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Originally published In Press as doi:10.1074/jbc.M405481200 on July 15, 2004

J. Biol. Chem., Vol. 279, Issue 38, 40112-40121, September 17, 2004
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Inhibition of JNK2 Disrupts Anaphase and Produces Aneuploidy in Mammalian Cells*

Rebecca A. MacCorkle and Tse-Hua Tan{ddagger}

From the Department of Immunology, Baylor College of Medicine, Houston, Texas 77030

Received for publication, May 17, 2004 , and in revised form, June 29, 2004.


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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
JNK11 and JNK2 are members of the mitogen-activated protein kinase family, which also includes the prototypical family members extracellular signal-regulated kinase and p38. Mitogen-activated protein kinases are components of signal transduction pathways that connect extracellular stimuli to intracellular responses such as modulation of cell viability, cell cycle regulation, and gene expression (1-3). JNK1 and JNK2 are ubiquitously expressed and are generally considered to share redundant functions in apoptosis and transformation and differential functions in T cell differentiation (3-6). However, recent studies have shown that the stress-induced proapoptotic function previously attributed to both isoforms is actually a function specific to JNK1 (7, 8). The function of JNK2, therefore, has become less clear. This suggests that JNK functions in transformation may also be isoform-specific and should be addressed in a way that differentiates between the contributions of JNK1 and JNK2.

Ras-activating mutations have been identified in close to 30% of human cancers (9). Transformation by Ras requires JNK activity, which has made JNK an attractive target for cancer therapy (10-12). Targeting JNK for cancer therapy is also supported by studies showing that JNK activity is elevated in human tumors and that loss of JNK function inhibits tumor growth in mice (13-16). The transforming mechanism downstream of JNK is not entirely clear, but it may work through the phosphorylation of c-Jun, which in turn regulates transcription of cell cycle regulators. In addition to this pathway, JNK may have a more direct function in cell cycle regulation. We have shown that JNK localizes to centrosomes and is active in this compartment from early S phase through late anaphase with peak activity at metaphase (17). While total soluble JNK activity also increases during mitosis (18, 19), neither JNK1 nor JNK2 has been shown to function in mitosis.

To address whether JNK1 or JNK2 functions in mitosis, we used three loss of function approaches: blocking JNK activation by ectopic overexpression of a JNK dominant-negative mutant, inhibiting JNK activity with the specific pharmacological inhibitor SP600125, and down-regulating JNK isoform expression with RNA interference. The mammalian cell lines used in this study include human cervical carcinoma (HeLa) cells, human small lung carcinoma (Calu-1), and Chinese hamster ovary (CHO) cells. All three methods produced an accumulation of cells with 4N DNA content. The cells with 4N DNA content were shown to be multinucleate cells with decondensed DNA rather than cells blocked in a particular stage of mitosis. Examination of the morphology of cells progressing through mitosis in the presence of JNK inhibitor suggests that polyploidy results from defects in chromosome segregation and central spindle formation during anaphase B. RNA interference showed that the loss of the JNK2 isoform, but not the JNK1 isoform, produced an accumulation of 4N cells. At later time points, apoptotic or polyploid populations were elevated depending on cell type, suggesting that whether multinucleate cells would be eliminated or reenter subsequent cell cycles was dependent on cellular context. We also found that while inhibition of JNK delayed mitotic progression, it did not delay cyclin B1 degradation or down-regulation of cyclin-dependent kinase 1 (Cdk1) activity. This suggests that JNK may regulate anaphase progression through a mechanism further downstream of or independent of Cdk1/cyclin B1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Derivation of Calu-1 7-5, 8-5, and 8-30 clones has been described previously (20). Additional clones were derived by limiting dilution of Calu-1 cells transiently transfected with FLAG-JNK(T183A,Y185F) dominant-negative mutant, abbreviated as JNK(APF), and selected in 0.5 mg/ml G418 for 2 weeks. CHO, human cervical carcinoma (HeLa), human small lung carcinoma (Calu-1), and human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 mM HEPES (pH 7.4), penicillin (100 units/ml), and streptomycin (100 µg/ml).

Synchronization—For the metaphase block, CHO cells were plated at 70% confluency on acid-etched poly-L-lysine-coated coverslips and allowed to attach for 4 h. Attached cells were synchronized by blocking in early S phase with 2.5 mM thymidine (Sigma) for 12 h, releasing in fresh media for 5 h, and then blocking in mitosis with 0.05 µg/ml nocodazole (Sigma) for 5 h. Synchronized cells were gently washed with fresh media, returned to 37 °C, and fixed at 15-min intervals. The JNK inhibitor SP600125 (Alexis Biochemicals, San Diego, CA) or Me2SO carrier alone was added to cultures 30 min prior to release from nocodazole treatment and to the fresh media following mitotic release as indicated. The final concentration of Me2SO in all samples was 0.1%.

For the double thymidine block, HeLa cells were incubated with 2 mM thymidine for 20 h, released into fresh media for 8 h, incubated with 2 mM thymidine for 15 h, and then released into fresh media for 4.5 h before addition of 0.1% Me2SO or 0.1% Me2SO with 15 µM SP600125.

