4N DNA Content Research Articles

Induction of Apoptosis by the Ste20-like Kinase SLK, a Germinal Center Kinase That Activates Apoptosis Signal-regulating Kinase and p38

Expression and activity of the germinal center kinase, Ste20-like kinase (SLK), are increased during kidney development and recovery from ischemic acute renal failure. In this study, we characterize the activation and functional role of SLK. SLK underwent dimerization via the C-terminal domain, and dimerization enhanced SLK activity. In contrast, the C-terminal domain of SLK did not dimerize with a related kinase, Mst1, and did not affect Mst1 activity. Phosphorylation/dephosphorylation of SLK were not associated with changes in kinase activity. SLK induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1) and increased ASK1 activity, indicating that ASK1 is a substrate of SLK. Moreover, SLK stimulated phosphorylation of p38 mitogen-activated protein kinase via ASK1, but not c-Jun N-terminal kinase nor extracellular signal-regulated kinase. Chemical anoxia and recovery during re-exposure to glucose (ischemia-reperfusion injury in cell culture) stimulated SLK activity. Overexpression of SLK enhanced anoxia/recovery-induced apoptosis, release of cytochrome c, and activities of caspase-8 and -9, and apoptosis was reduced significantly with p38 and caspase-9 inhibitors. Induction of the endoplasmic reticulum stress response by anoxia/recovery or tunicamycin (monitored by induction of Bip or Grp94 expression, phosphorylation of eukaryotic translation initiation factor 2alpha subunit, expression of CHOP, and activation of caspase-12) was attenuated in cells that overexpress SLK. Thus, SLK is an anoxia/recovery-dependent kinase that is activated via homodimerization and that signals via ASK1 and p38 to promote apoptosis. Attenuation of the protective aspects of the endoplasmic reticulum stress response by SLK may contribute to its proapoptotic effect.

Critical Function for SIP, a Ubiquitin E3 Ligase Component of the β-Catenin Degradation Pathway, for Thymocyte Development and G1 Checkpoint

Beta-catenin has been implicated in thymocyte development because of its function as a coactivator of Tcf/LEF-family transcription factors. Previously, we discovered a novel pathway for p53-induced beta-catenin degradation through a ubiquitin E3 ligase complex involving Siah1, SIP (CacyBP), Skp1, and Ebi. To gain insights into the physiological relevance of this new degradation pathway in vivo, we generated mutant mice lacking SIP. We demonstrate here that SIP-/- thymocytes have an impaired pre-TCR checkpoint with failure of TCRbeta gene rearrangement and increased apoptosis, resulting in reduced cellularity of the thymus. Moreover, the degradation of beta-catenin in response to DNA damage is significantly impaired in SIP-/- cells. SIP-/- embryonic fibroblasts show a growth-rate increase resulting from defects in G1 arrest. Thus, the beta-catenin degradation pathway mediated by SIP defines an essential checkpoint for thymocyte development and cell-cycle progression.

2-Methoxyestradiol Induces Cell Cycle Arrest and Mitotic Cell Apoptosis in Human Vascular Smooth Muscle Cells

It has been shown that 2-methoxyestradiol (2-ME) inhibits cell proliferation and DNA synthesis in human aortic smooth muscle cells. However, the cellular mechanisms underlying the antiproliferative activity of 2-ME are unclear. The present study was performed to explore the cellular mechanisms whereby 2-ME leads to growth inhibition and apoptosis of human smooth muscle cells. Our results demonstrate that at 1 hour of treatment, 1 micromol/L 2-ME induces multiple spindles, overamplified centrosomes, and multipolar cytokinesis, whereas 10 micromol/L 2-ME causes completely damaged spindle, disorientated centrosomes, and missegregated chromosomes. At 6 hours of treatment, the mitotic index was increased and reached a maximal level, and cells with 4N DNA content (4N cells) began to accumulate. The increased mitotic cells induced by 2-ME were apoptotic as detected by both annexin V and TUNEL staining. Blockage of cells in G(1/0) phase by thymidine prevented 2-ME-induced apoptosis. In addition, the increased mitotic index declined concurrently when even more 4N cells accumulated at 12 to 48 hours of treatment with 10 micromol/L 2-ME. Furthermore, in response to 2-ME, cells delayed entry into the next cell cycle and exhibited aneuploidy or micronuclei. Some aneuploidy cells continued to synthesize DNA. We conclude that 2-ME treatment not only arrests cells in mitosis and promotes mitotic cell apoptosis, but also causes cells to undergo "mitotic slippage" and endoreduplication. The induction of mitotic cell arrest and apoptosis may be a major cellular mechanism by which 2-ME inhibits proliferation of human smooth muscle cells.

