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Role of the Stress-activated/c-Jun NH2-terminal Protein Kinase Pathway in the Cellular Response to Adriamycin and Other Chemotherapeutic Drugs

Role of the Stress-activated/c-Jun NH2-terminal Protein Kinase Pathway in the Cellular Response... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 48, Issue of November 29, pp. 30950–30955, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Role of the Stress-activated/c-Jun NH -terminal Protein Kinase Pathway in the Cellular Response to Adriamycin and Other Chemotherapeutic Drugs* (Received for publication, July 19, 1996) Maudie T. Osborn and Timothy C. Chambers‡ From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199 transduce extracellular stimuli into intracellular responses. c-Jun NH -terminal protein kinase (JNK), a member of the mitogen-activated protein kinase family, is acti- Each MAPK cascade consists of a module of three kinases: a vated in response to many stressful stimuli including MAPK kinase kinase, which phosphorylates and activates a heat shock, UV irradiation, protein synthesis inhibitors, MAPK kinase, which in turn phosphorylates and activates a and inflammatory cytokines. In this study, we investi- MAPK. The classical MAPK module consists of Raf kinase, gated whether JNK plays a role in the cellular response MEK, and ERK and is activated in response to a variety of to different drugs commonly used in cancer chemother- mitogenic signals operating through different mechanisms. Ac- apy. Treatment of human KB-3 carcinoma cells with Ad- tivation by receptor tyrosine kinases is the best characterized riamycin resulted in a time- and dose-dependent activa- mechanism, but certain G-protein-coupled receptors and cyto- tion of JNK of up to 40-fold. Treatment with vinblastine kine receptors are also capable of activating the MAPK cascade or etoposide (VP-16) also activated JNK, with maximum (2). Downstream substrates regulated by ERK include tran- increases of 6.5- and 4.3-fold, respectively. Consistent scription factors such as Elk-1 and ATF2, protein kinases in- with these findings, increased c-Jun phosphorylation rsk cluding p90 , and several other target proteins (1). was observed after drug treatment of cells. In contrast, More recently, two other MAPK modules have been charac- none of the drugs significantly activated the extracellu- terized. One consists of MEKK, MKK4 (or SEK1), and c-Jun lar response kinase/mitogen-activated protein kinase NH -terminal kinase (JNK), which, like Raf/MEK/ERK, oper- pathway. Since these drugs are transport substrates for 2 ate in a phosphorylation cascade (3, 4). However, unlike the the MDR1 gene product, P-glycoprotein, JNK was as- classical MAPK pathway, the MEKK/SEK1/JNK module is sayed in two multidrug-resistant (MDR) KB cell lines, only modestly activated by growth factors and phorbol esters KB-A1 and KB-V1, selected for resistance to Adriamycin and vinblastine, respectively. Relative to KB-3 cells, ba- and is instead strongly activated by cellular stress including sal JNK activity was increased 7-fold in KB-A1 cells and heat shock, UV irradiation, protein synthesis inhibitors, and 4-fold in KB-V1 cells, with no change in JNK protein inflammatory cytokines (5). JNK is also termed stress-acti- expression, indicating that JNK is present in a more vated protein kinase (SAPK) (5), and two main forms (JNK1 of highly activated form in the MDR cell lines. Under con- 46 kDa and JNK2 of 55 kDa) have been described (6). An ditions optimal for JNK activation, Adriamycin, vinblas- important physiological substrate of JNK is c-Jun, and phos- tine, and VP-16 all induced MDR1 mRNA expression in phorylation of two sites in the NH -terminal transactivation KB-3 cells. Our findings suggest that JNK activation is domain (Ser-63 and Ser-73) regulates transcriptional activity an important component of the cellular response to sev- (6). eral structurally and functionally distinct anticancer A third MAPK isoform is p38, a homolog of the yeast HOG1 drugs and may also play a role in the MDR phenotype. (high-osmolarity glycerol response-1) kinase (7), also termed p40 (8), reactivating kinase (9), or cytokine-suppressive anti- inflammatory binding protein (10) in independent studies. Like Mitogen-activated protein kinases (MAPKs) are serine/thre- ERK and JNK, p38 is activated by dual phosphorylation on a onine kinases activated by dual phosphorylation on both a tyrosine and a threonine residue, and this is catalyzed by a tyrosine and a threonine (reviewed in Refs. 1 and 2). These MEK family member, MKK3 (11). p38 is activated by inflam- enzymes are important components of signaling pathways that matory cytokines and environmental stress including osmotic shock and UV irradiation (12). An important physiological sub- strate of p38 is MAPK-activated protein kinase-2, which phos- * This work was supported in part by the Mr. and Mrs. Sam Walton Research Award, University of Arkansas for Medical Sciences Medical phorylates heat shock protein hsp27 as part of the cellular Research Endowment Fund. response to stress (9). Although JNK and p38 appear to reside ‡ To whom correspondence should be addressed: Dept. of Biochemis- in distinct MAPK modules, a functional overlap is likely since try and Molecular Biology, University of Arkansas for Medical Sciences, each can complement the hog1-D1 yeast strain (7, 13). Mail Slot 516, 4301 W. Markham St., Little Rock, AR 72205-7199. Tel.: 501-686-5755; Fax: 501-686-8169. Recent data suggest that JNK is also activated in response to The abbreviations used are: MAPK, mitogen-activated protein ki- cellular stress induced by certain DNA-damaging agents. For nase; MEK, mitogen-activated/extracellular response kinase kinase; example, 1-b-D-arabinofuranosylcytosine (araC) (14), cis-plati- ERK, extracellular response kinase; MEKK, mitogen-activated/extra- num (15), and mitomycin C (15) activate JNK in NIH-3T3 cellular response kinase kinase kinase; SEK, stress-activated protein kinase kinase; JNK, c-Jun NH -terminal protein kinase; SAPK, stress- fibroblasts. In this study, we sought to determine whether activated protein kinase; araC, 1-b-D-arabinofuranosylcytosine; MDR, MAPKs play a role in the cellular stress response to other multidrug resistance or multidrug-resistant; GST, glutathione S-trans- cancer chemotherapeutic drugs. We examined JNK and ERK ferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MOPS, 4-morpho- activation in human carcinoma cells treated with Adriamycin, linepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction. vinblastine, or VP-16. These agents were chosen for study 30950 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Activation of SAPK/JNK by Chemotherapeutic Drugs 30951 Assay of ERK—Cells were harvested as described above and sus- because they represent widely utilized anticancer drugs with pended in 0.25 ml of ERK lysis buffer (50 mM Hepes, pH 7.5, 150 mM different mechanisms of action. Adriamycin has a complex NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium van- mechanism of cytotoxicity through its ability to intercalate adate, 50 mM sodium fluoride, 20 mM b-glycerophosphate, 0.1 mM oka- DNA and generate superoxide; vinblastine is a microtubule daic acid, and protease inhibitors as in JNK lysis buffer). After 15 min inhibitor; and VP-16 is a topoisomerase II inhibitor. Since