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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.007963
Research Article |
Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA
* Author for correspondence (e-mail: Jill.Lahti{at}stjude.org)
Accepted 15 May 2007
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Summary |
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Key words: CDK11p58, Cell cycle, Mitosis, Cyclin-dependent kinase, Sister chromatid cohesion, Cohesin, Mouse
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; Trembley et al., 2004; Chen et al., 2006). The human (CDC2L1 and CDC2L2) and mouse (cdc21) genes both produce two different protein products. The 110-kDa protein isoform of cyclin-dependent kinase 11 (CDK11p110), the major protein kinase isoform, is expressed throughout the cell cycle and is involved in transcriptional regulation and RNA processing (Trembley et al., 2002; Trembley et al., 2003; Hu et al., 2003). By contrast, the 58-kDa protein isoform of cyclin-dependent kinase 11 (CDK11p58) is specifically expressed in the G2/M phase of the cell cycle (Cornelis et al., 2000; Tinton et al., 2005; Wilker et al., 2007). This protein is generated by translation from an internal ribosome entry site located in the coding region of the CDK11 full-length mRNA (Cornelis et al., 2000). Previous studies have shown that minimal overexpression of CDK11p58 in Chinese hamster ovary (CHO) cells results in aneuploidy, increased numbers of cells that maintain postmitotic bridges or midbodies, and apoptosis (Lahti et al., 1995; Bunnell et al., 1990). A significant role for at least one CDK11 protein isoform in mitotic control in vivo was also identified through gene disruption. Deletion of the single CDK11 (cdc2l) gene in mice leads to embryonic death at E3.5 (Li et al., 2004). CDK11-deficient blastocysts exhibit both proliferative defects and mitotic arrest. Moreover, the involvement of CDK11p58 in centrosome maturation and bipolar spindle morphogenesis was recently reported (Petretti et al., 2006). Here we used a series of hypomorphic small interfering RNA (siRNA) expression constructs to downregulate CDK11 expression to varying extents. These studies revealed additional roles for CDK11 in mitotic progression and chromosome cohesion.
Sister chromatid cohesion is mediated by the cohesin complex. Establishment of chromosome cohesion occurs during DNA synthesis in S phase, and the association of paired sister chromatids must be maintained until anaphase to ensure accurate chromosome segregation. Chromosome arm cohesin is removed during prophase and prometaphase through a nonproteolytic process that requires Plk1 and aurora B kinase activity. Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, and the phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms (Sumara et al., 2002; Losada et al., 2002; Hauf et al., 2005). By contrast, centromeric cohesin is cleaved by separase specifically at the onset of anaphase (Gimenez-Abian et al., 2004; Hauf et al., 2001). Centromeric chromosome cohesion is protected from proteolysis before anaphase by Sgo1 (Kitajima et al., 2005; McGuinness et al., 2005). The centromeric localization of Sgo1 is regulated by Bub1 kinase and PP2A phosphatase (Kitajima et al., 2005; Tang et al., 2006). The cleavage of centromeric cohesin and initiation of sister chromatid separation is controlled by the spindle checkpoint. The spindle checkpoint monitors both the interactions between kinetochores and spindle microtubules and the tension across paired kinetochores, and it delays cohesin cleavage and anaphase onset until all chromosomes are properly attached to the spindle and aligned at the metaphase plate (Lew and Burke, 2003; Musacchio and Hardwick, 2002). Spindle checkpoint function involves protein phosphorylation and proteolysis (Cleveland et al., 2003). Indeed, several of the checkpoint proteins including Msp1, Bub1 and BubR1 are protein kinases. In addition to the spindle checkpoint kinases, several other known kinases such as CDK1, aurora kinases and Plk1 play important regulatory roles in mitosis (Peters, 2002; Tang et al., 2004a; Acquaviva et al., 2004; van Vugt et al., 2004; Jackman et al., 2003; Lampson et al., 2004; Sumara et al., 2004; Barr et al., 2004; van Vugt and Medema, 2005). These mitotic kinases also generate signals preventing the proteolysis of anaphase inhibitors and mitotic cyclins, thereby blocking the onset of anaphase (Peters, 2002). Here we show that the CDK11p58 kinase also plays a crucial role in mitotic progression and is required for the maintenance of sister chromatid cohesion and for the completion of mitosis in human cells.
