4N DNA Content Research Articles

Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells

Overexpression of Aurora-A kinase has been correlated with cancer susceptibility and poor prognosis in several human cancers. In this study, we evaluated the effect of inhibition of Aurora-A kinase on cell cycle progression and tumour cell survival after exposure to ionising radiation (IR). Combined IR and Aurora-A inhibition by short interfering RNA (siRNA) or by PHA680632 (a selective Aurora kinase inhibitor with submicromolar activity against Aurora-A) prior to IR led to an enhancement of radiation-induced annexin V positive cells, micronuclei formation, and Brca1 foci formation only in cells with deficient p53. However, the drug brought about additive to sub-additive interaction with radiation with regard to in vitro clonogenic survival. Cell cycle analysis revealed a high >4N DNA content 24 h after PHA680632 exposure. DNA content >4N was reduced dramatically when cells were irradiated combined with PHA680632 simultaneously. In vivo xenografts (p53−/− HCT116) of a mice study showed enhanced tumour growth delay (TGD) after the PHA680632−IR combinatorial treatment compared with IR alone. These results demonstrate that PHA680632 in association with radiation leads to an additive effect in cancer cells, especially in the p53-deficient cells, but does not act as a radiosensitiser in vitro or in vivo.

ETS2 and ERG1 Promote Megakaryopoiesis and Immortalize GATA1 Knockdown (ΔneoΔHS) Fetal Liver Progenitors: Implications for Leukemogenesis in Down Syndrome.

ETS2 and ERG are two ETS family transcription factors encoded on chromosome 21 that have been implicated in leukemia and other types of cancer. In the setting of trisomy 21 (e.g. in individuals with Down syndrome) there is a significant increase in the propensity for leukemia, including AMKL (acute megakaryoblastic leukemia). It has been theorized that genes located on chromosome 21 may contribute to the predisposition of children with Down syndrome for pediatric AMKL, possibly by collaborating with mutations in the transcriptional regulator GATA1. To test the oncogenic potential of these genes in the hematopoietic system, ETS2 and the ERG isoform, ERG1, were over-expressed in both wild-type and GATA1 knockdown (ΔneoΔHS) fetal liver hematopoietic progenitors and the effects on growth and differentiation were studied. We discovered that over-expression of either ETS2 or ERG1 in wild-type progenitors increased the formation of CD41 positive megakaryocytes by 2–3 fold in liquid culture. However, whereas ETS2 appeared to primarily promote the early stages of megakaryopoiesis, ERG1 over-expression led to a dramatic rise in the percentage of cells expressing CD42, a marker of more mature megakaryocytes. Ectopic ERG1 expression also affected the ploidy profile, with nearly all cells containing ≥4N DNA content. Quantitative real time RT-PCR analysis of lineage specific genes confirmed that expression of megakaryocyte genes, including GPIIb (CD41), platelet factor 4 (PF4), and ß1-tubulin, was significantly increased in undifferentiated cells over-expressing either ETS2 or ERG1. These results show that over-expression of ETS2 or ERG1 rapidly initiates the megakaryocyte program. Over-expression of either ETS2 or ERG1 in GATA1 knockdown fetal liver cells similarly led to a 2.5–5 fold increase in the formation of CD41 positive megakaryocytes in liquid culture; however, neither ETS2 or ERG1 could circumvent the differentiation block that is characteristic of the GATA1 knockdown strain, including the inability to form proplatelet extensions. We next assessed the capacity of ETS2 or ERG1 over-expression to alter the colony forming ability of wild-type and GATA1 knockdown fetal liver progenitors. Both ETS2 and ERG1 expression led to a significant increase in CFU-MK derived from wild-type progenitors, as evidenced by an increase in the number of acetylcholinesterase positive colonies. In contrast, neither gene altered the formation of CFU-MK when over-expressed in the GATA1 knockdown progenitors, which already show a prominent enhancement in megakaryocyte colony formation over wild-type fetal liver cells. Finally, serial re-plating assays performed with GATA1 knockdown fetal liver cells showed that ectopic expression of either ETS2 or ERG1 could immortalize hematopoietic progenitors, whereas only ERG1 could enhance colony re-plating of wild-type cells. These findings provide evidence for a specific synergy between GATA-1 deficiency and ETS2 over-expression and suggest a distinct oncogenic potential for each factor. Overall, these results in primary murine fetal liver cells demonstrate that ETS2 and ERG1 promote the megakaryocyte lineage and facilitate immortalization of GATA1 knockdown cells, consistent with a potential oncogenic role in Down syndrome AMKL for these two ETS family genes located on chromosome 21.