these on ice, insoluble material was removed by sedimentation for 20 min at drugs belong to the multidrug resistance (MDR) group of sub- 100,000 3 g, and ERK activity was determined by an immunocomplex assay with myelin basic protein as substrate as described (20). strates transported by P-glycoprotein (16), JNK activity was Preparation of Nuclear Extracts—Cell monolayers were washed in also evaluated in MDR variant carcinoma cell lines. Our find- phosphate-buffered saline, collected by scraping and sedimentation, ings suggest that JNK activation is an important component of and suspended in 0.3 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM the cellular response to mechanistically diverse cancer chemo- EDTA, 2 mM EGTA, 10 mM 2-mercaptoethanol, 20 mM b-glycerophos- therapeutic drugs and may also play a role in MDR. phate, 1 mM sodium vanadate, 0.1 mM okadaic acid, 1 mM phenylmeth- ylsulfonyl fluoride, 20 mg/ml aprotinin, 50 mg/ml leupeptin, and 10 mM EXPERIMENTAL PROCEDURES pepstatin). The cells were lysed by Dounce homogenization, and nuclei Materials—Polyclonal antibodies to JNK1 or ERK1 were obtained were collected by centrifugation (10 min, 1000 3 g). The samples, from Santa Cruz Biotechnology. Polyclonal antibodies specific for the adjusted to contain equal protein, were solubilized in SDS sample phosphorylated forms of ERK1/2 or c-Jun were obtained from New buffer with heating to 95 °C for 5 min. England Biolabs Inc. GST-c-Jun(79), a fusion protein of glutathione Immunoblot Analysis—Soluble cell lysates or nuclear suspensions S-transferase and residues 1–79 of human c-Jun, was either obtained (40–100 mg, as indicated in the figure legends) were fractionated by from Santa Cruz Biotechnology or purified from Escherichia coli cells SDS-PAGE; transferred to polyvinylidene difluoride membrane; and harboring the GST-c-Jun(79) expression plasmid (kindly provided by probed with antibodies, all of which were used at a dilution of 1:1000, Dr. Omar Coso, Molecular Signaling Unit, National Institutes of except anti-phospho-ERK1/2 antibody (1:2500). Primary antibody was Health). [g- P]ATP (3000 Ci/mmol) was obtained from Amersham In- detected by horseradish peroxidase-conjugated second antibody ternational. Tissue culture media and the SuperScript preamplification (1:5000), which in turn was visualized using enhanced chemilumines- system were purchased from Life Technologies, Inc. Taq polymerase cence (Amersham International). was from Fisher, and T4 polynucleotide kinase was from Promega. RNA Extraction and cDNA PCR Analysis of MDR1 Expression— 12-O-Tetradecanoylphorbol-13-acetate (TPA), Adriamycin, vinblastine, Total RNA from control and drug-treated cell populations (cultivated in etoposide (VP-16), araC, myelin basic protein, phenylmethylsulfonyl six-well plates) was extracted by a small-scale procedure using RNA fluoride, leupeptin, pepstatin, and aprotinin were obtained from Sigma. STAT-60 (Tel-Test “B”, Inc.). cDNA synthesis was carried out using the Okadaic acid was from LC Laboratories. SuperScript preamplification system as described by the manufacturer. Cell Culture and Treatment Conditions—Human KB-3 carcinoma Amplification with primers specific for MDR1 or b -microglobulin, the cells were maintained as monolayer cultures at 37 °C and 5% CO in latter as an internal control, was performed as described previously (21) Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose and with certain modifications. Briefly, PCR was performed with a Perkin- supplemented with 10% fetal calf serum, 2 mML-glutamine, 50 units/ml Elmer 2400 Thermocycler, and reaction mixtures contained 10 mM penicillin, and 50 mg/ml streptomycin. The MDR variant KB-V1 and Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl , 200 mM each dNTP, 200 ng KB-A1 cell lines were maintained in the same medium containing 1 of each gene-specific primer, 50 ng of RNA-equivalent cDNA, and 1 unit mg/ml vinblastine or 1 mg/ml Adriamycin, respectively. The MDR vari- of Taq polymerase. Sense primers were 59-end-labeled with [g- P]ATP ants, which overexpress MDR1/P-glycoprotein and were derived from for detection of the amplified product. After an initial denaturation at KB-3 cells by stepwise selection in medium containing vinblastine or 94 °C for 5 min, the following step-cycle program was initiated: dena- Adriamycin, respectively (17), were kindly provided by Dr. Michael turation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at Gottesman (National Cancer Institute, National Institutes of Health). 72 °C for 60 s. For MDR1, 37 cycles were performed, and for b -micro- Human K562 myelogenous leukemia cells were maintained as suspen- globulin, 27 cycles were performed, each followed by a final extension at sion cultures at 37 °C and 5% CO in RPMI 1640 medium supplemented 72 °C for 7 min. PCR products were analyzed by SDS-PAGE (12% with 10% fetal calf serum and were kindly provided by Dr. Jacki acrylamide) and autoradiography. Kornbluth (Arkansas Cancer Research Center). Cells were cultivated in 25-cm flasks; stock solutions of drugs were prepared in dimethyl sulf- RESULTS oxide; and treatment conditions were such that cell viability, deter- Activation of JNK by Adriamycin—To validate the JNK as- mined by trypan blue exclusion, was 90–95% in all experiments. say in KB-3 cell extracts and to examine possible effects on Cell Preparation and JNK Assay—Cells were washed in ice-cold phosphate-buffered saline, removed from the flask by gentle scraping activity of Adriamycin treatment, lysates were prepared from and sedimentation, and suspended in 0.25 ml of JNK lysis buffer (25 cells exposed to 500 ng/ml Adriamycin for either 1 or 16 h. As mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl , 0.2 mM EDTA, 0.5 mM controls, cells were untreated, exposed to vehicle (dimethyl dithiothreitol, 0.1% Triton X-100, 20 mM b-glycerophosphate, 1 mM sulfoxide) alone, heat-shocked, or stimulated with 100 nM TPA sodium vanadate, 0.1 mM okadaic acid, 1 mM phenylmethylsulfonyl for 30 min. JNK activity was determined by an immunocom- fluoride, 20 mg/ml aprotinin, 50 mg/ml leupeptin, and 10 mM pepstatin). After 15 min on ice, insoluble material was removed by sedimentation plex assay with GST-c-Jun(79) as substrate as described under for 20 min at 100,000 3 g. JNK activity was determined by an immu- “Experimental Procedures.” Consistent with previous reports nocomplex assay essentially as described (18). Briefly, each cell extract in other cell lines (e.g. Ref. 5), JNK was strongly activated by (400 mg) was mixed with 10 ml of anti-JNK antibody for 1 h, and then 30 heat shock, but was not significantly activated by TPA (Fig. 1). ml of 50% protein A-Sepharose in JNK lysis buffer was added for an Significantly increased JNK activity was also observed after additional 1 h. The immunocomplex was recovered by sedimentation for treatment of cells with Adriamycin for 16 h (Fig. 1). A more 5 min in a microcentrifuge; washed three times with 0.5 ml of phos- phate-buffered saline containing 1% Nonidet P-40 and 2 mM sodium detailed kinetic study demonstrated that JNK activity was vanadate; washed once with 0.1 M Tris-HCl, pH 7.5, and 0.5 M LiCl; and detectably increased after a 1-h exposure of cells to Adriamy- washed once with JNK reaction buffer (12.5 mM MOPS, pH 7.5, 20 mM cin, with a further increase after 4 h and a stimulation of