Epidermal transit of replication-arrested, undifferentiated keratinocytes in UV-exposed XPC mice: An alternative to in situ apoptosis

The interplay among nucleotide excision repair, cell-cycle regulation, and apoptosis in the UV-exposed epidermis is extremely important to avoid mutations and malignant transformation. In Xpc(-/-) mice deficient in global genome nucleotide excision repair (GGR), a cell-cycle arrest of epidermal cells in late S-phase [with near-double normal diploid (4N) DNA content] was observed 48-72 h after UV exposure. This arrest resolved without apoptosis (96-168 h). We surmised that these arrested keratinocytes with persistent DNA damage were removed by epidermal turnover. In vivo BrdUrd pulse-chase labeling (>17 h after UV exposure) showed that DNA replication after UV exposure was resumed in Xpc(-/-) mice, but it did not reveal any evidence of retained BrdUrd-labeled S-phase cells in the basal layer of the epidermis at 72 h. Interestingly, by this time a maximum number of cytokeratin 10-negative and cytokeratin 5-positive cells had appeared in the suprabasal epidermal cell layers of UV-exposed Xpc(-/-) mice. Accumulation of these "basal cell"-like keratinocytes in the suprabasal layers was clearly aberrant and was not observed in WT and heterozygous mice. Flow cytometric analyses of single-cell suspensions from UV-exposed Xpc(-/-) epidermis further showed that the "near-4N" arrested cells retained cytokeratin 5 and lacked cytokeratin 10. Hence, we conclude that the arrested near-4N cells became detached from the basal layer without entering a proper differentiation program and were indeed subsequently lost through the epidermal turnover. This expulsion apparently constitutes an alternative route, different from in situ apoptosis, to eliminate DNA-damaged arrested cells from the epidermis.

The Ste20-like Kinase SLK Is Required for Cell Cycle Progression through G2

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein that can regulate actin reorganization during cell adhesion and spreading (Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002) J. Biol. Chem. 277, 37685-37692). Because of its association with the microtubule network, we investigated whether SLK plays a role in cell cycle progression, a process that requires microtubule dynamics during mitosis. Consistent with microtubule association in exponentially growing cells, our results showed that SLK co-localizes with the mitotic spindle in cells undergoing mitosis. Expression of a kinase-inactive mutant or SLK small interfering RNAs inhibited cell proliferation and resulted in an accumulation of quiescent cells stimulated to re-enter the cell cycle in the G2 phase. Cultures expressing the mutant SLK displayed a normal pattern of cyclin D, E, and B expression but failed to down-regulate cyclin A levels, suggesting that they cannot proceed through M phase. In addition, these cultures displayed low levels of both phospho-H3 and active p34/cdc2 kinase. Overexpression of active SLK resulted in ectopic spindle assembly and the induction of cell cycle re-entry of Xenopus oocytes, suggesting that SLK is required for progression through G2 upstream of H1 kinase activation.

The Aurora Kinase Inhibitor AZD1152 Causes Perturbation of Cell Cycle Distribution in Cell Lines and Primary AML Samples.

