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Originally published In Press as doi:10.1074/jbc.M310413200 on October 27, 2003

J. Biol. Chem., Vol. 279, Issue 2, 1482-1490, January 9, 2004
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NF-{kappa}B Promotes Survival during Mitotic Cell Cycle Arrest*

Pratibha Mistry{ddagger}, Karl Deacon§, Sharad Mistry{ddagger}, Jonathan Blank§, and Rajnikant Patel{ddagger}||

From the {ddagger}Department of Biochemistry and the §Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom

Received for publication, September 22, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By activating the mitotic checkpoint, anti-microtubule drugs such as nocodazole cause mammalian cells to arrest in mitosis and then undergo apoptosis. Microtubule depolymerization is rapid and results in the activation of the transcription factor NF-{kappa}B and induction of NF-{kappa}B-dependent gene expression. However, the functional consequence of NF-{kappa}B activation has remained unclear. Evidence has accumulated to suggest that NF-{kappa}B transcriptional activity is required to suppress apoptosis. In the present study, we confirm and extend previous findings that microtubule depolymerization leads to the rapid activation of NF-{kappa}B and test the hypothesis that the induction of NF-{kappa}B regulates cell survival during mitotic cell cycle arrest in order to define its role. Using a range of functional assays, we have shown that microtubule depolymerization correlates with the activation of IKK{alpha} and IKK{beta}; the phosphorylation, ubiquitination, and degradation of I{kappa}B{alpha}; the translocation of native p65 (RelA) into the nucleus; and increased NF-{kappa}B transcriptional activity. By inhibiting either the activation of the IKKs or the degradation of I{kappa}B{alpha}, we find that the level of apoptosis is significantly increased in the mitotically arrested cells. Inhibition of NF-{kappa}B signaling in the nonmitotic cells did not affect their survival. We establish that although NF-{kappa}B is activated rapidly in response to microtubule depolymerization, its cell survival function is not required until mitotic cell cycle arrest, when the mitotic checkpoint is activated and apoptosis is triggered. We conclude that NF-{kappa}B may regulate the transcription of one or more antiapoptotic proteins that may regulate cell survival during mitotic cell cycle arrest.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By perturbing microtubule dynamics, certain anticancer drugs cause mammalian cells to arrest in mitosis through activation of the spindle checkpoint (or mitotic checkpoint) (1). Generally, mitotic arrest is then followed by apoptosis (2, 3). However, the biochemical mechanisms by which the anticancer drugs induce toxicity are not clearly understood. We have recently demonstrated that anticancer drugs such as nocodazole, vincristine, and vinblastine (microtubule-depolymerizing agents) and taxol (a microtubule-stabilizing agent) activate intracellular signaling pathways that have opposing effects on cell survival (4). Another signal transduction pathway that is activated by these anticancer drugs involves the transcription factor NF-{kappa}B (5-7). However, the functional consequence of NF-{kappa}B activation by either microtubule-depolymerizing or -stabilizing drugs remains unknown.

NF-{kappa}B belongs to the Rel family of transcription factors that regulate genes involved in immune and inflammatory responses, cell cycle progression, apoptosis, and oncogenesis (8, 9). In mammals, the Rel family comprises p50 (NF-{kappa}B1), p52 (NF-{kappa}B2), p65 (RelA), RelB, and c-Rel. The NF-{kappa}B subunits homo- and heterodimerize to form transcription factor complexes with a range of DNA binding and activation potentials. In unstimulated cells NF-{kappa}B exists in the cytoplasm as an inactive complex through association with one of several inhibitory molecules including I{kappa}B{alpha}, I{kappa}B{beta}, p105/I{kappa}B{gamma}, I{kappa}B{epsilon}, and p100. NF-{kappa}B can be activated by a range of stimuli including inflammatory cytokines, phorbol esters, lipopolysaccharide, viruses, UV light, and a variety of mitogens (10). These stimuli activate the I{kappa}B kinase (IKK)1 complex either directly or indirectly through activation of upstream protein kinases such as NF-{kappa}B-inducing kinase (11, 12), protein kinase B (13, 14), MEKK1, MEKK2, and MEKK3 (15-18). The IKK complex itself comprises two catalytic subunits, IKK{alpha} and IKK{beta}, and a structural component called IKK{gamma} (or NEMO (NF-{kappa}B essential modulator) (19-22). The activated IKK phosphorylates the inhibitory {kappa}B proteins on either serine residues 32 and 36 of I{kappa}B{alpha} or serine residues 19 and 23 of I{kappa}B{beta}. This phosphorylation targets the I{kappa}Bs for ubiquitin-dependent degradation through the 26 S proteasome complex, resulting in the release and nuclear translocation of NF-{kappa}B (23).

