Role of the stress-activated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs.

c-Jun NH2-terminal protein kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to many stressful stimuli including heat shock, UV irradiation, protein synthesis inhibitors, and inflammatory cytokines. In this study, we investigated whether JNK plays a role in the cellular response to different drugs commonly used in cancer chemotherapy. Treatment of human KB-3 carcinoma cells with Adriamycin resulted in a time- and dose-dependent activation of JNK of up to 40-fold. Treatment with vinblastine or etoposide (VP-16) also activated JNK, with maximum increases of 6.5- and 4.3-fold, respectively. Consistent with these findings, increased c-Jun phosphorylation was observed after drug treatment of cells. In contrast, none of the drugs significantly activated the extracellular response kinase/mitogen-activated protein kinase pathway. Since these drugs are transport substrates for the MDR1 gene product, P-glycoprotein, JNK was assayed in two multidrug-resistant (MDR) KB cell lines, KB-A1 and KB-V1, selected for resistance to Adriamycin and vinblastine, respectively. Relative to KB-3 cells, basal JNK activity was increased 7-fold in KB-A1 cells and 4-fold in KB-V1 cells, with no change in JNK protein expression, indicating that JNK is present in a more highly activated form in the MDR cell lines. Under conditions optimal for JNK activation, Adriamycin, vinblastine, and VP-16 all induced MDR1 mRNA expression in KB-3 cells. Our findings suggest that JNK activation is an important component of the cellular response to several structurally and functionally distinct anticancer drugs and may also play a role in the MDR phenotype.

Mitogen-activated protein kinases (MAPKs) 1 are serine/threonine kinases activated by dual phosphorylation on both a tyrosine and a threonine (reviewed in Refs. 1 and 2). These enzymes are important components of signaling pathways that transduce extracellular stimuli into intracellular responses. Each MAPK cascade consists of a module of three kinases: a MAPK kinase kinase, which phosphorylates and activates a MAPK kinase, which in turn phosphorylates and activates a MAPK. The classical MAPK module consists of Raf kinase, MEK, and ERK and is activated in response to a variety of mitogenic signals operating through different mechanisms. Activation by receptor tyrosine kinases is the best characterized mechanism, but certain G-protein-coupled receptors and cytokine receptors are also capable of activating the MAPK cascade (2). Downstream substrates regulated by ERK include transcription factors such as Elk-1 and ATF2, protein kinases including p90 rsk , and several other target proteins (1).
More recently, two other MAPK modules have been characterized. One consists of MEKK, MKK4 (or SEK1), and c-Jun NH 2 -terminal kinase (JNK), which, like Raf/MEK/ERK, operate in a phosphorylation cascade (3,4). However, unlike the classical MAPK pathway, the MEKK/SEK1/JNK module is only modestly activated by growth factors and phorbol esters and is instead strongly activated by cellular stress including heat shock, UV irradiation, protein synthesis inhibitors, and inflammatory cytokines (5). JNK is also termed stress-activated protein kinase (SAPK) (5), and two main forms (JNK1 of 46 kDa and JNK2 of 55 kDa) have been described (6). An important physiological substrate of JNK is c-Jun, and phosphorylation of two sites in the NH 2 -terminal transactivation domain (Ser-63 and Ser-73) regulates transcriptional activity (6).
A third MAPK isoform is p38, a homolog of the yeast HOG1 (high-osmolarity glycerol response-1) kinase (7), also termed p40 (8), reactivating kinase (9), or cytokine-suppressive antiinflammatory binding protein (10) in independent studies. Like ERK and JNK, p38 is activated by dual phosphorylation on a tyrosine and a threonine residue, and this is catalyzed by a MEK family member, MKK3 (11). p38 is activated by inflammatory cytokines and environmental stress including osmotic shock and UV irradiation (12). An important physiological substrate of p38 is MAPK-activated protein kinase-2, which phosphorylates heat shock protein hsp27 as part of the cellular response to stress (9). Although JNK and p38 appear to reside in distinct MAPK modules, a functional overlap is likely since each can complement the hog1-⌬1 yeast strain (7,13).