b-glycerophosphate, 7.5 mM MgCl , 0.5 mM EGTA, 0.5 mM sodium .40-fold (average of four experiments, as determined by den- fluoride, and 0.5 mM sodium vanadate). The immunoprecipitate was sitometric scanning) after a 16-h exposure to the drug (Fig. 2A, resuspended in 30 ml of JNK reaction buffer containing 1 mg of GST-c- upper panel). Immunoblotting of cell lysates with anti-JNK Jun(79), and the reaction was initiated by the addition of 5 ml of 0.1 mM [g- P]ATP (30,000 cpm/pmol). After incubation for 20 min at 30 °C, the antibody revealed that JNK1 protein levels remained constant reaction was terminated by the addition of 8 mlof5 3 SDS sample under these treatment conditions (Fig. 2A, lower panel). The buffer (19) and heating to 95 °C for 5 min. Samples were analyzed by concentration dependence of JNK activation by Adriamycin SDS-PAGE (12% acrylamide); gels were stained with Coomassie Blue (16-h exposure) was next determined; enzyme activation was and subjected to autoradiography. Quantitation was performed by den- observed in the range of 20–500 ng/ml Adriamycin, with an sitometric scanning of the autoradiographic film with a Model 300A increase of 40-fold at 500 ng/ml (Fig. 2B). laser densitometer and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Activation of JNK by Vinblastine and VP-16—We next ex- 30952 Activation of SAPK/JNK by Chemotherapeutic Drugs FIG.1. Activation of JNK by heat shock or Adriamycin. JNK activity in KB-3 cell lysates was determined by immunocomplex assay as described under “Experimental Procedures.” Phosphorylated GST-c- Jun(79) (GST-cJ) was detected by SDS-PAGE and autoradiography. Molecular mass standards (in kilodaltons) are indicated on the left. Lane 1, control (no addition); lane 2, control (dimethyl sulfoxide (DMSO) vehicle, 0.1% (v/v) final concentration); lane 3, heat shock (42 °C, 35 min); lane 4, TPA (100 nM, 30 min); lanes 5 and 6, Adriamycin (ADR; 500 ng/ml, 1 or 16 h). FIG.3. Concentration dependence of JNK activation by vin- blastine or VP-16. KB-3 cells were treated with the indicated concen- trations of vinblastine (VBL; A) or VP-16 (B) for 16 h. JNK activity was determined by immunocomplex assay (upper panels in A and B), and JNK protein levels by immunoblotting (lower panels in A and B)as described under “Experimental Procedures.” See the legend to Fig. 2 for other details. GST-cJ, GST-c-Jun(79). enzyme activity of 4.3-fold (average of two experiments) at a concentration of 1 mg/ml (Fig. 3B). We also tested araC, which, at 10 mM 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 mM 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 -terminal region of the nuclear substrate, c-Jun. NH -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- FIG.2. Time course and concentration dependence of JNK Ser-73 c-Jun immunoreactivity was observed in nuclei of cells activation by Adriamycin. A, time course of JNK activation and determination of JNK protein levels. JNK activity was determined by exposed to Adriamycin or vinblastine. Immunoreactivity was immunocomplex assay in lysates from KB-3 cells treated with 500 barely detectable in control or VP-16- or araC-treated cells ng/ml Adriamycin (ADR) for the times indicated. Upper panel, phos- under these conditions and was greater in vinblastine-treated phorylated GST-c-Jun(79) (GST-cJ) detected by SDS-PAGE and auto- versus Adriamycin-treated cells, despite the fact that Adriamy- radiography; lower panel, immunoblot of the respective cell lysates (40 mg of protein/lane) probed with anti-JNK antibody. The strongly immu- cin was a more potent activator of JNK. In Adriamycin-treated noreactive band corresponds to JNK1. B, concentration dependence of cells, activated JNK may preferentially phosphorylate another JNK activation. KB-3 cells were treated with the indicated concentra- substrate, or the time course of c-Jun phosphorylation may tions of Adriamycin for 16 h. Upper panel, JNK activity; lower panel, differ from that of JNK activation. Despite these caveats, the JNK immunoblot of the corresponding cell lysates (40 mg of protein/ lane). See “Experimental Procedures” for other details. increased abundance of c-Jun phosphorylated on Ser-73 is con- sistent with JNK activation in vivo in response to Adriamycin amined JNK activity in KB-3 cells treated for 16 h with two or vinblastine treatment. other important cancer chemotherapeutic drugs, vinblastine Effect of Drugs on ERK Activation—The results presented and VP-16. Activation of JNK was observed at vinblastine above indicated that certain chemotherapeutic drugs activate concentrations of 5 ng/ml and above, with a stimulation of the SAPK pathway in KB-3 cells. To determine whether the enzyme activity of 6.5-fold (average of three experiments) (Fig. drugs selectively activated specific MAPK cascades, ERK acti- 3A). VP-16 treatment also activated JNK, with a stimulation of vation was examined in KB-3 cells treated with drugs under Activation of SAPK/JNK by Chemotherapeutic Drugs 30953 FIG.4. Effect of drugs on c-Jun phosphorylation. KB-3 cells were untreated (Control) or were treated with Adriamycin (ADR; 500 ng/ml, 16 h), vinblastine (VBL; 20 ng/ml, 16 h), VP-16 (1 mg/ml, 16 h), or araC (10 mM, 40 h). Nuclear extracts (112 mg/lane) were examined for the presence of phosphorylated c-Jun by immunoblotting with a phos- pho-Ser-73-specific c-Jun antibody as described under “Experimental Procedures.” The immunoreactive band has an apparent molecular mass of 39 kDa. The results from a representative experiment are shown. 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. Some- what unexpectedly, ERK activity was increased .6-fold by heat shock, and this provided a convenient positive control for ERK activation in these cells (lane 3). TPA failed to signifi- cantly activate ERK in KB-3 cells (lane 9), but this appeared to be a cell type-specific phenomenon since TPA strongly acti- vated 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 FIG.5. Effect of drugs on ERK activation. A, ERK activity was observations with an independent assessment of ERK activa- determined by immunocomplex assay in lysates from K562 cells (lanes tion, cell lysates from similarly treated cells were subjected to 1 and 2) or KB-3 cells (lanes 3–9). Upper panel, phosphorylated myelin Western blot analysis with a phospho-ERK1/2 antibody (Fig. basic protein (MBP) detected by SDS-PAGE and autoradiography; lower panel, immunoblot of the respective cell lysates (50 mg of protein/ 5B). Phospho-ERK1/2 immunoreactivity was increased by heat lane) probed with anti-ERK1/2 antibody. Lanes 2 and 4, untreated cells; shock, but was not significantly affected by the drug treat- lanes 1 and 9, TPA (100 nM, 30 min); lane 3, heat shock (42 °C, 35 min); ments, consistent with the ERK immunocomplex assay data. lane 5, Adriamycin (ADR; 200 ng/ml, 16 h); lane 6, vinblastine (VBL;20 The specificity of the phospho-ERK antibody was confirmed by ng/ml, 16 h); lane 7, VP-16 (1 mg/ml, 16 h); lane 8, araC (10 mM,40h). Molecular mass standards (in kilodaltons) are indicated on the left. B, antibody recognition of a control protein consisting of bacte- KB-3 cells were untreated (Control), heat-shocked, or treated with rially expressed, purified ERK2 phosphorylated by MEK drugs as described in A. Extracts were prepared and subjected to (Fig. 5B). SDS-PAGE (100 mg of protein/lane) and immunoblotting with