Recent studies provide compelling evidence that NF-{kappa}B suppresses cell death in response to a variety of apoptotic stimuli (9). For example, the disruption of genes encoding components of the NF-{kappa}B signaling pathway results in early embryonic lethality, with the mouse embryos displaying increased apoptosis in various organs and tissues (9). Candidate antiapoptotic genes targeted by NF-{kappa}B include the mitochondrial membrane-stabilizing proteins Blf-1 (24) and Bcl-xL (25, 26), the caspase 8 inhibitor FLIP (27), the immediate early response gene IEX-1L (28), and the inhibitor of apoptosis proteins cIAP1, cIAP2, and XIAP (29, 30). Recent studies suggest that NF-{kappa}B can also inhibit tumor necrosis factor-{alpha}-induced apoptosis by inhibiting activation of the c-Jun NH2-terminal kinase via a poorly defined mechanism involving increased expression of either GADD{beta} or XIAP (31, 32). In this report, we show that NF-{kappa}B is activated in HeLa cells in response to both microtubule-depolymerizing and microtubule-stabilizing anticancer agents. We also show that inhibition of NF-{kappa}B using three independent approaches selectively increases the susceptibility of mitotically arrested cells to anticancer drug-induced apoptosis. Our data suggest that NF-{kappa}B activation is linked to cell survival through the activation of NF-{kappa}B-responsive antiapoptotic genes and may represent an important survival mechanism during mitotic cell cycle arrest.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Preparation of Cell Extracts—Human cervical carcinoma cells (HeLa) were cultured as described previously (33). To obtain mitotically arrested cells, an asynchronous population of HeLa cells was treated with either nocodazole (3 µM), taxol (1 µM), vincristine (1 µM), or vinblastine (1 µM) for various times (0-30 h). Mitotic cells were collected by mechanical shake-off, washed in Dulbecco's phosphate-buffered saline, and lysed in the appropriate buffer depending on the protein kinase being assayed as described previously (33). Radioimmunoprecipitation buffer containing 1% (v/v) Triton X-100 was used to prepare cell lysates for immunoblotting with the I{kappa}B{alpha} antibodies. Cells remaining attached to the plates after shake-off were washed three times with Dulbecco's phosphate-buffered saline (containing either nocodazole (3 µM), taxol (1 µM), vincristine (1 µM), or vinblastine (1 µM)) and lysed by scraping into the appropriate buffer. Cell lysates were centrifuged at 14,000 x g for 10 min at 4 °C, and the protein content of the supernatant was determined using the Coomassie protein assay reagent (Perbio Science UK Ltd., Cheshire, UK) before normalization and solubilization in 2x SDS-PAGE sample buffer.

Antibodies—IKK{alpha}, IKK{beta}, I{kappa}B{alpha}, anti-hemagglutinin epitope tag (HA), and phosphospecific histone H3 that was used as a marker for mitotic cells (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Anti-FLAG (M2), Cy3-conjugated anti-FLAG (M2), anti-{gamma}-tubulin, anti-{beta}-actin, horseradish peroxidase-conjugated goat anti-mouse, horseradish peroxidase-conjugated goat anti-rabbit, and horseradish peroxidase-conjugated mouse anti-goat were from Sigma. Anti-M30, which detects caspase-cleaved cytokeratin 18 (34) was from Roche Applied Science. Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 594 rabbit anti-mouse IgG were from Molecular Probes (Eugene, OR). Phosphospecific anti-I{kappa}B{alpha} was from New England Biolabs. Mouse anti-PkTag was from Serotec (Oxford, UK).

Plasmids—The plasmid expressing hemagglutinin (HA)-tagged ubiquitin was provided by Dirk Bohmann (Scripps, CA). Plasmids expressing both wild type PkTag I{kappa}B{alpha} and mutant (S32A/S36A) PkTag I{kappa}B{alpha} were provided by Ron Hay (University of St. Andrews, Scotland, UK). Mammalian constructs expressing the wild type IKKs (FLAG-tagged), the FLAG-tagged mutant IKKs (K44A), and the construct for expressing recombinant glutathione S-transferase-I{kappa}B{alpha} fusion protein (amino acids 0-200) were provided by David Goeddel (Tularik, CA) (19).

Immunoprecipitation and Western Blot Analysis—Immunoprecipitations were performed as described previously (35). The immunoprecipitated proteins and the cell extracts were resolved by SDS-PAGE and electroblotted onto Hybond-C nitrocellulose membrane using a Hoeffer semidry blotting apparatus (Amersham Biosciences). Immunoreactive proteins were visualized using ECL (Amersham Biosciences).

Protein Kinase Assays—Immunecomplex kinase assays for the IKKs were performed as described previously (35) using glutathione S-transferase-I{kappa}B{alpha} as substrate.

Transfection of HeLa Cells—HeLa cells were plated at a density of ~5 x 105 cells/well in a 6-well plate or at 1 x 105 cells/well for coverslips in an 8-well plate. 18-24 h later, the cells were transfected with 1-4 µg of DNA using FuGENE 6 according to the manufacturer's instructions (Roche Applied Sciences). At 18-24 h after transfection, the cells on the coverslips were fixed in cold (-20 °C) methanol, washed twice with Dulbecco's phosphate-buffered saline, and then processed for immunofluorescence microscopy as described below. The cells in the 6-well plates were either washed twice with Dulbecco's phosphate-buffered saline and lysed in the appropriate buffer to make cell extracts or collected (floating and attached cells) for the determination of apoptosis as described above.

Reporter Assays—For transient transfection of reporter plasmids, cells were plated in 12-well plates, and the following day, cells were co-transfected with a luciferase reporter plasmid containing three NF-{kappa}B binding sites (Stratagene) in combination with the internal control cytomegalovirus Renilla luciferase plasmid (provided by Anne Willis, University of Leicester). Luciferase activities of reporter plasmids were measured using the Dual-Luciferase Reporter Assay System (Promega UK, Southampton, UK).

Immunofluorescence Microscopy—Immunofluorescence microscopy was conducted as described previously (4).

Peptide Synthesis and Purification—The amino acid sequence of both the wild type and the mutant (W739A/W714A) NEMO-binding domain (NBD) peptide fused to the Antennapedia homeodomain was as described (36). Crude synthetic NBD peptides (wild type and mutant) were synthesized by Pepceuticals Ltd. (Leicester, UK). They were further purified using reverse-phase HPLC chromatography (Agilent Technologies UK Ltd., Stockport, Cheshire, UK) and concentrated by freeze-drying. The quality of the purified peptides was assessed using an analytical HPLC column (PerkinElmer Life Sciences) and by electrospray mass spectrometry on a Micromass Platform-II instrument (Waters Ltd., Manchester, UK).

