4N DNA Content Research Articles

Inhibition of Aurora-B kinase activity confers antitumor efficacy in preclinical mouse models of early and advanced gastrointestinal neoplasia

The Aurora family of kinases, play a fundamental role in cell division and are overexpressed in several cancers including colon. The activity of barasertib-hQPA, a selective inhibitor of Aurora-B kinase (ABK) was investigated in a range of preclinical models of gastrointestinal cancer. Treatment with barasertib-hQPA produced anti-proliferative and cytotoxic effects across a panel of human colorectal cancer (CRC) cell lines in vitro. Prodrug, barasertib [48-h subcutaneous (s.c.) infusion; 150 mg/kg/day] inhibited the growth of SW620, Colo205, HCT116 human colorectal tumor xenografts in nude mice significantly (Student's t-test, P<0.05, n=10-12 per group). Flow cytometric analysis of single cells from disaggregated barasertib-treated SW620 tumors revealed a decrease in phosphorylated histone H3 (phH3) and an increase in tumor cells with ≥4N DNA content P<0.05). The activity of barasertib was then examined in ApcMin/+ mice, a spontaneous model of early intestinal neoplasia. Macroscopic evaluation of the small intestine revealed that barasertib treatment [25 mg/kg intra-peritoneal (i.p.) Q1Dx4 each week for 3 weeks] of 8-week old ApcMin/+ mice produced a 39% reduction in macroadenoma number (P=0.02) and a 43% reduction in overall adenoma burden (P=0.02) compared with vehicle-treated controls. Quantification of microscopic adenomas revealed a >64% reduction in the number of adenomas spanning more than one villus. Histological analysis of these adenomas revealed a number of distinct changes in barasertib-treated ApcMin/+ mice, including a 94% reduction in the proportion of phospho-histone H3-positive cells (P<0.001) and a 53% reduction in the number of cells per adenoma (P=0.001). These results provide a scientific rationale for investigating ABK inhibitors as a treatment for intestinal cancer.

Replicatively senescent cells are arrested in G1 and G2 phases

Most human somatic cells do not divide indefinitely but enter a terminal growth arrest termed replicative senescence. Replicatively senescent cells are generally believed to arrest in G1 or G0 stage of the cell cycle. While doing cell cycle analysis on three different lines of normal human fibroblasts we observed that 36-60% of the replicatively senescent cells had 4N DNA content. Only up to 5% of senescent cells had more than one nucleus ruling out the possibility that the 4N cell population were G1-arrested bi-nucleated cells. Furthermore, it is unlikely that the 4N cells are tetraploids, because actively dividing pre-senescent cultures lacked the 8N tetraploid G2 population. Collectively these results suggest that the 4N population consists of G2 arrested cells. The notion that a large fraction of senescent cell population is arrested in G2 is important for understanding the biology of replicative senescence.

Targeting Sphingosine Kinase 1 in Carcinoma Cells Decreases Proliferation and Survival by Compromising PKC Activity and Cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

Novel DNA damage checkpoint in mitosis: Mitotic DNA damage induces re-replication without cell division in various cancer cells

DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair.

Azacitidine/Decitabine Synergism with the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML Models

Abstract 578MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials. Disclosures:Smith:Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.

Abstract A203: Identification of FoxM1/Bub1b signaling pathway as a molecular target in rhabdomyosarcoma.