anti- JNK Activity in Multidrug-resistant Cells—Adriamycin, vin- phospho-ERK1/2 antibody. Control Protein, shown in the last lane, was supplied by the manufacturer and consists of bacterially expressed blastine, and VP-16 share the common property of being sub- ERK2 phosphorylated by MEK and purified free of unphosphorylated strates for the drug efflux pump, P-glycoprotein, which is over- ERK2 protein. expressed 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 FIG.6. JNK activity in MDR cell lines. Extracts were prepared both MDR cell lines (4-fold for KB-V1 and 7-fold for KB-A1, from drug-sensitive KB-3 cells and the MDR variant KB-V1 and KB-A1 cell lines and subjected to JNK immunocomplex assay (left panel) and average of three independent experiments), while all three cell immunoblotting with anti-JNK antibody (right panel;40 mg of protein/ lines expressed similar levels of JNK protein. These results lane). GST-cJ, GST-c-Jun(79). suggest that JNK is present in a more highly activated form in the MDR variants. Basal ERK activity and ERK protein level (21), untreated cells expressed a barely detectable level of were also assessed and found to be similar for the KB-3, KB-V1, MDR1 mRNA. Treatment of cells with 200 ng/ml Adriamycin, and KB-A1 cell lines (data not shown). 5 ng/ml vinblastine, 1 mg/ml VP-16, or 10 mM araC induced Induction of MDR1 mRNA by Chemotherapeutic Drugs—An MDR1 expression (Fig. 7). These drug concentrations were earlier study found that treatment with chemotherapeutic found to be optimal for MDR1 induction in KB-3 cells. The drugs of certain drug-sensitive cancer cell lines, including MDR1-overexpressing KB-V1 cell line was utilized as a positive KB-3, K562, and H9 cells, resulted in the induction of MDR1 control, and b -microglobulin mRNA levels were determined as mRNA (21). It was suggested that MDR1 induction may be a an internal control and found to be unchanged (Fig. 7). general response to drug-induced cellular damage. We consid- DISCUSSION ered the possibility that JNK activation and MDR1 mRNA induction by chemotherapeutic drugs may be linked, particu- In this paper, we have shown that three structurally and larly in view of the results in Fig. 6 showing increased JNK mechanistically distinct anticancer drugs activate the stress- activity in MDR cells. We therefore analyzed MDR1 mRNA activated kinase, JNK, in human carcinoma cells. The most expression by reverse transcription-PCR in untreated and potent compound studied was Adriamycin, which maximally drug-treated KB-3 cells. In confirmation of the earlier findings activated JNK 40-fold; vinblastine and VP-16 maximally acti- 30954 Activation of SAPK/JNK by Chemotherapeutic Drugs tumor necrosis factor-a (34). Our finding that mechanistically distinct cytotoxic drugs activate the SAPK/JNK pathway sup- ports 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 FIG.7. Induction of MDR1 mRNA by chemotherapeutic drugs. between death and survival. One well characterized mecha- RNA was prepared, and MDR1-specific (167 base pairs) or b -micro- nism of protection against cytotoxic drugs is P-glycoprotein globulin (b M)-specific (120 base pairs) PCR products were amplified overexpression (16). Consistent with a previous report (21), we and analyzed as described under “Experimental Procedures.” Molecular size standards of 126 and 179 base pairs (bp) are indicated on the left. showed that Adriamycin, vinblastine, VP-16, and araC all in- Lanes 1 and 7, untreated KB-3 cells; lane 2, multidrug-resistant KB-V1 duced MDR1 mRNA expression in KB-3 cells (Fig. 7). With the cells; lanes 3–6, treated KB-3 cells (lane 3, Adriamycin (ADR; 200 exception of araC, which was a poor JNK activator in these ng/ml, 16 h); lane 4, vinblastine (VBL; 5 ng/ml, 16 h); lane 5, VP-16 (1 cells, the concentrations of the drugs optimal for MDR1 induc- mg/ml, 16 h); lane 6, araC (10 mM, 40 h)). tion were similar to those optimal for JNK activation. Although these results do not demonstrate a causal relationship between vated JNK 6.5- and 4-fold, respectively. JNK activation oc- JNK activation and MDR1 induction, they do suggest a possi- curred at clinically relevant drug concentrations. araC was also ble link between the two parameters. The fact that the MDR tested, but was a relatively poor JNK activator in this system. cell lines examined express a more highly activated form of In all cases, increased JNK enzyme activity occurred without a JNK is intriguing in this regard. The presence of activated JNK change in JNK protein expression. Consequences of activated could be due to activation of upstream regulators of JNK, JNK include phosphorylation of pre-existing c-Jun and an in- inactivation or down-regulation of a phosphatase acting on crease in c-jun transcription and synthesis of new c-Jun protein JNK, or both in the MDR cell lines. In the context of this study, (reviewed in Ref. 22). The increased abundance of c-Jun phos- it is interesting to note that JNK activation and MDR1 expres- phorylated on Ser-73 in cells treated with Adriamycin or vin- sion are induced by several other common stimuli. For exam- blastine provides further evidence that exposure to these ple, the MDR1 gene is induced by heat shock (35) and UV agents stimulates JNK activity in vivo. More detailed studies irradiation (36), both well established activators of JNK. The will be required to determine the temporal relationship be- presence of a non-canonical AP-1 consensus element in the tween c-Jun phosphorylation and JNK activation in response human MDR1 promoter (37) is perhaps significant since JNK to Adriamycin and vinblastine treatment and to determine enhances AP-1-dependent transcription through modulation of whether VP-16 treatment influences the phosphorylation of c-Jun phosphorylation and expression (22). Further investiga- c-Jun. The drugs examined failed to significantly activate the tion will be required to elucidate the role of JNK in the cellular ERK pathway relative to the JNK pathway, thereby displaying response to cytotoxic drugs and the relationship of this stress- specificity with regard to stimulation of different MAPK path- activated pathway to MDR1 expression. ways. It remains to be determined whether other SAPKs such MAPK Acknowledgments—We thank Dr. Michael Gottesman for the as p38 or the newly described SAPK3 (23) are also acti- KB-A1 and KB-V1 cell lines, Dr. Jacki Kornbluth for the K562 cell vated by these cytotoxic drugs and whether other JNK sub- line, Dr. Omar Coso for the GST-c-Jun(79) construct, Baiting Ning for strates such as ATF2 are affected. assistance with PCR, and Dr. Charlotte Peterson and Dr. Esther Dupont-Versteegden for helpful discussions and critical review of the The JNK pathway is likely activated as a result of drug- manuscript. induced cellular damage, but the intracellular signals linking cell damage to the stress response are incompletely defined. REFERENCES Many DNA-damaging drugs including Adriamycin and VP-16 1. Davis, R. J. (1993) J. Biol. Chem. 268, 14553–14556 induce expression of the nuclear phosphoprotein p53 (24, 25). 2. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. 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Role of the Stress-activated/c-Jun NH2-terminal Protein Kinase Pathway in the Cellular Response to Adriamycin and Other Chemotherapeutic Drugs

Osborn, Maudie T.; Chambers, Timothy C.