Densitometry—Autoradiograms were scanned and quantified using a {beta}-Imaging Computing Densitometer (Amersham Biosciences) with MD ImageQuant software (version 3.3).

Reagents—Nocodazole, taxol, vincristine, vinblastine, MG-132 (N-benzyloxycarbonyl-Leu-Leu-leucinal), and ALLN (N-acetyl-Leu-Leu-norleucinal) were all obtained from Sigma. All other reagents were of analytical grade and obtained from Sigma.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NF-{kappa}B Activation in Response to Anti-microtubule Drugs Is Mediated by the IKK and I{kappa}B Signaling Pathway—To investigate whether microtubule depolymerization activates NF-{kappa}B signaling, we treated exponentially growing HeLa cells with nocodazole, a reversible microtubule-depolymerizing compound (1). Cytoplasmic extracts were assayed for the activation of both IKK{alpha} and IKK{beta} using immune complex kinase assays. As shown in Fig. 1A, nocodazole treatment rapidly activated (within 15 min) both IKK{alpha} (1.8-fold activation above the basal level) and IKK{beta} (2.1-fold above the basal level). Both IKK{alpha} and IKK{beta} were increasingly activated for up to 6 h after nocodazole addition. From 12 h onward, we were able to separate the nocodazole-treated cells into a mitotic population (as assessed by increased phosphorylation of histone H3) (37) and an attached, nonmitotic population (as assessed by low phosphorylation of histone H3). As shown in Fig. 1A, both IKK{alpha} and IKK{beta} were highly activated in the mitotically arrested cells (~10-14-fold above basal levels, respectively) in comparison with the attached cell population (2.5-2.1-fold above basal levels, respectively). The activation of either IKK{alpha} or IKK{beta} could not be attributed to changes in their expression levels as indicated by immunoblots of the cytoplasmic extracts with the IKK-specific antibodies (Fig. 1A). The activation of both IKK{alpha} and IKK{beta} by nocodazole correlated with microtubule depolymerization as determined by immunocytochemistry with an {alpha}-tubulin antibody (Fig. 1B). Although considerable microtubule depolymerization was observed within 15 min of nocodazole addition, a more homogeneous distribution of {alpha}-tubulin was observed between 1 and 6 h after nocodazole addition (Fig. 1B). To determine whether the activation of both IKK{alpha} and IKK{beta} was nocodazole-specific, we additionally treated exponentially growing HeLa cells with either vincristine or vinblastine (to depolymerize microtubules) or with taxol (to stabilize microtubules). All four treatments caused activation of both IKK{alpha} and IKK{beta} in the attached cells and to a greater extent in the mitotic population (Fig. 1C), indicating that both microtubule depolymerization and microtubule stabilization can cause activation of the IKKs.



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  		FIG. 1.
Anti-microtubule drug-induced activation of the IKKs correlates with microtubule depolymerization. A, exponentially growing HeLa cells were treated with either Me2SO (t = 0 h represents asynchronous cells) or with nocodazole (3 µM) for the indicated times. 12 h after nocodazole treatment, the cells were separated into a mitotic (Mit) and an attached population (Att). Specific antibodies were used to immunoprecipitate (IP) IKK{alpha} and IKK{beta} from the cell lysates for either immune complex kinase assays or Western blots (WB) with anti-IKK{alpha}, anti-IKK{beta}, or anti-phosphohistone H3 (a marker of mitotic cells). A sample of the 12-h mitotic cell lysate was also incubated with protein A-Sepharose beads alone (lane C, control immunoprecipitation). The autoradiograms of the protein kinase assays were scanned by densitometry, and the -fold activation of the IKKs (compared with the asynchronous cells) is indicated. B, an asynchronous population of HeLa cells was treated with nocodazole for the indicated times, fixed, and stained with an {alpha}-tubulin antibody as described under "Materials and Methods." C, exponentially growing HeLa cells were treated with either nocodazole (3 µM), vincristine, vinblastine, or taxol (all 1 µM) for 12 h, and the cells were separated into mitotic and attached populations prior to performing immunocomplex kinase assays and Western blots. Asy, asynchronous cells. The -fold activation of IKK{alpha} and IKK{beta} is indicated.

 
The activation of both IKK{alpha} and IKK{beta} by nocodazole also correlated with the phosphorylation and degradation of I{kappa}B{alpha}. As shown in Fig. 2A, nocodazole treatment increased the phosphorylation of I{kappa}B{alpha} on serine 32. The increase in the phosphorylation of I{kappa}B{alpha} above basal levels was observed between 15 and 30 min after nocodazole treatment. The phosphorylation of I{kappa}B{alpha} was sustained for up to 6 h and correlated with the degradation of I{kappa}B{alpha}. Our results also show that although the IKKs are highly active in the mitotically blocked cells (Fig. 1A), the phosphorylation of I{kappa}B{alpha} was reduced in this cell population (Fig. 2A). As observed with nocodazole, treatment of HeLa cells with either vincristine, vinblastine, or taxol also resulted in the phosphorylation and degradation of I{kappa}B{alpha} (Fig. 2B), although I{kappa}B{alpha} appeared to be degraded slightly less efficiently in the presence of taxol.