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) expression constructs were used to allow us to vary the extent of CDK11 depletion. In general, the pSUPER expression constructs were more effective than the siRNAs in mediating CDK11 depletion. Direct comparison of siRNA expression construct pSUPER-CDK11-A1361 and oligonucleotide A1361, both of which target the same sequence used previously (Petretti et al., 2006), revealed that the expression construct silenced CDK11 much more effectively. Most of the studies described here were performed using HeLa cells and either the C-terminal pSUPER-CDK11-A2184 construct, which most efficiently downregulated both CDK11p110 and CDK11p58, or the N-terminal pSUPER-CDK11-A461 construct, because both of these constructs resulted in the most complete elimination of CDK11. However, the results were verified using constructs targeting different regions of CDK11 (A111, A188, A461, A910 and A1361) and different cell lines (293T, U2OS and SAOS2) (supplementary material Fig. S1 and data not shown). The specificity of these constructs was confirmed by analysis of several other proteins, including tubulin and actin. Experiments using SBI System Biosciences's inteferon response detection kit (# SI300A-1), which monitors induction of five genes (OAS1, OAS2, MX1, ISGF3
, IFITM1) whose expression are induced by activation of the interferon response pathway, were also performed to ensure that the phenotype was not because of induction of interferon-mediated responses to dsRNA (data not shown).
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). Abnormalities in centrosome numbers and positions were also present. The extent of these abnormalities and the number of cells exhibiting these aberrant phenotypes correlated with the extent of CDK11 depletion. Expression of green fluorescent protein (GFP)-tagged RNAi-resistant CDK11p58 protein rescued the centrosome number abnormality completely and corrected the centrosome positioning defects in >80% of the cells (data not shown). These findings are in agreement with those of previous studies (Petretti et al., 2006) and support a role for CDK11p58 in centrosome migration and spindle assembly or maintenance.
CDK11 depletion causes G2/M cell-cycle block and subsequent cell death
In addition to the spindle and centrosome abnormalities, we also noticed an accumulation of rounded cells and an increase in cells with an apoptotic appearance after treatment with the siRNAs that most effectively targeted CDK11. The number of cells exhibiting these morphologies correlated with the severity of CDK11 depletion. For example, cells treated with siRNA A1361 had a near-normal appearance, whereas treatment with expression construct A2184 caused the appearance of numerous rounded and dying cells (Fig. 1B). DNA content analysis verified an increase in the cell population in G2/M phase of the cell cycle and in the population of sub-G1 CDK11 RNAi cells in the cultures with the severe phenotype (Fig. 1C). This sub-G1 population was stained by annexin V, indicating that these cells were apoptotic (data not shown). The mitotic arrest and subsequent cell death resulting from CDK11 depletion were also shown by time-lapse microscopy of HeLa and HeLa H2B-GFP cells 48 hours after transfection (see supplementary material Fig. S2 and Movies 1 and 2).
G2/M accumulation is because of mitotic arrest of cells containing prematurely separated sister chromatids
To clarify the composition of the enhanced G2/M population resulting from CDK11 depletion (Fig. 1), chromosome analysis was performed. No obvious differences in chromosome condensation were visible upon staining with DAPI or antibodies that detect phosphorylation of serine 10 of histone H3, a marker that correlates with chromosome condensation (Wei et al., 1999) (data not shown). Interestingly, however, depletion of CDK11 with all of the constructs resulted in abnormalities in chromosome segregation. The extent of these abnormalities correlated with the level of CDK11 depletion. Moderate CDK11 depletion with the A1361 oligonucleotide and expression constructs did not prevent mitosis, although the cells exhibited an increase in the presence of misaligned and lagging chromosomes (Fig. 2A,B). By contrast, more complete depletion dramatically increased the population of cells with condensed chromosomes and slightly separated sister chromatids whereas mitotic cells with a typical anaphase or telophase appearance were absent (Fig. 2C,D). In addition, very few cells were observed with all chromosomes aligned on the metaphase plate. Rather, most cells exhibited incomplete chromosome congression. Expression of the pSUPER-A2184 (CDK11 RNAi-C) resulted in the strongest phenotype, which correlated directly with the level of CDK11 depletion (Fig. 1A). Of the total mitotic cells treated with the A2184 construct, 62% exhibited premature sister chromatid separation after CDK11 depletion, although most of the separated sister chromatids remained paired (Fig. 2C,D). This observation was confirmed by measuring the inter-kinetochore distances of 50-100 paired sister chromatids stained with CREST antibody at different points during G2/M. The interkinetochore distance between the sister chromatids of the control and CDK11 RNAi cells was identical in G2 (control 0.46±0.06 µm and CDK11 RNAi cells 0.43±0.08 µm) and in prophase (control 0.61±0.1 µm and CDK11 RNAi cells 0.61±0.04 µm). However, the distance between the kinetochores was significantly larger at later stages in mitosis. The distance between the sister chromatids of the postprophase-arrested CDK11 RNAi cells was 1.05±0.24 µm, whereas the average distance of the control cells at a comparable stage in mitosis was only 0.76±0.15 µm. In fact, the distance between the sister chromatids in CDK11 RNAi cells was larger than that of metaphase control cells (1.05±0.24 µm versus 0.89±0.14 µm). Importantly, overexpression of the RNAi-resistant form of CDK11p58-GFP, but not the RNAi-sensitive form, reduced the number of cells with prematurely separated sister chromatids by 
75% (Fig. 3).