Recent data suggest that JNK is also activated in response to cellular stress induced by certain DNA-damaging agents. For example, 1-␤-D-arabinofuranosylcytosine (araC) (14), cis-platinum (15), and mitomycin C (15) activate JNK in NIH-3T3 fibroblasts. In this study, we sought to determine whether MAPKs play a role in the cellular stress response to other cancer chemotherapeutic drugs. We examined JNK and ERK activation in human carcinoma cells treated with Adriamycin, vinblastine, or VP-16. These agents were chosen for study because they represent widely utilized anticancer drugs with different mechanisms of action. Adriamycin has a complex mechanism of cytotoxicity through its ability to intercalate DNA and generate superoxide; vinblastine is a microtubule inhibitor; and VP-16 is a topoisomerase II inhibitor. Since these drugs belong to the multidrug resistance (MDR) group of substrates transported by P-glycoprotein (16), JNK activity was also evaluated in MDR variant carcinoma cell lines. Our findings suggest that JNK activation is an important component of the cellular response to mechanistically diverse cancer chemotherapeutic drugs and may also play a role in MDR.

EXPERIMENTAL PROCEDURES
Materials-Polyclonal antibodies to JNK1 or ERK1 were obtained from Santa Cruz Biotechnology. Polyclonal antibodies specific for the phosphorylated forms of ERK1/2 or c-Jun were obtained from New England Biolabs Inc. GST-c-Jun(79), a fusion protein of glutathione S-transferase and residues 1-79 of human c-Jun, was either obtained from Santa Cruz Biotechnology or purified from Escherichia coli cells harboring the GST-c-Jun(79) expression plasmid (kindly provided by Dr. Omar Coso, Molecular Signaling Unit, National Institutes of Health). [␥-32 P]ATP (3000 Ci/mmol) was obtained from Amersham International. Tissue culture media and the SuperScript preamplification system were purchased from Life Technologies, Inc. Taq polymerase was from Fisher, and T4 polynucleotide kinase was from Promega. 12-O-Tetradecanoylphorbol-13-acetate (TPA), Adriamycin, vinblastine, etoposide (VP-16), araC, myelin basic protein, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin were obtained from Sigma. Okadaic acid was from LC Laboratories.
Cell Culture and Treatment Conditions-Human KB-3 carcinoma cells were maintained as monolayer cultures at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 g/ml streptomycin. The MDR variant KB-V1 and KB-A1 cell lines were maintained in the same medium containing 1 g/ml vinblastine or 1 g/ml Adriamycin, respectively. The MDR variants, which overexpress MDR1/P-glycoprotein and were derived from KB-3 cells by stepwise selection in medium containing vinblastine or Adriamycin, respectively (17), were kindly provided by Dr. Michael Gottesman (National Cancer Institute, National Institutes of Health). Human K562 myelogenous leukemia cells were maintained as suspension cultures at 37°C and 5% CO 2 in RPMI 1640 medium supplemented with 10% fetal calf serum and were kindly provided by Dr. Jacki Kornbluth (Arkansas Cancer Research Center). Cells were cultivated in 25-cm 2 flasks; stock solutions of drugs were prepared in dimethyl sulfoxide; and treatment conditions were such that cell viability, determined by trypan blue exclusion, was 90 -95% in all experiments.
Cell Preparation and JNK Assay-Cells were washed in ice-cold phosphate-buffered saline, removed from the flask by gentle scraping and sedimentation, and suspended in 0.25 ml of JNK lysis buffer (25 mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.1% Triton X-100, 20 mM ␤-glycerophosphate, 1 mM sodium vanadate, 0.1 M okadaic acid, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml aprotinin, 50 g/ml leupeptin, and 10 M pepstatin). After 15 min on ice, insoluble material was removed by sedimentation for 20 min at 100,000 ϫ g. JNK activity was determined by an immunocomplex assay essentially as described (18). Briefly, each cell extract (400 g) was mixed with 10 l of anti-JNK antibody for 1 h, and then 30 l of 50% protein A-Sepharose in JNK lysis buffer was added for an additional 1 h. The immunocomplex was recovered by sedimentation for 5 min in a microcentrifuge; washed three times with 0.5 ml of phosphate-buffered saline containing 1% Nonidet P-40 and 2 mM sodium vanadate; washed once with 0.1 M Tris-HCl, pH 7.5, and 0.5 M LiCl; and washed once with JNK reaction buffer (12.5 mM MOPS, pH 7.5, 20 mM ␤-glycerophosphate, 7.5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM sodium fluoride, and 0.5 mM sodium vanadate). The immunoprecipitate was resuspended in 30 l of JNK reaction buffer containing 1 g of GST-c-Jun(79), and the reaction was initiated by the addition of 5 l of 0.1 mM [␥-32 P]ATP (30,000 cpm/pmol). After incubation for 20 min at 30°C, the reaction was terminated by the addition of 8 l of 5 ϫ SDS sample buffer (19) and heating to 95°C for 5 min. Samples were analyzed by SDS-PAGE (12% acrylamide); gels were stained with Coomassie Blue and subjected to autoradiography. Quantitation was performed by densitometric scanning of the autoradiographic film with a Model 300A laser densitometer and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Assay of ERK-Cells were harvested as described above and suspended in 0.25 ml of ERK lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium vanadate, 50 mM sodium fluoride, 20 mM ␤-glycerophosphate, 0.1 M okadaic acid, and protease inhibitors as in JNK lysis buffer). After 15 min on ice, insoluble material was removed by sedimentation for 20 min at 100,000 ϫ g, and ERK activity was determined by an immunocomplex assay with myelin basic protein as substrate as described (20).
