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Experimental Therapeutics |
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199 [M. F., M. E. G., T. C. C.], and Department of Cell and Cancer Biology, Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland 20850 [M. J. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Bak, and several other genes in control but not in KB3-TAM67 cells, identifying these genes as putative targets of vinblastine-inducible AP-1. These results demonstrate that vinblastine-inducible AP-1 plays a destructive, proapoptotic role and may do so by regulating the expression of a specific subset of target genes that promotes efficient apoptotic cell death following mitotic arrest. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An important mediator of apoptotic signaling is the JNK3
subgroup of mitogen-activated protein kinases (11)
. JNKs are activated in response to proinflammatory cytokines and a variety of stressful stimuli, including UV- and 
-irradiation, heat shock, oxidative and osmotic stress, growth factor deprivation, metabolic inhibitors, and chemotherapeutic drugs (12)
. The JNK pathway is critical for certain forms of stress-induced apoptosis. For example, JNK signaling is essential for apoptosis of PC-12 cells deprived of NGF (13)
, and JNK3 is required for apoptosis of hippocampal neurons induced by glutamate-mediated excitotoxic injury (14)
. Transfection studies using dominant-negative approaches (15
, 16) , as well as studies of murine embryonic fibroblasts with simultaneous targeted disruptions of the jnk1 and jnk2 genes (17)
, have also strongly implicated JNK in stress-induced apoptosis of nonneuronal cells. However, opposing functions of JNK have also been reported, and evidence exists suggesting that in certain contexts JNK activation may play a protective role (18
, 19)
. In addition, inhibition of JNK2 expression has been shown to suppress growth and to induce apoptosis of human tumor cells (20)
, and JNK is responsive to activation by growth factors, suggesting an additional role in growth regulation (11)
.
A role for JNK signaling in the response of cells to microtubule inhibitors has been suggested. JNK activity is increased by microtubule inhibitors in a wide variety of cell types (21, 22, 23, 24, 25, 26) , and inhibition of JNK inhibits paclitaxel-induced apoptosis (24 , 26) . A major target of JNK signaling is the AP-1 transcription factor (27) . Activation of JNK by vinblastine and other microtubule inhibitors in KB3 cells leads to phosphorylation and stimulation of the transactivation domains of c-Jun and ATF-2 (22) . A critical role for c-Jun/AP-1 has been demonstrated in apoptosis of PC12 and neuronal cells after NGF withdrawal (13 , 28) , with the AP-1 target, Fas ligand, as a potential mediator (28) . However, the role of AP-1 in apoptosis caused by microtubule inhibitors has not been established, and recent results have suggested that the JNK pathway may influence the cell death process through nontranscriptional mechanisms. In KB3 cells, JNK mediates vinblastine-induced phosphorylation of the antiapoptotic proteins Bcl-2 and Bcl-XL (29) , consistent with an earlier study that also identified JNK as the kinase responsible for paclitaxel-induced Bcl-2 phosphorylation (30) . Evidence suggests that phosphorylation inhibits the antiapoptotic properties of Bcl-2 (31) . Thus, one way that JNK may promote apoptosis in response to microtubule inhibition may be to disable antiapoptotic proteins through phosphorylation, but whether such modifications are necessary and sufficient for cell death by microtubule inhibitors is not clear. This further emphasizes the need to elucidate the role of the other "arm" of JNK signaling, namely AP-1 activation, in the response to microtubule inhibition, especially to determine whether AP-1 induction opposes or complements direct effects of JNK on Bcl-2 proteins. We address this question in this study by generating KB3 cells that stably express the c-Jun dominant-negative mutant TAM67 (32) and by characterizing their response to vinblastine. Our results suggest that vinblastine-inducible AP-1 plays a destructive role by regulating the expression of a specific subset of genes required for efficient apoptotic cell death.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP was obtained from Amersham (Piscataway, NJ), and the dual luciferase reporter assay system was from Promega (Madison, WI). Fetal bovine serum was from Hyclone (Logan, UT) and other cell culture reagents were from Life Technologies, Inc. (Rockville, MD). Unless otherwise stated, other reagents were from Sigma Chemical Co. (St. Louis, MO).
Cell Culture.
The KB3 human carcinoma cell line was maintained in monolayer culture at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cell growth rates were measured by seeding 2 x 104 cells/well in 6-well dishes and at daily intervals determining the number of trypan blue-excluding intact cells.
Stable Transfection of TAM67.