Vorinostat Synergistically Potentiates MK-0457 Lethality in Chronic Myelogenous Leukemia (CML) Cells Sensitive and Resistant to Imatinib Mesylate.

MK-0457/VX-680 was developed as a pan-aurora kinase inhibitor that induces apoptosis in tumor cells and suppresses tumor growth in vivo. Recently, it was found that MK-0457 is also a potent inhibitor of both wild type and mutated Bcr/Abl kinases, including the T315I mutant which confers resistance to first- (i.e., imatinib mesylate/IM) and second-generation kinase inhibitors (e.g., dasatinib and nilotinib). Moreover, MK-0457 exhibits significant activity in patients with T315I phenotype-refractory CML or Ph-positive ALL. Here we examined interactions between MK-0457 and the histone deacetylase inhibitor (HDACI) vorinostat/SAHA in Bcr/Abl+ leukemia cells, including those resistant to IM through various mechanisms, particularly the T315I mutation. Co-administration of non- or subtoxic concentrations of vorinostat (0.5–2 μM) increased the lethality of MK-0457 (5–100 nM) in a highly synergistic manner in K562 and LAMA84 cells. Similar interactions were observed in primary CD34+ CML cells, whereas the combination largely spared normal bone marrow mononuclear cells. Moreover, the MK-0457/vorinostat regimen markedly induced cell death in IM-resistant K562 cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, a phenomenon accompanied by Lyn inactivation. Notably, the regimen was highly active against BaF/3 cells bearing various Bcr/Abl point mutations (i.e., T315I, E255K, and M351T) conferring resistance to IM. These events were associated with inactivation and downregulation of wild type and mutated Bcr/Abl (particularly T315I), reduced expression of phospho-CrkL, and vorinostat-mediated interruption of interactions between Bcr/Abl and Hsp90. Moreover, exposure of cells to marginally toxic doses of MK-0457 resulted in accumulation of cells with greater than 4N DNA content, resulting from induction of endoreduplication. Co-administration of vorinostat strikingly enhanced aurora kinase inhibition by MK-0457, reflected by markedly diminished expression of phospho-histone H3 (Ser 10), accompanied by preferential killing of polyploid cells. Interestingly, vorinostat also interacted with a selective inhibitor of aurora kinase A and B, at concentrations that inhibited histone H3 phosphorylation, to potentiate apoptosis in CML cells without modifying expression of phospho-Bcr/Abl or -CrkL. These results suggest that aurora kinase inhibition plays a functional role in synergistic interactions between HDACI and aurora kinase inhibitors. Finally, vorinostat markedly induced expression of the pro-apoptotic BH3-only protein Bim in various CML cell lines, including BaF/3 cells expressing the T315I Bcr/Abl mutation, as well as in primary CML cells. Knockdown of Bim by siRNA significantly protected K562 cells from apoptosis induced by vorinostat but not MK-0457. Furthermore, siRNA-mediated Bim downregulation blocked induction of Bim expression by vorinostat and dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. In addition, blockade of apoptosis by Bim siRNA did not modify downregulation of Bcr/Abl or phospho-histone H3 in cells exposed to the vorinostat/MK-0457 regimen, arguing against the possibility that the latter phenomena are secondary to activation of apoptotic cascade. Together, these findings indicate that vorinostat dramatically increases MK-0457 lethality in IM-sensitive and -resistant CML cells, and suggest that inactivation of Bcr/Abl kinase and aurora kinases, as well as Bim induction by vorinostat, contribute to this phenomenon.

Pre-Clinical Validation of Polo-Like Kinase 1 as a Therapeutic Target in Multiple Myeloma with the Selective Inhibitor BI2536.