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  		FIG. 2.
Anti-microtubule drugs induce the phosphorylation and degradation of I{kappa}B{alpha} A, exponentially growing HeLa cells were treated with nocodazole (3 µM), and cell extracts were prepared at the indicated times after nocodazole addition. After 12 h, the cells were separated into mitotic and attached populations as described under "Materials and Methods." The cell extracts were immunoblotted (WB) with a phosphospecific I{kappa}B{alpha} antibody, an I{kappa}B{alpha} antibody, a {gamma}-tubulin antibody, or a phosphospecific histone H3 antibody. B, exponentially growing HeLa cells were treated with either nocodazole (3 µM), vincristine, vinblastine, or taxol (all 1 µM) for 12 h, and the cells were separated into mitotic and attached populations prior to immunoblotting with a phosphospecific I{kappa}B{alpha} antibody, an I{kappa}B{alpha} antibody, a {gamma}-tubulin antibody, or a phosphospecific histone H3 antibody.

 
To determine whether the degradation of I{kappa}B{alpha} by nocodazole resulted in the nuclear translocation of NF-{kappa}B, we analyzed the intracellular distribution of native p65 (RelA) by immunocytochemistry. As shown in Fig. 3A, p65 was distributed uniformly throughout the cell prior to the addition of nocodazole but was found primarily in the nucleus following nocodazole treatment. We performed a time course analysis of the nuclear accumulation of p65 in the nonmitotic, attached cells following nocodazole treatment. Our results indicate that p65 translocated to the nucleus between 30 and 60 min after nocodazole treatment, with maximum nuclear localization occurring 6 h after nocodazole treatment (Fig. 3B). Between 6 and 24 h after nocodazole treatment, the number of cells showing a nuclear localization of p65 declined, although this phenotype remained above basal levels. Control cells treated with Me2SO alone did not show any change in the distribution of the p65 protein during the time course examined. In mitotic cells (collected at 12-24 h after nocodazole treatment), p65 was distributed uniformly throughout the cytoplasm (data not shown).



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  		FIG. 3.
Nocodazole induces the translocation of p65 (RelA) into the nucleus. A, exponentially growing HeLa cells were either treated with nocodazole (3 µM) for 6 h or Me2SO-treated. The attached, nonmitotic cells were then fixed and immunostained with a specific antibody to detect the localization of native p65 (RelA) as described under "Materials and Methods." Indirect immunofluorescence of p65 (left panels), DNA staining (middle panels), and the merged images (right panels) are shown. B, quantitation of the data shown in A at the indicated times following nocodazole treatment in the attached cell population. Control cells (Con) were treated with an equivalent volume of Me2SO. At least 100 cells were counted in randomly selected fields for each time point, and the bars represent the mean ± S.D. of three independent experiments.

 
To examine whether the nuclear accumulation of p65 (RelA) resulted in an increase in NF-{kappa}B-dependent transcription, HeLa cells were transfected with a luciferase reporter gene that is regulated by three tandem repeats of an NF-{kappa}B enhancer element (3x pNF-{kappa}B-Luc). As shown in Fig. 4, nocodazole treatment stimulated a time-dependent increase in luciferase activity in both the attached and the mitotically arrested cells. Taken together, our results suggest that the perturbation of microtubule dynamics leads to the activation of NF-{kappa}B-dependent gene expression through an IKK/I{kappa}B{alpha}-dependent signaling pathway.



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  		FIG. 4.
Nocodazole induces expression of an NF-{kappa}B-dependent reporter gene. Exponentially growing HeLa cells were co-transfected with an NF-{kappa}B/luciferase reporter construct (1 µg) and a Renilla luciferase reporter construct (1 µg) in duplicate. 24 h after transfection, the cells were treated with nocodazole (3 µM) for the indicated times. Cell extracts were prepared after separation of the attached and the mitotic populations at the indicated times, and the reporter activity was measured as described under "Materials and Methods." The -fold increase in NF-{kappa}B luciferase activity was calculated relative to the expression of Renilla luciferase in Me2SO-treated control cells at each time point. Each bar represents the mean ± S.D. of three independent experiments.

 
Dominant Negative IKK{alpha}, IKK{beta}, and Nondegradable I{kappa}B{alpha} Suppress Nocodazole-induced Activation of NF-{kappa}B and Reduce the Survival of Nocodazole-blocked Mitotic Cells—To determine whether NF-{kappa}B was involved in regulating the survival of nocodazole-arrested mitotic cells, signaling-defective mutants of both IKK{alpha} (K44A) and IKK{beta} (K44A) were used. Transfection of HeLa cells with MEKK1, a known activator of NF-{kappa}B (16), resulted in a 5-fold stimulation of luciferase activity in an NF-{kappa}B reporter assay (Fig. 5A). The MEKK1-stimulated luciferase activity was comparable with that observed with either wild type (Fig. 5, wt) IKK{alpha} (5.1-fold in the attached cells; 4.3-fold in the mitotic cells) or wild type IKK{beta} (5.4-fold in the attached cells; 6.3-fold in the mitotic cells) after nocodazole treatment. However, in the presence of either IKK{alpha} (K44A) or IKK{beta} (K44A), both basal and nocodazole-stimulated luciferase activity was reduced when compared with their respective wild type controls (IKK{alpha} (K44A) percentage reduction as follows: asynchronous cells, 90%; attached cells, 76%; mitotic cells, 64%; IKK{beta} (K44A) percentage reduction as follows: asynchronous cells, 90%; attached cells, 95%; mitotic cells, 90%). The reduction of luciferase activity by either dominant negative IKK{alpha} or IKK{beta} could not be attributed to variations in the expression levels of the recombinant proteins as shown by immunoblots of cell extracts with IKK-specific antibodies (Fig. 5B). To determine the function of NF-{kappa}B signaling following nocodazole treatment, HeLa cells were transfected with either wild type IKK{alpha} or IKK{beta} or with either dominant negative IKK{alpha} or IKK{beta}. As shown in Fig. 5C, treatment of control, mock-transfected cells with nocodazole caused 27% of the mitotically arrested cells to undergo apoptosis, whereas the attached cells remained viable (2.8% apoptotic cells). Similarly, in cells expressing either wild type IKK{alpha} or IKK{beta}, nocodazole treatment caused ~41 and 39.5% of the mitotically arrested cells to undergo apoptosis, respectively. However, in cells expressing either dominant negative IKK{alpha} or IKK{beta}, nocodazole treatment resulted in 71 and 74% of the mitotically arrested cells undergoing apoptosis. Neither the wild type nor the dominant negative IKKs greatly affected the survival of the nocodazole-treated attached cells.