Journal of Biological Chemistry – Nov 1, 1996
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10.1074/jbc.271.48.30950
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 48, Issue of November 29, pp. 30950–30955, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Role of the Stress-activated/c-Jun NH -terminal Protein Kinase Pathway in the Cellular Response to Adriamycin and Other Chemotherapeutic Drugs* (Received for publication, July 19, 1996) Maudie T. Osborn and Timothy C. Chambers‡ From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199 transduce extracellular stimuli into intracellular responses. c-Jun NH -terminal protein kinase (JNK), a member of the mitogen-activated protein kinase family, is acti- Each MAPK cascade consists of a module of three kinases: a vated in response to many stressful stimuli including MAPK kinase kinase, which phosphorylates and activates a heat shock, UV irradiation, protein synthesis inhibitors, MAPK kinase, which in turn phosphorylates and activates a and inflammatory cytokines. In this study, we investi- MAPK. The classical MAPK module consists of Raf kinase, gated whether JNK plays a role in the cellular response MEK, and ERK and is activated in response to a variety of to different drugs commonly used in cancer chemother- mitogenic signals operating through different mechanisms. Ac- apy. Treatment of human KB-3 carcinoma cells with Ad- tivation by receptor tyrosine kinases is the best characterized riamycin resulted in a time- and dose-dependent activa- mechanism, but certain G-protein-coupled receptors and cyto- tion of JNK of up to 40-fold. Treatment with vinblastine kine receptors are also capable of activating the MAPK cascade or etoposide (VP-16) also activated JNK, with maximum (2). Downstream substrates regulated by ERK include tran- increases of 6.5- and 4.3-fold, respectively. Consistent scription factors such as Elk-1 and ATF2, protein kinases in- with these findings, increased c-Jun phosphorylation rsk cluding p90 , and several other target proteins (1). was observed after drug treatment of cells. In contrast, More recently, two other MAPK modules have been charac- none of the drugs significantly activated the extracellu- terized. One consists of MEKK, MKK4 (or SEK1), and c-Jun lar response kinase/mitogen-activated protein kinase NH -terminal kinase (JNK), which, like Raf/MEK/ERK, oper- pathway. Since these drugs are transport substrates for 2 ate in a phosphorylation cascade (3, 4). However, unlike the the MDR1 gene product, P-glycoprotein, JNK was as- classical MAPK pathway, the MEKK/SEK1/JNK module is sayed in two multidrug-resistant (MDR) KB cell lines, only modestly activated by growth factors and phorbol esters KB-A1 and KB-V1, selected for resistance to Adriamycin and vinblastine, respectively. Relative to KB-3 cells, ba- and is instead strongly activated by cellular stress including sal JNK activity was increased 7-fold in KB-A1 cells and heat shock, UV irradiation, protein synthesis inhibitors, and 4-fold in KB-V1 cells, with no change in JNK protein inflammatory cytokines (5). JNK is also termed stress-acti- expression, indicating that JNK is present in a more vated protein kinase (SAPK) (5), and two main forms (JNK1 of highly activated form in the MDR cell lines. Under con- 46 kDa and JNK2 of 55 kDa) have been described (6). An ditions optimal for JNK activation, Adriamycin, vinblas- important physiological substrate of JNK is c-Jun, and phos- tine, and VP-16 all induced MDR1 mRNA expression in phorylation of two sites in the NH -terminal transactivation KB-3 cells. Our findings suggest that JNK activation is domain (Ser-63 and Ser-73) regulates transcriptional activity an important component of the cellular response to sev- (6). eral structurally and functionally distinct anticancer A third MAPK isoform is p38, a homolog of the yeast HOG1 drugs and may also play a role in the MDR phenotype. (high-osmolarity glycerol response-1) kinase (7), also termed p40 (8), reactivating kinase (9), or cytokine-suppressive anti- inflammatory binding protein (10) in independent studies. Like Mitogen-activated protein kinases (MAPKs) are serine/thre- ERK and JNK, p38 is activated by dual phosphorylation on a onine kinases activated by dual phosphorylation on both a tyrosine and a threonine residue, and this is catalyzed by a tyrosine and a threonine (reviewed in Refs. 1 and 2). These MEK family member, MKK3 (11). p38 is activated by inflam- enzymes are important components of signaling pathways that matory cytokines and environmental stress including osmotic shock and UV irradiation (12). An important physiological sub- strate of p38 is MAPK-activated protein kinase-2, which phos- * This work was supported in part by the Mr. and Mrs. Sam Walton Research Award, University of Arkansas for Medical Sciences Medical phorylates heat shock protein hsp27 as part of the cellular Research Endowment Fund. response to stress (9). Although JNK and p38 appear to reside ‡ To whom correspondence should be addressed: Dept. of Biochemis- in distinct MAPK modules, a functional overlap is likely since try and Molecular Biology, University of Arkansas for Medical Sciences, each can complement the hog1-D1 yeast strain (7, 13). Mail Slot 516, 4301 W. Markham St., Little Rock, AR 72205-7199. Tel.: 501-686-5755; Fax: 501-686-8169. Recent data suggest that JNK is also activated in response to The abbreviations used are: MAPK, mitogen-activated protein ki- cellular stress induced by certain DNA-damaging agents. For nase; MEK, mitogen-activated/extracellular response kinase kinase; example, 1-b-D-arabinofuranosylcytosine (araC) (14), cis-plati- ERK, extracellular response kinase; MEKK, mitogen-activated/extra- num (15), and mitomycin C (15) activate JNK in NIH-3T3 cellular response kinase kinase kinase; SEK, stress-activated protein kinase kinase; JNK, c-Jun NH -terminal protein kinase; SAPK, stress- fibroblasts. In this study, we sought to determine whether activated protein kinase; araC, 1-b-D-arabinofuranosylcytosine; MDR, MAPKs play a role in the cellular stress response to other multidrug resistance or multidrug-resistant; GST, glutathione S-trans- cancer chemotherapeutic drugs. We examined JNK and ERK ferase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MOPS, 4-morpho- activation in human carcinoma cells treated with Adriamycin, linepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction. vinblastine, or VP-16. These agents were chosen for study 30950 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Activation of SAPK/JNK by Chemotherapeutic Drugs 30951 Assay of ERK—Cells were harvested as described above and sus- because they represent widely utilized anticancer drugs with pended in 0.25 ml of ERK lysis buffer (50 mM Hepes, pH 7.5, 150 mM different mechanisms of action. Adriamycin has a complex NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium van- mechanism of cytotoxicity through its ability to intercalate adate, 50 mM sodium fluoride, 20 mM b-glycerophosphate, 0.1 mM oka- DNA and generate superoxide; vinblastine is