Di erential regulation of mitogen-activated protein kinases by ... - Nature

Oncogene (1999) 18, 377 ± 384 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc

Dierential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells Alexander A Shtil1, Sandhya Mandlekar2, Rong Yu2, Robert J Walter3, Karen Hagen1, Tse-Hua Tan4, Igor B Roninson1 and Ah-Ng Tony Kong2 1

Department of Molecular Genetics, College of Medicine, 2Department of Pharmaceutics and Pharmacodynamics, Center for Pharmaceutical Biotechnology, College of Pharmacy; University of Illinois at Chicago, Chicago, Illinois 60607; 3Department of Surgery, Cook County Hospital, Chicago, Illinois 60612; 4Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA

Drug design targeted at microtubules has led to the advent of some potent anti-cancer drugs. In the present study, we demonstrated that microtubule-binding agents (MBAs) taxol and colchicine induced immediate early gene (c-jun and ATF3) expression, cell cycle arrest, and apoptosis in the human breast cancer cell line MCF-7. To elucidate the signal transduction pathways that mediate such biological activities of MBAs, we studied the involvement of mitogen-activated protein (MAP) kinases. Treatment with taxol, colchicine, or other MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in MCF-7 cells. In contrast, p38 was activated only by taxol and none of the MBAs changed the activity of extracellular signal-regulated protein kinase 2 (ERK2). Activation of JNK1 or p38 by MBAs occurred subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds. Furthermore, baccatine III and b-lumicolchicine, inactive analogs of taxol and colchicine, respectively, did not activate JNK1 or p38. These results suggest that interactions between microtubules and MBAs are essential for the activation of these kinases. Pretreatment with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H2O2- or doxorubicininduced JNK1 activity, but had no eect on JNK1 activation by MBAs, excluding a role for oxidative stress. However, BAPTA/AM, a speci®c intracellular Ca2+ chelator, attenuated JNK1 activation by taxol but not by colchicine, and had no eect on microtubule changes induced by taxol. Thus, stabilization or depolymerization of microtubules may regulate JNK1 activity via distinct downstream signaling pathways. The dierential activation of MAP kinases opens up a new avenue for addressing the mechanism of action of antimicrotubule drugs. Keywords: taxol; colchicine; MAP kinases; microtubules; breast cancer

Correspondence: A-NT Kong The ®rst two authors contributed equally to this work Received 23 February 1998; revised 17 August 1998; accepted 17 August 1998

Introduction Microtubules, the cytoskeletal structures formed by the polymerization of tubulin heterodimers, play a crucial role in many biological processes including mitosis, cell-cell communication, intracellular transport, cell growth, and programmed cell death or apoptosis (Dustin, 1984). Given the pivotal importance of microtubules in cellular function, they have been the targets for the designing of eective anti-cancer drugs, such as taxol and the vinca alkaloids. By interfering with the dynamics of microtubule assembly, microtubule-binding agents (MBAs) exert profound eects on cellular processes. It has been shown that MBAs induce gene expression (Karin et al., 1995), cause cell cycle arrest (Jordan et al., 1991) and initiate apoptosis (Bhalla et al., 1993). However, the signal transduction pathways that mediate the cellular responses to MBAs remain unknown. Mitogen-activated protein kinases (MAPKs), characterized as proline-directed serine/threonine kinases (Cobb and Goldsmith, 1995), are important cellular signaling components which convert various extracellular signals into intracellular responses through serial phosphorylation cascades (Marshall, 1994). In mammalian cells, three distinct MAPK cascades have been identi®ed (Cano and Mahadevan, 1995). These include extracellular signal-regulated protein kinases (ERKs), c-jun N-terminal kinases or stress-activated protein kinases (JNKs/SAPKs), and p38. Each MAP kinase cascade consists of a module of three kinases: a MAPKKK (mitogen-activated protein kinase kinase kinase) which phosphorylates and activates a MAPKK (mitogen-activated protein kinase kinase), which, in turn, phosphorylates and activates a MAPK (Marshall, 1994). The ERK signaling cascade can be triggered by growth factors and phorbol esters through Rasdependent activation of Raf-MEK-ERK pathway (Stokoe et al., 1994). Activation of ERK can also be achieved via certain G-protein-coupled receptors and protein kinase C (Burgering and Bos, 1995). The JNK cascade is operated by a parallel signaling module consisting of MEKK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1), MKK4 (mitogen-activated protein kinase kinase 4) and JNK (Kyriakis et al., 1994). Unlike ERK, the JNK cascade is only modestly activated by growth factors or phorbol esters and instead is strongly activated by stress signals such as UV light (Hibi et al., 1993), g-irradiation (Chen et al., 1996), inhibitors of protein synthesis (Cano et al., 1994), ceramide (Verheij