BACKGROUND: The centrosome is the cellular organelle that nucleates the mitotic spindle. Polo-like kinase 1 (Plk1), a centrosome-associated serine/threonine kinase, serves as a key regulator of multiple steps in mitosis. Plk1 is overexpressed in a broad spectrum of tumor types, and its expression often correlates with poor patient prognosis. We have previously observed centrosome amplification in myeloma, and have validated other components of the myeloma centrosome and mitotic apparatus as therapeutic targets. The present study explored the expression of Plk1 in myeloma and the effect of BI2536, a potent and selective inhibitor of Plk1, on myeloma cells in the pre-clinical setting.METHODS: Plk1 expression was assayed in a panel of myeloma cell lines (MMCL) and AutoMACS-purified CD138+ patient bone marrow plasma cells (BMPC). The anti-myeloma effects of BI2536, alone or in combination with conventional agents, were assayed on myeloma cells with proliferation (MTS) and apoptosis (Annexin V/propidium iodide) assays. The phenotype of treated cells was examined with DNA content analysis and immunofluorescence microscopy. The efficacy of BI2536 monotherapy was evaluated in NOD/SCID mice bearing RPMI8226 xenografts.RESULTS: Plk1 is ubiquitously expressed in myeloma to varying degrees in both MMCL and BMPC. BI2536 inhibited the proliferation of MMCL (RPMI 8226, U266, LP-1 and KMS-11) and patient BMPC at nanomolar concentrations. The addition of BI2536 was able to overcome resistance to dexamethasone. Bortezomib in combination with BI2536 had significantly increased anti-myeloma effects compared to the use of either agent alone. BI2536-treated MMCL accumulated 4N DNA content prior to undergoing apoptosis. The phenotype of BI2536-treated cells is consistent with inhibition of Plk1, showing prometaphase arrest and monopolar mitotic spindles in a dose-dependent fashion. BI2536 induces regression of human myeloma xenografts in NOD/SCID mice. Taken together, BI2536 is a promising new agent for the treatment of multiple myeloma. This work provides further evidence that Plk1 and the amplified myeloma centrosome are targets for therapy.

A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle-dependent element (CDE)/cell cycle gene homology region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the negative control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest associated with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential molecular target for ATC treatment.

ATR Pathway Is the Primary Pathway for Activating G2/M Checkpoint Induction After Re-replication

DNA replication is tightly controlled to ensure accurate chromosome duplication and segregation in each cell cycle. Inactivation of Geminin, an inhibitor of origin licensing, leads to re-replication in human tumor cells within the same cell cycle and triggers a G(2)/M checkpoint. We find that the primary pathway to signal that re-replication has been detected is the ATR kinase and the Rad9-Rad1-Hus1 (9-1-1) clamp complex together with Rad17-RFC clamp loader. ATM kinase and the Mre11-Rad50-Nbs1 complex do not appear to play significant roles in the checkpoint. Chk1 activation occurs at early stages, whereas Chk2 activation occurs much later. Overall we conclude that ATR/Chk1 pathway is activated at an early time point after the loss of Geminin and contributes to checkpoint arrest essential for the accumulation of re-replicated cells, whereas activation of the ATM/Chk2 pathway is a by-product of DNA re-replication at a later period.

The FANCJ/MutLα interaction is required for correction of the cross-link response in FA-J cells

FANCJ also called BACH1/BRIP1 was first linked to hereditary breast cancer through its direct interaction with BRCA1. FANCJ was also recently identified as a Fanconi anemia (FA) gene product, establishing FANCJ as an essential tumor suppressor. Similar to other FA cells, FANCJ-null (FA-J) cells accumulate 4N DNA content in response to DNA interstrand crosslinks (ICLs). This accumulation is corrected by reintroduction of wild-type FANCJ. Here, we show that FANCJ interacts with the mismatch repair complex MutLalpha, composed of PMS2 and MLH1. Specifically, FANCJ directly interacts with MLH1 independent of BRCA1, through its helicase domain. Genetic studies reveal that FANCJ helicase activity and MLH1 binding, but not BRCA1 binding, are essential to correct the FA-J cells' ICL-induced 4N DNA accumulation and sensitivity to ICLs. These results suggest that the FANCJ/MutLalpha interaction, but not FANCJ/BRCA1 interaction, is essential for establishment of a normal ICL-induced response. The functional role of the FANCJ/MutLalpha complex demonstrates a novel link between FA and MMR, and predicts a broader role for FANCJ in DNA damage signaling independent of BRCA1.