Immunofluorescence—For all immunofluorescence experiments, cells were plated on acid-etched, polylysine-coated glass coverslips. Plated cells were maintained at 37 °C in 5% CO2. Coverslips were fixed in ice-cold methanol for 3 min followed by three 5-min washes in PEM buffer (80 mM K-PIPES (pH 7.6), 5 mM EGTA, 2 mM MgCl2) at room temperature. Cells were then permeabilized in 0.5% Triton X-100 in PEM buffer for 20 min and washed with TBST (50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20). Coverslips were then incubated for 1 h at 37 °C with primary antibodies diluted in TBST, washed in TBST, and incubated for 1 h at 37 °C with secondary antibodies diluted 1:400 in TBST. After washing in TBST, coverslips were counterstained with 0.4 µg/ml of 4,6-diamino-2-phenylindole (DAPI) (Molecular Probes, Eugene, OR) in TBST and mounted with ProLong antifade medium (Molecular Probes). The antibodies used for immunofluorescence were: anti-{beta}-tubulin (17), anti-pericentrin (BabCo/Covance, Denver, CO), goat anti-rabbit, and mouse IgG (heavy + light) conjugated to Alexa 488 or Alexa 594 (Molecular Probes). Images were obtained with a Zeiss AxioPlan2 Microscope (Zeiss, Thornwood, NY) and CoolSnap HQ CCD using MetaView software. Brightness adjustment, channel overlay, and figure arrangement was done with Adobe Photoshop software.

Western Blotting—The antibodies used for Western blotting were: anti-hemagglutinin (Sigma), anti-FLAG (Roche Applied Science), anti-JNK1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-JNK2 (Santa Cruz Biotechnology), anti-phospho-c-Jun (Ser-63) and anti-phospho-CREB (Ser-133) (New England Biolabs, Beverly, MA), anti-actin (Sigma), anti-aldolase (Biodesign International, Saco, ME), anti-cyclin B1 (Santa Cruz Biotechnology), and anti-Polo-like kinase 1 (Plk1) (Zymed Laboratories Inc.). Protein concentrations of samples were quantified by Bradford assay. Equal amounts of protein were analyzed by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked in 10% nonfat dry milk in TBST, incubated for 1 h in TBST containing primary antibody, washed three times (7 min each) in TBST, incubated for 1 h in TBST containing secondary antibody, and washed three times (7 min each) in TBST. Signal was developed using ECL reagents (Amersham Biosciences). Signal was quantified using Kodak 1D version 3.5.3 software (Scientific Imaging Systems, New Haven, CT).

Flow Cytometry—Cells were fixed in ice-cold 70% ethanol and incubated with 200 µg/ml RNase (Sigma) in phosphate-buffered saline for 30 min at 37 °C. A 4-fold volume of 50 µg/ml propidium iodide in phosphate-buffered saline was added, and samples were analyzed on a Beckman-Coulter EPICS XL-MCL flow cytometry system equipped with Beckman System II v3 flow analysis software. For determination of percentages of polyploid cells, gates were set to exclude nonviable cells based on forward and side scatter profiles. To quantitate sub-G0 events, samples were not gated on forward or side scatter. For determination of percentages of cells in S or 4N stages, gates were set to exclude nonviable and polyploid cells so that percentages could be accurately compared between samples. A minimum of 10,000 events were counted per sample.

Microtubule Nucleation Assay—CHO cells were plated on coverslips, cultured overnight, and treated with 5 x 10-6 M nocodazole in 37 °C preheated media for 1.5 h. Inhibitors or carrier alone was added to the media for an additional 30 min. Coverslips were transferred to fresh preheated media containing inhibitors or carrier alone for 5-15 min at 37 °C in 5% CO2 and then gently fixed in ice-cold methanol for 3 min.

Small Interfering RNA (siRNA)—The 2'-ACE-stabilized, duplexed siRNAs were synthesized by Dharmacon Research (Lafayette, CO) and desalted according to the manufacturer's instructions. Cells were transfected with siRNA using Oligofectamine (Invitrogen) or LipofectAMINE (transfection following plasmid transfection, Invitrogen) according to the manufacturer's protocol. For Western blots showing reduction of endogenous protein, the caspase inhibitor Z-VAD (Sigma) was included at a concentration of 100 mM in all cultures post-transfection.

Cdk1 Kinase Assay—Cells were lysed in lysis buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM ethylene glycol bis({beta}-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM glycerophosphate, 1% Triton X-100, 1 mM dithiothreitol, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) for 10 min on ice and cleared by centrifugation at 15,000 x g for 5 min. Lysate (100 µg) was incubated with anti-Cdk1 monoclonal antibody (Santa Cruz Biotechnology) for 1 h at 4 °C, and then protein A-agarose beads were added for an additional 30 min. Immunoprecipitates were washed with lysis buffer and incubated with 10 µg of histone H1 (Sigma), 15 µM ATP, 10 µCi of [{gamma}-32P]ATP in kinase buffer (25 mM HEPES (pH 7.6), 25 mM {beta}-glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol) for 30 min at 30 °C. Reactions were stopped by addition of SDS sample buffer and heating to 100 °C for 5 min. Sample proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To determine whether JNK activity is important for cell cycle progression, human small lung carcinoma (Calu-1), human cervical carcinoma (HeLa), and CHO cells were treated with SP600125, a pharmacological inhibitor of JNK (21). We focused on phenotypes that were common to all three cell lines and independent of differences in cellular background. The minimal effective dose of SP600125 was first determined in CHO cells to be 15 µM, which inhibited 80% of JNK activity (Fig. 1, A and B). In contrast, SP600125 showed no inhibition of the related kinase p38 below 15 µM in CHO cell culture as assessed by quantifying phosphorylation of its substrate, CREB (Fig. 1C). So that it could be used as a control, the effective dose of the p38 inhibitor SB202190 in CHO cell culture was determined. The IC50 for inhibition of p38 in CHO cell culture was 8.5 µM with 25 µM providing 70% inhibition of p38 and only 2% inhibition of JNK (Fig. 1C). Treatment of Calu-1, HeLa, and CHO cells with 15 µM SP600125 for 72 h caused a 2.0-fold increase in S phase HeLa cells but not Calu-1 or CHO cells (Table I). This accumulation of S phase cells is, therefore, dependent on cellular background. More interestingly, this treatment caused an accumulation of cells with 4N DNA content in all three cell lines. Specifically treatment of Calu-1, HeLa, and CHO cells with 15 µM SP600125 for 72 h caused 1.6-fold (p = 0.004), 3.5-fold (p = 0.009), and 3.0-fold (p = 0.000) increases in tetraploid cells, respectively (Fig. 1D and Table I). To further confirm this observation, we screened Calu-1 small lung carcinoma clones expressing a dominant-negative mutant, FLAG-human JNK(APF), for changes in cell cycle distribution (20). FLAG-JNK(APF) expression was detected by Western blotting (Fig. 2A). When plated at equal density and cultured for 18 h, JNK(APF)-expressing cultures had increased percentages of cells with 4N DNA content (Fig. 2B).



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  		FIG. 1.
In vivo specificity of SP600125 and effects on cell cycle progression. CHO cells were pretreated with the indicated concentrations of inhibitors for 30 min, exposed to 300 J/m2 UV irradiation, and incubated with inhibitors for an additional 30 min. Samples were Western blotted to detect phospho-c-Jun (A) or phospho-CREB and phospho-ATF1 (C), and actin as a loading control (A and B). B, signal was quantitated and normalized to actin loading controls. Relative -fold expression was plotted versus inhibitor concentration, and a best fit logarithmic curve was used to determine the IC50 and IC80 for SP600125 in this system. D, Calu-1, HeLa, and CHO cells were treated with 15 µM SP600125 for 72 h, stained with propidium iodide, and analyzed by flow cytometry. Quantification of flow cytometry with the mean ± S.D. for three data sets is shown. P-, phospho-.

 


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  		TABLE I
SP600125 effects on multiple cell lines

Cells were incubated with 0.1% Me2SO (untreated) and SP600125 for 72 h and analyzed by flow cytometry for DNA content. Percentage of events in S phase with 4N DNA content and with greater than 4N (4N+) DNA content are indicated.

 



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  		FIG. 2.
Inhibition of JNK produces tetraploid cell accumulation. A, stable Calu cell lines express FLAG-tagged JNK(T183A,Y185F) (also called FLAG-JNK(APF)) dominant-negative mutant. Whole cell lysates from Calu clones stably transfected with vector (7-5) or FLAG-JNK(APF) (8-5 and 8-30) were Western blotted with an anti-FLAG antibody. B, dominant-negative cells lines have increased percentages of cells with 4N DNA content. Calu clones were plated, incubated for 18 h, and analyzed by propidium iodide staining and flow cytometry for DNA content. C, HeLa cells were transiently transfected with vector (-), hemagglutinin-tagged JNK1 (HA-JNK1), or FLAG-tagged JNK2, incubated for 4 h, and then transfected with JNK1 or JNK2 siRNA. After an additional 20 h, anti-actin, anti-hemagglutinin, and anti-FLAG Western blotting was performed. D, HeLa cells were transfected with fluorescently labeled control siRNA (Fluor. siRNA), JNK1 siRNA, or JNK2 siRNA. After 48 h, anti-actin, anti-JNK1, and anti-JNK2 Western blotting was performed. E and F, reduction of JNK2 expression in HeLa and Calu cells causes accumulation of cells with 4N DNA content. HeLa and Calu cells were transfected with JNK1 or JNK2 siRNA for 96 or 120 h, respectively, stained with propidium iodide, and analyzed by flow cytometry for DNA content. 4N peaks are marked. Quantification of flow cytometry with the mean ± S.D. for three data sets shows that JNK2 but not JNK1 siRNA causes 4N accumulation.

 
To assess the relative contributions of JNK1 and JNK2 isoforms to the 4N population accumulation, we designed siRNA to block expression of human JNK1 and JNK2 in the human cells lines HeLa and Calu. The specificity of JNK1- and JNK2-targeting siRNAs was tested by transfecting HeLa cells with hemagglutinin epitope-tagged JNK1 or FLAG epitope-tagged JNK2 followed by transfection with JNK1 or JNK2 siRNA. After 24 h, expression of JNK1 and JNK2 was assessed by Western blotting against the epitope tags. Treatment of HeLa cells with JNK1 or JNK2 siRNAs specifically blocked greater than 95% of JNK1 and JNK2 expression, respectively (Fig. 2C). The effects of reducing JNK expression on cell cycle progression were determined by transfecting HeLa and Calu cells with isoform-specific siRNAs and analyzing their cell cycle profiles with propidium iodide staining and flow cytometry. Changes in cell cycle profiles were first observed at 72 h post-transfection. Down-regulation of JNK1 expression did not produce significant changes in the percentage of viable cells with 4N DNA content (Fig. 2, D and E). However, down-regulation of JNK2 produced a 2-fold accumulation of viable cells with 4N DNA content in both HeLa and Calu cells by 96 and 120 h, respectively (Fig. 2, D and E). These data show that JNK2, but not JNK1, is critical for proper mitotic progression in human cells.

The increase in the 4N cell population suggests that JNK inhibition causes a mitotic block or prolonged G2/M progression. However, the accumulation of 4N cells could also be the result of an accumulation of cells that have not undergone proper chromosome segregation during one cell cycle but have still exited mitosis and reentered G0/G1. In the latter case, subsequent rounds of the cell cycle would be expected to produce cells with greater than 4N DNA content. To test this hypothesis, polyploidy was assessed in HeLa, Calu-1, and CHO cells treated with SP600125. CHO cells were treated with increasing doses of SP600125 for 72 h. Accumulation of polyploid cells was observed with as little as 7 µM SP600125 with 90% of the cells becoming polyploid in the presence of 10 µM SP600125 (Fig. 3, A and B). In contrast, neither of the p38 inhibitors SB202190 and SB203580 nor the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 caused polyploidy (Fig. 3A). Treatment of Calu-1, HeLa, and CHO cells with 15 µM SP600125 for 72 h caused 3.5-fold (p = 0.000), 4.6-fold (p = 0.001), and 8.1-fold (p = 0.000) increases in polyploid cells, respectively (Table I). Similar results were seen with Calu clones expressing the dominant-negative JNK(APF) mutant. When the clones were assessed for DNA content by propidium iodide staining and flow cytometry, up to a 15-fold increase in polyploidy was observed in high expressing clones, and up to a 12-fold increase was observed in low expressing clones (Fig. 3, C-F). No significant increases in polyploidy were detected in JNK siRNA-treated cultures by propidium iodide staining and flow cytometry analysis (Fig. 2F). One possible reason that we did not observe polyploidy with the JNK siRNA is that the efficiency of JNK inhibition using siRNA may be below the threshold for detecting the polyploid phenotype. Alternatively the slower nature of this inhibition method, which requires degradation of pre-existing protein, could suggest that the polyploid cells do not survive in culture long enough to detect an accumulation. In support of this, JNK2 siRNA caused a significantly increased sub-G0 population, which is indicative of DNA fragmentation, a hallmark of apoptosis (Fig. 3G). Thus, it is possible that over longer time periods the fate of polyploid cells that result from specific JNK2 inhibition is apoptosis. This function of JNK2 in protecting cells from apoptosis contrasts with the known function of JNK1 in promoting apoptosis. The pharmacological JNK inhibitor SP600125 and the JNK(APF) dominant-negative mutant inhibit both the JNK1 and JNK2 isoforms. Thus, if the polyploid cells would have normally undergone apoptosis in a JNK1-dependent manner, SP600125 and the dominant-negative mutant would have blocked this process. This could also explain why the loss of polyploid cells and apoptosis are only detected when JNK2 function is inhibited without inhibiting JNK1.



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  		FIG. 3.
JNK inhibition produces polyploidy. A, CHO cells were treated for 48 h with 0.1% Me2SO (Untreated) and 10 µM SP600125, 25 µM SB202190, 25 µM SB203580, or 25 µM PD98059 and analyzed by propidium iodide staining and flow cytometry for DNA content. Percentages of events with greater than 4N DNA content are indicated (% Polyploidy). B, CHO cells were treated with the indicated concentrations of SP600125 for 72 h and analyzed by flow cytometry for DNA content. Percentages of cells with greater than 4N DNA content are shown. C and D, stable Calu cell lines expressing FLAG-tagged JNK(APF) dominant-negative mutant were stained with propidium iodide and analyzed by flow cytometry for DNA content. Percentages of 4N+ events are indicated. E, expression of FLAG-JNK(APF) in intermediate expressing clones, but not in Calu 7-5 (vector only), was detected by Western blotting with an anti-FLAG antibody. F, Calu clones were plated at the same densities, fixed at 18 h, and analyzed by flow cytometry for DNA content. Percentages of cells with greater than 4N DNA content as the mean ± S.D. for three data sets are shown. G, HeLa cells were transfected with JNK1 or JNK2 siRNA, incubated for 72 h, stained with propidium iodide, and analyzed by flow cytometry for DNA content. The average percentages of sub-G0 events with S.D. for three independent experiments are shown.

 
To determine which stage of mitosis was defective, cells were treated with carrier, 20 µM SP600125, 20 µM SB202190, or 20 µM SB203580 for 72 h and their mitotic morphology was assessed by DAPI DNA staining and microscopy. CHO cells treated with SP600125, did not appear to be blocked in any stage of mitosis. However, the treated cells had enlarged and multilobed nuclei, which were not seen in cells treated with carrier or the p38 inhibitors (Fig. 4A). SP600125 also produced multinuclei in HeLa and Calu-1 cells. Similar enlarged and multilobed nuclei were detected in the Calu clones expressing the JNK dominant-negative mutant (Fig. 4B).



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  		FIG. 4.
JNK2, but not JNK1, siRNA produces chromosome segregation defects. A, CHO cells were treated with 0.1% Me2SO (Untreated), 20 µM SP600125, 20 µM SB202190, or 20 µM SB203580 for 72 h and stained with DAPI DNA stain (white). B, Calu 7-5, 8-5, and 8-30 clones were plated at equal density on glass coverslips and grown for 18 h before staining with DAPI DNA stain (white). Representative fields are shown. Arrows mark enlarged nuclei. C-E, HeLa cells were transfected with JNK1 or JNK2 siRNA for 96 h and stained with anti-{beta}-tubulin (green) and DAPI DNA stain (blue). Representative cells are shown. Arrows mark binucleated and trinucleated cells. Cells in mitosis (D) or with enlarged or multilobed nuclei (E) were counted by visual inspection of morphology. The mean ± S.D. for three data sets of 300 cells each is shown for each condition.

 
To determine whether this was a specific effect of the JNK2 isoform, HeLa and Calu cells were treated with JNK1 or JNK2 siRNA and assessed for mitotic morphology by immunofluorescence. Cells were transfected with siRNA for 96 h and then stained with anti-{beta}-tubulin and DAPI DNA stain to visualize the microtubules and DNA, respectively. As expected, JNK2, but not JNK1, siRNA caused a 4-fold accumulation of HeLa cells with enlarged and multilobed nuclei (Fig. 4, C and E). This indicates that JNK2, but not JNK1, is required for proper chromosome segregation and cytokinesis in HeLa cells. These RNA interference-induced multinuclei, present after one cell cycle (HeLa and Calu-1) with reduced JNK expression, were more defined and less globular than the multinuclei seen with the long term pharmacological JNK inhibition (HeLa, Calu-1, and CHO) and the chronic suppression of JNK activity in the dominant-negative JNK-expressing Calu-derived clones (Fig. 4, A and B). However, all three methods produced cells with enlarged nuclei, showing that chromosome segregation and cytokinesis during mitosis did not occur when JNK was inhibited. Interestingly neither JNK1 nor JNK2 siRNA significantly altered the overall percentage of cells morphologically progressing through mitosis as assessed by microscopy (Fig. 4D). Therefore, because these cells were not blocked in a specific stage of mitosis, the nature of the defect leading to multinucleation was not clear. Although the inhibition of JNK appears to affect S phase progression in HeLa cells, CHO and Calu-1 cells progressed normally through S phase in the presence of JNK inhibitor (Table I). This suggests that the defect leading to multinucleation, which was common to all three cell lines, was occurring after S phase. To determine whether cells were properly progressing through G2 and mitosis when JNK was inhibited, analysis of cellular morphology by immunofluorescence was performed.

Several possible mechanisms may account for the multinucleation observed in cells depleted of JNK activity, including defects in chromosome condensation during G2, chromosome alignment to the metaphase plate, spindle formation, chromosome segregation, and cytokinesis. To determine whether JNK inhibition caused defects in global microtubule nucleation and/or growth, CHO cells were treated with nocodazole to depolymerize existing cytoskeletal microtubules. Nocodazole was then washed from the cell culture, and microtubules were allowed to nucleate and regrow at 37 °C in fresh cell culture media. Microtubule nucleation and regrowth were intact in the presence of 25 µM SP600125, suggesting that JNK is not required for these processes (Fig. 5A). More importantly, morphologically normal bipolar spindles with chromosomes aligned at the metaphase plate are present in HeLa, Calu-1, and CHO cells treated for 24 h with 15 µM SP600125. This indicates that in mammalian cells, chromosome condensation, chromosome alignment to the metaphase plate, and spindle formation do not require JNK activity (Fig. 5B and data not shown).



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  		FIG. 5.
Inhibition of JNK does not affect microtubule nucleation, growth, or spindle formation. A, CHO cells were treated with 5 µM nocodazole for 1.5 h and then 0.1% Me2SO and 0, 10, or 25 µM SP600125 was added for an additional 30 min. Cells were transferred to media containing 0.1% Me2SO (Untreated) or 0.1% Me2SO plus 25 µM SP600125 for 5-15 min and then fixed. Shown are microtubules stained with anti-{beta}-tubulin (green), centrosomes stained with anti-pericentrin (red), and DNA stained with DAPI (blue). B, CHO cells were treated with 0.1% Me2SO (Untreated) or 0.1% Me2SO plus 15 µM SP600125. Representative fields of cells with single bipolar spindles (inset) are shown.