Activation of MAP kinases by microtubule-binding agents AA Shtil et al

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et al., 1996), DNA-damaging drugs (Yu et al., 1996a), pro-in¯ammatory cytokines (Raingeaud et al., 1995), or chemopreventive agents (Yu et al., 1996b). Similar to JNK, p38 kinase is preferentially activated by many stress stimuli via the MEKK1-MKK3 cascade (Kyriakis and Avruch, 1996). Activation of MAPK pathways culminates in the phosphorylation of several transcription factors, such as c-Myc, p62TCF/Elk-1, cJun, ATF2, and ATF3 which ultimately leads to changes in the expression of various genes (Karin, 1995). Recent studies suggest that MAP kinases also regulate the cell cycle and apoptosis (Xia, 1995). In the present study, we demonstrate that MBAs activated JNK1 in human breast cancer cell lines in a time- and dose-dependent manner, whereas p38 MAP kinase was activated only by the microtubule-stabilizing agent taxol. The activation of both JNK1 and p38 required binding of the drugs to tubulin. Unlike JNK1 and p38, however, activity of ERK2 was not aected by MBAs. Thus, stabilization or depolymerization of microtubules by MBAs dierentially activated MAP kinase signaling pathways, which may be a novel mechanism for the regulation of MAP kinases in response to extracellular stress stimuli.

Results MBAs induce cell cycle arrest and apoptosis in the MCF-7 human breast cancer cell line Previous studies have shown that treatment with MBAs caused cell cycle arrest and apoptosis in some cancer cell lines (Jordan et al., 1991, Bhalla et al., 1993). To validate our system, we examined the eects of MBAs on the cell cycle and apoptosis in MCF-7 human breast cancer cells. As shown in Table 1, few apoptotic cells were detected after 12 h of treatment with taxol or colchicine. However, prolonged treatment (24 h) with 1 mM taxol and 2.5 mM colchicine induced a signi®cant amount of apoptotic cell death (41.7% in taxol- and 50.3% in colchicine-treated cells). Under these conditions, taxol and colchicine also caused signi®cant accumulation of cells with 4n DNA content, which may represent both G2/M-arrested cells and polyploid cells arrested in G1, as described most recently by Lanni and Jacks (1998). Similar results were obtained when cells were treated with low concentrations of taxol (100 nM) and colchicine (250 nM) for 24 h.

Taxol and colchicine up-regulate mRNA levels of c-jun and ATF3 To test whether changes in microtubule dynamics lead to alterations in gene expression, we examined the eects of taxol and colchicine on the induction of the immediate early genes, c-jun and ATF3, which are known to regulate many other genes. Treatment of MCF-7 cells with 1 mM taxol or 2.5 mM colchicine resulted in elevated steady-state levels of c-jun and ATF3 mRNAs, as determined by RT ± PCR (Figure 1a and b). The increase of c-jun and ATF3 mRNAs was time-dependent and maximal induction was seen between 1.5 and 3 h of treatment. MBAs dierentially activate MAP kinases To search for the potential signal transduction pathways that regulate the above biological activities of MBAs, we ®rst studied the involvement of JNK1, an important stress-response signaling kinase. Both concentration-response and kinetics of JNK1 activation by MBAs were determined in MCF-7 cells. Cells were harvested after treatment with MBAs and the endogenous activity of JNK1 was determined by using an immunocomplex kinase assay. As shown in Figure 2a, taxol activated JNK1 in a dose-dependent manner. The induced JNK1 activity appeared at a concentration of 0.5 mM and then gradually increased with increasing taxol concentrations. JNK1 activation by taxol was also time-dependent (Figure 2b). One micromolar taxol induced transient JNK1 activation such that JNK1 activity began to increase after 30 min of treatment, peaked at 3 h, and rapidly returned to basal levels after 4 h. Colchicine also activated JNK1 in MCF-7 cells (Figure 2c) but unlike taxol, colchicineinduced JNK1 activity was sustained and continued to increase even after 8 h of treatment (Figure 2d). When Western blotting was performed, no change in the level of JNK1 protein was observed throughout the treatment period, indicating that the sustained activation of JNK1 resulted from phosphorylation of preexisting JNK1 molecules rather than de novo protein synthesis (data not shown). Treatment of MCF-7 cells with other MBAs, including nocodazole, podophyllotoxin, or vincristine also caused an increase of JNK1 activity (Figure 2e). Furthermore, these agents induced JNK1 activation in BT-20, a human estrogen receptor-negative breast cancer cell line, to a similar extent as that seen in MCF-7 cells (data not