Predisposition to tetraploidy in pulmonary vascular smooth muscle cells derived from the Eker rats

Somatic mutations in the tuberous sclerosis complex-2 (TSC2) gene are associated with pulmonary lymphangioleiomyomatosis (LAM), a disorder characterized by benign lesions of smooth muscle and/or smooth muscle-like cells in the lung. However, the cellular mechanisms underlying LAM disease are largely unknown. Given that the TSC2 gene product tuberin is involved in the regulation of cell growth and proliferation, the present study was designed to investigate the potential roles of TSC2 in regulation of the cell cycle. We studied cell cycle profiles of pulmonary vascular smooth muscle cells (SMCs) derived from Eker rats (Tsc2(+/EK)), a genetic model carrying a germline insertional deletion in one copy of the Tsc2 gene, and the wild-type rats (Tsc2(+/+)), a noncarrier counterpart. We found that Tsc2(+/EK), but not Tsc2(+/+), SMCs displayed increases in cells with > or =4N DNA content (> or =4N cells) and in the bromodeoxyuridine (BrdU) incorporation of > or =4N cells. Centrosome number was also increased in Tsc2(+/EK) SMCs, but the mitotic index was comparable between Tsc2(+/+) and Tsc2(+/EK) SMCs. Furthermore, Tsc2(+/EK) SMCs showed elevated phosphorylation of p70S6K and increased expression of cell cycle regulatory proteins Cdk1, cyclin B, Cdk2, and cyclin E. Inhibition of the mammalian target of rapamycin (mTOR) pathway by rapamycin not only inhibited the phosphorylation of p70(S6K) and the expression of cell cycle regulatory proteins but also reduced accumulation of > or =4N cells and BrdU incorporation of >4N cells. Therefore, our results demonstrate that Tsc2(+/EK) SMCs are predisposed to undergo tetraploidization, involving activation of the mTOR pathway. These findings suggest an important role of Tsc2 in regulation of the cell cycle.

Potentiation of Kinesin Spindle Protein Inhibitor–Induced Cell Death by Modulation of Mitochondrial and Death Receptor Apoptotic Pathways

Targeting the mitotic motor kinesin kinesin spindle protein (KSP) is a new strategy for cancer therapy. We have examined the molecular events induced by KSP inhibition and explored possible mechanisms of resistance and sensitization of tumor cells to KSP inhibitors. We found that KSP inhibition induced cell death primarily via activation of the mitochondrial death pathway. In HeLa cells, inhibition of KSP by small-molecule inhibitor monastrol resulted in mitotic arrest and rapid caspase activation. BclXL phosphorylation and loss of mitochondrial membrane potential was detected before significant caspase activation, which was required to trigger the subsequent apoptotic pathway. In A549 cells, however, KSP inhibition did not induce mitochondrial damage, significant caspase activity, or cell death. A549 cells aberrantly exited mitosis, following a prolonged drug-induced arrest, and arrested in a G(1)-like state with 4N DNA content in a p53-dependent manner. Overexpression of BclXL provided a protective mechanism, and its depletion rescued the apoptotic response to monastrol. In addition, Fas receptor was up-regulated in A549 cells in response to monastrol. Treatment with Fas receptor agonists sensitized the cells to monastrol-induced cell death, following exit from mitosis. Thus, activation of the death receptor pathway offered another mechanism to enhance KSP inhibitor-induced apoptosis. This study has elucidated cellular responses induced by KSP inhibitors, and the results provide insights for a more effective cancer treatment with these agents.

Pim‐1 induced polyploidy but did not affect megakaryocytic differentiation of K562 cells and CD34+ cells from cord blood

In a previous study, we determined the gene expression profile of both megakaryocytic and non-megakaryocytic lineage cells via serial analysis of gene expression and microarray methods, and demonstrated that Pim-1 was expressed more abundantly in megakaryocytic lineage cells. In this study, we knocked down Pim-1 in K562 cells, as well as in CD34+ cells from cord blood, via RNA interference, in order to analyze the effects of Pim-1 expression on the megakaryocytic differentiation of these cells. We then additionally overexpressed the Pim-1 genes in K562 cells, and conducted a comparison of these effects with those of RNAi cells on the course of megakaryocytic differentiation. The results of this study revealed that Pim-1 knockdown exerted no effects on commitment or differentiation toward megakaryocytic lineage, as evidenced by the detected CD41+ or CD61+ cells, or on the number of megakaryocytic colony forming units. However, Pim-1 knockdown was found to elicit a reduction in CD41+ cells with >4n DNA content, and a concomitant increase in the fraction of cells achieving a ploidy of >4n in the Pim-1 overexpressing population of K562 cells. Collectively, the findings of these studies indicate that the expression of Pim-1 expression is both necessary and sufficient for polyploidization, but is not critical to cytoplasmic differentiation on megakaryopoiesis.