KB3 cells (106) were transfected with 10 µg of pcDNA-TAM67 (32)
or empty vector, using standard calcium phosphate transfection procedures. Transfected cells were selected in the presence of 1 mg/ml geneticin for 2 weeks, and drug-resistant colonies were chosen and maintained in growth medium containing 0.4 mg/ml geneticin. The relative number of clones obtained was similar for vector- and TAM67-transfected cells. The expression of TAM67 in the transfectant clones was detected by immunoblotting with an antibody that recognizes the COOH terminus of c-Jun.
Cell Viability and Microscopy.
Cells (2 x 105/well) were plated in a 6-well plate, treated with 30 nM vinblastine or vehicle (0.1% DMSO) for 1 h, and incubated in vinblastine-free medium. Cell viability was measured at daily intervals by counting the number of trypan blue-excluding intact cells. Cell morphology was visualized by differential interference contrast optics using transmitted light from an inverted Zeiss 410 confocal laser scanning microscope (Thornwood, NY).
Proliferation and Clonogenic Assays.
Inhibition of cell proliferation by vinblastine was measured by the MTT assay (33)
. Cells (5000/well) were plated in 96-well dishes and treated with either vinblastine (10–300 nM) or vehicle alone (0.1% DMSO) for 1 h. The medium was replaced with drug-free medium, and the MTT assay was performed 48 h after drug treatment. All treatments were performed in triplicate, and viability was expressed as a percentage of the untreated controls. Clonogenic assays were performed by plating 2 x 105 cells/well in a 6-well plate, treating with 30 nM vinblastine or vehicle (0.1% DMSO) for 1 h, and incubating in drug-free medium for another 23 h. Cells were then collected by trypsinization and replated into 60-mm dishes at a density of 1000/dish for vinblastine-treated cells and 200/dish for control cells. Cells were incubated for 8 days and stained with 0.5% methylene blue in 50% methanol. Colonies that contained >50 cells were scored, and cloning efficiency was expressed as (number of colonies/number of plated cells) x 100. All treatments were performed in triplicate, and results were expressed as mean ± SD.
Preparation of Cell Extracts and Immunoblotting.
Whole-cell extracts were prepared by suspending cells (
3 x 106) in 0.25 ml of lysis buffer [25 mM HEPES (pH 7.5), 0.3 M NaCl, 0.2% SDS, 0.5% sodium deoxycholate, 0.2 mM EDTA, 0.5 mM DTT, 20 mM ß-glycerophosphate, 1 mM Na3VO4, 0.1% Triton X-100, 20 µg/ml aprotinin, 50 µg/ml leupeptin, 10 µM pepstatin, 0.1 µM okadaic acid, and 1 mM phenylmethylsulfonyl fluoride]. After 15 min on ice, extracts were sonicated (three times 10 s each), insoluble material was removed by centrifugation (15 min at 12,000 x g), and the protein concentration in the supernatant was determined using the Bio-Rad protein assay. Immunoblotting was performed as described previously (22)
, using 50 µg protein/lane.
Transient Transfection and AP-1 Luciferase Assay.
Cells (2 x 105/well) were plated in a 6-well plate and transfected with 2 µg of TRE-Luc construct (firefly luciferase under control of two copies of TPA response element), together with 2 µg of pRL-TK-Luc construct (Renilla luciferase under control of TK promoter) to control for transfection efficiency, using 20 µl of Superfect Reagent (Qiagen, Valencia, CA). After 32 h, cells were treated with vehicle (0.1% DMSO) or 30 nM vinblastine for 1 h. After 15 h of incubation in drug-free medium, cells were harvested for determination of firefly and Renilla luciferase activities by the dual luciferase reporter assay system (Promega). The results were expressed as average relative firefly luciferase activity, normalized to Renilla luciferase activity, reported as mean ± SD (n = 6).
Flow Cytometry Analysis.
To determine cell cycle distribution, 5 x 105 cells were plated in 60-mm dishes, treated with 30 nM vinblastine or vehicle (0.1% DMSO) for 1 h, and incubated in the absence of drug for 24 or 40 h. Cells were then collected by trypsinization, fixed in 70% ethanol, washed in PBS, resuspended in 1 ml of PBS containing 1 mg/ml RNase and 50 µg/ml propidium iodide, incubated for 20 min in the dark at room temperature, and analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Mountain View, CA). The data were analyzed using the ModFit DNA analysis program (Verity Software House).
Caspase 3 Assay.