 
To determine whether defects occurred after metaphase during chromosome segregation of anaphase A and B, CHO and HeLa cells were first synchronized in metaphase with a single thymidine block followed by a nocodazole block. Synchronized cells were then released into fresh culture media, followed through anaphase in the presence or absence of JNK inhibitor by fixation every 15 min, and visualized by immunofluorescence imaging. In the absence of SP600125, central spindle formation, midbody formation, and cytokinesis were detected at 30, 45, and 60 min, respectively (Fig. 6A and data not shown). In the presence of SP600125, sister chromatid separation was initiated during anaphase A. However, central spindle formation during anaphase B, chromosome segregation, and cytokinesis were defective (Fig. 6, A and B, and data not shown). SP600125 produced chromosome segregation defects in over 90% of anaphase cells (Fig. 6C). In contrast, an inhibitor of the related mitogen-activated protein kinase p38 did not cause chromosome segregation defects (Fig. 6C). These results suggest that while JNK activity is dispensable for metaphase spindle formation, JNK activity is important for central spindle formation for cells to complete chromosome segregation during anaphase B and undergo proper cytokinesis.



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  		FIG. 6.
JNK inhibition produces chromosome segregation defects. A, metaphase-synchronized CHO cells were incubated with 0.1% Me2SO (Untreated) and 15 µM SP600125 or 20 µM SB203580 for 30 min, released from metaphase in the presence of 0.1% Me2SO (Untreated) and inhibitors as indicated, and fixed at 15-min intervals. Cells were stained with anti-{beta}-tubulin (green), anti-pericentrin (red), and DAPI (blue). Representative cells are shown. B, a field view of {beta}-tubulin staining from the 45-min time point is shown. Arrows indicate midbodies. C, cells were scored for anaphase defects by visual inspection. Percentages of 300 anaphase cells with normal (white bars) versus abnormal (black bars) chromosome segregation are shown for each sample.

 
Progression through anaphase and mitotic exit is regulated in part by a complex network of kinases that is just beginning to be understood in mammalian systems. One key central event is the destruction of cyclin B1 and the down-regulation of the kinase activity of its associated partner, Cdk1 (see Fig. 8A). The cyclin B1 degradation and the Cdk1 inactivation is initiated at the metaphase to anaphase transition and when complete allows mitotic exit (22). How cyclin B1/Cdk1 controls anaphase B and mitotic exit is still unclear. However, several downstream pathways have been implicated in this process, including the Plk1 pathway (23). Plk1 is also regulated by degradation during mitotic exit in human cells (24). To determine whether JNK activity is required for the down-regulation of Cdk1 activity, cyclin B1 protein degradation, and/or Plk1 protein degradation, HeLa cells were synchronized by double thymidine block, treated with JNK inhibitor just prior to entry into metaphase, and then assayed for Cdk1 kinase activity, cyclin B1 protein levels, and Plk1 protein levels as they progressed through mitosis. Mitotic exit was delayed by about 4 h in the presence of 15 µM SP600125 (Fig. 7A). In carrier-treated cells, the majority of 4N cells exited mitosis and entered G1 by 9 h postrelease from thymidine block (Fig. 7B). However, in the presence of SP600125, most cells were still in the 4N state by 9 h (Fig. 7B). Duplicate samples from this synchronization were assayed for Cdk1 kinase activity. While SP600125 increased the duration of the 4N state, it did not prolong Cdk1 kinase activity and instead showed a reduction in overall Cdk1 kinase activity (Fig. 7C). This shows that JNK is not prolonging mitotic cell turnover by inhibiting the Cdk1 down-regulation required for mitotic exit. Cyclin B and Plk1 protein down-regulation was also assessed in these samples by Western blotting (Fig. 7D). Neither cyclin B1 nor Plk1 down-regulation was blocked by SP600125 (Fig. 7D). This suggests that JNK acts further downstream than the Cdk1/cyclin B1 pathway through a mechanism independent of Plk1 protein degradation.



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  		FIG. 8.
JNK1 promotes apoptosis, while JNK2 regulates mitotic progression through mechanisms downstream of Cdk1/cyclin B1. A, JNK2 is not required for the down-regulation of Cdk1 kinase activity or cyclin B1 protein degradation. This suggests that JNK2 may function in pathways further downstream of the anaphase-promoting complex (APC). JNK2 may function by affecting the activity or localization of chromosomal passenger proteins such as Plk1 or other proteins necessary for anaphase progression. B, JNK1 has a well established role in promoting apoptosis induced by intracellular and extracellular stimuli. JNK2, however, plays a contrasting role in promoting cell viability. The JNK isoforms are further functionally distinguished from each other by the newfound function for JNK2 in mitotic progression, a function that is not shared by JNK1.