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Intensive multi-modal therapeutic regimens have increased overall survival in the last 25 years, but 25% of patients still succumb to disease, thus necessitating the identification of novel targets for therapy. In this study, we identified that Bub1b is necessary for the growth and survival of both ARMS and ERMS cells using a loss of function high-throughput shRNA screen. Using many additional Bub1b shRNA sequences, we were able to confirm that human RMS cells were more sensitive to Bub1b knockdown compared to human fibroblasts or human ovarian cancer cells. Knockdown of Bub1b also resulted in significant suppression of RMS xenografts growth in vivo. Mechanistically, flow cytometry analysis of knockdown Bub1b cells had an increase in &amp;gt;4N DNA content. Live-cell time-lapse microscopy studies provided direct evidence that knockdown of Bub1b promotes endoreduplication, eventually causing mitotic catastrophe. Further, ChIP assay demonstrated that Forkhead Box M1 (FoxM1), an essential transcriptional factor for G1/S transition and mitotic progression, directly binds to the Bub1b promoter. Luciferase reporter assay showed that FoxM1-binding region is critical for the activation of the Bub1b promoter. Suppression of FoxM1, either by shRNA or the inhibitor siomycin A, led to reduction of Bub1b expression and inhibition of cell growth and survival in vitro. These findings indicate that the FoxM1-Bub1b axis is important for the growth and survival of RMS cells and may be a target for RMS therapy in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A203.

Tasisulam Sodium, an Antitumor Agent That Inhibits Mitotic Progression and Induces Vascular Normalization

LY573636-sodium (tasisulam) is a small molecule antitumor agent with a novel mechanism of action currently being investigated in a variety of human cancers. In vitro, tasisulam induced apoptosis via the intrinsic pathway, resulting in cytochrome c release and caspase-dependent cell death. Using high content cellular imaging and subpopulation analysis of a wide range of in vitro and in vivo cancer models, tasisulam increased the proportion of cells with 4N DNA content and phospho-histone H3 expression, leading to G(2)-M accumulation and subsequent apoptosis. Tasisulam also blocked VEGF, epidermal growth factor, and fibroblast growth factor-induced endothelial cell cord formation but did not block acute growth factor receptor signaling (unlike sunitinib, which blocks VEGF-driven angiogenesis at the receptor kinase level) or induce apoptosis in primary endothelial cells. Importantly, in vivo phenocopying of in vitro effects were observed in multiple human tumor xenografts. Tasisulam was as effective as sunitinib at inhibiting neovascularization in a Matrigel plug angiogenesis assay in vivo and also caused reversible, non G(2)-M-dependent growth arrest in primary endothelial cells. Tasisulam also induced vascular normalization in vivo. Interestingly, the combination of tasisulam and sunitinib significantly delayed growth of the Caki-1 renal cell carcinoma model, whereas neither agent was active alone. These data show that tasisulam has a unique, dual-faceted mechanism of action involving mitotic catastrophe and antiangiogenesis, a phenotype distinct from conventional chemotherapies and published anticancer agents.

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

BackgroundThe Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets.MethodsThe expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy.ResultsThe results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar.ConclusionsWe demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment.

The nuclear receptor constitutive active/androstane receptor arrests DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase.

Here, we have demonstrated that xenobiotic activation of the nuclear receptor (CAR, NR1I3) can result in arresting DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase. Huh7 cells over-expressing CAR were either treated with dimethyl sulfoxide, the CAR activator TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; androstenol, 16,(5α)-androsten-3α-OL), or repressor androstenol; these treatments were then followed by adriamycin treatment to damage DNA. FACS analysis revealed that CAR-activation by TCPOBOP increased the rate of arrested Huh7 cells at the G2/M phase (4N DNA content) after DNA damage by adriamycin. This increase correlated with the increase of cell viability in TCPOBOP-treated Huh7 cells, as determined by MTT assays. Real-time polymerase chain reaction analysis determined that, as regulated by CAR, the growth arrest and DNA damage-inducible γ (GADD45γ) and Cyclin G2 genes increased and decreased, respectively, as TCPOBOP increased the number of Huh7 cells arrested at the G2/M phase. Thus, the results suggest that CAR regulates cell cycle, increasing G2/M arrest, and delaying the death of DNA-damaged cells.