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Depletion of CDK11 results in prometaphase-metaphase cell cycle arrest
To probe the nature of the mitotic arrest, we analyzed the response of CDK11-depleted cells to the microtubule poisons nocodazole and taxol. These agents trigger the spindle checkpoint, resulting in a prometaphase-metaphase mitotic arrest. As expected, the percentage of prometaphase-metaphase cells increased in control cultures during a six-hour exposure to these agents. By contrast, the number of prometaphase-metaphase CDK11 RNAi cells did not increase in response to either treatment, although there was a slight increase in the population of cells with prematurely separated sister chromatids in response to treatment with the microtubule-stabilizing drug taxol (Fig. 2D), suggesting that the spindle checkpoint can delay sister chromatid separation in the presence of tension or that sister chromatid tension per se plays a role in the retention of cohesion complexes.
As an additional means to determine the nature of the mitotic arrest in the CDK11-depleted cells, we performed immunofluorescence staining with antibodies to cyclins A and B1. Both of these cyclins are required for mitotic progression, although cyclin A is degraded during the prometaphase-metaphase transition, whereas cyclin B1 is degraded during the metaphase-anaphase transition (Lew and Kornbluth, 1996; Peters, 2002). Immunofluorescence staining of cyclins A and B1 in CDK11 RNAi cells revealed that most of the mitotic cells lacked detectable cyclin A but retained cyclin B1 (Fig. 4A). These findings support the conclusion that CDK11 depletion results in premature sister chromatid separation and mitotic arrest at the prometaphase-metaphase transition.
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; Nicklas et al., 1995). BubR1, a key component of the mitotic spindle checkpoint machinery, normally remains on the kinetochores until the onset of anaphase when sister chromatids start to segregate (Chen, 2002; Lampson and Kapor, 2005; Tang et al., 2001). Immunofluorescence staining with BubR1 revealed the presence of high levels of BubR1 on the kinetochore in all mitotic CDK11 RNAi cells, despite the presence of separated sister chromatids (Fig. 4B). These data suggest the spindle checkpoint is activated in CDK11 RNAi cells and that activation of the spindle checkpoint is at least partially responsible for the mitotic arrest observed in these cells. However, in CDK11 RNAi cells, the spindle checkpoint is not able to prevent the premature separation of the two chromatids.
CDK11 is required for maintenance of chromosome cohesion during mitosis
The premature separation of the sister chromatids suggested that diminished CDK11 results in defects in maintenance of cohesion and/or premature removal of cohesin complexes.
To investigate the effects of CDK11 on sister chromatid cohesion in more detail, we transfected HeLa cells expressing the Myc-tagged human Scc1 cohesin component with CDK11 RNAi constructs and monitored Scc1 by immunofluorescence. Our data indicated that the cohesin complexes were present on the chromosomes and kinetochores of CDK11-depleted cells at the early stage of mitosis; however, Scc1 was prematurely removed from post-prophase mitotic cells (Fig. 5). In fact, 64% of the CDK11-depleted mitotic cells were Myc-Scc1 negative, whereas only 25% of the control RNAi cells were Myc-Scc1 negative (Fig. 5) (McGuinness et al., 2005). The percentage of CDK11 RNAi cells exhibiting a premature loss of cohesion did not change in response to nocodazole treatment, suggesting that CDK11 plays a role in protecting and/or maintaining cohesion complexes prior to the metaphase.