Cells (5 x 105) were plated in 60-mm dishes, treated with 30 nM vinblastine or vehicle (0.1% DMSO) for 1 h, and incubated in drug-free medium for different periods. Cells were harvested, washed twice in PBS, and whole-cell extracts were prepared by sonication for 10 s with 0.3 ml of 20 mM HEPES (pH 7.5), 10% sucrose, 0.1% CHAPS, 2 mM DTT, 0.1% NP40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. The supernatants obtained after centrifugation (16,000 x g, 10 min) were used to determine the caspase 3 activity by fluorometric assay using the substrate Ac-Asp-Glu-Val-Asp-AMC (DEVD-AMC). Extracts containing 100 µg of protein were incubated in a final volume of 0.25 ml containing 100 mM HEPES (pH 7.5), 10% sucrose, 0.1% CHAPS, 10 mM DTT, and 50 µM substrate. After incubation for 30 min at 30°C, the liberated fluorescent group (AMC) was monitored using a spectrofluorometer (Perkin-Elmer) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
RNA Preparation, cDNA Synthesis, Hybridization, and Scanning of Microarray.
Cells (2 x 107) were treated with 30 nM vinblastine or DMSO (0.1%) for 1 h and harvested after 15 h incubation in drug-free medium. Total RNA preparation, [32P]cDNA synthesis, and array membrane hybridization were performed using the Atlas pure total RNA labeling system and the Atlas human apoptosis array kit, as specified by the manufacturer (Clontech). Details of the protocol and the full list of genes whose cDNAs are represented on the membranes are available from the manufacturer’s Web site.4
After hybridization and washing, array membranes were exposed on phosphorimager screens. The phosphorimager screens were scanned on a Model 445SI phosphorimager, and the scanned files were analyzed with the ImageQuaNT software (Molecular Dynamics, Inc.). The hybridization signals from different membranes were normalized using the average intensity of all nine housekeeping genes, and the background value subtracted. For presentation of the data in Fig. 9
, hybridized arrays were subjected to autoradiography with MS X-ray film (Kodak BioMax).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Several KB3-TAM67 clones were expanded, and growth rates were determined relative to control (untransfected and vector-transfected) KB3 cells. Representative examples are shown in Fig. 1B
. The majority (75%) of the TAM67-expressing clones showed growth rates identical to those for control cells, whereas others, such as clone 35, had a reduced proliferation rate. These results indicated that stable expression of TAM67 was tolerated without adverse effects on general growth properties in the majority of KB3 transfectants. For the remainder of the study, early-passage KB3-TAM67 stable transfectants that were unaffected in growth rate relative to control cells were used. All results are representative of experiments performed with two or more independent transfectants to ensure that the effects were attributable to TAM67 expression and not to clonal variation. Untransfected control cells and vector-transfected cells were indistinguishable and are termed KB3 for simplicity.
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, vinblastine stimulated phosphorylation of c-Jun in KB3 but not in KB3-TAM67 cells, as determined by immunoblotting with a phosphospecific (Ser-63) c-Jun antibody. In addition, both basal and vinblastine-induced expression of c-Jun was lower in KB3-TAM67 versus KB3 cells. Vinblastine-induced phosphorylation of ATF-2, evaluated with a phosphospecific antibody, was also reduced in KB3-TAM67, whereas total ATF-2 expression, evaluated with a phosphorylation-independent antibody, remained comparable (Fig. 2A)
. As a specificity control, we also examined Bcl-2 phosphorylation, which appears to occur universally in response to microtubule inhibition (34
, 35)
and which is mediated by the JNK signaling pathway (29
, 30)
. The extent and kinetics of vinblastine-induced phosphorylation of Bcl-2 were found to be similar in KB3 and KB3-TAM67 cells (Fig. 2A)
, confirming that TAM67 exhibits selective effects, interfering with the ability of JNK to phosphorylate c-Jun and ATF-2 but not Bcl-2. Fig. 2A7
also shows that TAM67 expression was unaffected by vinblastine treatment and that actin expression, as a loading control, was similar in all lanes.
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. AP-1 activity was significantly increased by vinblastine in KB3 cells, but vinblastine failed to induce AP-1 activity in KB3-TAM67 cells (Fig. 2B)
. Thus, TAM67 expression effectively blocked vinblastine-inducible AP-1 activation, consistent with the results shown in Fig. 2A
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Increased Resistance of KB3-TAM67 Cells to Vinblastine.