Immunoblot Analysis-Soluble cell lysates or nuclear suspensions (40 -100 g, as indicated in the figure legends) were fractionated by SDS-PAGE; transferred to polyvinylidene difluoride membrane; and probed with antibodies, all of which were used at a dilution of 1:1000, except anti-phospho-ERK1/2 antibody (1:2500). Primary antibody was detected by horseradish peroxidase-conjugated second antibody (1:5000), which in turn was visualized using enhanced chemiluminescence (Amersham International).
RNA Extraction and cDNA PCR Analysis of MDR1 Expression-Total RNA from control and drug-treated cell populations (cultivated in six-well plates) was extracted by a small-scale procedure using RNA STAT-60 (Tel-Test "B", Inc.). cDNA synthesis was carried out using the SuperScript preamplification system as described by the manufacturer. Amplification with primers specific for MDR1 or ␤ 2 -microglobulin, the latter as an internal control, was performed as described previously (21) with certain modifications. Briefly, PCR was performed with a Perkin-Elmer 2400 Thermocycler, and reaction mixtures contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 200 M each dNTP, 200 ng of each gene-specific primer, 50 ng of RNA-equivalent cDNA, and 1 unit of Taq polymerase. Sense primers were 5Ј-end-labeled with [␥-32 P]ATP for detection of the amplified product. After an initial denaturation at 94°C for 5 min, the following step-cycle program was initiated: denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 60 s. For MDR1, 37 cycles were performed, and for ␤ 2 -microglobulin, 27 cycles were performed, each followed by a final extension at 72°C for 7 min. PCR products were analyzed by SDS-PAGE (12% acrylamide) and autoradiography.

RESULTS
Activation of JNK by Adriamycin-To validate the JNK assay in KB-3 cell extracts and to examine possible effects on activity of Adriamycin treatment, lysates were prepared from cells exposed to 500 ng/ml Adriamycin for either 1 or 16 h. As controls, cells were untreated, exposed to vehicle (dimethyl sulfoxide) alone, heat-shocked, or stimulated with 100 nM TPA for 30 min. JNK activity was determined by an immunocomplex assay with GST-c-Jun(79) as substrate as described under "Experimental Procedures." Consistent with previous reports in other cell lines (e.g. Ref. 5), JNK was strongly activated by heat shock, but was not significantly activated by TPA (Fig. 1). Significantly increased JNK activity was also observed after treatment of cells with Adriamycin for 16 h (Fig. 1). A more detailed kinetic study demonstrated that JNK activity was detectably increased after a 1-h exposure of cells to Adriamycin, with a further increase after 4 h and a stimulation of Ͼ40-fold (average of four experiments, as determined by densitometric scanning) after a 16-h exposure to the drug ( Fig. 2A,  upper panel). Immunoblotting of cell lysates with anti-JNK antibody revealed that JNK1 protein levels remained constant under these treatment conditions ( Fig. 2A, lower panel). The concentration dependence of JNK activation by Adriamycin (16-h exposure) was next determined; enzyme activation was observed in the range of 20 -500 ng/ml Adriamycin, with an increase of 40-fold at 500 ng/ml (Fig. 2B).
Activation of JNK by Vinblastine and VP-16 -We next ex-amined JNK activity in KB-3 cells treated for 16 h with two other important cancer chemotherapeutic drugs, vinblastine and VP-16. Activation of JNK was observed at vinblastine concentrations of 5 ng/ml and above, with a stimulation of enzyme activity of 6.5-fold (average of three experiments) (Fig.  3A). VP-16 treatment also activated JNK, with a stimulation of enzyme activity of 4.3-fold (average of two experiments) at a concentration of 1 g/ml (Fig. 3B). We also tested araC, which, at 10 M for 2 h, has been reported to activate JNK in NIH-3T3 and 293 kidney cells (14). When KB-3 cells were exposed to 10 M araC for different periods up to 40 h, we observed a modest 2-fold activation of JNK activity (data not shown).