Table 1 Eect of microtubule-binding agents on cell cycle distribution Treatmenta Vehicle (control) Taxol, 1 mM, 12 h Taxol, 1 mM, 24 h Colchicine, 2.5 mM, 12 h Colchicine, 2.5 mM, 24 h Taxol, 0.1 mM, 24 h Colchicine, 0.25 mM, 24 h

C=2N 53.4 40.2 21.8 36.4 20.1 18.4 16.7

Cell DNA content (C)b CN5C54N C=4N 34.2 18.1 13.4 15.2 11.2 25.9 16.8

10.9 32.6 23.1 35.9 18.4 20.8 22.7

Apoptotic cells 1.5 9.1 41.7 12.5 50.3 34.9 43.8

a MCF-7 cells were treated for 12 or 24 h with taxol or colchicine as shown in the table. Cell cycle analysis was performed as described under Materials and methods. The values represent % of cells (mean of three experiments) with dierent DNA content. bCells with DNA content equal to 2N are G1 cells, cells with DNA content between 2N and 4N are S cells; cells with 4N DNA content may consist of G2/M cells and polyploids at G1 (Lanni and Jacks, 1998)


shown). These results suggest that JNK1 activation is a general response to MBAs. These data prompted us to investigate whether other members of the MAP kinase family are also activated by MBAs. MCF-7 cells were either treated with 1 mM taxol or 2.5 mM colchicine for the indicated time periods. The activities of p38 and ERK2 were determined by immunocomplex kinase assay. As shown in Figure 3a, taxol induced a time-dependent p38 activation, whereas colchicine had no eect on p38 activity. Furthermore, neither agent stimulated ERK2 activity in MCF-7 cells (Figure 3b). Taken together, these data suggest that MAP kinases are dierentially regulated by MBAs.

Figure 1 Taxol and colchicine increase the steady-state levels mRNAs of c-jun and ATF3. MCF-7 cells were treated with 1 mM taxol (a) or 2.5 mM colchicine (b) for indicated times. The induction of c-jun and ATF3 was determined by RT ± PCR. Data shown are the representative of three independent experiments with similar results

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JNK1 activation requires interaction of MBAs with microtubules To provide insight into the regulation of MAP kinases by MBAs, we tested whether interactions between microtubules and MBAs are critical for MAP kinase activation. To do so, we ®rst studied the time course of taxol- or colchicine-induced tubulin rearrangement. As shown in Figure 4b, treatment of MCF-7 cells with 2.5 mM colchicine for 30 min caused substantial shortening and disruption of microtubules as compared with untreated control cells (Figure 4a). With increasing treatment time, further microtubule disruption occurred until the microtubule network virtually collapsed (Figure 4c). In contrast to cells treated with colchicine, cells treated with 1 mM taxol exhibited long curving microtubules extending to the cell margins and dense bundles of microtubules coursing around the nucleus and out into the cytoplasm (Figure 4d and

Figure 2 Taxol, colchicine, and other mirotubule-binding agents stimulate JNK1 activity in a dose- and time-dependent manner in MCF-7 cells. Cells were treated with drugs as indicated. JNK1 activity, expressed as fold induction over control levels, was measured by in vitro immunocomplex kinase assay as described in Materials and methods. (a) Dose response of JNK1 activation by 2.5 h of treatment with taxol; (b) Time course of JNK1 activation by 1 mM taxol; (c) Dose response of JNK1 activation by 2.5 h of treatment with colchicine; (d) Time course of JNK1 activation by 2.5 mM colchicine; (e) JNK1 activation by treatment with 1 mM of nocodazole (NOC), podophyllotoxin (POD), vincristine (VIN) for 2 h. The blots shown are from one of three independent experiments with similar results