Abstract 3923: Constitutive Cdk2 activation results in endoreduplication, failed cytokinesis, and chromosomal instability

Abstract Breast cancer is the most common form of cancer and the second-leading cause of cancer-related deaths in women in the United States. While the causative factors and tumor characteristics are very heterogeneous between cases, Cyclin D1 has been shown to be expressed in approximately 50% of all breast cancers. While it may not be generally considered a canonical partner, Cyclin D1 has also been shown to form active complexes with Cdk2 and transient expression of Cyclin D1 causes irreparable chromosomal instability, the gain or loss of whole chromosomes, in a Cdk2 dependent manner. To study the role of Cyclin D1/Cdk2 complexes in this chromosomal instability, we are utilizing a Cyclin D1/Cdk2 (D1K2) fusion protein, which exhibits constitutive protein kinase activity. Mice with mammary expression of D1K2 under control of the MMTV promoter generate spontaneous tumors that exhibit aneuploidy and centrosome amplification. Karyotype analysis shows that the tumors consist of distinct populations clustered around 2N and 4N DNA content, suggesting mechanisms capable of doubling the DNA content of the cell in addition to causing small stochastic changes, likely through centrosome amplification. Live cell microscopy of a cell line derived from one such tumor shows the occurrence of failed cytokinesis. The cleavage furrow forms and the cells begin to pull apart but appear to fail abcission and merge back together to form a binucleate cell. The resultant cell is then tetraploid with an abnormal number of centrosomes. Following the cell further, it later reenters mitosis and completes division, providing a glimpse of what could be the underlying cause of the centrosome amplification and chromosomal instability seen in these cells. We have also engineered the MCF10A cell line, a normal human breast epithelial cell line, to express the D1K2 protein. Rb and NPM are hyperphosphorylated in these cells, providing potential mechanisms for loss of cell-cycle control and centrosome amplification, respectively. When treated with 1 μM paclitaxel for 72 hours, the cells expressing D1K2 show a distinct population with an 8N DNA content when analyzed by propidium iodide staining and flow cytometry, indicative of either endoreduplication or failed cytokinesis, whereas cells expressing only a hygromycin resistance vector do not. This effect is dependent on the kinase activity of the D1K2 as both cells expressing a kinase dead mutant of D1K2 and D1K2 cells in the presence of the specific Cdk2 inhibitor CVT313 yield results similar to the control cells. This implicates the constitutive Cdk2 activation in altering the spindle assembly checkpoint and/or the G1/S tetraploidy checkpoint since cells would need to both slip through the paclitaxel induced mitotic arrest and reenter S phase to attain an 8N ploidy. 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 3923. doi:10.1158/1538-7445.AM2011-3923

Abstract 2484: Aurora B silencing attenuates radiation resistance through PARP and identification of a missense mutation in aurora B as a determinant of radiosensitization in gliomas

Abstract Purpose: Aurora B (Aur B), a mitotic protein located on chromosome 17p13.1, is critical for proper chromosome segregation and changes in chromosome 17p are quite common in glioblastoma multiforme (GBM). Upregulation of Aur B induces multicellularity and fosters polyploidy. Aur B reaches maximum levels later in mitosis and regulates the chromosomal passenger complex progression. It has been reported that Aur B expression markedly correlates with short survival of GBM patients and may be a prognostic factor. Targeting Aur B is an ideal strategy, since it is active during mitosis and non-proliferating cells would not be adversely affected. Materials and Methods: The cell lines used for this study include both established and primary GBM cell lines. Radiation experiments were conducted using gammacell irradiator. DNA damage was estimated using comet assay. Apoptosis, cell cycle and multinucleated polyploid cells were estimated using flow cytometry. Gene silencing and overexpression were carried out using established protocols. Results and Discussion: Aur B expression increases after radiation in GBM cells and not in normal human astrocytes as determined both at transcriptional and translational levels. The radiosensitivity of cells lines was estimated by clonogenic survival assay. Through sequencing Aur B, we discovered mutations at the 885(C) and 893(T) residues in the GBM cell lines. The 885(C) was a silent mutation and 893(T) leads to an amino acid change from methionine (M) to threonine (T). This missense mutation leads to a new phospho site. The GBM cell lines tested had heterozygous and homozygous deletions. The radiosensitivity assay conducted on these cell lines shows a differential response with regards to the nature of mutation. The cell lines which had homozygous deletions on both sites 885(C) and 893(T) exhibit an increased radioresistance under in vitro conditions when compared to one of them being a heterozygote. Further, we show that Aur B silencing inhibits cytokinesis leading to 4N or 8N DNA content in p53 functional cell lines. The endoreduplicated cells due to Aur-B inhibition leads to the loss of viability in p53 mutant/null cell lines. Overexpression of Aur B increases the DNA repair enzyme PARP and scaffolding protein XRCC1 after radiation, which are critical for the activation and recruitment of Base Excision Repair. Whereas, silencing Aur B decreases the PARP expression levels. Conclusion: In GBM cell lines, we report that Aur B knockdown sensitizes cells to radiation and over expression of Aur B leads to radioresistance. Thus far, one of the prime targets in mediating this effect was found to be PARP, since silencing and forced induction of Aur B correlates with expression levels of PARP. For the first time, we have shown that the mutational status of Aur B alters the radiation sensitivity. Further in vivo studies are underway. 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 2484. doi:10.1158/1538-7445.AM2011-2484