Effect of Drugs on c-Jun Phosphorylation-An anticipated consequence of JNK activation is an increased phosphorylation of sites in the NH 2 -terminal region of the nuclear substrate, c-Jun. NH 2 -terminal c-Jun phosphorylation was monitored by gel electrophoresis of nuclear extracts and immunoblotting with an antibody specific for the phosphorylated Ser-73 form of c-Jun. As shown in Fig. 4, significantly increased phospho-Ser-73 c-Jun immunoreactivity was observed in nuclei of cells exposed to Adriamycin or vinblastine. Immunoreactivity was barely detectable in control or VP-16-or araC-treated cells under these conditions and was greater in vinblastine-treated versus Adriamycin-treated cells, despite the fact that Adriamycin was a more potent activator of JNK. In Adriamycin-treated cells, activated JNK may preferentially phosphorylate another substrate, or the time course of c-Jun phosphorylation may differ from that of JNK activation. Despite these caveats, the increased abundance of c-Jun phosphorylated on Ser-73 is consistent with JNK activation in vivo in response to Adriamycin or vinblastine treatment.
Effect of Drugs on ERK Activation-The results presented above indicated that certain chemotherapeutic drugs activate the SAPK pathway in KB-3 cells. To determine whether the drugs selectively activated specific MAPK cascades, ERK activation was examined in KB-3 cells treated with drugs under conditions established to activate JNK. As controls, cells were untreated, heat-shocked, or exposed to TPA. As shown in Fig.  5A (upper panel, lanes 4 -8), only weak stimulation of ERK activity (1.5-2-fold as determined by densitometric scanning) was observed for the chemotherapeutic drug treatments. Somewhat unexpectedly, ERK activity was increased Ͼ6-fold by heat shock, and this provided a convenient positive control for ERK activation in these cells (lane 3). TPA failed to significantly activate ERK in KB-3 cells (lane 9), but this appeared to be a cell type-specific phenomenon since TPA strongly activated ERK in K562 cells (lanes 1 and 2). In all cases, the expression level of ERK protein appeared unchanged as judged by immunoblot analysis (Fig. 5A, lower panel). To confirm these observations with an independent assessment of ERK activation, cell lysates from similarly treated cells were subjected to Western blot analysis with a phospho-ERK1/2 antibody (Fig.  5B). Phospho-ERK1/2 immunoreactivity was increased by heat shock, but was not significantly affected by the drug treatments, consistent with the ERK immunocomplex assay data. The specificity of the phospho-ERK antibody was confirmed by antibody recognition of a control protein consisting of bacterially expressed, purified ERK2 phosphorylated by MEK (Fig. 5B).
JNK Activity in Multidrug-resistant Cells-Adriamycin, vinblastine, and VP-16 share the common property of being substrates for the drug efflux pump, P-glycoprotein, which is overexpressed in many MDR cell lines (16). Since all three drugs activated JNK in KB-3 cells, it was of interest to evaluate JNK activity in the MDR derivative KB-V1 and KB-A1 cell lines, which were derived from KB-3 cells by selection for resistance to vinblastine and Adriamycin, respectively (17). Cell lysates were prepared and subjected to JNK immunocomplex assay and JNK immunoblot analysis (Fig. 6). Relative to KB-3 cells, basal JNK activity was found to be significantly increased in both MDR cell lines (4-fold for KB-V1 and 7-fold for KB-A1, average of three independent experiments), while all three cell lines expressed similar levels of JNK protein. These results suggest that JNK is present in a more highly activated form in the MDR variants. Basal ERK activity and ERK protein level were also assessed and found to be similar for the KB-3, KB-V1, and KB-A1 cell lines (data not shown).