Vinblastine sensitivity was first measured by the standard MTT assay as described in "Materials and Methods." Two controls (KB3 and KB3-vector) and three independent KB3-TAM67 cell lines were used. Cells were exposed to vehicle (0.1% DMSO) or vinblastine (10–300 nM) for 1 h and incubated for an additional 47 h in drug-free medium, and cell viability was determined. As shown in Fig. 3
, significantly increased cell survival was observed for all three KB3-TAM67 cell lines relative to control cell lines in the range 20–100 nM vinblastine. This same range of drug concentration was optimal for stimulation of c-Jun and ATF-2 phosphorylation (Fig. 2A
, and data not shown). At very high concentrations of vinblastine (
300 nM), however, TAM67 expression was not protective. Calculation of IC50 values indicated that KB3-TAM67 cell lines were 2–3-fold more resistant to vinblastine than controls (see legend for Fig. 3
).
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65% (Fig. 4)
. After exposure to 30 nM vinblastine for 1 h, the cloning efficiency of control cells was reduced to 10%, but that of KB3-TAM67 cells was reduced less than 2-fold to 37%. Representative culture dishes showing the stained colonies to visualize and highlight this marked difference are also shown in Fig. 4
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. After 24 h, the control cells were viable but not increased in number, and a slightly increased number of viable TAM67-expressing cells was observed, relative to time zero. However, a marked difference was observed at 48 h, consistent with the results of Fig. 3
. Thus, at 48 h posttreatment, the number of viable KB3 cells was decreased, whereas most KB3-TAM67 cells remained viable. Fig. 5B
shows cell morphology under these same conditions at 48 h. Vinblastine-treated KB3 cells exhibited an altered, apoptotic morphology, with cell shrinkage, membrane blebbing, and the appearance of apoptotic bodies. However, vinblastine-treated KB3-TAM67 cells, although in general somewhat enlarged and flattened, retained much of their original morphological features. From 48 h on, although viability was progressively lost in both KB3 and KB3-TAM67 cells, KB3-TAM67 cells maintained their survival advantage up to 96 h (Fig. 5A)
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, caspase 3 activity was detected in KB3 cells at 24 h and increased over the next 20 h. In contrast, at early time points, much lower caspase 3 activity was observed in KB3-TAM67 cells. By 40 and 48 h posttreatment, caspase 3 activity was comparable in KB3 and KB3-TAM67. These results indicate a marked delay in caspase 3 activation in response to vinblastine in KB3-TAM67 cells, consistent with the cell viability data.
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. At 24 h posttreatment, both cell lines gave similar DNA profiles, with cells arrested predominately at G2-M phase with 4N DNA, and a small subpopulation arrested with 8N DNA. At 40 h posttreatment, however, marked differences between KB3 and KB3-TAM67 were observed. A much greater percentage of KB3 cells contained "sub-G2" DNA, reflecting a higher proportion of apoptotic cells. In addition, a greater proportion of KB3 cells had DNA content intermediate between 4N and 8N, reflecting an increased tendency of KB3 cells to undergo DNA rereplication relative to KB3-TAM67 cells. These results indicate that KB3-TAM67 cells arrest normally at G2-M in response to vinblastine and that the influence of TAM67 expression on cell survival occurs downstream, affecting events subsequent to mitotic arrest.
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. In untreated KB3-TAM67 cells, basal expression of p53 was elevated versus untreated KB3 cells. Furthermore, vinblastine failed to cause down-regulation, and p53 remained elevated up to 24 h posttreatment. Examination of the p53 target gene product p21 revealed correspondingly higher expression at each time point in KB3-TAM67 versus KB3 cells (Fig. 8)
. These results suggest the intriguing possibility that the differential sensitivity of TAM67 cells to vinblastine may in part reside in the ability of c-Jun/AP-1 to down-regulate p53/p21 in KB3, but not KB3-TAM67 cells, which in turn may alter the fate of mitotically arrested cells.
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. These genes represent those affected either positively or negatively by expression of the c-Jun dominant-negative. Examples include c-Jun itself and cyclin D3, both of which were expressed at lower levels in TAM67 cells, and CD40 receptor-associated factor, which was up-regulated in TAM67 cells. The final group, and the one most pertinent to this study, included those genes whose expression was up-regulated by vinblastine in KB3 cells but not, or to a much lower extent, in KB3-TAM67 cells. These represent putative c-Jun/AP-1 target genes and included the proapoptotic protein Bak, the death receptor ligand TNF-, IGFBP4, and GST3 (Table 2
; Fig. 9
). Although p53 cDNA was represented on the array, its hybridization signal, and that of several other genes, including Fas L, was too weak to permit quantitation. The results presented in Fig. 9
and Tables 1
and 2
are representative of results obtained in two independent experiments. Increased expression of TNF-, Bak, and IGFBP4 mRNA in response to vinblastine has been independently confirmed by reverse transcription-PCR analysis (data not shown).