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; McGuinness et al., 2005; Watanabe and Kitajima, 2005; Kitajima et al., 2005). Therefore, we performed experiments to determine whether Sgo1 and Bub1 were present at the centromeres in CDK11 RNAi cells. Immunofluorescence examination of Sgo1 localization in CDK11-depleted cells showed that Sgo1 proteins were present at the centromeres, although the position of the protein was altered relative to CREST (Fig. 6A). In the control cells, the Sgo1 signal was flanked by CREST signals, whereas the Sgo1 signal appeared to be colocalized with the CREST signal on kinetochores in CDK11 RNAi cells. In addition, Bub1 was prematurely removed from kinetochores in post-prophase CDK11 siRNA-treated cells, although the prophase Bub1 staining pattern of these cells was similar to that of the controls (Fig. 6B).
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; van Vugt and Medema, 2005; Kitajima et al., 2005; Salic et al., 2004). As reported previously, 88% of the mitotic chromosomes in Plk1 RNAi-treated cells exhibited closed arms (Fig. 7). Interestingly, codepletion of Plk1 and CDK11 caused a loss of both arm and centromeric cohesion in 32% of the mitotic cells. This phenotype is similar to, although less severe than, that produced by codepletion of Sgo1 and Plk1, which caused a loss of chromosome cohesion in 71% of the cells. Recently, data has been published demonstrating that centromeric Plk1 staining is reduced in CDK11 RNAi cells (Petretti et al., 2006). Therefore, it is somewhat surprising that CDK11-Plk1 codepletion has milder effects on premature sister chromatid separation than Sgo1-Plk1 depletion. However, it is possible that the reduction in Plk1 kinetochore binding is not sufficient to compromise cohesin complex removal or that other kinases, such as aurora B, can compensate for reduced chromosomal Plk1 in CDK11 RNAi cells. Nonetheless, these findings suggest that CDK11, like Sgo1, plays roles in protecting or maintaining chromosome cohesion both along the arms and at the kinetochore regions during mitosis.
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; Blagosklonny, 2007), frequently results in aneuploidy and/or multinucleated cells. By contrast, severe depletion of CDK11 resulted in more dramatic mitotic abnormalities. Under these conditions, mitotic chromosomes underwent normal chromatin condensation and appeared to progress normally through prophase and prometaphase; however, the condensed chromosomes failed to congress properly at the metaphase plate, and the cells underwent a permanent mitotic arrest followed by the premature loss of sister chromatid cohesion and cell death. Based on the observations that dying cells were stained by annexin V, we believe that the loss of CDK11 leads to mitotic failure followed by apoptosis. However, because we have not examined other markers of apoptosis, such as activation of effector caspases, cytochrome C release or DNA ladder formation, it remains possible that these severely depleted CDK11 cells also die from mitotic catastrophe.