Aurora kinases (AK) participate in chromosome separation during mitosis and are essential for mitotic processes and completion of cytokinesis. AK-A is found at the centrosome and spindle apparatus during prophase to telophase, while AK-B is seen in the midzone during anaphase of the cell cycle. Inhibition of these kinases may be a viable therapeutic strategy. AZD1152 is a specific AK inhibitor, designed to target cell division in proliferating tumour cells, with potential for activity in a wide range of tumours. In order to evaluate the potential utility of the AK inhibitor AZD1152 in acute myeloid leukaemia (AML), the expression levels of AK-A, -B, and -C were evaluated in a panel of myeloid cell lines (HL-60, NB4, NB4R2, U937, KG1, and K562) and in cells from 10 normal bone marrows and 240 AML patients using the Affymetrix gene expression system (Affymetrix Inc.) validated by real-time quantitative polymerase chain reaction in 105 patients. AK-C was absent in all samples, while AK-A and -B were present in 5% and 34%, respectively, of the AML patients and both were present in normal bone marrow samples. Expression levels did not correlate with age, sex, French-American-British classification or cytogenetic risk group. AML cell lines were incubated with a variable concentration of AK inhibitor (0.01 μM to 10 μM) for up to 72 h and the effects on viability, cell growth, cell cycle, and DNA content measured after 24 and 48 h exposure. This resulted in the accumulation of cells with 4N DNA content, indicating an increased proportion of cells in G2, and development of cells with DNA content greater than G2/M phase cells, on occasions showing 8N ploidy. This effect was time and dose dependent. At 24 h AK inhibitor caused a significant increase (p≤0.05) in the number of cells with 4N DNA content, suggestive of G2, or with greater than 4N DNA content at concentrations as low as 0.01 μM. Growth of HL-60 and NB4 cells was inhibited, following 72 h treatment with AK inhibitor (0.01 μM), by 50% and 90% (p<0.05), respectively. Incubation of AML samples (n=28) with AK inhibitor also showed an effect on the cell cycle, with a significant increase in G2 population (p≤0.05), and occasionally an increase of DNA content (p≤0.05) after 24 and 48 h at a minimum concentration of 1.0 μM. Flow cytometric analysis showed histone H3 phosphorylation was decreased, from around 3–5% in untreated cells, only in a proportion (~35%) of primary samples. The growth inhibition effects seen are combined with increased DNA content, which is consistent with the endoreduplication effects associated with these agents. AK-B is expressed in around one-third of AML patients, and agents targeted against this kinase may have therapeutic potential in AML.

The Activity of the Novel Aurora Kinase B Inhibitor AZD1152 in Acute Myeloid Leukaemia Cells.

Background: Aurora kinases (A, B and C) play a critical role in regulating mitosis and cell division. Aurora kinase A is required for correct spindle assembly while aurora kinase B is involved in histone H3 phosphorylation, chromosome segregation, and cytokinesis. Emerging data suggest these proteins are implicated in the survival and proliferation of both haematologic and solid malignancies, and therefore represent novel therapeutic targets. Several aurora kinase inhibitors have been described, mainly inhibiting both A and B kinases, although inhibition of aurora kinase B is more closely associated with the effects seen with these compounds. We have therefore investigated the activity of AZD1152, a novel, specific inhibitor of aurora kinase activity, with selectivity for aurora B.Methods: Drug activity was studied in a panel of leukaemic cell lines (HL-60, MV411, THP-1, U937) and in primary AML cells (n=4) at concentrations of 0–1000 nM AZD1152 for up to 168 h. Cell number and percent viability were determined using a Viacount assay on a Guava PCA-96 analyser, cell cycle distribution (including apoptotic and polyploid (>4n) cells) by PI staining with flow cytometry, and the phosphorylation of an aurora kinase B substrate histone H3 by fluorescence immunocytochemistry using a phospho-specific antibody.Results: AZD1152 displayed time- and concentration-dependent activity in cell lines and primary cells. A clear decrease in phospho histone H3 positive cells was apparent by 48 h, with <1% positive cells at 50 nM AZD1152 in cell lines compared with 8–10% in control cells and <0.5% positive cells in primary cultures at 100 nM AZD1152 compared with 2 - 4.5% in control cultures. In all cell lines studied a cell population with >4n DNA content by flow cytometry was apparent at 48 h (18–60% cells), confirmed by karyotype analysis which showed a variable but marked increase in chromosome number. This population had typically reduced, with a corresponding increase in apoptotic cells, by 96 h. In THP-1 cells, in which little effect on cell viability was observed, this >4n population increased to 80% by 96 h and persisted out to 168 h, with little apoptosis. In primary cells an increase in the G2/M population was evident in some cultures at 48 h, with only small changes in cells with >4n DNA content. In cell lines 10 nM AZD1152 resulted in a 28–65% reduction in cell number and a 6–35% reduction in cell viability after 48 h. At 100 nM these values increased to 65–78% and 6–42%, respectively. Notably little further increase in activity was observed at 1000 nM. By 96 h 100 nM AZD1152 reduced cell number by >80% in all cell lines, and cell viability by >67% in 3 lines (17% reduction in THP-1 cells). In primary AML cells AZD1152 over 48 h had little effect on cell number, but by 120 h this had decreased by >50% at 100 nM AZD1152, with a small effect on cell viability. Activity with 48 h exposure to drug followed by 72 h drug free was similar to that seen with a continuous 120 h exposure.Conclusions: The specific aurora kinase inhibitor AZD1152 demonstrates antiproliferative and apoptotic activity in leukaemic cell lines and primary cells at concentrations associated with changes in the phosphorylation of an aurora kinase B substrate protein.

