 proteins. (Note that Bcl-2 expression was low to undetectable in both DLD-1 and HT-29 cells compared to KB-3 cells.) These results support the hypothesis that death in mitosis results from sustained Cdk1 activation and high levels of phosphorylation and subsequent inactivation of anti-apoptotic Bcl-2 proteins. Further, our findings suggest that the robustness of these signaling events may be useful as a marker to define tumor cell susceptibility to anti-mitotic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 14. doi:10.1158/1538-7445.AM2011-14

Cell Cycle-Associated Kinases as Targets for Therapy in Lung Cancer

Cell Cycle-Associated Kinases as Targets for Therapy in Lung Cancer

A critical role of integrin-linked kinase, ch-TOG and TACC3 in centrosome clustering in cancer cells

Many cancer cells contain more than two centrosomes, which imposes a potential for multipolar mitoses, leading to cell death. To circumvent this, cancer cells develop mechanisms to cluster supernumerary centrosomes to form bipolar spindles, enabling successful mitosis. Disruption of centrosome clustering thus provides a selective means of killing supernumerary centrosome-harboring cancer cells. Although the mechanisms of centrosome clustering are poorly understood, recent genetic analyses have identified requirements for both actin and tubulin regulating proteins. In this study, we demonstrate that the integrin-linked kinase (ILK), a protein critically involved in actin and mitotic microtubule organization, is required for centrosome clustering. Inhibition of ILK expression or activity inhibits centrosome clustering in several breast and prostate cancer cell lines that have centrosome amplification. Furthermore, cancer cells with supernumerary centrosomes are significantly more sensitive to ILK inhibition than cells with two centrosomes, demonstrating that inhibiting ILK offers a selective means of targeting cancer cells. Live cell analysis shows ILK perturbation leads cancer cells to undergo multipolar anaphases, mitotic arrest and cell death in mitosis. We also show that ILK performs its centrosome clustering activity in a focal adhesion-independent, but centrosome-dependent, manner through the microtubule regulating proteins TACC3 and ch-TOG. In addition, we identify a specific TACC3 phosphorylation site that is required for centrosome clustering and demonstrate that ILK regulates this phosphorylation in an Aurora-A-dependent manner.

Abstract 3892: Chromosome instability mediated by impaired AuroraB kinase activity on kinetochores in CIN cancer cells

Abstract Aneuploidy and chromosome instability (CIN) are hallmarks of solid tumors. Recent studies showed that chromosome missegregation caused by defective kinetochore microtubule attachments during division can give rise to the CIN phenotype. However, the underlying molecular defect that is responsible for the CIN phenotype is poorly understood. On the other hand, increasing evidence suggests that aneuploidy is detrimental to cell growth. Similarly, how CIN cancer cells tolerate a continual state of aneuploidy also remains to be clarified. These are critical questions, given that CIN provides tumor cells a rapid way to adapt to stressful conditions such as chemotherapy. In this study, we examined a panel of ovarian tumor cell lines and report that they exhibit the CIN phenotype. Timelapse movies showed chromosomes in these cell lines were able to align at the spindle equator but exhibited high rates (numbers) of lagging chromosomes, indicative of aberrant kinetochore: microtubule attachments. This observation was confirmed by the finding that a large fraction (numbers) of kinetochores in these cell lines exhibited a variety of attachment defects relative to Hela cells. As the role of Aurora B kinase at kinetochores is to monitor and repair defective microtubule attachments, we quantitated the levels of Aurora B at kinetochores of these ovarian tumor cell lines. We report that Aurora B levels are reduced relative to Hela cells that do not exhibit attachment defects. Consistent with this finding, FRET studies to monitor the real-time Aurora B kinase activity at the kinetochores of two of the three ovarian cell lines showed that kinase activity was weak. This defect was localized to kinetochores, as the overall activity of Aurora B kinase, as monitored by the western blotting of phospho-Histone H3, was similar to that in Hela cells. These findings provide a molecular explanation for kinetochore attachment defects in these ovarian tumor cell lines and, as a consequence, their chromosome instability. We also analyzed the response of these cell lines to treatment with various anti-mitotic agents. We found no obvious growth advantage of these ovarian CIN cell lines in response to drugs, relative to Hela cells. Sensitivity to mitotic death may reflect the variation of the genetic background of a given cell line. Also, the mitotic checkpoint appeared to be required for death in mitosis. Although apoptosis clearly played an important role in mitotic cell death in our studies, other death pathways could participate in this event, when apoptosis was blocked. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3892.