 



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  		FIG. 7.
JNK activity is not required for CDK1/cyclin B1 inactivation or Plk1 degradation during mitotic exit. A, HeLa cells were synchronized in early S phase by incubating in 2 mM thymidine for 20 h, fresh culture media for 8 h, and then 2 mM thymidine for an additional 15 h. Cells were released by incubating in fresh media for 4.5 h, then 0.1% Me2SO (open circles) or 15 µM SP600125 JNK inhibitor (closed squares) was added, and samples were fixed every hour for propidium iodide staining and flow cytometry of cell cycle distributions. Time indicates hours post-release from second thymidine incubation. B, cell cycle profiles of samples 10 h postrelease show mitotic exit delay in the presence of JNK inhibitor. C, duplicate samples from A were assayed for Cdk1 kinase activity. Cdk1 was immunoprecipitated with an anti-Cdk1 antibody and incubated with histone H1 for 30 min in the presence of [{gamma}-32P]ATP. D, duplicate samples from A were assayed for cyclin B1 expression and Plk1 expression. Lysates were Western blotted with anti-cyclin B1, anti-Plk1, and anti-actin antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identified a new function for JNK in cell cycle control. We showed that while JNK activity was dispensable for chromosome condensation, chromosome alignment to the metaphase plate, and spindle formation, loss of JNK function interfered with chromosome segregation and central spindle formation in mammalian cell lines. In three different cell lines (HeLa, Calu-1, and CHO) the loss of JNK activity also led to an accumulation of polyploid cells, indicating that there is a breakdown in genomic segregation and either a direct or an indirect block in cytokinesis. Accumulation of polyploid cells after total JNK inhibition has also been detected in breast cancer cells (25). JNK1 and JNK2 are ubiquitously expressed in mammalian cells and have long been thought to have redundant functions. However, we have found that inhibition of specific JNK1 or JNK2 isoforms produces different effects on the mammalian cell cycle. Down-regulation of JNK1 did not affect cell cycle progression or viability of either human cell line (HeLa or Calu-1). In contrast, inhibition of JNK2 produced an accumulation of cells with 4N DNA content in both cell lines, showing that this function is specific to the JNK2 isoform. It is interesting to note that while down-regulation of JNK2 also caused an accumulation of polyploid Calu-1 cells, it did not produce an accumulation of polyploid HeLa cells. Instead down-regulation of JNK2 appeared to cause substantial apoptosis in HeLa cells, which may be the final fate of cells with mitotic defects in this cellular background when JNK1 activity remains intact. JNK1 has a well established role in stress-induced apoptosis, and it is interesting to speculate that it may also be involved in apoptosis induced by mitotic defects. These functions contrast with those of the related mitogen-activated protein kinases extracellular signal-regulated kinase and p38 in spindle microtubule attachment to kinetochores and in the spindle assembly checkpoint, respectively (26, 27).

Previous studies have suggested that JNK functions in either S phase or G2 phase of the mammalian cell cycle (20, 25). In contrast, we found that JNK function in S phase is cell line-specific. Inhibition of JNK resulted in S phase delay in only one of three cell lines tested. However, we found that JNK produced mitotic defects in all three cell lines tested. We followed cells through mitosis to determine where the defect occurred and found that while cells progressed through G2 to metaphase without apparent defects, chromosome segregation during anaphase was disrupted. As previously mentioned, JNK2 does not appear to be required for passing the spindle assembly checkpoint or for initiation of sister chromatid separation during anaphase A. Sister chromatid separation appears to be initiated when JNK is inhibited, although whether it is completed is still under investigation. In either case, central spindle formation, poleward movement of the chromatids during anaphase B, and cytokinesis are blocked. Sister chromatid separation is initiated by activation of the anaphase-promoting complex, which in turn initiates cyclin B degradation/Cdk1 inactivation. However, we observed that cyclin B1 degradation and Cdk1 kinase activity down-regulation are intact in the absence of JNK activity. Thus, anaphase-promoting complex activation would be expected to occur independently of JNK2 activity (Fig. 8A).

JNK may be regulating central spindle formation and/or chromosome segregation by participating in further downstream signaling pathways (Fig. 8A). For example, inhibition of Cdk1 activity is required for the activation and translocation of chromosomal passenger proteins to the central spindle, which are essential events for cytokinesis in mammalian cells (28). Future studies will determine whether the activation or movement of chromosomal passenger proteins such as Aurora B, inner centromere protein, and/or survivin are disrupted in JNK-deficient cells. If all of these events are intact, JNK may be working through a so far unidentified pathway that regulates anaphase progression and/or mitotic exit in mammalian cells.

Several alternative mechanisms that contribute to the regulation of anaphase B and cytokinesis may be regulated by JNK. In mammalian cells, actin depolymerization is often accompanied by failure of central spindle formation, implicating actin polymerization in the initiation of anaphase B (29). Interestingly JNK is required for proper actin dynamics in Drosophila embryos and therefore may contribute to central spindle formation through the regulation of actin dynamics (30). Proper regulation of actin dynamics is also necessary for cytokinesis. It is known that degradation of cyclin B during anaphase activates a Rho GTPase, called Pebble in Drosophila, which is required for actin ring assembly and cytokinesis (31-33). If JNK regulates cytokinesis through the regulation of actin dynamics, this study shows that it is most likely acting independently of this Rho/CyclinB1/Cdk1 pathway. Further study is necessary to determine whether JNK is acting downstream of this Rho pathway to regulate actin dynamics during central spindle formation and/or cytokinesis.