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Figure 3 Taxol and colchicine dierentially activate p38 but have no eect on ERK2 activity. After treatment with 1 mM taxol or 2.5 mM colchicine for indicated time periods, cells were harvested and the activities of p38 (a) and ERK2 (b) were measured by in vitro immunocomplex kinase assay as described in Materials and methods. The experiments were repeated three times

e). However, in both cases, signi®cant rearrangement of microtubule structure was evident within 30 min of treatment, preceding drug-induced JNK1 activation as seen in Figure 2b and d. In addition, doxorubicin, a DNA damaging agent, did not aect the integrity of microtubules (Figure 4f). To provide further evidence into the role of interactions between microtubules and MBAs in JNK1 or p38 activation, we measured the JNK1 or p38 activity in MCF-7 cells treated with 1 mM baccatine III, an inactive analog of taxol, or 2.5 mM b-lumicolchicine, a colchicine analog which lacks tubulin-binding activity. As shown in Figure 5a and b, neither agent induced JNK1 or p38 activation. In preliminary experiments, we found that pretreatment of MCF-7 cells with 100 nM taxol prevented microtubule depolymerization by 2.5 mM colchicine (data not shown). Accordingly, we examined the eect of pretreatment with 100 nM taxol on colchicine-induced JNK1 activity. As expected, pretreatment with taxol attenuated JNK1 activation by colchicine (Figure 6). Interestingly, at 100 nM, taxol alone caused microtubule stabilization, but did not activate JNK1 (Figure 7a). Further morphological studies revealed that the microtubule rearrangement induced by 100 nM taxol was delayed and mild as compared with that induced by 1 mM taxol. Similar results were also obtained with colchicine, which, at low concentration (0.25 mM), induced a delayed and modest change in microtubule structure and did not activate JNK1 even up to 24 h (Figure 7b). Taken together, these data suggest that tubulin-binding and disruption of the normal microtubule cytoskeleton are crucial for JNK1 or p38 activation by MBAs.

Figure 4 Colchicine and taxol induce morphological changes of the microtubule network in a time-dependent fashion. Following culture on coverslips for 40 h, MCF-7 cells were either untreated (a) or treated with 2.5 mM colchicine for 30 min (b) and 1.5 h (c), or with 1 mM taxol for 30 min (d) and 1.5 h (e), or treated with doxorubicin (3.2 mM) for 2 h (f). After treatment, the morphological changes of microtubules were visualized by indirect immuno¯uorescence assay as detailed in Materials and methods. Each treatment was done in duplicate. Ten ®elds were examined in each group. Magni®cation bar, 10 mm

Intracellular calcium chelator blocks JNK1 activation by taxol, whereas antioxidants have no eect JNK1 activation by various stress stimuli has been shown to be mediated by the generation of reactive oxygen intermediates (Cadwallader et al., 1997; Assefa et al., 1997). To determine the role of oxidative stress in MBA-induced JNK1 activation, we used several potent antioxidants, N-acetyl-L-cysteine (NAC), ascorbic acid, and vitamin E. NAC is a precursor of glutathione, an abundant intracellular antioxidant, and is also a scavenger of many dierent types of reactive free radicals. Ascorbic acid is a more potent free radical scavenger than NAC, because of its lower reduction potential. Vitamin E is a lipid soluble antioxidant, which eectively scavenges lipid-related free radicals. Thus, the inhibitory eect of these antioxidants would implicate the involvement of oxidative stress. As shown in Figure 8a, pretreatment of MCF-7 cells with NAC (20 mM),


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Figure 7 Low concentrations of taxol and colchicine do not activate JNK1. MCF-7 cells were treated with 100 nM taxol (a) or 100 nM colchicine (b) for dierent times. The JNK1 activity was determined by in vitro immunocomplex kinase assay. The experiment was repeated twice Figure 5 Inactive analogs of taxol and colchicine do not activate JNK1 or p38. After treatment of MCF-7 cells with 1 mM baccatineIII (inactive analog of taxol) or 2.5 mM b-lumicolchicine (inactive analog of colchicine) for indicated time periods, the activity of JNK1 (a) or p38 (b) was determined by in vitro immunocomplex kinase assay. Data shown are representative of three independent experiments with similar results