Defects in Management of Labile Iron and Oxidative Stress Underpin Red Cell Enucleation Defects in Rb Null Mice.

The Rb tumor suppressor is critically required for end-stage red cell maturation under conditions of oxidative stress, including in the developing fetal liver, in the bone marrow of aging mice, in the spleen and bone marrow of young mice treated with phenylhydrazine to induce hemolytic anemia, and in lethally irradiated mice reconstituted with donor tissue [1]. Loss of Rb resulted in a failure of end-stage red cells to enucleate, accumulation of red cells with a 4N DNA content and aberrant chromatin structure [1]. The molecular basis of these defects is not defined nor do we understand the reasons why pRb should be required under stress conditions, but not during normal “steady-state” erythropoiesis. The work presented will address both of these questions.In determining why pRb is critically required for stress erythropoiesis but not for steady-state erythropoiesis, we have demonstrated increased levels of reactive oxygen species (ROS) and labile iron in Rb null erythroblasts relative to wild-type control erythroblasts derived from E12.5 fetal liver. Furthermore, we show that quenching of ROS in Rb null erythroblasts by treatment of mice with the anti-oxidant N-acetyl cysteine (NAC) rescued aspects of the erythroid defect, including red cell enucleation and also extended the lifespan of Rb null mice. Similarly, chelation of labile iron with desferroxiamine restored enucleation capacity to Rb null erythroblasts. Furthermore, we show that the transferrin receptor (CD71) is transcriptionally repressed by pRb/E2F and examine whether deregulated expression of CD71 contributes to increased labile iron and oxidative stress in Rb null erythroblasts. These results suggest that loss of pRb limits the ability of erythroblasts to manage labile iron and oxidative stress, in part through deregulated expression of CD71, and that this contributes to the enucleation defect observed in Rb null mice. Given that pRb is itself regulated by ROS, we present a model in which the timely induction and repression of the CD71 receptor in differentiating erythroblasts is required to manage labile iron, oxidative stress and to coordinate cell cycle exit with end-stage maturation.

Depletion of Chk1 Leads to Premature Activation of Cdc2-cyclin B and Mitotic Catastrophe

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.

Cyclin-dependent Kinase Inhibitors Uncouple Cell Cycle Progression from Mitochondrial Apoptotic Functions in DNA-damaged Cancer Cells

DNA damage results in transcriptional induction of p53 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (CDKN1A) and the proapoptotic Bcl-2 family member p53 up-regulated modulator of apoptosis (PUMA). Depending on the cellular context, p21(Cip1) and PUMA mediate cell cycle arrest and apoptosis, respectively. By imposing cell cycle arrest at the expense of apoptosis, p21(Cip1) can sharply reduce the effectiveness of DNA-damaging anticancer agents in colorectal cancer cells. We investigated the link between cell cycle progression and the onset of apoptosis in DNA-damaged cells by analyzing the activation of the apoptotic cascade in p21(Cip1)-deficient HCT116 colorectal cancer cells. DNA damage induced a similar level of p53 activation and PUMA induction in p21(Cip1)-deficient cells compared with wild-type isogenic counterparts. p21(Cip1) did not act as a direct blocker of PUMA. However, only p21(Cip1)-deficient cells showed extensive cytochrome c release, mitochondrial membrane depolarization, and caspase activation. An increase in caspase activation occurred as these cells reached M-phase and incurred polyploidy. When ectopically expressed in p21(Cip1)-deficient HCT116 cells, p21(Cip1), its family member p27(Kip1), and the structurally unrelated CDK inhibitor p16(Ink4a) were similarly effective at causing cell cycle arrest and inhibiting DNA damage-induced apoptotic events such as cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These observations suggest that by blocking dysregulated cell cycle progression, CDK inhibitors can influence the sensitivity of the mitochondria to proapoptotic signals in DNA damage-induced cancer cells.