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  		FIG. 5.
Dominant negative IKKs inhibit the nocodazole-induced activation of NF-{kappa}B and potentiate nocodazole-induced apoptosis in mitotically arrested cells. A, exponentially growing HeLa cells were transfected with either the wild type (wt) or dominant negative (K44A) IKK{alpha} or IKK{beta} or wild type MEKK1 (all 1 µg) together with an NF-{kappa}B/luciferase reporter construct (1 µg) and a Renilla luciferase reporter construct (1 µg) in duplicate. 24 h after transfection, the cells were treated with either nocodazole (3 µM) or an equivalent volume of Me2SO (Asy) for 12 h. Cell extracts were prepared after separation of the attached (Att) and the mitotic (Mit) populations, and the reporter activity was measured as described under "Materials and Methods." Each bar represents the mean ± S.D. of three independent experiments. B, cell extracts prepared in A were immunoblotted (WB) with either a FLAG antibody to assess expression of the IKKs or a {gamma}-tubulin antibody. Lane C, mock-transfected cells. C, HeLa cells were transfected with either the wild type or K44A IKK{alpha} or IKK{beta}. 24 h after transfection, the cells were treated with nocodazole (3 µM) for 12 h. The mitotic cells were removed by "shake-off" and reattached to poly-D-lysine-coated coverslips. The mitotic and attached cells were fixed and immunostained with a Cy3-conjugated anti-FLAG antibody to identify the transfected cells and with the M30 antibody to identify the apoptotic cells as described under "Materials and Methods." At least 100 FLAG-positive cells were counted in randomly selected fields, and the bars represent the mean ± S.D. of three independent experiments. Control, mock-transfected cells.

 
To further investigate the relationship between NF-{kappa}B activation and the survival of nocodazole-arrested mitotic cells, we transfected cells with either wild type I{kappa}B{alpha} or a mutant, nondegradable I{kappa}B{alpha} (S32A/S36A) (38). As shown in Fig. 6A, nondegradable I{kappa}B{alpha} reduced nocodazole-stimulated luciferase activity by 82% in the attached cells and by 78% in the mitotically arrested cells when compared with wild type I{kappa}B{alpha}. The reduction in luciferase activity by the nondegradable I{kappa}B{alpha} could not be attributed to variations in the level of expression of the mutant I{kappa}B{alpha} when compared with the wild type protein (Fig. 6B). Expression of nondegradable I{kappa}B{alpha} selectively increased the level of apoptosis in the nocodazole-arrested mitotic cells (79% apoptosis compared with 41% apoptosis in cells expressing wild type I{kappa}B{alpha}), without affecting the survival of the nocodazole-treated attached cells (Fig. 6C).



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  		FIG. 6.
Dominant negative I{kappa}B{alpha} inhibits the nocodazole-induced activation of NF-{kappa}B and potentiates nocodazole-induced apoptosis in mitotically arrested cells. A, exponentially growing HeLa cells were transfected with either the wild type (wt) or nondegradable (S32A/S36A) I{kappa}B{alpha} (both 1 µg) together with an NF-{kappa}B/luciferase reporter construct (1 µg) and a Renilla luciferase reporter construct (1 µg) in duplicate. 24 h after transfection, the cells were treated with either nocodazole (3 µM) or with an equivalent volume of Me2SO (Asy) for 12 h. Cell extracts were prepared after separation of the attached (Att) and the mitotic (Mit) populations, and the reporter activity was measured as described under "Materials and Methods." Each bar represents the mean ± S.D. of three independent experiments. B, cell extracts prepared in A were immunoblotted (WB) with either a PkTag antibody to assess expression of I{kappa}B{alpha} or with a {gamma}-tubulin antibody. C, HeLa cells were transfected with either the wild type or S32A/S36A I{kappa}B{alpha}. 24 h after transfection, the cells were treated with nocodazole (3 µM) for 12 h. The mitotic cells were removed by shake-off and reattached to poly-D-lysine-coated coverslips. The mitotic and attached cells were fixed and immunostained with the PkTag antibody to identify the transfected cells and with the M30 antibody to identify the apoptotic cells as described under "Materials and Methods." At least 100 FLAG-positive cells were counted in randomly selected fields, and the bars represent the mean ± S.D. of three independent experiments. Control, mock-transfected cells.

 
The NBD Peptide Inhibits Nocodazole-induced Activation of NF-{kappa}B and Reduces the Survival of Nocodazole-blocked Mitotic Cells—As a second approach to suppress nocodazole-induced activation of NF-{kappa}B, we used the NBD peptide, a known inhibitor of the IKKs (36). Treatment of exponentially growing HeLa cells with nocodazole for 3 h caused p65 to translocate to the nucleus in ~32% of the cells (Fig. 7A). However, treatment of cells with 100 µM NBD peptide reduced nocodazole-induced p65 translocation into the nucleus by 80% to near basal levels, whereas treatment of cells with a mutant NBD peptide caused a small but statistically significant (p < 0.05) increase in nocodazole-induced p65 translocation into the nucleus (Fig. 7A). The inhibition of p65 nuclear translocation by the NBD peptide was not restricted to nocodazole treatment but was also observed with other NF-{kappa}B inducers such as tumor necrosis factor-{alpha} (data not shown). To determine the effect of the NBD peptide on the survival of the nocodazole-treated cells, exponentially growing HeLa cells were treated with nocodazole in the presence of varying concentrations of either the wild type or the mutant NBD peptide. As shown in Fig. 7B, the wild type NBD peptide, but not the mutant peptide, caused a dose-dependent increase in apoptosis in the mitotically arrested cells but not in the attached cell population.



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  		FIG. 7.
The NBD peptide inhibits nocodazole-induced translocation of p65 (RelA) into the nucleus and potentiates nocodazole-induced apoptosis in the mitotically arrested cells. A, exponentially growing HeLa cells were pretreated with a 100 µM concentration of either the wild type (wt) or mutant NBD peptide or with an equivalent volume of Me2SO (Control) for 2 h prior to nocodazole treatment (3 µM) for 6 h. The cells were then fixed and immunostained with a specific antibody to detect the localization of native p65 (RelA) as described under "Materials and Methods." At least 100 cells were counted in randomly selected fields for each time point, and the bars represent the mean ± S.D. of three independent experiments. *, p < 0.05 (analysis of variance) compared with control nocodazole-treated cells. B, graph showing the effect of the NBD peptide on nocodazole-induced apoptosis in the mitotically arrested cells. Exponentially growing HeLa cells were pretreated with the indicated concentrations of either the wild type or mutant NBD peptide or with an equivalent volume of Me2SO (Control) for 2 h prior to nocodazole treatment (3 µM) for 12 h. The mitotic cells were removed by shake-off and reattached to poly-D-lysine-coated coverslips. The mitotic and attached cells were fixed and immunostained with the M30 antibody to identify the apoptotic cells as described under "Materials and Methods." At least 100 cells were counted in randomly selected fields, and the bars represent the mean ± S.D. of three independent experiments. The percentage of apoptotic cells (mean ± S.D., n = 3) following treatment with the NBD peptide alone for 12 h was as follows: Me2SO control, 2.4 ± 0.8%; 200 µM NBD (wild type), 4.4 ± 2.3%; 200 µM NBD (mutant), 4.1 ± 2.1%.

 
Proteasome Inhibitors Block Nocodazole-induced Activation of NF-{kappa}B and Reduce the Survival of Nocodazole-blocked Mitotic Cells—Finally, we have used inhibitors of the 26 S proteasome to inhibit nocodazole-induced degradation of I{kappa}B{alpha} and suppress activation of NF-{kappa}B (38-41). As shown in Fig. 8A, treatment of exponentially growing HeLa cells with nocodazole resulted in the time-dependent degradation of I{kappa}B{alpha}. The nocodazole-induced degradation of I{kappa}B{alpha} was inhibited by the proteasome inhibitors MG132 or ALLN (42) but not the protease inhibitors leupeptin or PMSF (Fig. 8A). The inhibition of I{kappa}B{alpha} degradation by MG132 either in the absence (Fig. 8B, lane 4) or presence of nocodazole (Fig. 8B, lane 3) correlated with the appearance of higher molecular weight forms of I{kappa}B{alpha} when compared with control, untreated cells (Fig. 8B, lane 1)or following treatment of cells with nocodazole only (Fig. 8B, lane 2). The low molecular weight bands (indicated by an asterisk) observed in the I{kappa}B{alpha} immunoblot have also been observed in a previous study (38) and are thought to represent intermediates in the degradation of I{kappa}B{alpha}. These intermediate products of I{kappa}B{alpha} degradation were lost following nocodazole treatment (Fig. 8B, lane 2), but their levels were maintained in cells treated with MG132 either in the presence or absence of nocodazole (Fig. 8B, lanes 3 and 4). To further demonstrate that MG132 inhibits degradation of ubiquitinated I{kappa}B{alpha}, we co-transfected HeLa cells with HA-ubiquitin and PkTag-I{kappa}B{alpha}. Immunoblots of the anti-HA immunoprecipitates from control, untreated cells (Fig. 8C, lane 1) indicated the presence of monoubiquitinated I{kappa}B{alpha} (~48 kDa) when compared with native I{kappa}B{alpha} (~40 kDa) (Fig. 8C, lane marked cell lysate). Anti-HA immunoprecipitates from cells transfected with PkTag-I{kappa}B{alpha} alone indicated the presence of IgG bands only (Fig. 8C, lane 5). Treatment with nocodazole resulted in the degradation of ubiquitinated I{kappa}B{alpha} (Fig. 8C, lane 2) that was suppressed by MG132 (Fig. 8C, lanes 3 and 4).