Dual Regulation of Cdc25A by Chk1 and p53-ATF3 in DNA Replication Checkpoint Control

Eukaryotic cells respond to DNA damage and stalled replication forks by activating signaling pathways that promote cell cycle arrest and DNA repair. A systematic screening of the protein kinase small interfering RNA library reveals that Chk1 and ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) are the main kinases responsible for intra-S-phase checkpoint upon topoisomerase I inhibitor camptothecin-induced DNA damage. It is well known that ATR-Chk1-mediated protein degradation of Cdc25A protein phosphatase is a crucial mechanism conferring this checkpoint activation. Here we describe another mechanism underlying Cdc25A down-regulation in response to DNA damage that occurs at the transcriptional level. We show that activation of tumor suppressor p53 by DNA damage results in inhibition of Cdc25A transcription as a result of activation of transcriptional repressor ATF3 that directly binds to the Cdc25A promoter. In cells deficient in both Chk1 and p53, Cdc25A down-regulation upon camptothecin-induced DNA damage is completely abolished, leading to severe defects in cell cycle checkpoints and remarkable cell death in mitosis. Our findings reveal two independent mechanisms acting in concert in regulation of Cdc25A in DNA damage response. Although Chk1 affects Cdc25A via rapid phosphorylation and protein turnover, inhibition of Cdc25A transcription by p53-ATF3 is required for the maintenance of cell cycle arrest.

Epothilones and new analogues of the microtubule modulators in taxane-resistant disease

Background: Microtubule-stabilising agents typified by the epothilone class of drug have demonstrated promising activity in Phase II and III clinical trials. Objective: Data supporting the efficacy of these agents are reviewed and their potential use in taxane-refractory disease assessed. Methods: Preclinical evidence assessing the role of the spindle assembly checkpoint in determining the cellular response to microtubule stabilization are presented together with clinical data documenting the efficacy of non-taxane microtubule modulators. Results/conclusions: Evidence suggests that microtubule-stabilising agents prolong activation of the spindle assembly checkpoint which may promote cancer cell death in mitosis or following mitotic exit. A weakened spindle assembly checkpoint is associated with altered sensitivity to agents targeting the microtubule and therefore pathways of drug resistance may be shared by these cytotoxic therapies. Preliminary clinical trial data do suggest modest activity of epothilones in truly taxane-resistant patient cohorts, indicating the potential niche for these agents in a molecularly undefined patient group, potentially implicating the role of P-glycoprotein in the acquisition of taxane-resistant disease. Trial data of these antimitotic agents will be presented together with their potential role in taxane-resistant disease and the implications for future clinical trial design.

PKC inhibitor Go6976 induces mitosis and enhances doxorubicin-paclitaxel cytotoxicity in urinary bladder carcinoma cells

Protein kinase C (PKC) α/βI isoenzyme inhibitor Go6976 has been suggested to be a G2 checkpoint abrogator by direct Chk1 inhibition. In the present study, we demonstrate that Go6976 induces mitosis in doxorubicin treated G2-arrested 5637 urinary bladder transitional cell carcinoma cells and interestingly also in non-synchronized 5637 cells. Importantly, the results demonstrated that both doxorubicin treated and non-synchronized cancer cells are forced to mitosis by Go6976. However, part of the cells avoid the death in mitosis and continue in the cell cycle which may increase the probability of genomic instability. Cytotoxicity of Go6976 alone and in combination with chemotherapeutic agents was further studied. Go6976 treatment alone induced apoptotic cell death. Cytostatic doxorubicin pre-treatment induced G2 arrest and inhibited the cytotoxic effects of mitosis specific drug paclitaxel. Cytotoxicities of doxorubicin-paclitaxel and doxorubicin-Go6976 sequences could be markedly enhanced by combining Go6976 with paclitaxel after doxorubicin pre-treatment. In doxorubicin-Go6976+paclitaxel sequence, paclitaxel arrested the cells to mitosis and unfavourable progression of the cell cycle was inhibited. Analyzes of the molecular mechanisms underlying Go6976 induced mitosis showed that PKC inhibiting concentrations of Go6976 induced cdc2 activation concentration-dependently in non-synchronized and in DNA damaged cells. Simultaneously, Chk1/2 became deactivated and cdc25C activated in DNA damaged cells, indicating regulatory events upstream. In non-synchronized cells, activation of cdc25C, but not Chk1/2, was observed, suggesting inactivation of c-TAK1. The results of the current study suggest that Go6976 has a synergistic cytotoxic effect when combined with doxorubicin and paclitaxel.

Targeting Aurora Kinases as Therapy in Multiple Myeloma.