ZM447439, a Novel Aurora Kinase Inhibitor, Induces Growth Arrest and Apoptosis of Human Leukemia Cells.

The Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. Aberrant expression of these kinases have been shown in solid tumors and considered to associate with aneuploidy and carcinogenesis. This study found that Aurora kinase A and B were aberrantly expressed in human leukemia cell lines (n=15, e.g., PALL-1, -2, HL-60, NB4, MV4-11, etc.) as well as freshly isolated leukemia cells from individuals with acute myeloid leukemia (n=44) compared to peripheral blood mononuclear cells from healthy volunteers (n=12), as measured by real-time PCR. In addition, ZM447439, a novel selective Aurora kinase inhibitor, induced growth inhibition, accumulation of cells with 4N DNA content and apoptosis in human leukemia cells as measured by thymidine-uptake, cell cycle analysis and annexin V staining, respectively. Especially, profound growth inhibition occurred in PALL-1 and -2 cells which possess wild-type p53 gene. Of note, ZM447439 did not inhibit clonogenic growth of myeloid committed stem cells harvested from healthy volunteers. Taken together, inhibition of Aurora kinase may be a promising treatment strategy for the individuals with leukemia.

Inhibition of Kinesin Spindle Protein Induces Apoptosis and Overcomes Drug Resistance in Models of Multiple Myeloma.

Introduction: Kinesin spindle protein is a mitotic kinesin that is expressed only in proliferating cells and plays a key role in spindle pole separation, formation of a bipolar mitotic spindle, and thus in satisfaction of the mitotic checkpoint. Ispinesib (SB-715992) is a potent and selective inhibitor of kinesin spindle protein with a Ki of 0.6 nM, has cytotoxic activity at less than 10 nM in a spectrum of tumor cell lines, and disrupts the assembly of functional bipolar mitotic spindles.Methods: This study sought to examine whether spindle disruption by inhibition of kinesin spindle protein with ispinesib may have therapeutic potential in the treatment of multiple myeloma.Results: Ispinesib reduced cell viability in both interleukin-6-independent (RPMI 8226 and U266) and interleukin-6-dependent (ANBL-6 and KAS-6/1) models of multiple myeloma in a time- and concentration-dependent fashion. The average IC50 for ispinesib against these cell lines was 3.0 nM, 1.7 nM, 1.8 nM, and 1.8 nM, respectively. Cell cycle analysis showed that ispinesib induced growth arrest of myeloma cells with 4N DNA content (in M phase) within 24-hours. Two days after treatment at a 1 nM concentration, cells were able to recover from M phase arrest and resume normal cycling but, after exposure to 10 nM, treated cells could not escape M phase arrest, and instead entered apoptosis as determined by an increased sub-G1 population. Ispinesib was able to overcome resistance to melphalan in that the IC50 in melphalan-resistant RPMI 8226/LR5 cells (1.6 ± 0.2 nM) was comparable to that in parental RPMI 8226 controls (3.0 ± 0.9 nM). Similarly, ispinesib was also able to overcome dexamethasone resistance and bortezomib resistance. In regard to the latter, KAS-6/VR5 bortezomib-resistant cells (IC50 of 12.5 nM for bortezomib) retained sensitivity to ispinesib (IC50 1.6 ± 0.2 nM). Combination therapy with ispinesib and bortezomib in these cells resulted in enhanced levels of specific apoptosis (24%) that were greater than the sum of either agents alone (7% for ispinesib and 1% for bortezomib), suggesting synergy. Importantly, ispinesib was also active against freshly isolated CD138+ patient-derived multiple myeloma cells, while relatively sparing CD138− cells.Conclusions: Taken together, these studies demonstrate that kinesin spindle protein inhibition with ispinesib was able to induce growth arrest and apoptosis in myeloma cells, and overcome resistance to both conventional drugs and novel agents such as bortezomib. Moreover, the preferential activity against transformed plasma cells with sparing of normal bone marrow cells provides a strong rationale for translation of this agent into the clinic to combat relapsed/refractory multiple myeloma.