The presence of prematurely separated sister chromatids after downregulation of CDK11p58 suggests that the kinase plays a role in maintenance or protection of chromosome cohesion. To test this possibility, we examined the effects of simultaneous depletion of CDK11 and two proteins with known roles in chromosome cohesion, Plk1 and Sgo1. Codepletion of CDK11 and Sgo1 increased the extent of the sister chromatid separation beyond that caused by the depletion of CDK11 alone. Depletion of Plk1 alone resulted in mitotic cells with hypercondensed chromosomes with closed arms. When the loss of Plk1 was combined with the loss of CDK11, the chromosomes remained hypercondensed, but the chromatids were separated. These findings support a role for CDK11 in the maintenance of chromosome cohesion but not in chromosome condensation. A similar phenotype has also been observed upon codepletion of Sgo1 and Plk1 (McGuinness et al., 2005). The similarities in the codepletion phenotype suggest that CDK11, like Sgo1, is required for maintenance of both centromere and arm cohesion and that CDK11 and Sgo1 function in the same or redundant pathways. This theory is supported by the similar appearance of chromosomes from Sgo1 and CDK11 RNAi cells and by the alterations in the localization of Sgo1 in CDK11-depleted cells. This hypothesis is also supported by other similarities in the phenotypes of CDK11 and Sgo1 RNAi cells. RNAi-mediated depletion of Sgo1 causes premature centromeric sister chromatid separation and a permanent mitotic arrest comparable to that observed in CDK11 RNAi cells (Tang et al., 2004b; Salic et al., 2004; McGuinness et al., 2005; Kitajima et al., 2005). In addition, both CDK11- and Sgo1-depleted cells retain cyclin B expression (McGuinness et al., 2005). Sister chromatid separation in Sgo1 RNAi cells does not appear to be mediated by separase but rather occurs through another mechanism, possibly involving phosphorylation of cohesin subunits (Tang et al., 2004b; McGuinness et al., 2005). Our analysis of CDK11-depleted cells suggests that removal of cohesin complexes from CDK11 and Sgo1 RNAi cells may occur through similar mechanisms, because CDK11 RNAi cells have high levels of cyclin B and a premature loss of Scc1 without a significant increase in Scc1 cleavage (data not shown). Thus, it is possible that CDK11 may function coordinately with Sgo1. Alternatively, CDK11 may be required for proper localization, binding or function of Sgo1 and Bub1 or PP2A; because these two other proteins are required for proper Sgo1 localization and function and for centromeric cohesion (Tang et al., 2004b; Tang et al., 2006). The observation that centromeric Bub1 is not properly retained at the centromere in post-prophase CDK11 RNAi cells is consistent with this possibility. The localization of PP2A has been reported to be Bub1-dependent and proper localization of Sgo1 is also dependent on PP2A (Tang et al., 2006), thus the diminished binding of Bub1 in the CDK11 cells might explain the abnormal localization of Sgo1. A model depicting the role of CDK11 in chromosome cohesion is shown in Fig. 8. In this model, the effects of CDK11 on chromosome cohesion could result from direct interactions between CDK11 and Sgo1, Bub1 or PP2A. Alternatively, we propose that these interactions are transient and/or CDK11 phosphorylates one or more of these proteins prior to their localization at the kinetochore. We currently favor the second proposition because we have not been able to detect either interaction between CDK11 and the Sgo1 complex or kinetochore-associated CDK11. Although this may be because of technical problems with antibody recognition or CDK11 accessibility, these data indicate that further studies are needed to address the exact role of CDK11 in the maintenance of sister chromatid cohesion.
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Although the spindle checkpoint in CDK11-depleted cells appears to be intact as these cells arrest in mitosis, it is not clear whether the loss of sister chromatid cohesion occurs before or after mitotic arrest. Cohesion defects because of premature dissociation of cohesion complexes caused by the loss of CDK11 could lead to checkpoint activation and cell cycle arrest. This model is consistent with recent data from studies with Xenopus demonstrating that antibody-mediated depletion of cohesin causes defects in chromosome congression, spindle abnormalities, changes in kinetochore structure, and aberrant kinetochore attachment and chromosome movement that trigger the spindle checkpoint (Kenney and Heald, 2006). Alternatively, dissociation of sister chromatid cohesion could occur after mitotic progression was halted by the spindle checkpoint. In this scenario, induction of the spindle checkpoint could result from defects in spindle-kinetochore attachment, faulty spindle structure, and/or improper function of the spindle checkpoint because of aberrant phosphorylation resulting from the loss of CDK11. The absence of CDK11 would compromise the ability of Sgo1 to protect the arrested cells from a loss of centromeric cohesion and premature sister chromatid separation. Importantly, the inability to override the spindle checkpoint and proceed through mitosis in either model could result from a combination of spindle checkpoint activity, aberrations in the structure of the mitotic spindle, defects in kinetochore attachment, and/or a requirement for CDK11 kinase activity for the completion of anaphase.