Checkpoint Kinase 1 Regulates Diallyl Trisulfide-induced Mitotic Arrest in Human Prostate Cancer Cells

We have shown previously that diallyl trisulfide (DATS), a constituent of processed garlic, inhibits proliferation of PC-3 and DU145 human prostate cancer cells by causing G(2)-M phase cell cycle arrest in association with inhibition of cyclin-dependent kinase 1 activity and hyperphosphorylation of Cdc25C at Ser(216). Here, we report that DATS-treated PC-3 and DU145 cells are also arrested in mitosis as judged by microscopy following staining with anti-alpha-tubulin antibody and 4',6-diamidino-2-phenylindole and flow cytometric analysis of Ser(10) phosphorylation of histone H3. The DATS treatment caused activation of checkpoint kinase 1 and checkpoint kinase 2, which are intermediaries of DNA damage checkpoints and implicated in Ser(216) phosphorylation of Cdc25C. The diallyl trisulfide-induced Ser(216) phosphorylation of Cdc25C as well as mitotic arrest were significantly attenuated by knockdown of check-point kinase 1 protein in both PC-3 and DU145 cells. On the other hand, depletion of checkpoint kinase 2 protein did not have any appreciable effect on G(2) or M phase arrest or Cdc25C phosphorylation caused by diallyl trisulfide. The lack of a role of checkpoint kinase 2 in diallyl trisulfide-induced phosphorylation of Cdc25C or G(2)-M phase cell cycle arrest was confirmed using HCT-15 cells stably transfected with phosphorylation-deficient mutant (T68A mutant) of checkpoint kinase 2. In conclusion, the results of the present study suggest existence of a checkpoint kinase 1-dependent mechanism for diallyl trisulfide-induced mitotic arrest in human prostate cancer cells.

CCT Chaperonin Complex Is Required for the Biogenesis of Functional Plk1

Experiments from several different organisms have demonstrated that polo-like kinases are involved in many aspects of mitosis and cytokinesis. Here, we provide evidence to show that Plk1 associates with chaperonin-containing TCP1 complex (CCT) both in vitro and in vivo. Silencing of CCT by use of RNA interference (RNAi) in mammalian cells inhibits cell proliferation, decreases cell viability, causes cell cycle arrest with 4N DNA content, and leads to apoptosis. Depletion of CCT in well-synchronized HeLa cells causes cell cycle arrest at G(2), as demonstrated by a low mitotic index and Cdc2 activity. Complete depletion of Plk1 in well-synchronized cells also leads to G(2) block, suggesting that misfolded Plk1 might be responsible for the failure of CCT-depleted cells to enter mitosis. Moreover, partial depletion of CCT or Plk1 leads to mitotic arrest. Finally, the CCT-depleted cells reenter the cell cycle upon reintroduction of the purified constitutively active form of Plk1, indicating that Plk1 might be a CCT substrate.

A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation

Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.

Viral Transport of DNA Damage That Mimics a Stalled Replication Fork

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

Effects of SB559457, a Novel Small Molecule Thrombopoietin Receptor (TpoR) Agonist, on Human Hematopoietic Cell Growth and Differentiation.