Induction of MDR1 mRNA by Chemotherapeutic Drugs-An earlier study found that treatment with chemotherapeutic drugs of certain drug-sensitive cancer cell lines, including KB-3, K562, and H9 cells, resulted in the induction of MDR1 mRNA (21). It was suggested that MDR1 induction may be a general response to drug-induced cellular damage. We considered the possibility that JNK activation and MDR1 mRNA induction by chemotherapeutic drugs may be linked, particularly in view of the results in Fig. 6 showing increased JNK activity in MDR cells. We therefore analyzed MDR1 mRNA expression by reverse transcription-PCR in untreated and drug-treated KB-3 cells. In confirmation of the earlier findings (21), untreated cells expressed a barely detectable level of MDR1 mRNA. Treatment of cells with 200 ng/ml Adriamycin, 5 ng/ml vinblastine, 1 g/ml VP-16, or 10 M araC induced MDR1 expression (Fig. 7). These drug concentrations were found to be optimal for MDR1 induction in KB-3 cells. The MDR1-overexpressing KB-V1 cell line was utilized as a positive control, and ␤ 2 -microglobulin mRNA levels were determined as an internal control and found to be unchanged (Fig. 7). DISCUSSION In this paper, we have shown that three structurally and mechanistically distinct anticancer drugs activate the stressactivated kinase, JNK, in human carcinoma cells. The most potent compound studied was Adriamycin, which maximally activated JNK 40-fold; vinblastine and VP-16 maximally acti- vated JNK 6.5-and 4-fold, respectively. JNK activation occurred at clinically relevant drug concentrations. araC was also tested, but was a relatively poor JNK activator in this system. In all cases, increased JNK enzyme activity occurred without a change in JNK protein expression. Consequences of activated JNK include phosphorylation of pre-existing c-Jun and an increase in c-jun transcription and synthesis of new c-Jun protein (reviewed in Ref. 22). The increased abundance of c-Jun phosphorylated on Ser-73 in cells treated with Adriamycin or vinblastine provides further evidence that exposure to these agents stimulates JNK activity in vivo. More detailed studies will be required to determine the temporal relationship between c-Jun phosphorylation and JNK activation in response to Adriamycin and vinblastine treatment and to determine whether VP-16 treatment influences the phosphorylation of c-Jun. The drugs examined failed to significantly activate the ERK pathway relative to the JNK pathway, thereby displaying specificity with regard to stimulation of different MAPK pathways. It remains to be determined whether other SAPKs such as p38 MAPK or the newly described SAPK3 (23) are also activated by these cytotoxic drugs and whether other JNK substrates such as ATF2 are affected.
The JNK pathway is likely activated as a result of druginduced cellular damage, but the intracellular signals linking cell damage to the stress response are incompletely defined. Many DNA-damaging drugs including Adriamycin and VP-16 induce expression of the nuclear phosphoprotein p53 (24,25). Vinblastine, which does not directly damage DNA, has also been shown to increase the levels of transcriptionally active p53 in NIH-3T3 fibroblasts (26). Downstream events of p53 activation include cell cycle arrest at G 1 and apoptosis in many cell systems (reviewed in Ref. 27 (33) reported that stable expression of a dominant inhibiting SEK1 mutant blocked SAPK activation and conferred increased resistance to cell death induced by several different stressful stimuli including heat shock, UV irradiation, and cis-platinum. In addition, transient expression of a dominant-negative kinase-inactive SEK1 mutant reduced apoptosis induced by several JNK activators including H 2 O 2 , UV irradiation, heat shock, or treatment with tumor necrosis factor-␣ (34). Our finding that mechanistically distinct cytotoxic drugs activate the SAPK/JNK pathway supports the concept that JNK may play a role in the cell death pathways induced by these agents.
Although evidence has accumulated suggesting a role for the SAPK pathway in cell death, there may be other consequences of JNK activation. It is possible that, in some circumstances, SAPK/JNK activation is part of a protective mechanism to support cell survival. The degree of damage and the capacity for repair may be important determinants dictating the choice between death and survival. One well characterized mechanism of protection against cytotoxic drugs is P-glycoprotein overexpression (16). Consistent with a previous report (21), we showed that Adriamycin, vinblastine, VP-16, and araC all induced MDR1 mRNA expression in KB-3 cells (Fig. 7). With the exception of araC, which was a poor JNK activator in these cells, the concentrations of the drugs optimal for MDR1 induction were similar to those optimal for JNK activation. Although these results do not demonstrate a causal relationship between JNK activation and MDR1 induction, they do suggest a possible link between the two parameters. The fact that the MDR cell lines examined express a more highly activated form of JNK is intriguing in this regard. The presence of activated JNK could be due to activation of upstream regulators of JNK, inactivation or down-regulation of a phosphatase acting on JNK, or both in the MDR cell lines. In the context of this study, it is interesting to note that JNK activation and MDR1 expression are induced by several other common stimuli. For example, the MDR1 gene is induced by heat shock (35) and UV irradiation (36), both well established activators of JNK. The presence of a non-canonical AP-1 consensus element in the human MDR1 promoter (37) is perhaps significant since JNK enhances AP-1-dependent transcription through modulation of c-Jun phosphorylation and expression (22). Further investigation will be required to elucidate the role of JNK in the cellular response to cytotoxic drugs and the relationship of this stressactivated pathway to MDR1 expression.