The data from this study demonstrates that CDK11p58 is required for mitotic progression. The severe depletion data imply that complete loss of CDK11 in vivo would prevent mitotic progression and would be lethal to cells and to survival of an organism. The observation that CDK11-null embryos die immediately after depletion of maternal CDK11 protein stores (Li et al., 2004) is consistent with this prediction. Importantly, CDK11-null embryos have an increased percentage of mitotic and apoptotic cells, suggesting that loss of CDK11 results in a mitotic arrest followed by apoptotic death similar to that observed in severely depleted CDK11 RNAi cells. By contrast, HeLa cells treated with hypomorphic siRNA constructs, which caused only partial loss of CDK11 protein, completed mitosis despite the presence of spindle defects and lagging chromosomes. These data suggest that loss of a single CDK11 allele because of deletion of chromosome 1p36, which is frequently observed in tumors such as neuroblastoma, melanoma, breast cancer and several types of leukemia and lymphoma (Brodeur et al., 1977; Poetsch et al., 2003; Mori et al., 2003; Melendez et al., 2003; Bieche et al., 1993), could increase the number of aneuploid cells, thereby contributing to tumorigenesis. This conclusion is supported by recent studies examining the responses of CDK11 haploinsufficient mice to carcinogens known to cause skin cancer (Chandramouli et al., 2007). Mice with a single functional CDK11 allele exhibited an increase in tumor size and number in this skin cancer model system (Chandramouli et al., 2007). Importantly, however, the rate of tumor formation was not increased in this model system nor have we observed any increase in the frequency of spontaneous tumors in our CDK11 heterozygous mice, suggesting that other cooperating genetic events are required for tumor formation and progression.
The plethora of mitotic effects observed in CDK11 RNAi cells probably results from alteration of phosphorylation of CDK11 substrates. Recently, WAPL (Kueng et al., 2006; Gandhi et al., 2006), Haspin (Dai et al., 2006) and PICH (Baumann et al., 2007) have been found to play roles in chromosome cohesion. It will be of interest to determine whether any of these proteins are phosphorylated directly by CDK11p58. However, it is very likely that many of the mitotic abnormalities we have identified in this study are indirect and arise from defective kinase cascades. The observation that CDK11 RNAi expression decreased Plk1 localization at the centromere (Petretti et al., 2006) strongly supports this conclusion. Indeed, multiple kinase cascades involving kinases that regulate CDK11 or are regulated by CDK11, such as BubR1, Mps1, Plk1, aurora, CDK1, MAPK and others, are likely to exist. In addition, the CDK11p58 amino acid sequence has strong homology to CDK1 and possesses all of the sites and motifs that regulate the mitotic activity of CDK1 (Trembley et al., 2004), suggesting the mitotic kinase activity of CDK11 might be regulated by posttranslational modifications. It remains to be determined whether modification of these sites regulates the activity of CDK11. Nonetheless, the collection of mitotic abnormalities resulting from CDK11 depletion clearly establishes a role for CDK11 as a mitotic kinase.
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), cyclin A (NeoMarkers), cyclin B1 (Santa Cruz and Abcam), -tubulin (Sigma), 
-tubulin (Cell Skeleton), anti-Myc monoclonal antibody (mAb) (Upstate-Millipore), Bub1 (Chemicon International), Sgo1 (kindly provided by H. Yu, University of Texas Southwestern Medical Center, Dallas) and actin (Santa Cruz). Secondary antibodies included anti-mouse IgG-Texas Red (Amersham Biosciences), anti-rabbit IgG-FITC (Amersham Biosciences), anti-mouse IgG-Cy3 (Jackson ImmunoResearch), anti-rabbit IgG-Cy3 (Jackson ImmunoResearch) and anti-human IgG-FITC (Pierce). For studies using microtubule toxin treatment to examine the spindle checkpoint, nocodazole (0.1 µg/ml) or taxol (33 nM) were added to the culture media, and the cells were harvested at the indicated times for further analysis.