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DISCUSSION |
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In the assay using the AP-1-dependent luciferase reporter, AP-1 transcriptional activity was stimulated by vinblastine in KB3 cells, which is consistent with the ability of the drug to stimulate phosphorylation of the transactivation domains of c-Jun and ATF-2 (Fig. 2)
. The time course of c-Jun/ATF-2 phosphorylation, beginning at 8 h with a maximum at 16–20 h, paralleled the kinetics of JNK activation reported previously (22)
. In contrast, in KB3-TAM67 cells, vinblastine-induced phosphorylation of c-Jun and ATF-2 was inhibited, and in parallel, vinblastine failed to increase AP-1 activity much above basal level. Thus, TAM67 appeared to function as an effective inhibitor of JNK-mediated c-Jun and ATF-2 phosphorylation and consequently effectively blocked vinblastine-inducible AP-1. The ability of TAM67 to inhibit phosphorylation of c-Jun and ATF-2 is not unexpected, given that TAM67 forms defective heterodimers with endogenous AP-1 proteins (32)
and that efficient phosphorylation of the transactivation domains of Jun and ATF-2 proteins by JNK requires functional homo- or heterodimers (42)
. The effects of TAM67 on downstream JNK signaling were specific in that the overall extent and kinetics of vinblastine-induced phosphorylation of Bcl-2, also catalyzed by JNK (29
, 30)
, were unaffected. Vinblastine also induced transient c-Jun expression at 4–8 h in KB3 cells and, to a lesser extent, in KB3-TAM67 cells (Fig. 2A)
. However, because c-Jun induction occurred earlier than c-Jun/ATF-2 phosphorylation in KB3 cells and occurred in the apparent absence of c-Jun phosphorylation in KB3-TAM67 cells, the mechanism responsible appears distinct from the well-established pathway whereby activated c-Jun/ATF-2 heterodimers transcriptionally activate an AP-1-like site in the c-Jun promoter (27)
. Recent work has suggested that the c-Jun promoter is under complex regulation by both JNK-dependent and JNK-independent pathways (43)
, and the increased protein expression we observed could also reflect regulation at posttranscriptional or posttranslational levels. Additional experiments will be required to define this distinct mechanism of early c-Jun induction by vinblastine as well as its functional significance.
The kinetics of cell death are highly dependent on the apoptotic stimulus and on the cell type. To ascertain relative resistance or sensitivity of two or more cell lines to a toxic insult, a wide range of assays are available. Short-term cell viability assays are useful for examining the rate of cell death, but they can underestimate the final extent of cell killing (44) . On the other hand, clonogenic assays quantitate the proportion of cells in a population that can divide in an unlimited fashion to form a colony. In this study, we used a broad range of assays to clearly show that KB3-TAM67 cell lines were significantly more resistant to vinblastine in both kinetic and absolute terms. Thus, assays examining proliferation rate, cell viability, cell morphology, caspase 3 activity, and apoptotic DNA content all revealed that KB3-TAM67 cells exhibited a delay in apoptotic cell death following exposure to vinblastine. In addition, KB3-TAM67 cells exhibited greater clonogenic efficiency following exposure to vinblastine, suggesting that not only was the rate of cell death reduced but that overall cell killing was also reduced.
An alternative explanation for the vinblastine-resistant phenotype of the KB3-TAM67 cell lines is that an adaptation to overexpression of the AP-1 dominant-negative has occurred. As we noted, the basal expression of p21 (Fig. 8)
and mRNA for a subgroup of genes, including p16 (Table 1)
, was altered. However, untreated KB3-TAM67 cells were largely unaffected in growth rate, in cell cycle distribution, in cloning efficiency, in morphology, and, in response to vinblastine, in metaphase arrest. In addition, the altered pattern of gene expression in response to vinblastine is clearly consistent with their resistant phenotype (Table 2
and Fig. 9
). Therefore, the resistant phenotype is likely related to defects in c-Jun signaling in response to vinblastine, rather than an adaptation process.