Figure 6 Pretreatment with low concentration of taxol (100 nM) prevents JNK1 activation by colchicine. MCF-7 cells were pretreated with 100 nM taxol for 30 min and then challenged with 2.5 mM colchicine for 2 h. JNK1 activity was measured by in vitro immunocomplex kinase assay. The experiment was repeated three times

ascorbic acid (100 mM) and vitamin E (1 mM), which by themselves had no eect on JNK1 basal activity, strongly inhibited JNK1 activation by hydrogen peroxide (H2O2) as well as by doxorubicin, which is known to generate reactive oxygen species (Bachur et al., 1979) and activate JNK1 (Yu et al., 1996a; Osborn and Chambers, 1996). However, these antioxidants had no eect on taxol- or colchicine-induced JNK1 activity, suggesting that microtubule-binding drugs activate JNK1 via an oxidative stress-independent mechanism (Figure 8b). When cells were pretreated with a speci®c intracellular calcium chelator, BAPTA/AM, taxol-induced JNK1 activity was greatly reduced, but JNK1 activation by colchicine remained intact (Figure 8c). Furthermore, the presence of BAPTA/AM did not aect microtubule rearrangement induced by both agents (data not shown). Thus, it is tempting to speculate that drug-induced microtubule stabilization or depolymerization may regulate JNK1 activity via distinct downstream signaling pathways. Discussion Microtubules are highly labile cellular structures, which can undergo rapid assembly and disassembly and this

dynamic behavior strongly in¯uences microtubule function. Pharmacological or other modulation of the structure-function dynamics of microtubules may result in cellular stress. In the present study, we demonstrated that both microtubule-stabilizing (taxol) and -depolymerizing (colchicine, vincristine, nocodadazale, and podophyllotoxin) agents stimulated JNK1 activity in human breast cancer cells. However, p38 was activated only by the stabilizing drug, taxol, and the activity of ERK2 was not aected by any of the agents tested. Thus, microtubule-binding agents dierentially activated MAP kinases, which provides a molecular basis for understanding the mode of action of antimicrotubule drugs. Activation of JNK1 or p38 by MBAs requires interactions between the microtubules and MBAs. This is evidenced by the following results: ®rst, drug-induced morphological changes of microtubules preceded JNK1 or p38 activation. Second, MBAs did not interact with kinase molecules directly, because JNK1 or p38 was not activated by these agents in a cell-free system (data not shown). Furthermore, pretreatment of cells with a low concentration of taxol prevented colchicine-induced depolymerization of microtubules, and also inhibited JNK1 activation by colchicine. Most importantly, baccatine III and b-lumicolchicine, non-binding analogs of taxol and colchicine, respectively, had no eect on JNK1 or p38 activity. However, the question remains as to how interfering with microtubule dynamics triggers downstream signaling events leading to the activation of JNK1 and p38. Previous studies suggested that reactive oxygen species (ROS) might serve as general mediators of JNK activation by many environmental stresses and physiological stimuli, such as UV-C (Adler et al., 1995), g-irradiation (Chen et al., 1996), arsenite (Liu et al., 1996), and in¯ammatory cytokines (Sluss et al., 1994). In this study, we showed that ROS also mediated JNK1 activation by a potent chemotherapeutic drug, doxorubicin, as evidenced by the inhibitory eects of antioxidants, NAC, ascorbic acid and vitamin E. However, these antioxidants had no eect on JNK1 activation by taxol or colchicine. Therefore, MBAs may activate JNK1 signaling pathway through a distinct mechanism. It is also intriguing to note that, although both taxol and colchicine activate JNK1, they may regulate JNK1 activity through dierent pathways


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Figure 8 Antioxidants do not inhibit JNK1 activation by taxol and colchicine, whereas intracellular calcium chelator preferentially blocks taxol-induced JNK1 activity. (a) Eect of antioxidants on JNK1 activation by Doxorubicin (Dox) and H2O2. Cells were pretreated with NAC (20 mM), ascorbic acid (100 mM), or vitamin E (1 mM) for 1 h and then challenged with 3.2 mM Dox or 800 mM H2O2 for 2 h. JNK1 activity was assayed as described above; (b) Eect of antioxidants on JNK1 activation by taxol and colchicine. Cells were pretreated with antioxidants as above and challenged with 1 mM taxol or 2.5 mM colchicine for 2 h before assay for JNK1 activity; (c) Eect of intracellular calcium chelator BAPTA/AM on JNK1 activation by taxol and colchicine. Cells were pretreated with 10 mM BAPTA/AM for 1 h, and then challenged with 1 mM taxol or 2.5 mM colchicine for 2 h. JNK1 activity was assayed as described in Materials and methods. The experiments were performed three times with similar results