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  		FIG. 8.
Proteasome inhibitors suppress the nocodazole-induced degradation of I{kappa}B{alpha} and potentiate nocodazole-induced apoptosis in the mitotically arrested cells. A, HeLa cells were pretreated with Me2SO, 100 µM ALLN, 5 µM MG132, 100 µM PMSF, or 250 µM leupeptin for 1 h prior to nocodazole treatment (3 µM) for the indicated times. The cell lysates were then immunoblotted (WB) with an I{kappa}B{alpha} antibody. B, HeLa cells were pretreated with 5 µM MG132 or an equivalent volume of Me2SO for 1 h prior to nocodazole treatment (3 µM) for 6h. The cell lysates were then immunoblotted with either an I{kappa}B{alpha} antibody or a {gamma}-tubulin antibody. The positions of the molecular mass markers (kDa) are indicated on the left. The slower migrating and faster migrating forms of I{kappa}B{alpha} are indicated by the bracket and asterisk, respectively. C, HeLa cells were co-transfected with PkTag-I{kappa}B{alpha} (1 µg) and HA-ubiquitin (1 µg) (lanes 1-4) or with PkTag-I{kappa}B{alpha} alone (lane 5). 24 h after transfection, the cells were pretreated with 5 µM MG132 or an equivalent volume of Me2SO for 1 h prior to nocodazole treatment (3 µM) for 6 h. The cell lysates were immunoprecipitated with an anti-HA antibody, and the immunoprecipitated proteins were immunoblotted with the PkTag antibody. The positions of the molecular mass markers (kDa) are indicated on the left. The positions of the native and ubiquitinated I{kappa}B{alpha} and the cross-reacting IgG bands are indicated on the right. Cell lysate, cell lysate prepared from mock-transfected cells. D, HeLa cells were pretreated with 5 µM MG132 or an equivalent volume of Me2SO for 1 h prior to nocodazole treatment (3 µM) for 12 h. The mitotic cells were recovered by shake-off and reattached onto poly-D-lysine-coated coverslips. The mitotic cells were fixed and immunostained with the M30 antibody to identify the apoptotic cells as described under "Materials and Methods." Shown are indirect immunofluorescence showing M30 staining (upper panels) and DNA staining (middle panels) and the merged images (lower panels). E, a graph showing the effect of proteasome inhibitors on the survival of nocodazole-arrested mitotic cells. HeLa cells were pretreated with either Me2SO or with the indicated concentrations of ALLN, MG132, PMSF, or leupeptin for 1 h prior to nocodazole treatment (3 µM) for 12 h. The mitotic cells were removed by shake-off and reattached to poly-D-lysine-coated coverslips. The mitotic and attached cells were fixed and immunostained with the M30 antibody to identify the apoptotic cells as described under "Materials and Methods." At least 100 cells were counted in randomly selected fields, and the bars represent the mean ± S.D. of three independent experiments. In cells treated with the inhibitors alone for 12 h, the percentage of apoptotic cells was as follows (mean ± S.D., n = 3): Me2SO control, 1.9 ± 1.1%; 10 µM MG132, 3.8 ± 1.5%; 200 µM ALLN, 4.6 ± 0.9%; 200 µM PMSF, 3.0 ± 0.5%; 500 µM leupeptin, 3.3 ± 0.4%.

 
To determine the effect of MG132 on nocodazole-induced apoptosis, HeLa cells were treated with either nocodazole or with both nocodazole and MG132. As shown in Fig. 8D, MG132 enhanced nocodazole-induced apoptosis as assessed by cleavage of cytokeratin 18. Quantitation of the data shown in Fig. 8D indicated that MG132 caused a dose-dependent increase in nocodazole-induced cell death selectively in the mitotically arrested cells and not in the attached, nonmitotic cells (Fig. 8E). Similarly, ALLN, but not PMSF or leupeptin, also enhanced nocodazole-induced apoptosis preferentially in the mitotic cell population. Treatment of cells with the inhibitors alone did not increase apoptosis significantly above basal levels. Together, these results suggest that activation of NF-{kappa}B by microtubule depolymerizing agents and the corresponding increase in NF-{kappa}B-dependent gene expression may be a mechanism by which NF-{kappa}B contributes to the survival of mitotically arrested cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous work has clearly demonstrated that microtubule depolymerization leads to the activation of NF-{kappa}B and NF-{kappa}B-dependent gene expression (5). What has remained unclear is the functional consequence of activating this particular signaling pathway. In this study, we have identified a number of components in the NF-{kappa}B signaling pathway that are activated by anti-microtubule drugs and demonstrate that one functional consequence of activating this signaling pathway is to selectively aid the survival of mitotically arrested cells.

We have shown in this study that a range of compounds that either depolymerize or stabilize microtubules can activate the NF-{kappa}B signaling pathway. Consistent with previous work (5), we have shown that the phosphorylation, ubiquitination, and degradation of I{kappa}B{alpha} by the anti-microtubule drugs accompany the activation of NF-{kappa}B. Furthermore, we now demonstrate that the activation of NF-{kappa}B by the anti-microtubule drugs is also dependent on the activation of the IKKs. Recent studies have shown that whereas both IKK{alpha} and IKK{beta} are critical for NF-{kappa}B-mediated gene expression, only IKK{beta} appears to be critical for I{kappa}B degradation (43-45). IKK{alpha} appears to regulate NF-{kappa}B-mediated gene expression independently of I{kappa}B through direct interaction with the promoters of NF-{kappa}B-responsive genes and phosphorylation of histone H3 (46, 47). Our analysis of IKK activation following nocodazole treatment indicates that both IKK{alpha} and IKK{beta} are activated to an equal extent and that this activation is biphasic. The early activation of both IKKs is readily reconciled with microtubule depolymerization and their role in NF-{kappa}B-mediated gene expression through either an I{kappa}B-dependent or -independent pathway. The function of the late activation of the IKKs that we observe specifically in the mitotically arrested cells is more difficult to reconcile with changes in gene expression, since both transcription and translation are generally suppressed during mitosis (48, 49). The identity of the protein kinase(s) that activate the IKKs in mitotically arrested cells and their potential function remains to be identified, although there are data to suggest that the activation of the IKKs in mitotically arrested cells may be mediated by caspases. In mammalian cells, the three death domain- containing proteins, caspase-8, caspase-10, and MRIT (Mach-related inducer of toxicity) have been shown to activate IKK by a mechanism that involves an interaction between the prodomain of caspases and the IKKs (50). This caspase-dependent mechanism of IKK activation would be consistent with the observation that caspase 8 is known to be active in taxol-arrested mitotic cells (51, 52).