The vinblastine-resistant phenotype of the KB3-TAM67 cell lines indicates that the JNK/AP-1 pathway is a critical mediator of apoptosis in this system. TAM67 overexpression has been used in other systems to probe the role of AP-1 in cellular responses to stressful stimuli and anticancer drugs that activate JNK/AP-1. In some systems, TAM67 overexpression has been found to be protective. For example, TAM67 overexpression protected U937 monoblastic leukemia cells from several forms of stress, including X-rays, UV-C, H2O2, and heat shock (45)
, and protected neurons from apoptosis induced by MAPK kinase kinase 1 overexpression or NGF withdrawal (13)
. In addition, kainate-induced neuronal apoptosis (46)
and apoptosis of cerebellar granule neurons after survival factor withdrawal (28
, 47)
are both inhibited by a mutant c-Jun (JunAA) lacking major JNK sites, suggesting that JNK-mediated phosphorylation is required. These results implicate c-Jun/AP-1 as a positive mediator of cell death, as we found in this study. However, TAM67 overexpression (18)
or overexpression of JunAA (48)
has been found to sensitize glioblastoma or ovarian carcinoma cells, respectively, to cisplatin. In these cases, the JNK/AP-1 pathway has been implicated as a protective response involved in DNA repair. In another study, TAM67 expression did not affect apoptosis of U937 cells by etoposide (49)
. Taken together, these results suggest that AP-1 may have opposing or even neutral functions, depending on the cell type and stimulus. However, important parameters that perhaps should receive more attention are the concentration and duration of the stress stimulus. In our studies, TAM67 was protective over a specific range of vinblastine concentrations, but was not protective at higher concentrations (Fig. 3)
. Therefore, AP-1-dependent pathways may dominate under certain conditions, but may be overridden by AP-1-independent death pathways under more extreme conditions, emphasizing the need to examine the effects of expression of TAM67 and other AP-1 inhibitors over a range of drug concentrations.
Recent results have shown that c-jun-/- fibroblasts exhibit a severe proliferation defect because of inefficient progression through G1 to S phase related to increased p53 and p21 levels (39) . Further studies demonstrated that c-Jun negatively regulates an AP-1-like site in the p53 promoter and that cell cycle progression is dependent on the ability of c-Jun to down-regulate p53 (39) . These results are of interest in the context of the present work because p53 has also been shown to play a key role in a DNA rereplication checkpoint after aberrant mitotic exit (6, 7, 8, 9, 10) . Thus, cells that exit an abnormal mitosis require p53 induction of p21 to arrest in G1 and prevent DNA rereplication, ensuring that S phase can follow only a successful mitosis.
Because of the links between c-Jun and p53, and between p53 and the postmitotic checkpoint, p53 and p21 levels were examined. p53 was found to be down-regulated by vinblastine in KB3 cells, and p21 levels were relatively low, whereas in KB3-TAM67 cells, p53 levels remained high and p21 levels were increased (Fig. 8)
. Taken together, these results provide a potential mechanistic basis for vinblastine resistance of KB3-TAM67 cells. As part of a working model, we envision that one role of AP-1 activation by vinblastine in KB3 cells is to down-regulate p53/p21 to prevent cell cycle arrest and promote apoptotic cell death. In contrast, vinblastine-induced c-Jun/AP-1 activation is blocked in KB3-TAM67 cells, p53 down-regulation is consequently prevented, and p21 levels are elevated. This may divert cells into an arrested state following mitotic exit, avoiding the fate of control cells. The arrested KB3-TAM67 cells may undergo a delayed cell death, consistent with their vinblastine resistance in short-term viability assays, or may recover and be competent to proliferate, consistent with increased clonogenicity.
cDNA microarray analysis provided a unique opportunity to further investigate, at the level of gene expression, the mechanism of AP-1 in vinblastine-induced cell death and the basis for resistance of KB3-TAM67 cells. Putative vinblastine-inducible AP-1 target genes in KB3 cells included TNF-, Bak, IGFBP4, and GST3 (Fig. 9
and Table 2
). The identification of GST3 as a potential AP-1 target is consistent with the presence of AP-1 elements in genes encoding GST isozymes (50)
. GST3 induction may represent a defense mechanism against electrophiles generated in vinblastine-treated cells. TNF- is a pleiotropic cytokine that mediates immune regulation and can induce diverse responses, including differentiation, proliferation, and apoptosis (51)
. The human TNF- promoter has a consensus AP-1-binding site as well as a site preferentially recognized by c-Jun/ATF-2 dimers (52)
, and mutation of the AP-1 site diminished both basal and TPA-activated promoter activity in macrophage cell lines (53)
. Interestingly, paclitaxel has long been known to stimulate TNF- secretion from murine macrophages (54)
, and the released TNF- can induce apoptosis of transformed mouse embryonic fibroblasts (55)
. In both these studies, however, extremely high concentrations of paclitaxel (3–30 µM) were required to induce TNF- production. Our results suggest that TNF- may be induced by low but lethal concentrations of vinblastine in nonmacrophage cell lines. Upstream regions of the human Bak gene have not been characterized sufficiently to indicate whether AP-1 sites might be present (56)
. However, potential binding sites for AP-1 and ATF/cAMP-responsive element-binding protein have been identified in the 5' flanking region of the human IGFBP4 gene (57)
, the product of which has the ability to sequester IGFs and inhibit proliferative signaling (58)
.