Treatment of thrombocytopenia resulting from chemotherapy, radiation, or stem cell transplantation remains problematic. Hopes that TPO, the major regulator of megakaryocyte maturation, might prove clinically useful for this purpose have not been fulfilled, in part because of untoward reactions to the molecule. To avoid undesirable side effects, small molecule TpoR agonists are being developed. We have evaluated the biological effects of one such molecule, SB559457, a hydrazone compound, on hematopoietic cells. We first examined SB559457's ability to stimulate proliferation of the recombinant human TPO (rhTPO) dependent cell line UT-7/TPO. We found a dose dependent augmentation of cell proliferation at doses between 1 and 10 μM, with maximal effects, compared to 100ng/ml of rhTPO at 10μM. We then evaluated SB559457's growth promoting effects on normal human CD34+ cells. We again found growth stimulation to be dose dependent between 1 and 10 μM and also noted enhancement when SB559457 was combined with IL-3 and SCF. Specifically, incubation of CD34 cells in 10 μM SB559457 for 7 days lead to a 2 fold increase in cell number; incubation of cells with IL3 and SCF in addition to SB559457 lead to a 12 fold increase in cell number after 7 days. Both results were comparable to those seen with 100 ng/ml of rhTPO. Curiously, the addition of IL-6 to cultures containing IL-3 and SCF compromised the ability of SB559457, but not Tpo, to stimulate CD34+ cell growth. To determine if SB559457 stimulates megakaryocyte differentiation directly, CD34+ cells were incubated in cytokines with rhTPO or SB559457 for periods of 7–10 days, after which time cells in culture were examined for degree of polyploidization, and expression of megakaryocyte lineage markers CD41 and CD61. 14% of cells grown in SB559457 showed greater than 4N DNA content compared to 8% in rhTPO. These same cells expressed the megakaryocyte markers CD41 and CD61on 35% of cells, comparable to the results with rhTPO. Preliminary results demonstrate that incubation of CD34 cells in SB559457 maintained stem cells numbers, as assayed by NOD/SCID engraftment, for at least 4 days at a level comparable to the levels maintained by rhTPO. These results demonstrate that SB559457 is a bona fide Tpo R agonist that likely acts through activation of c-mpl, the receptor for TPO. These experiments provide a rationale for the evaluation of SB559457, or SB559457 congeners, in patients with thrombocytopenia.

G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium

Because proliferation of eukaryotic cells requires cell cycle-regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr(288) phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr(288) phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.

Inhibition of JNK2 Disrupts Anaphase and Produces Aneuploidy in Mammalian Cells

The JNK family members JNK1 and JNK2 regulate tumor growth and are essential for transformation by oncogenes such as constitutively activated Ras. The mechanisms downstream of JNK that regulate cell cycle progression and transformation are unclear. Here we show that inhibition of JNK2, but not JNK1, with either a dominant-negative mutant, a pharmacological inhibitor, or RNA interference caused an accumulation of mammalian cells with 4N DNA content. When observed by immunofluorescence, these cells progressed to metaphase without apparent defects in spindle formation or chromosome alignment to the metaphase plate, suggesting that the 4N accumulation is a result of postmetaphase defects. Consistent with this prediction, when JNK activity was suppressed, we observed defects in central spindle formation and chromosome segregation during anaphase. In contrast, cyclin-dependent kinase 1 activity, cyclin B1 protein, and Polo-like kinase 1 protein turnover remained intact when JNK was inhibited. In addition, continued inhibition of JNK activity did not block reentry into subsequent cell cycles but instead resulted in polyploidy. This evidence suggests that JNK2 functions in maintaining the genomic stability of mammalian cells by signaling that is independent of cyclin-dependent kinase 1/cyclin B1 down-regulation.

Heliocoverpa armigera single nucleocapsid nucleopolyhedrovirus induces Hz-AM1 cell cycle arrest at the G 2 phase with accumulation of cyclin B 1

The cell cycle phase distributions of Hz-AM1 cells grown in monolayer culture were G 1 = 49.7 ± 3.3%, S = 22.7 ± 3.8% and G 2/ M = 27.8 ± 4.2% without >4N DNA content. The culture doubling time was about 40 h and the duration of the G 1, S and G 2/ M phases was estimated to be 10, 14 and 16 h, respectively. HaSNPV infection of Hz-AM1 cells resulted in both unsynchronized and synchronized G 1 phase arrest at G 2/ M phase. HaSNPV infection also resulted in the appearance of more than 4N DNA content, which accumulated to the highest levels 72 h post-infection. We also found that the expression level of cyclin B 1 increased significantly after 16 h post-infection, while cyclin A did not show any change. This observation supports the Hz-AM1-infected arrest at the G 2/ M phage. Cytoplasm location of cyclin B 1 indicated that the Hz-AM1 cell cycle arrest was at the G 2 phase.

Intra-S-phase checkpoint activation by direct CDK2 inhibition.

To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.

Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats.

Vertebrate telomeres consist of tandem repeats of T2AG3 and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression.