CDK11 Promotes Cytokine-Induced Apoptosis in Pancreatic Beta Cells Independently of Glucose Concentration and Is Regulated by Inflammation in the NOD Mouse Model.

Background: Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the Cdk11(Cyclin Dependent Kinase 11) is downregulated by inflammation in the T1D prone NOD (non-obese diabetic) mouse model. The aim of this study is to determine the role of CDK11 in the pathogenesis of T1D and to assess the hierarchical relationship between CDK11 and Cyclin D3 in beta cell viability, since Cyclin D3, a natural ligand for CDK11, promotes beta cell viability and fitness in front of glucose. Methods: We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (Chop, Atf4 and Bip) was also determined. Results: N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency. Conclusions: This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D.

Roles of ChlR1 DNA helicase in replication recovery from DNA damage.

The ChlR1 DNA helicase is mutated in Warsaw breakage syndrome characterized by developmental anomalies, chromosomal breakage, and sister chromatid cohesion defects. However, the mechanism by which ChlR1 preserves genomic integrity is largely unknown. Here, we describe the roles of ChlR1 in DNA replication recovery. We show that ChlR1 depletion renders human cells highly sensitive to cisplatin; an interstrand-crosslinking agent that causes stalled replication forks. ChlR1 depletion also causes accumulation of DNA damage in response to cisplatin, leading to a significant delay in resolution of DNA damage. We also report that ChlR1-depleted cells display defects in the repair of double-strand breaks induced by the I-PpoI endonuclease and bleomycin. Furthermore, we demonstrate that ChlR1-depeleted cells show significant delays in replication recovery after cisplatin treatment. Taken together, our results indicate that ChlR1 plays an important role in efficient DNA repair during DNA replication, which may facilitate efficient establishment of sister chromatid cohesion.

Th-MYCN mice with caspase-8 deficiency develop advanced neuroblastoma with bone marrow metastasis.

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow. Advances in therapy and understanding of the metastatic process have been limited due, in part, to the lack of animal models harboring bone marrow disease. The widely used transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here, we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the bone marrow. This model recapitulates 2 frequent alterations in metastatic neuroblastoma, overexpression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. Although overexpression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced bone marrow metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation, and downregulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine TGF-ß pathway in various tumor models. Cytokine TGF-ß can preferentially promote single cell motility and blood-borne metastasis and therefore activation of this pathway may explain the enhanced bone marrow metastasis observed in this animal model.

Tetraspanin CD82 inhibits protrusion and retraction in cell movement by attenuating the plasma membrane-dependent actin organization.

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.

Tetraspanin CD151 maintains vascular stability by balancing the forces of cell adhesion and cytoskeletal tension.

Tetraspanin CD151 is highly expressed in endothelial cells and regulates pathologic angiogenesis. However, the mechanism by which CD151 promotes vascular morphogenesis and whether CD151 engages other vascular functions are unclear. Here we report that CD151 is required for maintaining endothelial capillary-like structures formed in vitro and the integrity of endothelial cell-cell and cell-matrix contacts in vivo. In addition, vascular permeability is markedly enhanced in the absence of CD151. As a global regulator of endothelial cell-cell and cell-matrix adhesions, CD151 is needed for the optimal functions of various cell adhesion proteins. The loss of CD151 elevates actin cytoskeletal traction by up-regulating RhoA signaling and diminishes actin cortical meshwork by down-regulating Rac1 activity. The inhibition of RhoA or activation of cAMP signaling stabilizes CD151-silenced or -null endothelial structure in vascular morphogenesis. Together, our data demonstrate that CD151 maintains vascular stability by promoting endothelial cell adhesions, especially cell-cell adhesion, and confining cytoskeletal tension.

Mammalian ChlR1 has a role in heterochromatin organization.

The ChlR1 DNA helicase, encoded by DDX11 gene, which is responsible for Warsaw breakage syndrome (WABS), has a role in sister-chromatid cohesion. In this study, we show that human ChlR1 deficient cells exhibit abnormal heterochromatin organization. While constitutive heterochromatin is discretely localized at perinuclear and perinucleolar regions in control HeLa cells, ChlR1-depleted cells showed dispersed localization of constitutive heterochromatin accompanied by disrupted centromere clustering. Cells isolated from Ddx11(-/-) embryos also exhibited diffuse localization of centromeres and heterochromatin foci. Similar abnormalities were found in HeLa cells depleted of combinations of HP1α and HP1ß. Immunofluorescence and chromatin immunoprecipitation showed a decreased level of HP1α at pericentric regions in ChlR1-depleted cells. Trimethyl-histone H3 at lysine 9 (H3K9-me3) was also modestly decreased at pericentric sequences. The abnormality in pericentric heterochromatin was further supported by decreased DNA methylation within major satellite repeats of Ddx11(-/-) embryos. Furthermore, micrococcal nuclease (MNase) assay revealed a decreased chromatin density at the telomeres. These data suggest that in addition to a role in sister-chromatid cohesion, ChlR1 is also involved in the proper formation of heterochromatin, which in turn contributes to global nuclear organization and pleiotropic effects.

Preclinical models for neuroblastoma: establishing a baseline for treatment.

BACKGROUND: Preclinical models of pediatric cancers are essential for testing new chemotherapeutic combinations for clinical trials. The most widely used genetic model for preclinical testing of neuroblastoma is the TH-MYCN mouse. This neuroblastoma-prone mouse recapitulates many of the features of human neuroblastoma. Limitations of this model include the low frequency of bone marrow metastasis, the lack of information on whether the gene expression patterns in this system parallels human neuroblastomas, the relatively slow rate of tumor formation and variability in tumor penetrance on different genetic backgrounds. As an alternative, preclinical studies are frequently performed using human cell lines xenografted into immunocompromised mice, either as flank implant or orthtotopically. Drawbacks of this system include the use of cell lines that have been in culture for years, the inappropriate microenvironment of the flank or difficult, time consuming surgery for orthotopic transplants and the absence of an intact immune system. PRINCIPAL FINDINGS: Here we characterize and optimize both systems to increase their utility for preclinical studies. We show that TH-MYCN mice develop tumors in the paraspinal ganglia, but not in the adrenal, with cellular and gene expression patterns similar to human NB. In addition, we present a new ultrasound guided, minimally invasive orthotopic xenograft method. This injection technique is rapid, provides accurate targeting of the injected cells and leads to efficient engraftment. We also demonstrate that tumors can be detected, monitored and quantified prior to visualization using ultrasound, MRI and bioluminescence. Finally we develop and test a "standard of care" chemotherapy regimen. This protocol, which is based on current treatments for neuroblastoma, provides a baseline for comparison of new therapeutic agents. SIGNIFICANCE: The studies suggest that use of both the TH-NMYC model of neuroblastoma and the orthotopic xenograft model provide the optimal combination for testing new chemotherapies for this devastating childhood cancer.

Chromosome damage and repair in children with sickle cell anaemia and long-term hydroxycarbamide exposure.

Hydroxycarbamide (hydroxyurea) provides laboratory and clinical benefits for adults and children with sickle cell anaemia (SCA). Given its mechanism of action and prior reports of genotoxicity, concern exists regarding long-term toxicities and possible carcinogenicity. We performed cross-sectional analyses of chromosome stability using peripheral blood mononuclear cells (PBMC) from 51 children with SCA and 3-12 years of hydroxycarbamide exposure (mean age 13·2 ± 4·1 years), compared to 28 children before treatment (9·4 ± 4·7 years). Chromosome damage was less for children receiving hydroxycarbamide than untreated patients (0·8 ± 1·2 vs. 1·9 ± 1·5 breaks per 100 cells, P = 0·004). There were no differences in repairing chromosome breaks after in vitro radiation; PBMC from children taking hydroxycarbamide had equivalent 2 Gy-induced chromosome breaks compared to untreated patients (30·8 ± 16·1 vs. 31·7 ± 8·9 per 100 cells, P = not significant). Radiation plus hydroxycarbamide resulted in similar numbers of unrepaired breaks in cells from children on hydroxycarbamide compared to untreated patients (95·8 ± 44·2 vs. 76·1 ± 23·1 per 100 cells, P = 0·08), but no differences were noted with longer exposure (97·9 ± 42·8 breaks per 100 cells for 3-6 years of hydroxycarbamide exposure vs. 91·2 ± 48·4 for 9-12 years of exposure). These observations provide important safety data regarding long-term risks of hydroxycarbamide exposure for children with SCA, and suggest low in vivo mutagenicity and carcinogenicity.

The connections between neural crest development and neuroblastoma.

Neuroblastoma (NB), the most common extracranial solid tumor in childhood, is an extremely heterogeneous disease both biologically and clinically. Although significant progress has been made in identifying molecular and genetic markers for NB, this disease remains an enigmatic challenge. Since NB is thought to be an embryonal tumor that is derived from precursor cells of the peripheral (sympathetic) nervous system, understanding the development of normal sympathetic nervous system may highlight abnormal events that contribute to NB initiation. Therefore, this review focuses on the development of the peripheral trunk neural crest, the current understanding of how developmental factors may contribute to NB and on recent advances in the identification of important genetic lesions and signaling pathways involved in NB tumorigenesis and metastasis. Finally, we discuss how future advances in identification of molecular alterations in NB may lead to more effective, less toxic therapies, and improve the prognosis for NB patients.

The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing.

Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo.

Phase I study of decitabine with doxorubicin and cyclophosphamide in children with neuroblastoma and other solid tumors: a Children's Oncology Group study.

BACKGROUND: Demethylating agents may alter the expression of genes involved in chemotherapy resistance. We conducted a phase I trial to determine the toxicity and molecular effects of the demethylating agent, decitabine, followed by doxorubicin and cyclophosphamide in children with refractory solid tumors. PROCEDURE: Stratum A included children with any solid tumor; Stratum B included neuroblastoma patients only. Patients received a 1-hr decitabine infusion for 7 days, followed by doxorubicin (45 mg/m(2)) and cyclophosphamide (1 g/m(2)) on day 7. Pharmacokinetic studies were performed after the first dose of decitabine. Biological studies included methylation and gene expression analyses of caspase-8, MAGE-1 and fetal hemoglobin (HbF), and expression profiling of pre- and post-treatment peripheral blood and bone marrow cells. RESULTS: The maximum-tolerated dose of decitabine was 5 mg/m(2)/day for 7 days. Dose-limiting toxicities at 10 mg/m(2)/day were neutropenia and thrombocytopenia. Decitabine exhibited rapid clearance from plasma. Three of 9 patients in Stratum A and 4/12 patients in Stratum B had stable disease for > or = 4 months. Sustained MAGE-1 demethylation and increased HbF expression were observed in the majority of patients post-treatment (12/20 and 14/16, respectively). Caspase-8 promoter demethylation and gene expression were seen in 2/7 bone marrow samples. Differentially expressed genes were identified by microarray analysis. CONCLUSION: Low-dose decitabine when combined with doxorubicin/cyclophosphamide has tolerable toxicity in children. However, doses of decitabine capable of producing clinically relevant biologic effects were not well tolerated with this combination. Alternative strategies of combining demethylating agents with non-cytotoxic, biologically targeted agents such as histone deacetylase inhibitors should be explored.

Synthetic mRNA splicing modulator compounds with in vivo antitumor activity.

We report our progress on the development of new synthetic anticancer lead compounds that modulate the splicing of mRNA. We also report the synthesis and evaluation of new biologically active ester and carbamate analogues. Further, we describe initial animal studies demonstrating the antitumor efficacy of compound 5 in vivo. Additionally, we report the enantioselective and diastereospecific synthesis of a new 1,3-dioxane series of active analogues. We confirm that compound 5 inhibits the splicing of mRNA in cell-free nuclear extracts and in a cell-based dual-reporter mRNA splicing assay. In summary, we have developed totally synthetic novel spliceosome modulators as therapeutic lead compounds for a number of highly aggressive cancers. Future efforts will be directed toward the more complete optimization of these compounds as potential human therapeutics.

Caspase-8 association with the focal adhesion complex promotes tumor cell migration and metastasis.

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

Perturbation of HP1 localization and chromatin binding ability causes defects in sister-chromatid cohesion.

Sister-chromatid cohesion, the machinery used in eukaryote organisms to prevent aneuploidy, tethers sister chromatids together after their replication in S phase until mitosis. Previous studies in fission yeast, Drosophila and mammals have demonstrated the requirement for the heterochromatin formation pathway for proper centromeric cohesion. However, the exact role of heterochromatin protein 1 (HP1) in sister-chromatid cohesion in mammals is still unknown. In this study, we disrupted endogenous HP1 expression in HeLa cells using a dominant-negative mutant of HP1beta and wild-type or mutant forms of HP1alpha. We then examined their effects on chromosome alignment, segregation and cohesion. Enforced expression of these constructs leads to frequent chromosome misalignment and missegregation. Mitotic chromosomes from these cells also exhibit a loosened primary constriction and separated sister chromatids. We further demonstrate that alignment of the cohesin proteins around kinetochores was also aberrant and that cohesin complexes bound less tightly in these cells. Unexpectedly, we observed a "wavy" chromosome morphology resembling that seen upon depletion of condensin proteins in cells with over-expression of HP1alpha, but not in cells expressing the HP1beta mutant. These results indicate that proper HP1 status is required for sister-chromatid cohesion in mammalian cells, and suggest that HP1alpha might be required for chromosome condensation.

Retinoic acid induces caspase-8 transcription via phospho-CREB and increases apoptotic responses to death stimuli in neuroblastoma cells.

Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-gamma. Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA-protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, mutations of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2-5 days and were more sensitive to doxorubicin and TNFalpha. Thus, RA treatment in conjunction with TNFalpha and/or subsets of cytotoxic agents may have therapeutic benefits.

The microenvironmental determinants for kidney epithelial cyst morphogenesis.

Although epithelial morphogenesis is tightly controlled by intrinsic genetic programs, the microenvironment in which epithelial cells proliferate and differentiate also contributes to the morphogenetic process. The roles of the physical microenvironment in epithelial morphogenesis, however, have not been well dissected. In this study, we assessed the impact of the microenvironment on epithelial cyst formation, which often marks the beginning or end step of morphogenesis of epithelial tissues and the pathological characteristic of some diseases. Previous studies have demonstrated that Madin-Darby canine kidney (MDCK) epithelial cells form cysts when grown in a three-dimensional (3D) extracellullar matrix (ECM) environment. We have now further demonstrated that the presence of ECM in the 3D scaffold is required for the formation of properly polarized cysts. Also, we have found that the full interface of epithelial cells with the ECM environment (in-3D) is not essential for cyst formation, since partial contact (on-3D) is sufficient to induce cystogenesis. In addition, we have defined the minimal ECM environment or the physical threshold for cystogenesis under the on-3D condition. Only above the threshold can the morphological cues from the ECM environment induce cyst formation. Moreover, cyst formation under the on-3D condition described in this study defines a novel and more feasible model to analyze in vitro morphogenesis. Finally, we have found that, during cystogenesis, MDCK cells generate basal microprotrusions and produce vesicle-like structures to the basal extracellular space, which are specific to and correlated with cyst formation. For the first time, we have systematically and quantitatively elucidated the microenvironmental determinants for epithelial cystogenesis.

Characterization of cyclin L1 and L2 interactions with CDK11 and splicing factors: influence of cyclin L isoforms on splice site selection.