a microtubule daic acid, and protease inhibitors as in JNK lysis buffer). After 15 min inhibitor; and VP-16 is a topoisomerase II inhibitor. Since these on ice, insoluble material was removed by sedimentation for 20 min at drugs belong to the multidrug resistance (MDR) group of sub- 100,000 3 g, and ERK activity was determined by an immunocomplex assay with myelin basic protein as substrate as described (20). strates transported by P-glycoprotein (16), JNK activity was Preparation of Nuclear Extracts—Cell monolayers were washed in also evaluated in MDR variant carcinoma cell lines. Our find- phosphate-buffered saline, collected by scraping and sedimentation, ings suggest that JNK activation is an important component of and suspended in 0.3 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM the cellular response to mechanistically diverse cancer chemo- EDTA, 2 mM EGTA, 10 mM 2-mercaptoethanol, 20 mM b-glycerophos- therapeutic drugs and may also play a role in MDR. phate, 1 mM sodium vanadate, 0.1 mM okadaic acid, 1 mM phenylmeth- ylsulfonyl fluoride, 20 mg/ml aprotinin, 50 mg/ml leupeptin, and 10 mM EXPERIMENTAL PROCEDURES pepstatin). The cells were lysed by Dounce homogenization, and nuclei Materials—Polyclonal antibodies to JNK1 or ERK1 were obtained were collected by centrifugation (10 min, 1000 3 g). The samples, from Santa Cruz Biotechnology. Polyclonal antibodies specific for the adjusted to contain equal protein, were solubilized in SDS sample phosphorylated forms of ERK1/2 or c-Jun were obtained from New buffer with heating to 95 °C for 5 min. England Biolabs Inc. GST-c-Jun(79), a fusion protein of glutathione Immunoblot Analysis—Soluble cell lysates or nuclear suspensions S-transferase and residues 1–79 of human c-Jun, was either obtained (40–100 mg, as indicated in the figure legends) were fractionated by from Santa Cruz Biotechnology or purified from Escherichia coli cells SDS-PAGE; transferred to polyvinylidene difluoride membrane; and harboring the GST-c-Jun(79) expression plasmid (kindly provided by probed with antibodies, all of which were used at a dilution of 1:1000, Dr. Omar Coso, Molecular Signaling Unit, National Institutes of except anti-phospho-ERK1/2 antibody (1:2500). Primary antibody was Health). [g- P]ATP (3000 Ci/mmol) was obtained from Amersham In- detected by horseradish peroxidase-conjugated second antibody ternational. Tissue culture media and the SuperScript preamplification (1:5000), which in turn was visualized using enhanced chemilumines- system were purchased from Life Technologies, Inc. Taq polymerase cence (Amersham International). was from Fisher, and T4 polynucleotide kinase was from Promega. RNA Extraction and cDNA PCR Analysis of MDR1 Expression— 12-O-Tetradecanoylphorbol-13-acetate (TPA), Adriamycin, vinblastine, Total RNA from control and drug-treated cell populations (cultivated in etoposide (VP-16), araC, myelin basic protein, phenylmethylsulfonyl six-well plates) was extracted by a small-scale procedure using RNA fluoride, leupeptin, pepstatin, and aprotinin were obtained from Sigma. STAT-60 (Tel-Test “B”, Inc.). cDNA synthesis was carried out using the Okadaic acid was from LC Laboratories. SuperScript preamplification system as described by the manufacturer. Cell Culture and Treatment Conditions—Human KB-3 carcinoma Amplification with primers specific for MDR1 or b -microglobulin, the cells were maintained as monolayer cultures at 37 °C and 5% CO in latter as an internal control, was performed as described previously (21) Dulbecco’s modified Eagle’s medium containing 4.5 g/liter glucose and with certain modifications. Briefly, PCR was performed with a Perkin- supplemented with 10% fetal calf serum, 2 mML-glutamine, 50 units/ml Elmer 2400 Thermocycler, and reaction mixtures contained 10 mM penicillin, and 50 mg/ml streptomycin. The MDR variant KB-V1 and Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl , 200 mM each dNTP, 200 ng KB-A1 cell lines were maintained in the same medium containing 1 of each gene-specific primer, 50 ng of RNA-equivalent cDNA, and 1 unit mg/ml vinblastine or 1 mg/ml Adriamycin, respectively. The MDR vari- of Taq polymerase. Sense primers were 59-end-labeled with [g- P]ATP ants, which overexpress MDR1/P-glycoprotein and were derived from for detection of the amplified product. After an initial denaturation at KB-3 cells by stepwise selection in medium containing vinblastine or 94 °C for 5 min, the following step-cycle program was initiated: dena- Adriamycin, respectively (17), were kindly provided by Dr. Michael turation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at Gottesman (National Cancer Institute, National Institutes of Health). 72 °C for 60 s. For MDR1, 37 cycles were performed, and for b -micro- Human K562 myelogenous leukemia cells were maintained as suspen- globulin, 27 cycles were performed, each followed by a final extension at sion cultures at 37 °C and 5% CO in RPMI 1640 medium supplemented 72 °C for 7 min. PCR products were analyzed by SDS-PAGE (12% with 10% fetal calf serum and were kindly provided by Dr. Jacki acrylamide) and autoradiography. Kornbluth (Arkansas Cancer Research Center). Cells were cultivated in 25-cm flasks; stock solutions of drugs were prepared in dimethyl sulf- RESULTS oxide; and treatment conditions were such that cell viability, deter- Activation of JNK by Adriamycin—To validate the JNK as- mined by trypan blue exclusion, was 90–95% in all experiments. say in KB-3 cell extracts and to examine possible effects on Cell Preparation and JNK Assay—Cells were washed in ice-cold phosphate-buffered saline, removed from the flask by gentle scraping activity of Adriamycin treatment, lysates were prepared from and sedimentation, and suspended in 0.25 ml of JNK lysis buffer (25 cells exposed to 500 ng/ml Adriamycin for either 1 or 16 h. As mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl , 0.2 mM EDTA, 0.5 mM controls, cells were untreated, exposed to vehicle (dimethyl dithiothreitol, 0.1% Triton X-100, 20 mM b-glycerophosphate, 1 mM sulfoxide) alone, heat-shocked, or stimulated with 100 nM TPA sodium vanadate, 0.1 mM okadaic acid, 1 mM phenylmethylsulfonyl for 30 min. JNK activity was determined by an immunocom- fluoride, 20 mg/ml aprotinin, 50 mg/ml leupeptin, and 10 mM pepstatin). After 15 min on ice, insoluble material was removed by sedimentation plex assay with GST-c-Jun(79) as substrate as described under for 20 min at 100,000 3 g. JNK activity was determined by an immu- “Experimental Procedures.” Consistent with previous reports nocomplex assay essentially as described (18). Briefly, each cell extract in other cell lines (e.g. Ref. 5), JNK was strongly activated by (400 mg) was mixed with 10 ml of anti-JNK antibody for 1 h, and then 30 heat shock, but was not significantly activated by TPA (Fig. 1). ml of 50% protein A-Sepharose in JNK lysis buffer was added for an Significantly increased JNK activity was also observed after additional 1 h. The immunocomplex was recovered by sedimentation for treatment of cells with Adriamycin for 16 h (Fig. 1). A more 