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J. Biol. Chem., Vol. 277, Issue 46, 43648-43658, November 15, 2002

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Nocodazole-induced p53-dependent c-Jun N-terminal Kinase Activation Reduces Apoptosis in Human Colon Carcinoma HCT116 Cells^*

Hong Zhang, Xiaoqing Shi, Qian-Jin Zhang^§, Maggie Hampong, Harry Paddon, Dewi Wahyuningsih^¶, and Steven Pelech^¶

From the  Department of Medicine and the ^§ Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, and ^¶ Kinexus Bioinformatics Corporation, Vancouver, British Columbia V6T 1Z4, Canada

Received for publication, April 4, 2002, and in revised form, August 30, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53/ cells in response to short term nocodazole treatment through application of Kinetworks^TM immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53/ cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53/ cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53/ cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Given the pivotal roles of microtubules in numerous biological processes such as mitotic spindle formation, treatment of cells with nocodazole and other microtubule-interfering agents evokes the activation of stress response pathways, cell cycle arrest, and the induction of apoptosis. This accounts for the extensive use of microtubule-interfering agents in tumor chemotherapy. In recent years, a great deal of effort has been devoted to elucidating the signaling pathways that mediate the biological activities of microtubule-interfering agents (1).

The p53 tumor suppressor protein is a short lived transcription factor that serves as a key player in the cellular response to a variety of extra- and intracellular insults, such as DNA damage, oncogenic activation, and microtubule disruption (2, 3). It is known that p53 exerts its function mainly through transcriptional activation of target genes such as the CDK¹ inhibitor, p21^Waf1/Cip1, for arresting the cell cycle and the proapoptotic protein, Bax, for inducing apoptosis (4, 5). Similar to other stresses, microtubule disruption results in an increase of p53 phosphorylation at multiple sites in a drug- and cell-specific manner, with resultant accumulation of transcriptionally active protein (6, 7). Recently, we have demonstrated that nocodazole-induced phosphorylation of p53 at Ser-392, one of its key activating sites, is mediated through direct p38 MAP kinase stimulation of casein kinase 2 (CK2) in the HeLa cervical and HCT116 colon carcinoma cell lines (8, 9).

To explore downstream p53-dependent regulation of signaling proteins in the early response of cells to nocodazole treatment, we applied three of our Kinetworks^TM screens to track quantitatively the expressions of 75 protein kinases and 25 protein phosphatases and the states of 31 known phosphorylation sites in these and other phosphoproteins. This was accomplished by comparing the expression and phosphorylation profiles of these proteins in a human colon carcinoma cell line HCT116 p53+/+ and its derivative HCT116 p53/, where the p53 gene was disrupted through homologous recombination (10, 11). Among the known phosphoproteins tracked, the p53-dependent increase in the phosphorylation and activation of the c-Jun N-terminal kinase (JNK) was the only significant difference between p53+/+ and p53/ cells. Even though JNK is well known to play an important role in coordinating the cellular response to stress by phosphorylating the transcription factors c-Jun and p53 (12, 13), this report is the first time that a p53-mediated JNK activity has been unambiguously identified. In addition, we provide evidence for the possible involvement of the p53-mediated JNK activity in a protective response elicited by the stress of microtubule disruption.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines and Culture Conditions-- HCT116 p53 wild-type (p53+/+) and knockout derivative (p53/) (11) cells were kindly provided by Dr. Bert Vogelstein (Howard Hughes Medical Institute, Baltimore, MD). Human breast carcinoma MCF-7 and MCF-7 p53-deficient derivative, and human lung carcinoma A549 and A549 p53-deficient derivative were from Dr. Michel Roberge (University of British Columbia, Vancouver, BC, Canada). Both MCF-7 and A549 p53-deficient cell lines are derived from E6 oncogene overexpression. Cells were maintained in monolayer culture in a humidified 5% CO₂ atmosphere at 37 °C in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin.

Antibodies and Chemicals-- Anti-JNK1, p53, MEK4, MKP1, MKP2, -actin, and horseradish peroxidase)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-phospho-[Thr-183, Tyr-185]JNK polyclonal antibody was purchased from Promega (Madison, WI). The phospho-[Ser-63]c-Jun antibody was obtained from New England Biolabs (Beverly, MA). GST-c-Jun(1-79)-agarose conjugate was purchased from StressGen Biotechnologies (Victoria, BC, Canada). GST-JNK2 agarose conjugate was obtained from Upstate Biotechnology (Lake Placid, NY). Nocodazole, vinblastine, colchicine, taxol, and sodium arsenate were purchased from Sigma. The JNK inhibitor SP600125 was from Tocris Cookson Ltd. (Bristol, UK). Other reagents were all from commercial sources, unless otherwise stated.

Oligonucleotides, Plasmids, and Cell Transfection-- Fluorescein-labeled phosphorothioate p53 antisense (5'-CCCTGCTCCCCCCTGGCTCC-3') and control nonsense (5'-CGGTGATCTCCAGAGTATGC-3') oligonucleotides were synthesized by the NAPS unit of University of British Columbia on a 349 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). The antisense oligonucleotide is complementary to nucleotides 1071-1090 of exon 10 of the p53 gene, which is located in the C-terminal region that is required for oligomerization of p53 (14). The oligonucleotides were purified twice by ethanol precipitation. Cells were transfected with oligonucleotides at the concentrations indicated using Lipofectin transfection reagent from Invitrogen according to the manufacturer's protocol. The pcDNA3-HA-MEK4(AL) dominant negative mutant plasmid was provided by Dr. Jim Woodgett (Ontario Cancer Institute, ON, Canada), and pLNCX vectors containing wild-type and dominant negative mutants (APF) of JNKs, HAp40JNK1, , HAp40JNK2, , were gifts from Dr. Lynn Heasley (University of Colorado, Denver) (15). Transfections of HCT116 cells with these constructs were performed using LipofectAMINE Plus reagent (Invitrogen).