Fluorescence kinetics in HeLa cells after treatment with cell cycle arrest inducers visualized with Fucci (fluorescent ubiquitination-based cell cycle indicator)

Fucci (fluorescent ubiquitination-based cell cycle indicator) is able to visualize dynamics of cell cycle progression in live cells; G1- and S-/G2-/M-phase cells expressing Fucci emit red and green fluorescence, respectively. This system could be applied to cell kinetic analysis of tumour cells in the field of cancer therapy; however, it is still unclear how fluorescence kinetics change after various treatments, including exposure to anticancer agents. To explore this, we arrested live HeLa cells expressing the Fucci probes at various cell cycle stages and observed the fluorescence, in conjunction with flow cytometric analysis. X-irradiation, HU (hydroxyurea) and nocodazole arrest cells at G2/M boundary, early S-phase and early M-phase, respectively. Although X-irradiation and HU treatment induced similar accumulation kinetics of green fluorescent cells, nocodazole treatment induced an abnormal red fluorescence at M phase, followed by accumulation of both red and green fluorescent cells with 4N DNA content. We conclude that certain agents that disrupt normal cell cycle regulation could cause unexpected fluorescence kinetics in the Fucci system.

SCH 1473759, a novel Aurora inhibitor, demonstrates enhanced anti-tumor activity in combination with taxanes and KSP inhibitors

Aurora kinases are required for orderly progression of cells through mitosis, and inhibition of these kinases by siRNA or small molecule inhibitors results in cell death. We previously reported the synthesis of SCH 1473759, a novel sub-nanomolar Aurora A/B inhibitor. We utilized SCH 1473759 and a panel of tumor cell lines and xenograft models to gain knowledge about optimal dosing schedule and chemotherapeutic combinations for Aurora A/B inhibitors. SCH 1473759 was active against a large panel of tumor cell lines from different tissue origin and genetic backgrounds. Asynchronous cells required 24-h exposure to SCH 1473759 for maximal induction of >4N DNA content and inhibition of cell growth. However, following taxane- or KSP inhibitor-induced mitotic arrest, less than 4-h exposure induced >4N DNA content. This finding correlated with the ability of SCH 1473759 to accelerate exit from mitosis in response to taxane- and KSP inhibitor-induced arrest. We tested various dosing schedules in vivo and demonstrated SCH 1473759 dose- and schedule-dependent anti-tumor activity in four human tumor xenograft models. Further, the efficacy was enhanced in combination with taxanes and found to be most efficacious when SCH 1473759 was dosed 12-h post-taxane treatment. SCH 1473759 demonstrated potent mechanism-based activity, and activity was shown to be enhanced in combination with taxanes and KSP inhibitors. This information may be useful for optimizing the clinical efficacy of Aurora inhibitors.

TopBP1 Deficiency Causes an Early Embryonic Lethality and Induces Cellular Senescence in Primary Cells

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.