5 min in a microcentrifuge; washed three times with 0.5 ml of phos- phate-buffered saline containing 1% Nonidet P-40 and 2 mM sodium detailed kinetic study demonstrated that JNK activity was vanadate; washed once with 0.1 M Tris-HCl, pH 7.5, and 0.5 M LiCl; and detectably increased after a 1-h exposure of cells to Adriamy- washed once with JNK reaction buffer (12.5 mM MOPS, pH 7.5, 20 mM cin, with a further increase after 4 h and a stimulation of b-glycerophosphate, 7.5 mM MgCl , 0.5 mM EGTA, 0.5 mM sodium .40-fold (average of four experiments, as determined by den- fluoride, and 0.5 mM sodium vanadate). The immunoprecipitate was sitometric scanning) after a 16-h exposure to the drug (Fig. 2A, resuspended in 30 ml of JNK reaction buffer containing 1 mg of GST-c- upper panel). Immunoblotting of cell lysates with anti-JNK Jun(79), and the reaction was initiated by the addition of 5 ml of 0.1 mM [g- P]ATP (30,000 cpm/pmol). After incubation for 20 min at 30 °C, the antibody revealed that JNK1 protein levels remained constant reaction was terminated by the addition of 8 mlof5 3 SDS sample under these treatment conditions (Fig. 2A, lower panel). The buffer (19) and heating to 95 °C for 5 min. Samples were analyzed by concentration dependence of JNK activation by Adriamycin SDS-PAGE (12% acrylamide); gels were stained with Coomassie Blue (16-h exposure) was next determined; enzyme activation was and subjected to autoradiography. Quantitation was performed by den- observed in the range of 20–500 ng/ml Adriamycin, with an sitometric scanning of the autoradiographic film with a Model 300A increase of 40-fold at 500 ng/ml (Fig. 2B). laser densitometer and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Activation of JNK by Vinblastine and VP-16—We next ex- 30952 Activation of SAPK/JNK by Chemotherapeutic Drugs FIG.1. Activation of JNK by heat shock or Adriamycin. JNK activity in KB-3 cell lysates was determined by immunocomplex assay as described under “Experimental Procedures.” Phosphorylated GST-c- Jun(79) (GST-cJ) was detected by SDS-PAGE and autoradiography. Molecular mass standards (in kilodaltons) are indicated on the left. Lane 1, control (no addition); lane 2, control (dimethyl sulfoxide (DMSO) vehicle, 0.1% (v/v) final concentration); lane 3, heat shock (42 °C, 35 min); lane 4, TPA (100 nM, 30 min); lanes 5 and 6, Adriamycin (ADR; 500 ng/ml, 1 or 16 h). FIG.3. Concentration dependence of JNK activation by vin- blastine or VP-16. KB-3 cells were treated with the indicated concen- trations of vinblastine (VBL; A) or VP-16 (B) for 16 h. JNK activity was determined by immunocomplex assay (upper panels in A and B), and JNK protein levels by immunoblotting (lower panels in A and B)as described under “Experimental Procedures.” See the legend to Fig. 2 for other details. GST-cJ, GST-c-Jun(79). enzyme activity of 4.3-fold (average of two experiments) at a concentration of 1 mg/ml (Fig. 3B). We also tested araC, which, at 10 mM 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 mM 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 -terminal region of the nuclear substrate, c-Jun. NH -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- FIG.2. Time course and concentration dependence of JNK Ser-73 c-Jun immunoreactivity was observed in nuclei of cells activation by Adriamycin. A, time course of JNK activation and determination of JNK protein levels. JNK activity was determined by exposed to Adriamycin or vinblastine. Immunoreactivity was immunocomplex assay in lysates from KB-3 cells treated with 500 barely detectable in control or VP-16- or araC-treated cells ng/ml Adriamycin (ADR) for the times indicated. Upper panel, phos- under these conditions and was greater in vinblastine-treated phorylated GST-c-Jun(79) (GST-cJ) detected by SDS-PAGE and auto- versus Adriamycin-treated cells, despite the fact that Adriamy- radiography; lower panel, immunoblot of the respective cell lysates (40 mg of protein/lane) probed with anti-JNK antibody. The strongly immu- cin was a more potent activator of JNK. In Adriamycin-treated noreactive band corresponds to JNK1. B, concentration dependence of cells, activated JNK may preferentially phosphorylate another JNK activation. KB-3 cells were treated with the indicated concentra- substrate, or the time course of c-Jun phosphorylation may tions of Adriamycin for 16 h. Upper panel, JNK activity; lower panel, differ from that of JNK activation. Despite these caveats, the JNK immunoblot of the corresponding cell lysates (40 mg of protein/ lane). See “Experimental Procedures” for other details. increased abundance of c-Jun phosphorylated on Ser-73 is con- sistent with JNK activation in vivo in response to Adriamycin amined JNK activity in KB-3 cells treated for 16 h with two or vinblastine treatment. other important cancer chemotherapeutic drugs, vinblastine Effect of Drugs on ERK Activation—The results presented and VP-16. Activation of JNK was observed at vinblastine above indicated that certain chemotherapeutic drugs activate concentrations of 5 ng/ml and above, with a stimulation of the SAPK pathway in KB-3 cells. To determine whether the enzyme activity of 6.5-fold (average of three experiments) (Fig. drugs selectively activated specific MAPK cascades, ERK acti- 3A). VP-16 treatment also activated JNK, with a stimulation of vation was examined in KB-3 cells treated with drugs under Activation of SAPK/JNK by Chemotherapeutic Drugs 30953 FIG.4. Effect of drugs on c-Jun phosphorylation. KB-3 cells were untreated (Control) or were treated with Adriamycin (ADR; 500 ng/ml, 16 h), vinblastine (VBL; 20 ng/ml, 16 h), VP-16 (1 mg/ml, 16 h), or araC (10 mM, 40 h). Nuclear extracts (112 mg/lane) were examined for the presence of phosphorylated c-Jun by immunoblotting with a phos- pho-Ser-73-specific c-Jun antibody as described under “Experimental Procedures.” The immunoreactive band has an apparent molecular mass of 39 kDa. The results from a representative experiment are shown. 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. Some- what unexpectedly, ERK activity was increased .6-fold by heat shock, and this provided a convenient positive control for ERK activation in these cells (lane 3). TPA failed to signifi- cantly activate ERK in KB-3 cells (lane 9), but this appeared to be a cell type-specific phenomenon since TPA strongly acti- vated 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 FIG.5. Effect of drugs on ERK activation. A, ERK activity was observations with an independent assessment of ERK activa- determined by immunocomplex assay in lysates from K562 cells (lanes tion, cell lysates from similarly treated cells were subjected to 1 and 2) or KB-3 cells (lanes 3–9). Upper panel, phosphorylated myelin Western blot analysis with a phospho-ERK1/2 antibody (Fig. basic protein (MBP) detected by SDS-PAGE and autoradiography; lower panel, immunoblot of the respective cell lysates (50 mg of protein/ 5B). Phospho-ERK1/2 immunoreactivity was increased by heat lane) probed with anti-ERK1/2 antibody. Lanes 2 and 4, untreated cells; shock, but was not