because taxol and colchicine stimulated JNK1 activity with dierent kinetics. Activation of JNK1 by taxol was transient, whereas the activation of JNK1 by colchicine was sustained. Furthermore, a speci®c intracellular calcium inhibitor, BAPTA/AM, inhibited JNK1 activation by taxol but not by colchicine. Identi®cation of such downstream eectors of signaling cascades initiated through stabilization and depolymerization of microtubules, which lead to JNK1 activation warrants further study. Activation of MAP kinases results in phosphorylation of many cytosolic as well as nuclear substrates (Cobb and Goddsmith, 1995). Activated JNK1 is known to phosphorylate transcription factors, c-jun, ATF2, and Elk-1, leading to transactivation of many genes, including c-jun itself (Karin, 1995). Activated p38 can also phosphorylate many transcription factors, such as ATF2 and Elk-1, thereby enhancing their transcriptional activity (Karin, 1995). Activation of JNK1 or p38 by MBAs would, therefore, be expected to induce gene expression. Indeed, treatment of MCF-7 cells with taxol or colchicine increased mRNA levels of the immediate early genes, c-jun and ATF3. The dose response and the

time course of c-jun and ATF3 induction correlated well with those of JNK1 activation by these two agents. Consistent with our results, previous studies have shown that rearrangement of microtubules induced phosphorylation of c-jun and transcriptional activity of AP-1 (Manie et al., 1993), which may contribute to the upregulation of AP-1-dependent genes, such as urokinasetype plasminogen activator, stromelysin, collagenase, and a- and b-actin (Unemori and Werb, 1986; Matrisian et al., 1986; Buckler et al., 1988; Sympson and Geoghegan, 1990). Recently, nuclear factor kappa B (NF-kB) has been shown to be activated by JNK1 or its upstream activator MEKK1 (Meyer et al., 1996; Lee et al., 1997). Thus, activation of JNK1 may also provide a plausible mechanism for the induction of NF-kBdependent genes by microtubule-depolymerizing agents (Rosette and Karin, 1995). Activation of JNK1 has been shown to be a common phenomenon in apoptotic cell death. This activation is required for apoptosis induction by growth factor withdrawal (Xia et al., 1995), radiation (Chen et al., 1996), heat shock (Zanke et al., 1996), and ceramide (Verheij et al., 1996). Recently, Yang et al. (1997) reported that JNK1 activation also mediated Fas-induced apoptosis. On the other hand, JNK may not be essential for tumor necrosis factor- or dexamethasone-mediated apoptosis (Liu et al., 1996; Chauhan et al., 1997). Thus, the importance of JNK1 activation seems to vary, depending on the nature of apoptosis-inducing agents. In the present study, we found that higher concentrations of taxol or colchicine were required to activate JNK than to induce cell death. For example, treatment of cells with 100 nM of taxol or colchicine caused a signi®cant amount of cell death (Table 1), but did not activate JNK1 even after 24 h of treatment (Figure 7a and b). This suggests that taxol or colchicine can induce cell death without activating JNK. Since the action of these drugs is highly dose-dependent, and we have seen enhanced cell death upon treatment with 1 mM taxol or with 2.5 M colchicine, it would be interesting to determine whether JNK1 activation at such concentrations potentiates taxol- or colchicine-induced cell death. In summary, our ®ndings suggest that an intracellular signaling machinery may be involved in sensing the integrity of the microtubule cytoskeleton and in initiating cellular responses in reaction to changes in microtubule dynamics. Tubulin is known to have GTPase activity and to act as a GTP-binding protein. Therefore, it is possible that tubulin itself may function as an activator of the JNK pathway as seen with Rac1/ Cdc42 (Coso et al., 1995). Alternatively, microtubules may interact with other GTP-binding proteins, as actin does (Symons, 1996), to initiate downstream signaling. Future studies focusing on the downstream eectors and the physiological consequences of JNK1 or p38 activation may well unravel a novel mechanism for the regulation of MAP kinases and for the chemotherapeutic action of anti-microtubule drugs. Materials and methods Cell culture and drugs MCF-7 and BT-20 human breast cancer cells (American Type Culture Collection, Rockville, MD, USA) were