Although it has been reported that cyclin L1alpha and L2alpha proteins interact with CDK11(p110), the nature of the cyclin L transcripts, the formation of complexes between the five cyclin L and the three CDK11 protein isoforms, and the influence of these complexes on splicing have not been thoroughly investigated. Here we report that cyclin L1 and L2 genes generate 14 mRNA variants encoding six cyclin L proteins, one of which has not been described previously. Using cyclin L gene-specific antibodies, we demonstrate expression of multiple endogenous cyclin L proteins in human cell lines and mouse tissues. Moreover, we characterize interactions between CDK11(p110), mitosis-specific CDK11(p58), and apoptosis-specific CDK11(p46) with both cyclin Lalpha and -beta proteins and the co-elution of these proteins following size exclusion chromatography. We further establish that CDK11(p110) and associated cyclin Lalpha/beta proteins localize to splicing factor compartments and nucleoplasm and interact with serine/arginine-rich proteins. Importantly, we also determine the effect of CDK11-cyclin L complexes on pre-mRNA splicing. Preincubation of nuclear extracts with purified cyclin Lalpha and -beta isoforms depletes the extract of in vitro splicing activity. Ectopic expression of cyclin L1alpha, L1beta, L2alpha, or L2beta or active CDK11(p110) individually enhances intracellular intron splicing activity, whereas expression of CDK11(p58/p46) or kinase-dead CDK11(p110)represses splicing activity. Finally, we demonstrate that expression of cyclins Lalpha and -beta and CDK11(p110) strongly and differentially affects alternative splicing in vivo. Together, these data establish that CDK11(p110) interacts physically and functionally with cyclin Lalpha and -beta isoforms and SR proteins to regulate splicing.

Loss of ChlR1 helicase in mouse causes lethality due to the accumulation of aneuploid cells generated by cohesion defects and placental malformation.

Human DDX11 and DDX12 are closely related genes encoding the helicases ChlR1 and ChlR2, which belong to the CHL1 DNA helicase family. Recently, it was shown that human ChlR1 interacts with components of the cohesin complex and is required for proper centromeric cohesion. To establish the function of ChlR1 in development we made a mutant mouse lacking Ddx11, the single mouse ChlR gene. The absence of Ddx11 resulted in embryonic lethality at E10.5. The mutant embryos were smaller in size, malformed and exhibited sparse cellularity in comparison to normal or heterozygous litter mates. Importantly, loss of Ddx11 resulted in the inability to form a proper placenta, indicating that ChlR1 is essential for placental formation. Detailed analysis of cells isolated from Ddx11-/- embryos revealed a G2/M cell cycle delay, an increased frequency of chromosome missegregation, decreased chromosome cohesion, and increased aneuploidy. To examine whether ChlR proteins are required for arm cohesion and for loading of the cohesin complex, further studies were preformed in ChlR1 siRNA treated cells. These studies revealed that ChlR1 is required for proper sister chromatid arm cohesion and that cohesin complexes bind more loosely to chromatin in the absence of ChlR1. Taken together, these studies provide the first data indicating that the ChlR1 helicase is essential for proper binding of the cohesin complex to both the centromere and the chromosome arms, and indicate that ChlR1 is essential for embryonic development and the prevention of aneuploidy in mammals.

CDK11(p58) is required for the maintenance of sister chromatid cohesion.

Cyclin-dependent kinase 11 (CDK11) mRNA produces a 110-kDa protein (CDK11(p110)) throughout the cell cycle and a 58-kDa protein (CDK11(p58)) that is specifically translated from an internal ribosome entry site sequence during G2/M. CDK11(p110) is involved in transcription and RNA processing, and CDK11(p58) is involved in centrosome maturation and spindle morphogenesis. Deletion of the CDK11 gene in mice leads to embryonic lethality at E3.5, and CDK11-deficient blastocysts exhibit both proliferative defects and mitotic arrest. Here we used hypomorphic small interfering RNAs (siRNAs) to demonstrate that, in addition to playing a role in spindle formation and structure, CDK11(p58) is also required for sister chromatid cohesion and the completion of mitosis. Moderate depletion of CDK11 causes misaligned and lagging chromosomes but does not prevent mitotic progression. Further diminution of CDK11 caused defective chromosome congression, premature sister chromatid separation, permanent mitotic arrest and cell death. These cells exhibited altered Sgo1 localization and premature dissociation of cohesion complexes. This severe phenotype was not corrected by codepletion of CDK11 and either Plk1 or Sgo1, but it was rescued by CDK11(p58). These findings are consistent with the mitotic arrest we observed in CDK11-deficient mouse embryos and establish that CDK11(p58) is required for the maintenance of chromosome cohesion and the completion of mitosis.