Fas L has been implicated as a proapoptotic target of JNK/AP-1 signaling. For example, using c-jun-/- fibroblasts, Kolbus et al. (59)
provided evidence that c-Jun-dependent expression of Fas L represents a rate-limiting step in apoptosis induced by methyl methanesulfonate. Apoptosis of neuronal cells deprived of NGF is triggered by AP-1-dependent Fas L transcription (28)
, and thymineless death of colon cancer cells also involves transcriptional regulation of the Fas L promoter by AP-1 and nuclear factor 
B (60)
. However, in our studies, the hybridization signal of Fas L was too weak to permit quantitation, and additional experiments will be required to determine whether Fas L represents an AP-1 target in this system.
As part of a working model, we propose that increased expression of TNF-, Bak, and IGFBP4, through JNK/AP-1, and decreased p53/p21 may represent elements of a coordinated response to promote efficient apoptosis in response to microtubule inhibition. Independent studies will be required to demonstrate that these gene products act as cellular intermediates in vinblastine-induced apoptosis. Nonetheless, given that a major limitation in defining a role for AP-1 in apoptosis has been difficulties in identification of the relevant target genes (61)
, the present results represent an important advancement and provide a sound basis for further exploration. Finally, the use of paired cell lines, where one cell line has a definable signaling defect, as described here, together with microarray technology represents a powerful and generally applicable approach for initial identification of genes acting at the distal ends of signal transduction pathways.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant CA-75577 (to T. C. C.). 
2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Mail Slot 516, 4301 West Markham Street, Little Rock, AR 72205-7199. Phone: (501) 686-5755; Fax: (501) 686-8169; E-mail: chamberstimothyc{at}uams.edu
3 The abbreviations used are: JNK, c-Jun NH2-terminal protein kinase; NGF, nerve growth factor; AP-1, activating protein 1; ATF-2, activating transcription factor-2; Bak, Bcl-2 homologous antagonist/killer protein; TNF-, tumor necrosis factor ; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA response element; AMC, amino-4-methylcoumarin; IGFBP4, insulin-like growth factor-binding protein 4; GST3, glutathione S-transferase 3.
Received 11/15/00. Accepted 3/28/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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radiation. Duration of JNK activation may determine cell death and proliferation. J. Biol. Chem., 271: 31929-31936, 1996.
-class glutathione S-transferase gene. Biochem. J., 324: 91-95, 1997.
promoter in primary human T lymphocytes. Gene. Expr., 8: 115-127, 1999.[Medline]
promoter region in macrophage. T-cell and B-cell lines. J. Biol. Chem., 267: 22102-22107, 1992.B and AP-1 in Fas-dependent thymineless death of human colon carcinoma cells. J. Biol. Chem., 275: 10023-10029, 2000.This article has been cited by other articles:
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  S. Singh, S. M. Manda, D. Sikder, M. J. Birrer, B. A. Rothermel, D. J. Garry, and P. P. A. Mammen Calcineurin Activates Cytoglobin Transcription in Hypoxic Myocytes J. Biol. Chem., April 17, 2009; 284(16): 10409 - 10421. [Abstract] [Full Text] [PDF]
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 S. N. Kolomeichuk, A. Bene, M. Upreti, R. A. Dennis, C. S. Lyle, M. Rajasekaran, and T. C. Chambers Induction of Apoptosis by Vinblastine via c-Jun Autoamplification and p53-Independent Down-Regulation of p21WAF1/CIP1 Mol. Pharmacol., January 1, 2008; 73(1): 128 - 136. [Abstract] [Full Text] [PDF]
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 M. Ammirante, R. Di Giacomo, L. De Martino, A. Rosati, M. Festa, A. Gentilella, M. C. Pascale, M. A. Belisario, A. Leone, M. Caterina Turco, et al. 1-Methoxy-Canthin-6-One Induces c-Jun NH2-Terminal Kinase-Dependent Apoptosis and Synergizes with Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Activity in Human Neoplastic Cells of Hematopoietic or Endodermal Origin. Cancer Res., April 15, 2006; 66(8): 4385 - 4393. [Abstract] [Full Text] [PDF]