ENMD-981693 Is an Orally-Active Kinase Inhibitor with Activity towards Human Hematologic Cancers In Vitro and In Vivo.

The clinical success of Gleevec (imatinib) for chronic myelogenous leukemia has demonstrated that small-molecule inhibitors of specific kinases can be developed into effective oncology therapies. However, the rapid development of resistance in leukemic cells to drugs such as Gleevec and the limited therapeutic indications addressed with these molecules suggests that considerable opportunity exists for new inhibitors with a distinctive spectrum of activities against multiple kinase targets. ENMD-981693 is a novel, orally-active molecule that was discovered through a screening effort directed towards Aurora kinases, a family of serine/threonine kinases that are essential for mitotic progression. ENMD-981693 is selective for the Aurora A isoform, with an IC50 value of 25 nM, compared to an IC50 value of ~700 nM for Aurora B. The activity of ENMD-981693 was evaluated against a panel of 100 recombinant kinases, and the compound was shown to inhibit a broad range of tyrosine kinase targets including Flt3, CSF1R, Lck, JAK2, and c-Kit. ENMD-981693 inhibited the in vitro growth of human hematopoietic cancer lines including MV4;11, K562, THP-1, Jurkat, TF-1, U937, and HL-60 with IC50 values ranging from 0.04 – 21 μM. ENMD-981693 was shown to induce G2/M cell cycle arrest followed by apoptosis in U937 cells, without induction of the endo-reduplication phenotype (≥4N DNA content) associated with Aurora B-acting inhibitors such as MK-0457 (VX-680) and AZD1152. Primary cells derived from AML patients were sensitive to treatment with ENMD-981693 in vitro, resulting in IC50 values from 0.2 – 6.0 μM. Sensitivity of primary CML samples was more variable in in vitro cytotoxicity assays, with IC50s in the range of 0.1 – 40 μM. ENMD-981693 shows significant antitumor activity and is well tolerated in xenograft studies, with no weight loss or morbidity observed in administration schedules of up to 100 mg/kg bid or 200 mg/kg qd, given continuously for more than 30 days. Results from in vivo efficacy studies with ENMD-981693 using the MV4;11 xenograft model will be described. In conclusion, ENMD-981693 is a novel kinase inhibitor with potent activity towards a number of targets important in hematologic cancers.

Endoreduplication of human smooth muscle cells induced by 2-methoxyestradiol: a role for cyclin-dependent kinase 2

Endoreduplication has been suggested to contribute to the development of hypertrophy of smooth muscle cells (SMCs) in hypertension. However, endoreduplication in vascular SMCs and the underlying molecular mechanisms are not clear. Treatment of human SMCs with 10 microM 2-methoxyestradiol (2-ME) for 24 h induces accumulation of cells with > or =4N DNA content, and some polyploid/aneuploid cells actively synthesize their DNA, suggesting the occurrence of endoreduplication. In addition, 2-ME treatment upregulates the expression of cyclin-dependent kinase 2 (Cdk2). The present study was designed to characterize endoreduplication of human SMCs and explore the potential roles of Cdk2 in endoreduplication induced by 2-ME. Treatment with 2-ME (10 microM) for 2-4 days not only caused increases in >4N cells and their reentry into S phase but also induced overduplication of chromosomes. Furthermore, 2-ME increased the kinase activity of Cdk2 and its interaction with cyclin E. Inducible overexpression of dominant-negative Cdk2 in human SMCs inhibited both DNA synthesis of >4N cells and the accumulation of >4N cells induced by 2-ME. We conclude that 2-ME induces endoreduplication of human SMCs and Cdk2 plays an important role in endoreduplication in response to 2-ME.