Apoptosis Assays-- For flow cytometry analyses of DNA staining profile, transfected or nontransfected cells in 100-mm dishes at ~60-80% confluence were treated with 200 ng/ml nocodazole. At various times as indicated under "Results," the cells were harvested by trypsin treatment, combined with floating cells in the medium, washed once in phosphate-buffered saline, and fixed in methanol for 30 min at 20 °C. After three washes in phosphate-buffered saline, the cells were resuspended in phosphate-buffered saline containing 25 µg/ml RNase A and 25 µg/ml propidium iodine at 37 °C for 30 min. The DNA fluorescence was measured using a BD Biosciences FACScan; data acquisition and analysis were performed with the Cell Quest software. DNA fragmentation assays were performed as described by Huang et al. (16).

Western Blot Analysis-- Total cell lysates were prepared as described previously (17). Briefly, cells were washed with ice-cold phosphate-buffered saline, scraped in lysis buffer (20 mM Tris, 20 mM -glycerophosphate, 150 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM Na₃VO₄, 0.5% Nonidet P-40, 1 mM dithiothreitol) supplemented with 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 4 µg/ml aprotinin, and 1 µg/ml pepstatin A, sonicated for 15 s. Cell debris was removed by centrifugation at 13,000 rpm for 15 min at 4 °C. Protein concentration was determined by the method of Bradford (18). Aliquots of cell lysates were resolved on SDS-PAGE (13% gel), transferred to nitrocellulose membranes, and incubated with various primary antibodies followed by relevant horseradish peroxidase-conjugated secondary antibodies. The blots were developed with ECL Plus reagent (Amersham Biosciences), and signals were then captured by Fluor-S MultiImager and quantified using Quantity One software (Bio-Rad).

Kinetworks^TM Analyses-- For the preparation of cytosolic and particulate fractions for Kinetworks^TM analyses, cells were homogenized using a Dounce homogenizer in the above lysis buffer without NaCl and Nonidet P-40. After ultracentrifugation at 100,000 rpm for 30 min at 4 °C, the supernatant was collected as a cytosolic fraction. The pellet fraction was then rehomogenized in lysis buffer with NaCl and 0.5% Nonidet P-40. After ultracentrifugation, the detergent-solubilized supernatant was saved as a particulate fraction. Kinetworks^TM analyses were performed on 300-600 µg of protein/sample. The Kinetworks^TM analyses carried out included KPKS 1.0 for 75 protein kinases, KPPS 1.1 for 25 protein phosphatases, and KPSS 1.0 for 33 phosphoproteins. The immunoblotting analyses involved probing with mixes of in-house validated primary antibodies from commercial sources and the application of each mix into a separate lane of a 20-lane multiblotter (Immunetics). Detailed protocols for the Kinetworks^TM analyses can be found at the Kinexus Bioinformatics website (www.kinexus.ca).

In Vitro Kinase Activity Assay-- For JNK kinase assay, endogenous JNK was immunoprecipitated from cell lysate using anti-JNK1 antibody. In vitro JNK kinase assay was performed as described (19). The JNK activity was assayed by phosphorylation of GST-c-Jun(1-79), as revealed by Western blot analysis using anti-phospho-[Ser-63]c-Jun antibody. MEK4 kinase activity was assayed similarly except using GST-JNK2 as substrate, instead of GST-c-Jun, whose phosphorylation was detected by Western blotting with anti-phospho-[Thr-183,Tyr-185]JNK antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Different Phosphorylation Profiles of Signaling Proteins Revealed by Kinetworks^TM Analysis May Account for the Different Sensitivities of p53+/+ and p53/ Cells to Nocodazole-induced Apoptosis-- Previous studies have revealed a correlation between p53 status and sensitivity of tumor cells to chemotherapeutic drugs (20-22). The difference in drug response between tumors with wild-type p53 and those harboring p53 loss-of-function mutations can be explained in part by p53-mediated apoptosis (23). Treatment of HCT116 cells with 200 ng/ml nocodazole induced a much stronger apoptotic response in p53+/+ cells than in p53/ cells. As shown in Fig. 1, endonucleolytic cleavage of genomic DNA, an indicator of apoptosis, was much more evident in p53+/+ than in p53/ cells at 72 h (Fig. 1A). Consistent with this, flow cytometry revealed that most cells from both cell lines were arrested at G₂/M transition (4 n DNA) after a 72-h nocodazole treatment (Fig. 1, C and D). However, most of these cells were in G₁ phase (2 n DNA) when cultured in the absence of this microtubule disrupter (Fig. 1B). About 30% of cells contained less than 2 n DNA content (sub-G₁) characteristic of apoptotic cells in p53+/+ cells (Fig. 1C) compared with less than 13% in p53/ cells 72 h after nocodazole treatment (Fig. 1D).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Different sensitivities to nocodazole-induced apoptosis by HCT116 p53+/+ and p53/ cells, as demonstrated by genomic DNA fragmentation assay (A) and flow cytometry (B-D). A, genomic DNAs were extracted from cells treated with 200 ng/ml nocodazole for 0, 24, 48, 72, and 96 h and analyzed by electrophoresis on a 1.8% agarose gel. The p53+/+ cells exhibited a higher extent of DNA fragmentation at 72 h than the p53/ cells. The 1-kb DNA ladder from Invitrogen is indicated on the left. Flow cytometry was performed to assess the percentage of apoptotic cells after treatment of p53+/+ cells in the absence (B) and presence of 200 ng/ml nocodazole (C) for 72 h. D, p53/ cells were also examined by flow cytometry after incubation with 200 ng/ml nocodazole for 72 h and treatment with propidium iodide. DNA content is represented on the x axis; the number of cells counted is represented on the y axis. M1, the fraction of apoptotic cells containing less than 2n DNA.

To examine the signaling pathways downstream of p53 which might account for the difference in apoptosis induction, we undertook an unbiased proteomics-based approach to focus on signaling proteins that may be important in mediating the actions of the tumor suppressor protein p53 in response to nocodazole. In preliminary studies, we applied three Kinetworks^TM screens to analyze the expression profiles of up to 75 protein kinases and 25 protein phosphatases, as well as the phosphorylation states of 25 of these protein kinases (Table I) and 10 other known phosphoproteins (data not shown) from p53+/+ and p53/ cells treated with 200 ng/ml nocodazole for 1 h.

View this table: [in this window] [in a new window]   Table I Expression levels of protein kinases and protein phosphatases and phosphorylation levels of protein kinases in HCT-116 p53+/+ and p53/ cells The trace quantity units are arbitrary based on the intensity of ECL fluorescence detection for target immunoreactive proteins recorded with a Bio-Rad Fluor-S MultiImager and Quantity One software. The target proteins were tracked using the KinetworksTM KPKS 1.0, KPPS 1.1 and KPSS 1.1 screens performed by Kinexus Bioinformatics Corporation. The phosphorylation sites listed are based on human protein sequences. With the exceptions of MEK6 and Src, only the results for those protein kinases and protein phosphatases that yielded signals in excess of 2,500 with pan-specific antibodies are shown. Backgrounds were less than 500 for these analyses. The actual trace quantity values are provided for the untreated p53+/+ cells, whereas the other values reflect the percent differences in the recorded signals relative to the untreated cytosolic and particulate p53+/+ control cells. Values are the average of duplicate determinations. The reproducibility of these signal transduction protein screens was typically within 15%.

48 distinct protein kinases isoforms were clearly evident in the HCT116 cell lines (listed in Table I), and weaker signals were recorded for 14 other protein kinases (calmodulin-dependent kinase 1, calmodulin-dependent kinase kinase, casein kinase 1, cGMP-dependent protein kinase, cyclin-dependent kinases 2, 6, and 9, death-associated kinase 1, JAK2, Lyn, MST1, protein kinase C, RhoA kinase, and Syk; data not shown) of 75 kinases that can be detected by the Kinetworks^TM KPKS 1.0 screen. Of these 48 kinases, 18 protein kinases revealed expression differences of 49% or greater between the untreated p53+/+ and p53/ cell lines when either the cytosolic or particulate fractions of these cells were investigated separately. In particular, loss of p53 function was associated with increases in cytosolic CK2, ERK6, MEK6, MEK7, Pim1, and RafB; increases in particulate MEK1, MEK2, MEK6, Pim1, PKC-, RafB, and S6 kinase; decreases in cytosolic CDK1, PKC-1, Rsk1 and Yes; and reductions in particulate Hpk1, MosI and p40 JNK (SAPK-).² The 3.8-fold or greater increased productions of Pim1 and MEK6 in the p53/ cells were the most striking. These findings indicated extensive alterations in protein kinase regulation as a function of p53 status.

There were also marked differences in the responses of the p53+/+ and p53/ cells to a 1-h exposure to nocodazole. In the p53+/+ cells, nocodazole led to 1.5-1.7-fold increases in cytosolic CK2 and Csk, and particulate MEK7, and it was associated with 53-74% reductions in the levels of cytosolic PKC-1 and particulate ERK2, ERK3, and Yes (the Yes change is not likely to be significant because of low signal:noise ratio). In the p53/ cells, the nocodazole treatment caused 1.5-3-fold increases in cytosolic Csk, CDK1, CDK7, GCK, PKA, PKC-1, Rsk1, Yes, ZAP70, and ZIP kinase, and particulate ERK1, PKC-, and p40 JNK; and 57-100% reductions in cytosolic Pim1, and particulate Csk1 and Fyn. MosI expression was elevated 5-fold in the particulate fraction of nocodazole-treated p53/ cells but unaffected in the p53+/+ cells.

Of 25 different protein phosphatases that could be potentially detected with the Kinetworks^TM KPPS 1.1 screen, 15 were clearly detected in the HCT116 cell lines (Table I), and 5 others were weakly detected (i.e. LAR, MKP1, MKP3, protein phosphatase 2A catalytic subunit and protein phosphatase X A'2 subunit; data not shown). In the p53/ cells compared with the p53+/+ cells, there were 1.5-2-fold elevated protein levels of particulate PTP-1B and cytosolic protein phosphatase 1 catalytic subunit, PTP-PEST, and PTP-1C, and 48-83% reductions in the expressions of particulate MKP2, P5/PPT, and protein phosphatase 1 catalytic subunit. The elevation of cytosolic PTP-1C and reduction of particulate PTP-1C in the p53/ cells may reflect translocation of this protein-tyrosine phosphatase.

Nocodazole caused 2.1-2.5-fold increases in the cytosolic levels of protein phosphatase V catalytic subunit in both the p53/ and p53+/+ cells, and this might also arise from redistribution of this phosphatase away from the particulate fraction in at least the p53/ cells. However, nocodazole treatment also had differential effects on other protein phosphatases in the two HCT116 cell lines. It selectively evoked a 2.7-fold increase in cytosolic KAP and 4-fold more particulate MKP2 in p53/ cells, and 1.8-2.5-fold increased expressions of particulate KAP, protein phosphatase 1C and PTP-1B in p53+/+ cells.

The extensive p53-dependent changes in the levels of the protein kinases and protein phosphatases in the HCT116 cells were also accompanied by many altered states of phosphorylation of protein kinases as detected with phosphorylation site-specific antibodies employed in the Kinetworks^TM KPSS 1.1 screen (Fig. 2 and Table I). 19 distinct phosphorylation sites in 17 different protein kinases were observed to undergo phosphorylation, and 12 of these kinases demonstrated at least 50% increases or decreases in their phosphorylation signals between the p53+/+ and p53/ cell lines in the absence of nocodazole treatment. After exposure to nocodazole, there were also p53-dependent changes in phosphorylation states of 11 protein kinases which exceeded 50%. In several cases, the altered states of phosphorylation reflected at least in part changes in the protein levels of the various protein kinases. This was observed for the CDK1 inhibitory Tyr-14, ERK2-activating Thr-185/Tyr-187, GSK3-activating Tyr-216, PKB-- activating Ser-473, Raf1 p72 Ser-259, Rsk1-activating Thr-360/Ser-364, and Src inhibitory Tyr-529 phosphorylation changes.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   KinetworksTM KPSS 1.0 phosphoprotein analysis of the phosphorylation states of up to 33 known signaling proteins in p53+/+ (A) and p53/ HCT116 cells (B) after a 1-h treatment with 200 ng/ml nocodazole. Known phosphoproteins are identified below as numbered bands. Changes in the intensity of phosphorylation signals that were recorded with Quantity One software from Bio-Rad are provided as a percent increase or decrease in the p53/ cells compared with the p53+/+ cells. The major increase in phosphorylation associated with p53/ was observed for an unidentified 115-kDa protein that migrated below band 22.

The "specific phosphorylation" of a protein takes into account the magnitude of the phosphorylation signal relative to the amount of that protein in a sample. Therefore, the specific phosphorylations of the aforementioned protein kinases were not affected markedly by the p53 status or by nocodazole treatment. However, there were cases where loss of p53 function was associated with increased specific phosphorylation of protein kinases, and although the specific phosphorylation of these kinases could be stimulated further by nocodazole in the p53+/+ cells, this was not evident in the p53/ cells. For example, the specific phosphorylation of cytosolic MEK1/MEK2-activating Ser-217/Ser221 was enhanced in untreated p53/ cells compared with p53 +/+ cells (percent change in phosphorylation state versus percent change in protein level: 384/79 = 4.9-fold),³ and after nocodazole exposure it was increased (265/103 = 2.6-fold) in p53+/+ cells, but not further (122/138 = 0.9-fold) in p53/ cells. Likewise, the specific phosphorylation of cytosolic PKC-1 Thr-638/Ser-657 (128/38 = 3.4-fold) and Thr-641 (149/38 = 3.9-fold) were increased in untreated p53/ cells compared with p53+/+ cells. After nocodazole treatment, these specific phosphorylations of cytosolic PKC -1 were reduced in p53/ cells (Thr-638/Ser-657 (66/188 = 0.35-fold) and Thr-641 (69/188 = 0.37-fold)) but were enhanced in p53+/+ cells (Thr-638/Ser-657 (111/47 = 2.4-fold) and Thr-641 (91/47 = 1.9-fold)). By contrast, there was a complete loss of detectable Ser-259 specific phosphorylation of both cytosolic and particulate p62 Raf1 in untreated p53/ cells compared with p53+/+ cells.

Of greatest interest from the Kinetworks^TM analyses was the p53-dependent differential regulation of p40 JNK and p47 JNK phosphorylation at their activation sites. In the untreated p53/ cells compared with p53+/+ cells, there were marked reductions of the specific phosphorylations of cytosolic p40 JNK (56/111 = 0.5-fold), particulate p40 JNK (1/51 = 0.02-fold), and cytosolic p47 JNK phosphorylation (1/109 = 0.01-fold). Although nocodazole treatment evoked clear stimulations of the specific phosphorylations of cytosolic (247/116 = 2.1-fold) and particulate (336/90 = 3.7-fold) p40 JNK, and cytosolic (258/100 = 2.6-fold) and particulate (134/100 = 1.3-fold) p47 JNK in p53+/+ cells, no increases in JNK specific phosphorylation occurred after exposure of the p53/ cells to nocodazole. These p53 loss of function-associated reductions in basal phosphorylation of JNK along with the abrogation of nocodazole-induced phosphorylation at these activating sites, revealed that JNK acts downstream of p53 in a signaling cascade in the HCT116 p53+/+ cells. The remainder of this study focuses on confirming this finding and establishing its physiological significance.

Nocodazole-induced JNK Activation Is p53-dependent-- To confirm the results of the Kinetworks^TM analysis with respect to JNK regulation in the two HCT116 cell lines in response to nocodazole, we monitored p40 JNK protein and phosphorylation levels by Western blot analysis. Maximum phosphorylation of p40 JNK was induced with 200 ng/ml nocodazole in the p53+/+ cells in 1 h (Fig. 3, A and C). By contrast, there was a slightly higher level of expression of p40 JNK in the p53/ cells even before nocodazole treatment. However, the protein level of p40 JNK remained relatively constant during the course of treatment (Fig. 3B). These findings further indicate a marked loss in the ability of this microtubule-interfering agent to activate p40 JNK in p53/ cells. In correlation with the higher levels of immunoreactive phosphorylated JNK, the JNK immunoprecipitated from p53+/+ cells treated with nocodazole exhibited much higher phosphotransferase activity toward GST-c-Jun when assayed in vitro compared with p53/ counterparts (Fig. 3D). Therefore, the difference observed after nocodazole treatment in these two cell lines in p40 JNK phosphorylation was caused by the differential activation of the kinase.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Nocodazole activates JNK differently in p53+/+ and p53/ HCT116, MCF-7, and A549 cells. Cells were treated with 0-500 ng/ml nocodazole for 1 h (A) or with 200 ng/ml nocodazole for 0-2 h (B and C) before harvesting. MCF-7 (E and F) and A549 (G and H) parental cells and their p53-deficient derivatives, in which the p53 protein was inactivated by E6 overexpression, were treated with 200 ng/ml nocodazole for 1 h. JNK activation was determined indirectly by immunoblotting with anti-phospho-[Thr-183,Tyr-185]JNK antibody (A, C, E, and G). Expression of JNK protein was determined by immunoblotting of the respective cell lysates with anti-JNK1 antibody (B, F, and H). JNK activation in the cells treated with 200 ng/ml nocodazole for 1 h was confirmed further by immunocomplex assay using GST-c-Jun(1-79) as substrate (D). Phosphorylated GST-c-Jun was examined by immunoblotting with phospho-[Ser-63]c-Jun antibody.

p53-dependent JNK Activation Is a General Response to Microtubule Depolymerization-- To examine further the dependence of nocodazole-induced JNK activation on p53 status, we investigated the effect of nocodazole on JNK activity in p53-deficient derivatives of two other cell lines, MCF-7 and A549, in which p53 function was abrogated through expression of viral E6 oncoprotein. Although MCF-7 p53-deficient cells displayed a much higher level of p40 JNK protein expression, MCF-7 parental cells exhibited a marked increase of phosphorylated p40 JNK compared with their p53-deficient counterparts in response to nocodazole treatment (Fig. 3, E and F). Similarly, a higher degree of JNK phosphorylation was observed in A549 parental cells upon nocodazole treatment than in A549 p53-deficient cells, although they exhibited similar JNK protein expression levels (Fig. 3, G and H). Therefore, it is evident that the p53-dependent JNK activation upon nocodazole treatment is not limited to HCT116 cells. However, because of the possible complication of other activities of E6 oncoprotein, we chose to focus subsequent work on the HCT116 cells.

Furthermore, to ascertain whether the p53-dependent JNK activation was a general response to microtubule interference, we examined the effects of other microtubule-interfering agents including vinblastine, colchicine, and taxol on JNK activity in HCT116 cells. The higher degree of JNK phosphorylation in HCT116 p53+/+ cells was observed after treatment with microtubule-depolymerizing drugs, vinblastine and colchicine (Fig. 4, A-D), but not with microtubule-stabilizing drug, taxol (data not shown), indicating that the p53-dependent JNK activation might be related to the microtubule-depolymerizing activity of nocodazole, vinblastine, and colchicine.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   p53 dependence of microtubule-depolymerizing drugs and arsenate on JNK activation in HCT116 cells. HCT116 p53+/+ and p53/ cells were treated with 1 µM vinblastine for 1 h (A and B) or colchicine for 1 h (C and D), or 0.5 µM sodium arsenate for 30 min (E and F). Cells treated with dimethyl sulfoxide and H2O were used as controls for vinblastine and colchicine, respectively. Phospho-JNK (A, C, and E) and JNK protein (B, D, and F) were monitored by Western blot analyses.

Arsenate and Nocodazole Induce JNK Activation through Different Pathways-- It was conceivable that the lack of significant activation of p40 JNK in p53/ cells upon nocodazole treatment may result from defects in the JNK signaling pathway in p53/ cells. To examine this possibility, we used sodium arsenate, a potent activator of stress response pathways involving p38 MAP kinase and JNK, to treat HCT116 cells. Treatment with 0.5 mM sodium arsenate for 0.5 h increased the phospho- p40 JNK levels in p53/ cells, equivalent to, if not better than that observed in the p53+/+ cells (Fig. 4, E and F). This result indicated that the stress response JNK pathways were functional in both cell lines and were independent of the p53 status. Moreover, based on these observations, it is also reasonable to speculate that sodium arsenate and nocodazole may regulate JNK activity via distinct upstream signaling pathways. It further supports the idea that the lack of significant activation of JNK in p53/ cells after nocodazole treatment may be caused by the absence of a functional p53 gene.

p53 Mediates JNK Activation in Response to Nocodazole Treatment-- To explore further the specific role of p53 for nocodazole-induced JNK activation, we examined the effect of the p53 antisense oligonucleotide treatment on p40 JNK activation in p53+/+ cells in response to nocodazole treatment. In p53+/+ cells, treatment with an antisense oligonucleotide targeted at the p53 oligomerization domain located near its C terminus at 140 and 500 nM resulted in 48 and 59% reduction at its protein level, respectively (Fig. 5A). No significant difference was observed between nontransfected and nonsense oligonucleotide-transfected cells in p53 protein levels, indicating that the reduction of p53 protein level was specific for p53 antisense oligonucleotide treatment (Fig. 5A).

View larger version (51K): [in this window] [in a new window]   Fig. 5.   p53 antisense oligonucleotide treatment attenuates JNK activation in p53+/+ cells treated with nocodazole. HCT116 p53+/+ cells were transfected with a p53-specific antisense oligonucleotide at the indicated concentrations before nocodazole treatment. Cells treated with a random oligonucleotide at 140 nM were used as control. The p53 protein levels were monitored by immunoblotting of total lysates with anti-p53 antibody (A). JNK activity was determined by immunoblotting with anti-phospho-[Thr-183,Tyr-185]JNK antibody (B). The -actin level as shown by immunoblotting with an antibody for -actin was used as control for equal loading (C). JNK activity in antisense oligonucleotide-treated cells was further determined by immunocomplex kinase assay using GST-c-Jun(1-79) as substrate and detection with phospho-[Ser-63]c-Jun antibody (D). Numbers below each band indicate the percentage of intensity relative to control.

Correlated to the p53 protein level, the levels of active p40 JNK in the p53 antisense oligonucleotide-treated p53+/+ cells also decreased by 47 and 53%, respectively, in a dose-dependent fashion (Fig. 5B). -Actin was used as an equal loading control (Fig. 5C). In vitro kinase assay of JNK immunoprecipitates further confirmed the decrease of JNK phosphotransferase activity after antisense oligonucleotide treatment (Fig. 5D).

Based on the above results, we conclude that p53 is required for p40 JNK activation, and it may act upstream of JNK pathway to mediate the biological activities of nocodazole in HCT116 cells.

Nocodazole Activates JNK by Up-regulating Its Upstream Kinase MEK4-- The increased phosphorylation and activation of p40 JNK may arise from increased MEK4 and/or MEK7 phosphotransferase activity and/or decreased MKP1 and MKP2 phosphatase activity. The levels of MKP1 and MKP2 were similar in both HCT116 cell lines and relatively unaffected by nocodazole (Fig. 6, A and B).⁴ Although there was about 1.6 times more MEK4 protein in anti-MEK4 immunoprecipitates from p53/ than from p53+/+ cells, the protein level of MEK4 remained relatively constant over the course of nocodazole treatment (Fig. 6D). However, there was a marked 5-fold activation by nocodazole of immunoprecipitated MEK4 phosphotransferase activity toward GST-JNK in p53+/+ cells (Fig. 6C). By contrast, the phosphotransferase activity of the MEK4 could not be stimulated in the p53/ cells (Fig. 6C). Therefore, p53 induced activation of p40 JNK at least in part through stimulation of MEK4.

View larger version (73K): [in this window] [in a new window]   Fig. 6.   Activation of MEK4 is at least in part responsible for JNK activation in p53+/+ cells treated with nocodazole. The MPK-1 (A) and MPK-2 (B) protein phosphatase levels in p53+/+ and p53/ treated for 0-2 h with 200 ng/ml nocodazole were monitored by immunoblotting with their respective antibodies. MEK4 phosphotransferase activity was determined by immunocomplex kinase assay using GST-JNK2 as substrate and detection with anti-phospho-[Thr-183,Tyr-185]JNK antibody (C). The level of MEK4 proteins in the MEK4 immunoprecipitates used for the kinase assays in C were determined by immunoblotting with anti-MEK4 antibody (D).

Inhibition of Endogenous p53-dependent JNK Activity Enhances Apoptosis in p53+/+ cells-- Given the roles of JNK associated with induction of apoptosis in response to microtubule disruption, as demonstrated by a number of recent studies (24, 25), an interesting question arises as to whether the differential JNK activation in response to nocodazole may account for the different sensitivity of p53+/+ and p53/ cells to nocodazole-induced cytotoxicity.

To address this question, we transfected p53+/+ cells with dominant negative mutants of four different p40 JNK isoforms, JNK1(APF), JNK1(APF), JNK2(APF), and JNK2(APF), individually, followed by a 1-h nocodazole treatment to evaluate whether any of these treatments could inhibit nocodazole-induced apoptosis. No significant difference in p40 JNK activation was observed between empty vector-transfected control cells and JNK(APF)-transfected cells upon nocodazole treatment (Fig. 7A). Correlated with this, no apparent effect was seen on apoptosis induction after a 72-h nocodazole treatment in the cells transfected with each individual JNK(APF) mutant, as assessed by flow cytometry.

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Inhibition of endogenous JNK activity resulted in increase of apoptosis in p53+/+ cells in response to nocodazole treatment. Transfection of HCT116 p53+/+ cells with dominant negative mutants of p40 JNK individually showed no effect on either endogenous JNK activity or apoptosis in response to nocodazole treatment (A). Cotransfection of p53+/+ cells with dominant negative mutants of all four JNK isoforms resulted in a decrease in JNK phosphotransferase activity (B) but an increase in the percentage of apoptotic cells as measured by flow cytometry. Incubation of HCT116 p53+/+ cells with 0.5 µM SP600125 and 200 ng/ml nocodazole for 1 h resulted in the inhibition of JNK and c-Jun phosphorylation as well as increased apoptotic response 72 h after treatment (C, D, and E). Endogenous JNK phosphotransferase activity in cells was monitored indirectly by immunoblotting with anti-phospho-[Thr-183,Tyr-185]JNK antibody (C) and anti-phospho-[Ser-63]c-Jun antibody (E). Expression of JNK was determined by immunoblotting with anti-JNK antibody (D). Results from one representative experiment are shown here. The experiment was repeated three times with similar results.

However, when cotransfected with dominant negative mutants of all four different p40 JNK isoforms, p53+/+ cells exhibited a 38% decrease in the phosphorylation of endogenous p40 JNK 1 h after nocodazole treatment compared with vector-transfected control cells (Fig. 7B). Surprisingly, a 1.7-fold increase in the number of apoptotic cells within 72 h after nocodazole treatment was detected in the p53+/+ cells cotransfected with all four dominant negative mutants.

SP600125 is a newly identified JNK inhibitor and exhibits significant selectivity for JNKs, leading to inhibition of both phosphorylation of c-Jun and JNKs (26). When a short term treatment with both nocodazole and SP600125 was administrated, we observed inhibition of JNK and c-Jun phosphorylation in p53+/+ cells in response to a 1-h SP600125 treatment compared with nocodazole only treatment (Fig. 7, C-E). When treated with a combination of nocodazole and SP600125 for 72 h, a ~25% increase in apoptosis was observed in HCT116 p53+/+ cells, whereas no effect was found when SP600125 was used alone. This is consistent with the inhibitory effect of JNK activity on apoptosis observed above. Taken together, our observations indicated that the p53- dependent JNK activation initially induced by nocodazole treatment is more likely to be anti-apoptotic rather than pro-apoptotic.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The extensive use of microtubule-interfering agents in chemotherapeutic treatment of a variety of human tumors has justified a great deal of effort devoted to identifying the signaling pathways that mediate the cellular responses to microtubule disruption. By taking a broad analysis of signaling pathways through the use of Kinetworks^TM proteomics screens, we observed complex changes in the levels of expression and phosphorylation of many protein kinases and phosphatases in association with loss of p53 function and in response to nocodazole treatment (Table I and Fig. 2). Our knowledge of the precise roles of these regulatory enzymes is insufficient at this juncture to permit proper interpretation of all of these findings. However, the most profound difference in nocodazole effects which was dependent upon p53 status was noted for the regulation of JNK. Although both p53 and JNK have previously been shown to mediate cellular responses to the actions of microtubule-interfering agents, their interrelationships have been far from clear.

The dependence of nocodaozle-induced JNK activation on p53 status was examined in three cell lines and their p53-deficient derivatives, in which the p53 function was disrupted through two distinct approaches. A transient JNK activation was observed in all three parental cells but not in their p53-deficient counterparts, indicating that the p53-dependent JNK activity may be a general response to nocodazole-induced microtubule disruption and is not just restricted to a particular cell line. In addition to nocodazole, other microtubule-depolymerizing agents including vinblastine and colchicine, but not microtubule-stabilizing taxol, could also elicit similar JNK activation in p53-dependent manner, which implied that it is the effects of microtubule-depolymerizing drugs that trigger the p53-dependent JNK activation. The underlying mechanism for this effect remains to be elucidated.

The failure of JNK activation in nocodazole-treated p53/ cells could not be attributed to defects in the stress response pathways mediated by JNK because arsenate was able to activate JNK in both types of cell. Furthermore, treatment of p53+/+ cells with p53 antisense oligonucleotide revealed that the differential activation of JNK in response to nocodazole resulted from the difference in the p53 status in these two cell lines, indicating that the p53 tumor suppressor can act upstream of JNK. This is in sharp contrast to the conclusions drawn from most published studies that p53 is a downstream effector of JNK. It has been shown that JNK phosphorylates p53 specifically at Ser-34 and Thr-81 in response to UV irradiation and anisomycin treatment (12, 13, 27), leading to stabilization of p53, which in turn induces expression of p21^Waf1/Cip1 and proapoptotic members of Bcl2 family such as Bax. Similar to our observations, the correlation between p53 status and JNK activation has been observed recently in LNCaP (p53+/+) and PC-3 (p53/) cells treated with N-(4-hydroxyphenyl)retinamide. However, conclusions drawn from a study involving two distinct cell lines with different p53 status cannot be considered conclusive (28).

Even though it is still unclear how p53 mediates JNK activation in response to nocodazole, we have shown here that at least in part, it acts through up-regulating the activity of the JNK upstream kinase MEK4 without affecting known phosphatases that down-regulate JNK activity. Moreover, our preliminary data indicate that the p53-mediated JNK activation is independent of p53 transcriptional activity because pifithrin , an inhibitor of p53 transcriptional activity (29),⁵ cannot block the JNK activation upon nocodazole treatment. This is not totally surprising because transcriptionally independent activities of p53 have also been documented in several recent studies (3). It would be very interesting to delineate the signaling pathway that mediates signals from p53 to JNK by examining the effects of dominant negative mutants of each component of JNK as well as p53 pathways on the p53-dependent JNK activity.

There is increasing evidence showing that activation of the JNK signaling pathways is implicated in regulation of apoptosis (24, 30, 31). However, the exact roles of JNK in promoting or preventing apoptosis differ and depend on the cell type, apoptosis-triggering signals, and even the duration of JNK activation (32, 33). The correlation between apoptotic response and JNK activation in p53+/+ cells prompted us to speculate that the p53-dependent JNK activity might be responsible for inducing apoptosis in response to nocodazole treatment. Overexpression of nonphosphorylatable dominant negative mutants of JNK isoforms individually exhibited no effect on endogenous JNK activity and apoptosis in p53+/+ treated with nocodazole. This may be caused by functional redundancy among different isoforms of JNKs. In agreement with this, cotransfection of p53+/+ cells with dominant negative mutants of all four JNK1/2 isoforms reduced JNK activation induced by nocodazole treatment. However, to our surprise, instead of an attenuation of the apoptotic response, we observed a significant increase in the number of apoptotic cells in cotransfected cells. The results demonstrated that the p53-dependent JNK activity induced by nocodazole is anti-apoptotic in the HCT116 cells. Consistent with this, inhibiting JNK activity by treating p53+/+ cells with nocodazole for 72 h along with a JNK inhibitor, SP600125, also resulted in an enhanced apoptotic response compared with those treated with nocodazole only. Furthermore, our preliminary experiments have shown that cells expressing MEK4 dominant negative mutant MEK4(AL) also exhibit increased apoptosis relative to vector controls.⁵

Although little is known about the detailed mechanisms, the role of JNK in protecting cells from stress-induced apoptosis has been documented in many studies. Based on our observations, we propose a hypothesis for the role of anti-apoptotic JNK activity in p53+/+ cells in response to nocodazole treatment. Given the important roles of p53 in the cellular response to stress, treating HCT116 cells with nocodazole elicits a p53-mediated microtubule damage signal that is expected to be stronger in p53+/+ than in p53/ cells. When the damage signal reaches a certain threshold, cells will activate their defense system, which is mediated by the p53-dependent JNK activity, in an effort to repair the damage. However, the prolonged nocodazole treatment in our study in the end may result in massive destruction of microtubule structures in p53+/+ cells, which may render the repairing efforts futile.

Several lines of our observations and those of others are in favor of this hypothesis. First, Chen et al. (32) showed that the JNK activation in T-cell apoptosis and T-cell activation was distinguished by the different activation kinetics, persistent versus transient, respectively. Given the transient nature of the p53-dependent JNK activity, our data indeed support a survival role for the JNK activity in the stress response to nocodazole treatment. Second, the roles of MEKK1, a MAP kinase kinase kinase upstream of MEK4, and its dependent JNK activation, in cell survival have been defined by targeted gene disruption in mouse embryonic stem cell line in response to microtubule disruption by nocodazole (34). Third, no apparent difference in apoptosis was observed between p53+/+ and p53/ cells being treated with nocodazole for a shorter time (up to 48 h), which indirectly supports a role of JNK in protecting cells from stress-induced apoptosis in p53+/+ cells.⁵ The early JNK activation relative to the delayed occurrence of massive apoptosis implicated the p53-dependent JNK activity as an initial response to treatment of microtubule-depolymerizing drugs. However, how this early response event triggers the subsequent apoptosis signaling cascade is still unclear. Finally, our preliminary observations have indicated that there is no increase of apoptosis in p53+/+ cells transfected with JNK wild-type constructs at 72 h after nocodazole treatment,⁵ which is also in agreement with our hypothesis.

A growth inhibitory effect of JNK was reported by Potapova et al. (19) in HCT116 p53/ and other p53-deficient cell lines upon JNK antisense oligonucleotide treatment, which supports the protective role of JNK in human tumor cells lacking functional p53. The two seemly paradoxical observations in the same cell line could be resolved if one considers the fact that it is the JNK basal protein level in nonstressed cells that was the focus of their study, not stress-induced JNK activity as in our study reported here.

In conclusion, our study provides new insights into the mechanisms of the cellular response to microtubule disruption. It is the first time that the p53-dependent JNK activity has been clearly documented and shown to be associated with cell protection in stress response to microtubule disruption. Further study will be required to elucidate the mechanisms by which the JNK exerts its protective role during this process. Our study may also have clinical implications for optimizing cancer therapies involving microtubule-interfering drugs in future.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Bert Vogelstein for providing HCT116 p53+/+ and p53/ cell lines, Drs. Lynn Heasley and Jim Woodgett for JNK constructs, Dr. Liren Tang for p53 antibody, and Dr. Michel Roberge for helpful discussions.

	FOOTNOTES

^* The study was supported in part by an operating grant from the Canadian Institutes of Health Research (to S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medicine, University of British Columbia, The Brain Research Centre, 1st Floor, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-9966; Fax: 604-822-9964; E-mail: spelech@kinexus.ca.

Published, JBC Papers in Press, September 6, 2002, DOI 10.1074/jbc.M203214200

² JNK1 and JNK2 have been reported previously to have at least four different isoforms, p46, p46, p54, and p54, because of alternative splicing (34-36). From our study we believe that p54 JNK corresponds to p47 JNK, and p46 JNK corresponds to p40 JNK. The difference in the apparent molecular masses may result from different electrophoresis conditions used in the Kinetworks^TM analysis.

³ The -fold change in specific phosphorylation of each protein = (100 + % change in phosphorylation state)/(100 + % change in total protein level).

⁴ Table I shows a reduction in particulate MKP2 without nocodazole treatment and an increase with nocodazole treatment in p53/ cells.

⁵ H. Zhang, X. Shi, and S. Pelech, unpublished data.

	ABBREVIATIONS

The abbreviations used are: CDK, cyclin-dependent kinase; CK, casein kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKP, MAP kinase phosphatase; PKA, protein kinase A; PKC, protein kinase C; PTP, protein-tyrosine kinase. For additional abbreviations, see Table I.

	REFERENCES

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