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R. Aneja, M. Lopus, J. Zhou, S. N. Vangapandu, A. Ghaleb, J. Yao, J. H. Nettles, B. Zhou, M. Gupta, D. Panda, et al. Rational Design of the Microtubule-Targeting Anti-Breast Cancer Drug EM015. Cancer Res., April 1, 2006; 66(7): 3782 - 3791. [Abstract] [Full Text] [PDF]
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Y.-M. Yang, M. Jhanwar-Uniyal, J. Schwartz, C. C. Conaway, H. D. Halicka, F. Traganos, and F.-L. Chung N-Acetylcysteine Conjugate of Phenethyl Isothiocyanate Enhances Apoptosis in Growth-Stimulated Human Lung Cells Cancer Res., September 15, 2005; 65(18): 8538 - 8547. [Abstract] [Full Text] [PDF]
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 A. Bene, R. C. Kurten, and T. C. Chambers Subcellular localization as a limiting factor for utilization of decoy oligonucleotides Nucleic Acids Res., October 21, 2004; 32(19): e142 - e142. [Abstract] [Full Text] [PDF]
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 Y. Huang, Y. Fang, J. Wu, J. M. Dziadyk, X. Zhu, M. Sui, and W. Fan Regulation of Vinca alkaloid-induced apoptosis by NF-{kappa}B/I{kappa}B pathway in human tumor cells Mol. Cancer Ther., March 1, 2004; 3(3): 271 - 277. [Abstract] [Full Text]
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X. Qi, S. Borowicz, R. Pramanik, R. M. Schultz, J. Han, and G. Chen Estrogen Receptor Inhibits c-Jun-dependent Stress-induced Cell Death by Binding and Modifying c-Jun Activity in Human Breast Cancer Cells J. Biol. Chem., February 20, 2004; 279(8): 6769 - 6777. [Abstract] [Full Text] [PDF]
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 J. A. Choudhury, C. L. Russell, S. Randhawa, L. S. Young, D. H. Adams, and S. C. Afford Differential Induction of Nuclear Factor-kappa B and Activator Protein-1 Activity after CD40 Ligation Is Associated with Primary Human Hepatocyte Apoptosis or Intrahepatic Endothelial Cell Proliferation Mol. Biol. Cell, April 1, 2003; 14(4): 1334 - 1345. [Abstract] [Full Text] [PDF]
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A. Cuadrado, L. F. Garcia-Fernandez, L. Gonzalez, Y. Suarez, A. Losada, V. Alcaide, T. Martinez, J. M. Fernandez-Sousa, J. M. Sanchez-Puelles, and A. Munoz AplidinTM Induces Apoptosis in Human Cancer Cells via Glutathione Depletion and Sustained Activation of the Epidermal Growth Factor Receptor, Src, JNK, and p38 MAPK J. Biol. Chem., January 3, 2003; 278(1): 241 - 250. [Abstract] [Full Text] [PDF]
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J. Zhou, K. Gupta, J. Yao, K. Ye, D. Panda, P. Giannakakou, and H. C. Joshi Paclitaxel-resistant Human Ovarian Cancer Cells Undergo c-Jun NH2-terminal Kinase-mediated Apoptosis in Response to Noscapine J. Biol. Chem., October 11, 2002; 277(42): 39777 - 39785. [Abstract] [Full Text] [PDF]
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 D. E. Frigo, B. N. Duong, L. I. Melnik, L. S. Schief, B. M. Collins-Burow, D. K. Pace, J. A. McLachlan, and M. E. Burow Flavonoid Phytochemicals Regulate Activator Protein-1 Signal Transduction Pathways in Endometrial and Kidney Stable Cell Lines J. Nutr., July 1, 2002; 132(7): 1848 - 1853. [Abstract] [Full Text] [PDF]
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Q. Yu, M. He, N. H. Lee, and E. T. Liu Identification of Myc-mediated Death Response Pathways by Microarray Analysis J. Biol. Chem., April 5, 2002; 277(15): 13059 - 13066. [Abstract] [Full Text] [PDF]
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B. Comin-Anduix, N. Agell, O. Bachs, J. Ovadi, and M. Cascante A New Bis-Indole, KARs, Induces Selective M Arrest with Specific Spindle Aberration in Neuroblastoma Cell Line SH-SY5Y Mol. Pharmacol., December 1, 2001; 60(6): 1235 - 1242. [Abstract] [Full Text] [PDF]
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