Induction of apoptosis by monastrol, an inhibitor of the mitotic kinesin Eg5, is independent of the spindle checkpoint

Spindle poisons such as paclitaxel are widely used as cancer therapeutics. By interfering with microtubule dynamics, paclitaxel induces mitotic arrest and apoptosis. Targeting the kinesin Eg5, which is required for the formation of a bipolar spindle, is a promising therapeutic alternative to drugs that interfere with microtubule dynamics. Recent data suggest that the spindle checkpoint can determine the response of tumor cells to microtubule poisons. The relationship between checkpoint function and Eg5 inhibition, however, has not yet been fully investigated. Here, we used time-lapse video microscopy and biochemical analysis to study the effect of spindle checkpoint abrogation on the response of HeLa cells to monastrol, a selective Eg5 inhibitor. In HeLa cells, monastrol activated the spindle checkpoint, leading to mitotic arrest and apoptosis. Small interfering RNA-mediated depletion of the spindle checkpoint proteins BubR1 or Mad2 significantly shortened drug-induced arrest, causing premature mitotic exit without cell division. Time-lapse microscopy as well as analysis of caspase activation shows that these checkpoint-deficient cells initiate apoptosis after mitotic exit in response to monastrol. Checkpoint-deficient cells treated with paclitaxel, on the other hand, yielded a higher frequency of cells with >4N DNA content and a decreased incidence of apoptotic events, particularly in Mad2-depleted cells. These results indicate that the immediate fate of postmitotic cells is influenced by both the nature of the checkpoint defect and the type of drug used. Furthermore, these results show that inactivation of the kinesin Eg5 can induce apoptosis in tumor cells in the absence of critical spindle checkpoint components.

Human Immunodeficiency Virus Type 1 Vpr Induces DNA Replication Stress InVitro and In Vivo

The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.

Vertebrate POT1 Restricts G-Overhang Length and Prevents Activation of a Telomeric DNA Damage Checkpoint but Is Dispensable for Overhang Protection

Although vertebrate POT1 is thought to play a role in both telomere capping and length regulation, its function has proved difficult to analyze. We therefore generated a conditional cell line that lacks wild-type POT1 but expresses an estrogen receptor-POT1 fusion. The cells grow normally in tamoxifen, but drug removal causes loss of POT1 from the telomere, rapid cell cycle arrest, and eventual cell death. The arrested cells have a 4N DNA content, and addition of caffeine causes immediate entry into mitosis, suggesting a G(2) arrest due to an ATM- and/or ATR-mediated checkpoint. gammaH2AX accumulates at telomeres, indicating a telomeric DNA damage response, the likely cause of the checkpoint. However, POT1 loss does not cause degradation of the G-strand overhang. Instead, the amount of G overhang increases two- to threefold. Some cells eventually escape the cell cycle arrest and enter mitosis. They rarely exhibit telomere fusions but show severe chromosome segregation defects due to centrosome amplification. Our data indicate that vertebrate POT1 is required for telomere capping but that it functions quite differently from TRF2. Instead of being required for G-overhang protection, POT1 is required to suppress a telomeric DNA damage response. Our results also indicate significant functional similarities between POT1 and Cdc13 from budding yeast (Saccharomyces cerevisiae).

Radiation clastogenesis and cell cycle checkpoint function as functional markers of breast cancer risk

Familial breast cancer is associated with mutations in several genes (BRCA1, BRCA2, p53, ATM) whose protein products protect against radiation-induced genotoxicity. This study tested whether sporadic breast cancer was associated with constitutive radiation hypersensitivity. Blood lymphocytes and EBV-transformed lymphoblasts from patients with newly diagnosed breast cancer and controls without cancer were evaluated for ionizing radiation (IR)-induced chromosomal aberrations and cell cycle delays. Lymphoblasts from patients with ataxia telangiectasia (AT) and heterozygous AT carriers were tested as positive controls for radiation hypersensitivity. Lymphoblasts from AT patients and AT carriers displayed G2-irradiation, chromosomal hypersensitivity (GICH). Irradiated G2 phase lymphocytes from breast cancer cases and controls displayed 3-fold inter-individual variation in frequencies of chromatid damage. However, the percentage of breast cancer cases with damage frequencies in excess of 2 SD of the control mean (8/102 or 8%) was not significantly elevated compared to controls (2/48 or 4%, P=0.5). Lymphoblasts sampled 24 h after 3 Gy of IR also varied in the ratios of cells with 4N and 2N DNA content (4N/2N ratio), as a measure of cell cycle checkpoint function. 4N/2N ratios in irradiated lymphoblasts were strongly correlated with the fractions of S phase cells in un-irradiated control cultures (Pearson's correlation coefficient, r=0.87). After normalization to S fraction, the radiation-induced increment in the 4N/2N ratio was significantly elevated in AT lymphoblasts but not in lymphoblasts from AT carriers. The fraction of breast cancer cases with reduced checkpoint function (2/45 or 4%) was equal to the control fraction (2/45 or 4%). For breast cancer cases and controls, GICH in primary lymphocytes was not associated with reduced cell cycle checkpoint function in lymphoblasts. Constitutive radiation hypersensitivity in blood lymphocytes and lymphoblasts was not a useful biomarker for identifying women at increased risk of breast cancer.