Once formed, elongation of the anaphase B central spindle is regulated by opposing forces of the kinesin and dynein families of motor proteins on central spindle microtubules (34). Kinesins regulate the sliding of central spindle microtubules to push spindle poles apart, while dyneins regulate astral microtubule dynamics to pull spindle poles apart. JNK interacts with the Kif3 kinesin on cytoplasmic microtubules (35), raising the possibility that JNK may also interact with mitotic kinesin family members. In addition, JNK associates with the JNK-interacting protein family of scaffolding proteins, which in turn associate with kinesins (36, 37). Yet another possible link between JNK and dynein regulation is the kinase Fyn. Fyn interacts with dynein and is an upstream regulator of the JNK pathway (38-41). It is therefore possible that interactions between JNK and kinesin motor proteins or participation of JNK in a dynein regulatory pathway with Fyn may function to ensure the completion of the mitosis and the maintenance of genomic stability.

This new function for JNK in the regulation of mitosis is a possible mechanism for how JNK acts in transformation and tumor progression signaling pathways. Loss of JNK2 function may result in an inhibition of cell growth by blocking chromosome segregation and cytokinesis. If the loss of JNK2 function leads to slowed cell cycle progression in nontransformed contexts, as it does in the transformed cells line models presented here, then loss of JNK2 may be a useful target for treating hyperproliferative disorders such as cancer. This would suggest that the loss of JNK2 function would have little effect on tumor incidence and instead would reduce tumor progression. This model is supported by the result that JNK2-null mice are resistant to mitogen-induced tumor growth and malignant transformation (16). This is further supported by a recent finding that JNK2 is constitutively activated in primary glial tumors (42).

The presence of MKK4-inactivating mutations (MKK4 is a kinase directly upstream of JNK activation) in some human tumors (43) is inconsistent with this model. However, MKK4 mutation-associated tumorigenesis would most likely be related to the loss of JNK1 functions and therefore the loss of DNA damage-induced apoptosis pathways (Fig. 8B). Reduced apoptosis of cells with DNA damage would lead to an accumulation of mutations in cells, thus sensitizing organisms to tumorigenesis. This model is supported by a report that JNK1-null mice are sensitized to mitogen-induced tumor incidence (44).

Several JNK inhibitors are being developed and tested in clinical trials for the treatment of neurological diseases, metabolic disorders, inflammatory conditions, and cancer (11). However, these inhibitors are not specific for JNK isoforms. Our studies would suggest that this could reduce the efficacy of these inhibitors and increase unwanted side effects in therapeutic applications. The data presented here show that JNK2 participates in the essential process of chromosome segregation during anaphase in mammalian cells. In a transformation background, the down-regulation of JNK2 would be expected to reduce tumor cell growth. In addition, one would predict that the genetic loss of JNK2 function would create a tumorigenesis-resistant phenotype. In contrast, inhibition of JNK1 would be predicted to lead to an increased frequency of survival of cells with potentially oncogenic mutations, thereby sensitizing organisms to tumorigenesis. It is, therefore, essential to consider the distinct functions of JNK isoforms during rational drug design (Fig. 8B). In view of our newfound understanding of the differential roles of JNK1 and JNK2 in differentiation, apoptosis, and proliferation, specific targeting of JNK1 or JNK2 may produce more precise and effective therapies by reducing harmful or unwanted side effects that result from inhibiting a cellular pathway unrelated to disease progression.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01-CA87076 and R01-AI42532 and an American Institute for Cancer Research Grant (to T.-H. T.) and Department of Defense Breast Cancer Research Program Fellowship DAMD 17-00-1-0141 and National Institutes of Health Training Grant T32-AI07495 (to R. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Immunology, Baylor College of Medicine, One Baylor Plaza, M929, Houston, TX 77030. Tel.: 713-798-4665; Fax: 713-798-3033; E-mail: ttan{at}bcm.tmc.edu.

1 The abbreviations used are: JNK, c-Jun N-terminal kinase; Cdk1, cyclin-dependent kinase 1; Plk1, Polo-like kinase 1; siRNA, small interfering RNA; CHO, Chinese hamster ovary; PIPES, 1,4-piperazine-diethanesulfonic acid; DAPI, 4,6-diamino-2-phenylindole; CREB, cAMP-response element-binding protein; MKK4, mitogen-activated protein kinase kinase 4. Back


   ACKNOWLEDGMENTS
 
We thank B. R. Brinkley, R. J. Davis, and K. M. Tchou-Wong for the generous gifts of anti-{beta}-tubulin antibody; JNK(APF) construct; and Calu 7-5, 8-5, and 8-30 clones, respectively.



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 DISCUSSION
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