Targeting Non-Histone Protein Acetylation Impairs Platelet Production During Normal Megakaryocytopoiesis.

AbstractAbstract 2610Megakaryocytopoiesis consists of a succession of events in which MK progenitors initially proliferate and acquire lineage-specific markers, followed by polyploidization and cytoplasmic maturation. MK maturation culminates in the formation of cytoplasmic extensions (i.e. proplatelets) that leads to platelet shedding into the circulation. Panobinostat (LBH589) is a histone deacetylase inhibitor that has antiproliferative and cytotoxic effects on several types of cancer cells including blood cells from patients with hematological malignancies. One of the major adverse events associated with LBH589 treatment is thrombocytopenia. In this study, we hypothesize that the effects of LBH589 on thrombopoiesis might occur by targeting acetylation of histone and/or non-histone proteins resulting in defective platelet production. To test this hypothesis we investigated the effects of LBH589 on megakaryocytopoiesis in MK cell lines (i.e. HEL JAK2V617F positive cells) and in primary human MK. First, we tested the effects of LBH589 on the ability of human CD34+ cells to generate MK colony forming units (CFU-MKs). Neither CFU-MK or CFU-MIX derived colony formation was reduced in the presence of LBH589. To evaluate the effects of LBH589 on parameters of MK maturation, MK were generated in vitro from peripheral blood-derived CD34+ cells by employing an expansion culture system containing SCF and TPO for 6 days followed by 8 additional days incubation in the presence of TPO. These studies were pursued in the presence or absence of LBH589. Treatment with LBH589 did not significantly influence the number of CD61+ MK (i.e. control = 55.8%; 2.5nM LBH589 = 45.2%, p value=0.109; 5nM LBH589=38.5%, p value=0.095, of viable 7-AAD−/CD61+ cells) or the degree of polyploidization (i.e. control = 17.4%; 2.5nM LBH589 = 14.4%86.7, p value=0.157; 5nM LBH 589=12.8%, p value=0.116, cells with >4N DNA content). Culture-derived platelets were analyzed phenotypically and quantitated by means of dual labeling with anti-CD41 antibodies and with thiazole orange (TO) in order to identify new reticulated platelets. The percentage of CD41+/TO+ platelets derived from MK generated in the presence of LBH589 was significantly reduced (i.e. 2.5nM LBH589=11%, p value 0.046 and 5nM LBH589=9%, p value=0.011, CD41+/TO+ cells) as compared with MK generated in the absence of LBH589 (18.5% CD41+/TO+ cells). These findings were consistent with the observation of significant numbers of proplatelet-bearing MKs in control cultures but not in LBH 589-treated cultures. Collectively, these data suggest that LBH589 impairs platelet production while having a minimal effect on MK commitment, cytoplasmic maturation or polyploidization. To better understand the mechanisms responsible for such effects on thrombopoiesis, RNA extracted from control MK and from MK treated in vitro with LBH589 was analyzed by real time quantitative PCR to evaluate GATA-1 and NF-E2 expression. GATA-1 and NF-E2 mRNA levels were unchanged after treatment with LBH589. We found, however, that LBH589 induced a 4.8 to 7.5-fold increase in histone H3 acetylation. These data suggest that the negative impact of LBH589 on MK maturation was not mediated by its effects on chromatin but rather was possibly due to its effects on acetylation of nonhistone proteins. We demonstrated that LBH589 treatment increased acetylation of tubulin, a non-histone cytoplasmic protein that is a component of the microtubule (MT) cytoskeleton. The later stages of MK maturation are highly dependent on MT which represent the structural scaffold for proplatelet extension and enables the transport of cytoplasmic organelles into nascent platelets. The changes in the acetylation status of tubulin are critical for proper MT function and are mediated by HDAC6 which we found by Western blot analysis to be inhibited by LBH589 treatment. Based on these findings we suggest that LBH589-induced changes in tubulin acetylation result in aberrant MT function which in turn, leads to defective proplatelet and platelet formation. These nonhistone protein modifications might serve as a drug target for the development of novel agents (LBH589) to treat patients with extreme thrombocytosis due to underlying myeloproliferative neoplasms. Disclosures:Iancu-Rubin:Novartis: Research Funding. Hoffman:Novartis: Research Funding.