significantly affected by the drug treat- lanes 1 and 9, TPA (100 nM, 30 min); lane 3, heat shock (42 °C, 35 min); ments, consistent with the ERK immunocomplex assay data. lane 5, Adriamycin (ADR; 200 ng/ml, 16 h); lane 6, vinblastine (VBL;20 The specificity of the phospho-ERK antibody was confirmed by ng/ml, 16 h); lane 7, VP-16 (1 mg/ml, 16 h); lane 8, araC (10 mM,40h). Molecular mass standards (in kilodaltons) are indicated on the left. B, antibody recognition of a control protein consisting of bacte- KB-3 cells were untreated (Control), heat-shocked, or treated with rially expressed, purified ERK2 phosphorylated by MEK drugs as described in A. Extracts were prepared and subjected to (Fig. 5B). SDS-PAGE (100 mg of protein/lane) and immunoblotting with anti- JNK Activity in Multidrug-resistant Cells—Adriamycin, vin- phospho-ERK1/2 antibody. Control Protein, shown in the last lane, was supplied by the manufacturer and consists of bacterially expressed blastine, and VP-16 share the common property of being sub- ERK2 phosphorylated by MEK and purified free of unphosphorylated strates for the drug efflux pump, P-glycoprotein, which is over- ERK2 protein. expressed 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 FIG.6. JNK activity in MDR cell lines. Extracts were prepared both MDR cell lines (4-fold for KB-V1 and 7-fold for KB-A1, from drug-sensitive KB-3 cells and the MDR variant KB-V1 and KB-A1 cell lines and subjected to JNK immunocomplex assay (left panel) and average of three independent experiments), while all three cell immunoblotting with anti-JNK antibody (right panel;40 mg of protein/ lines expressed similar levels of JNK protein. These results lane). GST-cJ, GST-c-Jun(79). suggest that JNK is present in a more highly activated form in the MDR variants. Basal ERK activity and ERK protein level (21), untreated cells expressed a barely detectable level of were also assessed and found to be similar for the KB-3, KB-V1, MDR1 mRNA. Treatment of cells with 200 ng/ml Adriamycin, and KB-A1 cell lines (data not shown). 5 ng/ml vinblastine, 1 mg/ml VP-16, or 10 mM araC induced Induction of MDR1 mRNA by Chemotherapeutic Drugs—An MDR1 expression (Fig. 7). These drug concentrations were earlier study found that treatment with chemotherapeutic found to be optimal for MDR1 induction in KB-3 cells. The drugs of certain drug-sensitive cancer cell lines, including MDR1-overexpressing KB-V1 cell line was utilized as a positive KB-3, K562, and H9 cells, resulted in the induction of MDR1 control, and b -microglobulin mRNA levels were determined as mRNA (21). It was suggested that MDR1 induction may be a an internal control and found to be unchanged (Fig. 7). general response to drug-induced cellular damage. We consid- DISCUSSION ered the possibility that JNK activation and MDR1 mRNA induction by chemotherapeutic drugs may be linked, particu- In this paper, we have shown that three structurally and larly in view of the results in Fig. 6 showing increased JNK mechanistically distinct anticancer drugs activate the stress- activity in MDR cells. We therefore analyzed MDR1 mRNA activated kinase, JNK, in human carcinoma cells. The most expression by reverse transcription-PCR in untreated and potent compound studied was Adriamycin, which maximally drug-treated KB-3 cells. In confirmation of the earlier findings activated JNK 40-fold; vinblastine and VP-16 maximally acti- 30954 Activation of SAPK/JNK by Chemotherapeutic Drugs tumor necrosis factor-a (34). Our finding that mechanistically distinct cytotoxic drugs activate the SAPK/JNK pathway sup- ports 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 FIG.7. Induction of MDR1 mRNA by chemotherapeutic drugs. between death and survival. One well characterized mecha- RNA was prepared, and MDR1-specific (167 base pairs) or b -micro- nism of protection against cytotoxic drugs is P-glycoprotein globulin (b M)-specific (120 base pairs) PCR products were amplified overexpression (16). Consistent with a previous report (21), we and analyzed as described under “Experimental Procedures.” Molecular size standards of 126 and 179 base pairs (bp) are indicated on the left. showed that Adriamycin, vinblastine, VP-16, and araC all in- Lanes 1 and 7, untreated KB-3 cells; lane 2, multidrug-resistant KB-V1 duced MDR1 mRNA expression in KB-3 cells (Fig. 7). With the cells; lanes 3–6, treated KB-3 cells (lane 3, Adriamycin (ADR; 200 exception of araC, which was a poor JNK activator in these ng/ml, 16 h); lane 4, vinblastine (VBL; 5 ng/ml, 16 h); lane 5, VP-16 (1 cells, the concentrations of the drugs optimal for MDR1 induc- mg/ml, 16 h); lane 6, araC (10 mM, 40 h)). tion were similar to those optimal for JNK activation. Although these results do not demonstrate a causal relationship between vated JNK 6.5- and 4-fold, respectively. JNK activation oc- JNK activation and MDR1 induction, they do suggest a possi- curred at clinically relevant drug concentrations. araC was also ble link between the two parameters. The fact that the MDR tested, but was a relatively poor JNK activator in this system. cell lines examined express a more highly activated form of In all cases, increased JNK enzyme activity occurred without a JNK is intriguing in this regard. The presence of activated JNK change in JNK protein expression. Consequences of activated could be due to activation of upstream regulators of JNK, JNK include phosphorylation of pre-existing c-Jun and an in- inactivation or down-regulation of a phosphatase acting on crease in c-jun transcription and synthesis of new c-Jun protein JNK, or both in the MDR cell lines. In the context of this study, (reviewed in Ref. 22). The increased abundance of c-Jun phos- it is interesting to note that JNK activation and MDR1 expres- phorylated on Ser-73 in cells treated with Adriamycin or vin- sion are induced by several other common stimuli. For exam- blastine provides further evidence that exposure to these ple, the MDR1 gene is induced by heat shock (35) and UV agents stimulates JNK activity in vivo. More detailed studies irradiation (36), both well established activators of JNK. The will be required to determine the temporal relationship be- presence of a non-canonical AP-1 consensus element in the tween c-Jun phosphorylation and JNK activation in response human MDR1 promoter (37) is perhaps significant since JNK to Adriamycin and vinblastine treatment and to determine enhances AP-1-dependent transcription through modulation of whether VP-16 treatment influences the phosphorylation of c-Jun phosphorylation and expression (22). Further investiga- c-Jun. The drugs examined failed to significantly activate the tion will be required to elucidate the role of JNK in the cellular ERK pathway relative to the JNK pathway, thereby displaying response to cytotoxic drugs and the relationship of this stress- specificity with regard to stimulation of different MAPK path- activated pathway to MDR1 expression. ways. 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Published: Nov 1, 1996

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