The Aurora Kinase Inhibitor VX-680 Induces Endoreduplication and Apoptosis Preferentially in Cells with Compromised p53-Dependent Postmitotic Checkpoint Function

VX-680 is a potent inhibitor of Aurora kinases that induces the accumulation of cells with > or =4N DNA content, followed by cell death. Here, we define the role of p53 and p21(Waf1/Cip1) in cell cycle perturbations following exposure to VX-680. Endoreduplication and apoptosis in response to VX-680 are limited in A549 and MCF-7 cells expressing wild-type p53, and markedly enhanced in cells lacking p53, including those engineered to express the HPV16-E6 oncoprotein or short interfering RNA pools targeting p53. In contrast, endoreduplication and apoptosis occur in the p53 wild-type cell lines, RKO and U2OS. The difference in response to VX-680 among these cell lines correlates with the timing of induction of p21(Waf1/Cip1) and its ability to inhibit cyclin E-cdk2 activity. In A549 cells, VX-680 induces the expression of p53 and p21(Waf1/Cip1) within 24 hours, with consequent inhibition of cyclin E-cdk2, and reduction of retinoblastoma protein phosphorylation, limiting endoreduplication. In RKO and U2OS cells, the induction of p21(Waf1/Cip1) is delayed and associated with higher residual cyclin E-cdk2 kinase activity and retinoblastoma protein phosphorylation, followed by progressive endoreduplication and apoptosis. Abrogation of p21(Waf1/Cip1) expression by short interfering RNA targeting in A549 cells results in a substantial increase in the degree of endoreduplication, whereas inducible expression of p21(Waf1/Cip1) in p53-negative NCI-H1299 cells inhibits VX-680-induced endoreduplication and cell death. These data suggest that the integrity of the p53-p21(Waf1/Cip1)-dependent postmitotic checkpoint governs the response to Aurora kinase inhibition. Although cells with intact checkpoint function arrest with 4N DNA content, those with compromised checkpoint function are more likely to undergo endoreduplication followed by eventual apoptosis.

Normal Cells, but Not Cancer Cells, Survive Severe Plk1 Depletion

We previously reported the phenotype of depletion of polo-like kinase 1 (Plk1) using RNA interference (RNAi) and showed that p53 is stabilized in Plk1-depleted cancer cells. In this study, we further analyzed the Plk1 depletion-induced phenotype in both cancer cells and primary cells. The vector-based RNAi approach was used to evaluate the role of the p53 pathway in Plk1 depletion-induced apoptosis in cancer cells with different p53 backgrounds. Although DNA damage and cell death can occur independently of p53, p53-deficient cancer cells were much more sensitive to Plk1 depletion than cancer cells with functional p53. Next, the lentivirus-based RNAi approach was used to generate a series of Plk1 hypomorphs. In HeLa cells, two weak hypomorphs showed only slight G2/M arrest, a medium hypomorph arrested with 4N DNA content, followed later by apoptosis, and a strong Plk1 hypomorph underwent serious mitotic catastrophe. In well-synchronized HeLa cells, a medium level of Plk1 depletion caused a 2-h delay of mitotic progression, and a high degree of Plk1 depletion significantly delayed mitotic entry and completely blocked cells at mitosis. In striking contrast, normal hTERT-RPE1 and MCF10A cells were much less sensitive to Plk1 depletion than HeLa cells; no apparent cell proliferation defect or cell cycle arrest was observed after Plk1 depletion in these cells. Therefore, these data further support suggestions that Plk1 may be a feasible cancer therapy target.