Context-Dependent Cell Cycle Checkpoint Abrogation by a Novel Kinase Inhibitor

BackgroundCheckpoint kinase 1 and 2 (Chk1/Chk2), and the Aurora kinases play a critical role in the activation of the DNA damage response and mitotic spindle checkpoints. We have identified a novel inhibitor of these kinases and utilized this molecule to probe the functional interplay between these two checkpoints.Principal FindingsFragment screening, structure guided design, and kinase cross screening resulted in the identification of a novel, potent small molecule kinase inhibitor (VER-150548) of Chk1 and Chk2 kinases with IC50s of 35 and 34 nM as well as the Aurora A and Aurora B kinases with IC50s of 101 and 38 nM. The structural rationale for this kinase specificity could be clearly elucidated through the X-ray crystal structure. In human carcinoma cells, VER-150548 induced reduplication and the accumulation of cells with >4N DNA content, inhibited histone H3 phosphorylation and ultimately gave way to cell death after 120 hour exposure; a phenotype consistent with cellular Aurora inhibition. In the presence of DNA damage induced by cytotoxic chemotherapeutic drugs, VER-150548 abrogated DNA damage induced cell cycle checkpoints. Abrogation of these checkpoints correlated with increased DNA damage and rapid cell death in p53 defective HT29 cells. In the presence of DNA damage, reduplication could not be observed. These observations are consistent with the Chk1 and Chk2 inhibitory activity of this molecule.ConclusionsIn the presence of DNA damage, we suggest that VER-150548 abrogates the DNA damage induced checkpoints forcing cells to undergo a lethal mitosis. The timing of this premature cell death induced by Chk1 inhibition negates Aurora inhibition thereby preventing re-entry into the cell cycle and subsequent DNA reduplication. This novel kinase inhibitor therefore serves as a useful chemical probe to further understand the temporal relationship between cell cycle checkpoint pathways, chemotherapeutic agent induced DNA damage and cell death.

Alteration of mtDNA copy number, mitochondrial gene expression and extracellular DNA content in mice after irradiation at lethal dose

High steady-state transcriptional activity is essential for normal mitochondrial function. The requisite transcription rate is satisfied in part by high copy number of mitochondrial DNA (mtDNA). In the present study, we analyze mtDNA copy number by real-time PCR in nucleated blood cells from control mice and mice exposed to 1- or 10-Gy X-radiation. Transcription of the oxidative phosphorylation-associated genes cytb, atp6, nd4, nd2 and d-loop region was monitored in these nucleated blood cells similarly by real-time PCR. We observed a 50% decrease in the ratio of mitochondrial to nuclear DNA (mtDNA/nDNA) in blood cells, while the mtDNA/nDNA ratio in serum increased. After a lethal 10-Gy dose of X-irradiation, we observed an 80% decrease in the number of circulating lymphocytes. In response to a 10-Gy radiation dose, we observed transiently increased mtDNA/nDNA ratio and transcription within the initial 5 h post-treatment. At 24-72 h, the mtDNA/nDNA ratio in surviving cells was reduced to the level observed in blood cells irradiated with 1 Gy. We observed a decrease in the serum mtDNA/nDNA ratio due to an increase in nDNA content rather than a decrease in mtDNA. Taken together, results presented herein suggest that the mtDNA/nDNA ratio may be of clinical value potentially as a diagnostic tool, particularly in oncology patients undergoing radiation therapy.

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.

Functional evaluation of missense variations in the human MAD1L1 and MAD2L1 genes and their impact on susceptibility to lung cancer

BackgroundHuman MAD1 mitotic arrest deficient-like 1 (MAD1L1) and MAD2 mitotic arrest deficient-like 1 (MAD2L1) are two interactive proteins playing important roles in maintaining spindle checkpoint function. This study examined the...

Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest

Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.