Although microtubule depolymerization and not microtubule stabilization was originally reported to be the stimulus for NF-{kappa}B activation (5), subsequent studies have shown that the microtubule-stabilizing drug taxol can also activate NF-{kappa}B in mammalian cells (6, 7, 53, 54). In the present study, we have also shown that taxol can activate NF-{kappa}B, as assessed by the increase in the phosphorylation and degradation of I{kappa}B{alpha}. Therefore, we suggest that it is not microtubule depolymerization or stabilization per se that may lead to NF-{kappa}B activation but rather a perturbation of microtubule dynamics. Another explanation for the activation of NF-{kappa}B by both microtubule-depolymerizing and microtubule-stabilizing drugs is that these compounds are thought to activate a common enzyme, such as protein kinase C (6), that subsequently activates NF-{kappa}B. The mechanism by which protein kinase C stimulates NF-{kappa}B is unclear but may involve the activation of the IKKs (55).

The reported interaction between I{kappa}B{alpha} and a microtubule-associated dynein light chain protein (56) may provide the link between changes in microtubule dynamics and activation of NF-{kappa}B. It can be envisaged that the pool of microtubule-bound I{kappa}B{alpha} may act as a sensor of microtubule dynamics that is phosphorylated and degraded upon microtubule depolymerization/stabilization. It is less clear how anti-microtubule drugs stimulate the activation of the IKKs. NF-{kappa}B inducers such as tumor necrosis factor-{alpha}, interleukin-1, or Fas activate the IKKs through upstream protein kinases such as NF-{kappa}B-inducing kinase (12) and the MAP3K kinases including MEKK1, -2, and -3 (15-18). There are data to indicate that MEKK1 is activated in response to microtubule depolymerization (57, 58). However, using immunocomplex kinase assays, we have not observed the activation of MEKK1, MEKK2, or MEKK3 in HeLa cells upon nocodazole treatment (data not shown). Whether it is NF-{kappa}B-inducing kinase or other protein kinases that activate the IKKs in response to the anti-microtubule drugs remains to be determined.

Whereas the precise biochemical mechanism by which perturbation of microtubule dynamics leads to the activation of NF-{kappa}B remains unclear, the current study has established a role for NF-{kappa}B in regulating cell survival during mitotic cell cycle arrest. As outlined in the Introduction, the prevalent hypothesis is that NF-{kappa}B functions as an antiapoptotic factor. In some instances, however, NF-{kappa}B is reported to have a proapoptotic function (53, 54, 59-62), suggesting that the precise effect of NF-{kappa}B may be cell and stimulus-specific. Our data are consistent with the majority view and assign a survival function for NF-{kappa}B. Anti-microtubule drugs cause cells to arrest in mitosis through activation of the mitotic checkpoint, and as demonstrated in this study and in earlier work (1-4), the mitotically arrested cells then undergo apoptosis. Cells that have been treated with the anti-microtubule drugs for an equivalent time but that are not in M phase do not undergo apoptosis, suggesting that activation of the mitotic checkpoint initiates the apoptotic response. The mechanism by which activation of the mitotic checkpoint then leads to cell death is not yet understood. We have recently demonstrated that both proapoptotic signals, such as p38 MAP kinase, and antiapoptotic signals, such as p21-activated kinase, are preferentially activated in the mitotically arrested cells (4). Other work has shown that the antiapoptotic protein Bcl-2 becomes hyperphosphorylated during mitotic cell cycle arrest and may constitute a proapoptotic signal (63). In contrast, the rapid activation of NF-{kappa}B in response to microtubule perturbation may represent a survival signal that is activated prior to activation of the mitotic checkpoint. Whether the activation of NF-{kappa}B and the resulting transcription of antiapoptotic genes are fortuitous or a programmed response to microtubule perturbation that preempts the requirement for survival factors during subsequent mitotic arrest is presently unclear. As outlined in the Introduction, there are a number of antiapoptotic genes that are targeted by NF-{kappa}B. The identity of the NF-{kappa}B-regulated antiapoptotic gene product(s) that contribute to cell survival in response to anti-microtubule drugs is currently under investigation.


   FOOTNOTES
 
* 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

Present address: AstraZeneca R&D Charnwood, Bakewell Rd., Loughborough, Leicestershire LE11 5RH, United Kingdom. Back

|| To whom correspondence should be addressed: Dept. of Biochemistry, University of Leicester, University Rd., Leicester LE1 7RH, United Kingdom. Tel.: 44-0116-252-2740; Fax: 44-0116-252-3699; E-mail: rp31{at}le.ac.uk.

1 The abbreviations used are: IKK, I{kappa}B kinase; NBD, NEMO-binding domain; HPLC, high pressure liquid chromatography; PMSF, phenylmethylsulfonyl fluoride; HA, hemagglutinin. Back



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