The aurora kinases A and B are critical for facilitating cell cycle transit from G2 through to cytokinesis. As important regulators of the mitotic event, they are being tested as potential targets in cancer therapy. Multiple myeloma (MM) is a B cell malignancy characterized by progressive genetic instability, suggesting a disruption of cell cycle checkpoints has occurred that normally arrest cells at G2M or within mitosis when injury to the mitotic machinery occurs. Since these deficient checkpoints would prevent cell cycle arrest and potential repair and may render MM cells susceptible to apoptotic death in mitosis, we tested the anti-myeloma effecy of two separate agents that inhibit aurora kinases. Both agents induced cytoreduction of MM cell lines and primary myeloma samples at nM concentrations while normal peripheral blood lymphocytes and CLL cells were not affected. MM cells were not protected by IL-6 or activating mutations of RAS. Anti-myeloma effects were characterized by induction of tetraploidy followed by apoptosis. The myeloma apoptotic effect correlated well with the inhibition of aurora kinase activity as shown by reduction of histone 3B phosphorylation (substrate of auroras). Furthermore, stable ectopic over-expression of aurora kinase A significantly protected MM cells against aurora inhibitors but had no effect on apoptosis induced by velcade. As expression of the centrosomal protein RHAMM in MM cells may contribute to genetic instability, we tested effects of RHAMM over-expression on the sensitivity to aurora inhibitors. Although RHAMM over-expression in transfected MM cells was very modest, it significantly enhanced sensitivity to apoptosis induced by aurora kinase inhibitors. These results suggest the potential for aurora kinase inhibitors in multiple myeloma especially in patients where RHAMM is over-expressed.

Aberrant expression of cell cycle regulators in Hodgkin and Reed–Sternberg cells of classical Hodgkin's lymphoma

The characteristic Hodgkin and Reed–Sternberg cells of classical Hodgkin's lymphoma, although highly positive for proliferation markers, do not accumulate to excessive cell numbers. These cells are characterized by abortive mitotic cycles, leading to multinucleation or cell death in mitosis. We have previously described high expression of G1-phase cyclins in classical Hodgkin's lymphoma, which could explain the high percentage of cells staining for proliferation markers. To further our understanding of proliferation control in classical Hodgkin's lymphoma, we extended our immunohistochemical analysis to the main S-phase cyclin, cyclin A, and its regulators p21CIP1 and p27KIP1. Expression of proliferating cell nuclear antigen (PCNA) was used as an additional marker for cells being in either S- or G2-phase. In 47% (112/239) of classical Hodgkin's lymphoma cases p21CIP1 was detected within a mean frequency of 15% positive Hodgkin's and Reed–Sternberg cells per case. Similarly, 47% (116/249) of the cases stained positively for p27KIP1 with a mean frequency of expression in Hodgkin's and Reed–Sternberg cells of 12%. In contrast, 90% of the cells in all 246 evaluable classical Hodgkin's lymphoma cases were positive for PCNA. In addition, 98% of Hodgkin's and Reed–Sternberg cells in 99% (250/253) of the cases stained strongly positive for cyclin A. These findings further corroborate the hypothesis that Hodgkin and Reed–Sternberg cells exhibit a disturbed cell cycle with an abnormally short or even absent G1-phase. In contrast to other tumors, expression of PCNA or cyclin A had no prognostic value for patient survival.

Radiation-induced heart disease: review of experimental data on dose response and pathogenesis.

Clinical and experimental heart irradiation can cause a variety of sequelae. A single dose of greater than or equal to 15 Gy leads to a reversible exudative pericarditis, occurring in dogs, rabbits or rats at around 100 days. Its time-course is very similar in all species investigated, but there are considerable species and strain differences in severity and incidence. After longer, dose-dependent latency times chronic congestive myocardial failure develops. At histological examination myocardial degeneration and necrosis is observed, which in some species is accompanied by a variable degree of interstitial fibrosis. In rabbits and rats, myocardial degeneration becomes apparent at around 70 days after 20 Gy and is preceded by a marked reduction in capillary density as well as ultrastructural endothelial cell degeneration. Simultaneously to structural capillary damage, a focal loss of the endothelial marker enzyme alkaline phosphatase was observed in rats in areas with subsequent myocardial degeneration. Cell kinetic studies in rabbits and rats revealed a radiation-induced wave of increased endothelial cell proliferation at 30-100 days postirradiation. In the rat it is exclusively seen in conjunction with alteration of endothelial cell marker enzymes. The temporal and spatial pattern of proliferative response exludes endothelial cell death in mitosis as the sole pathogenetic mechanism causing capillary loss and myocardial degeneration. Parallel to development of morphological damage, haemodynamic studies in various rats strains revealed a drop in cardiac output and left ventricular ejection fraction to about 64% of normal values after 20 Gy. In vivo, this slightly reduced cardiac function was then maintained in a steady state for many weeks, probably due to a compensatory up-regulation of cardiac beta-adrenergic receptors. In denervated working heart preparations in vitro, however, these compensatory mechanisms are not effective and stroke volume as well as cardiac contractility show a rapid and steady deterioration. In many respects radiation-induced heart disease conforms to radiobiological concepts of late-responding tissues, showing a chronic progressive time-course and a very pronounced fractionation effect. However, pathogenesis cannot be understood in terms of target cell depletion alone, and experimental evidence indicates the importance of alterations of regulatory mechanisms.