CDK11 RNAi
The pSUPER siRNA expression vector (Brummelkamp et al., 2002), kindly provided by Reuven Agami (Netherlands Cancer Institute, Amsterdam), was used for construction of CDK11 RNAi constructs containing 19-nucleotide sequences designed to target various regions of CDK11, as previously described (Brummelkamp et al., 2002). The 19-nucleotide CDK11-targeting sequences were A461: 5'-GGGAGATGGCAAGGGAGCA-3' (CDK11 RNAi-N); A2184: 5'-GAGCGAGCAGCAGCGTGTG-3' (CDK11 RNAi-C); A910: 5'-GGGAGCACCAGTGAAGAAT-3'; A111: 5'-TTCTGATGACCGGGATTCC-3'; A188: 5'-GGAACTCCCCGTATAGAAG-3'; and A1361: 5'-AGCGGCTGAAGATGGAGAA-3'. For electroporation delivery of pSUPER RNAi constructs, cells were grown to 85% confluence, harvested, and suspended at a density of approximately 107 cells/ml. Cells (400 µl of the suspension) were then added to a 4-mm electroporation cuvette (BioRad), along with 25 µg of plasmid DNA. Electroporation was performed using an Electro Cell Manipulator (BTX ECM 630, BTX Instruments) at 220 V and 960 µF capacitance. After electroporation, cells were transferred to plates with complete DMEM for cultivation. Apoptosis and cell-cycle analysis of the CDK11 RNAi-treated cells were examined by annexin V staining and flow cytometry, respectively. Immunobloting with cell lysates prepared from CDK11 RNAi-treated cells and control cells was performed with the indicated antibodies used as described throughout this study. The pSUPER Plk1 and Sgo1 siRNA expression constructs were constructed similarly using the following oligonucleotides: Sgo1, 5'-CAGUAGAACCUGCUCAGAA-3'; and Plk1, 5'-CGAGCUGCUUAAUGACGAG-3') (McGuinness et al., 2005). The A1361 CDK11 siRNA was synthesized from Dharmacon and transfected into HeLa cells using 4 µl of Dharmafect1 and 50 nM siRNA oligonucleotides in each well of a six-well dish according to the manufacturer's instructions (Dharmacon).
CDK11p58-GFP cell lines
The plasmids used for generation of stable HeLa tranfectants were constructed by cloning the CDK11p58 cDNA or a CDK11p58 cDNA with a silent mutation in the CDK11 RNAi targeting region (the mutated CDK11 siRNA targeting sequence: 5'-AAGTGAACAACAGCGTGTG-3') into pEGFP-N3 vector, whose CMV promoter was replaced with an SV40 promoter taken from a pSVL plasmid by PCR to yield a C-terminal p58-GFP fusion protein. The resulting plasmids were transfected into HeLa cells, and single cells were cloned under neomycin selection and GFP sorting.
Immunofluorescence analysis
HeLa cells, cultured on coverslips for 48 hours after being transfected with RNAi constructs with or without microtubule toxin treatment, were fixed for 10 minutes with 4% formaldehyde and permeabilized for 3 minutes with 0.3% Triton X-100/phosphate-buffered saline (PBS) and then blocked with 10% fetal bovine serum (FBS) for 30 minutes followed by incubation with various primary antibodies for 1 hour at 37°C. The coverslips were then washed and incubated with the fluorchrome-conjugated secondary antibodies for 1 hour at 37°C, washed with PBS and mounted with Vectashield mounting medium containing 0.05 µg/ml DAPI. Samples were analyzed either on a Nikon C1si Eclipse TE2000-E using a 60x objective (NA 1.45) with images acquired using EZ C1 software or on a Nikon Eclipse E-80i using a 100x objective (NA 1.35) with images acquired with a DS-SM-L digital camera using the Nikon DS-L1 software. TIFF image files were further manipulated using Adobe Photoshop 7.0.
FISH analysis
FISH analysis was performed as described previously (Gururajan et al., 1998) with a BAC clone containing the human histone cluster located at 6p21 (RPCI-11-300H3) and a plasmid containing human chromosome 1 heterochromatin repeats (pUC 1.77). Specific hybridization signals were detected with anti-digoxigenin FITC (Roche Biochemicals) followed by counter-staining with DAPI. The distance between sister chromatids of 25 G2 cells from each RNAi-treated group was determined by measuring the distance between paired FISH signals from the same cell for multiple sets of signals; 25 independent G2 cell measurements were made for each G2-synchronized RNAi group from photomicrographs.
Mitotic chromosome analysis and mitotic index analysis
Cells transfected with RNAi constructs were harvested with trypsin and subjected to a hypotonic treatment for chromosome mitotic spread and Giemsa staining. For each sample, a mitotic index was determined by dividing the number of mitotic cells by the total number of cells examined in a sample. More than 1000 cells were counted in each experiment and the experiments were performed multiple times with similar data. Chromosome appearance and occurrence frequency were determined by analyzing more than 100 mitotic cells for each RNAi condition in a single experiment.
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