propagated in Dulbecco's modi®ed Eagle's medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine (Gibco BRL, Grand Island, NY, USA), 100 U/ml penicillin and 50 U/ml streptomycin at 378C, in a humidi®ed atmosphere containing 5% CO2. Cells were routinely tested and found to be negative for Mycoplasma. For experiments with MAP kinase activation, cells were incubated overnight in medium without serum and then treated with the pharmacological agents. The experiments with BAPTA/AM (Calbiochem, La Jolla, CA, USA) were performed in Ca2+-free Eagle's medium (Sigma Chemical Co., St Louis, MO, USA). Taxol (paclitaxel), colchicine, vincristine, podophyllotoxin, nocodazole, and blumicolchicine (Sigma) were dissolved in ethanol as 10006 stock solutions and kept at 7208C. NAC (Sigma) was dissolved in culture medium prior to the experiments and pH was adjusted to 7.2. In vitro immunocomplex assay of JNK1, ERK and p38 kinase activity After treatment with drugs, cells were harvested, washed three times with ice-cold PBS, pH 7.2 and lysed in 0.7 ml of lysis buer (10 mM Tris-HCl, pH 7.1, 50 mM NaCl, 30 mM inorganic sodium pyrophosphate, 50 mM NaF, 100 mM sodium orthovanadate, 2 mM iodoacetic acid, 5 mM ZnCl2, 1 mM phenylmethylsulfonyl ¯uoride, and 0.5% Triton X-100). The lysates were homogenized by passing through a 23 G needle three times, incubated on ice for 15 min. and centrifuged (12 000 r.p.m., 10 min at 48C). The supernatants were transferred to fresh tubes and JNK1 activity was determined as described (Yu et al., 1996a). Brie¯y, equal amounts of total protein, as determined by the Bio-Rad Protein Assay, were incubated with rabbit anti-JNK1 serum (Ab 101) (Meyer et al., 1996) pre-bound to protein A- Sepharose 4B (Zymed Laboratories, San Francisco, CA, USA) for 2 h at 48C. The immunocomplexes were then washed twice with lysis buer and twice with kinase assay buer (20 mM HEPES, pH 7.9, 10 mM MgCl2, 2 mM MnCl2, 50 mM b-glycerophosphate, 10 mM pnitrophenyl phosphate, and 100 mM sodium orthovanadate). The kinase reaction was initiated by resuspending the immunocomplexes in 30 ml of kinase assay buer containing 10 mg of GST-c-jun fusion protein (amino acids 1 ± 79) (Hibi et al., 1993), 2 mCi [g-32P]ATP (3000 Ci/mmol, NEN-Dupont, Boston, MA, USA) and 20 mM ATP. After incubation for 30 min. at 308C the reaction was terminated by adding 10 ml of 46 Laemmli buer and heating to 958C for 5 min. The samples were resolved in 10% SDS ± PAGE. The phosphorylation of GST-c-jun fusion protein was visualized by autoradiography and quantitated on a PhosphoImager (AMBIS, Inc., San Diego, CA, USA). The assays for ERK2 and p38 kinase activities were performed according to the procedures described above for the JNK1 assay with the following modi®cations. After immunoprecipitation of the lysates with anti-ERK2 or p38 antibody (Santa Cruz Biotech., Santa Cruz, CA, USA), the kinase activity was determined using 10 mg of myelin basic protein (Sigma) or 2 mg GST-ATF2 as the substrate for ERK2 and p38, respectively. Reactions were performed at 308C for 15 min and the phosphorylated products were resolved in 13.5% SDS ± PAGE and quantitated as described above. RNA isolation and RT ± PCR Total RNA was isolated with TRIzol reagent (Gibco) as recommended by the manufacturer. RNA (0.5 ± 1 mg) was treated with 1 U of RNase-free DNase I (Epicentre Tech., Madison, WI, USA) for 30 min at 378C followed by inactivation of the enzyme at 708C for 15 min. RNA was then reverse transcribed using the Superscript Preamplifi-

cation System (Gibco). Complementary DNA was treated with 0.1 U/ml E.coli RNase H at 378C for 20 min. PCR was performed with amounts of cDNAs corresponding to 50 ng of total RNA, 200 ng/ml of each amplimer (Integrated DNA Tech., Coralville, IA, USA), 200 nM of each deoxynucleotide triphosphate, 1 mCi [a-32P]deoxycytidine triphosphate (Amersham, Arlington Heights, IL, USA, 3000 Ci/mmol), 1.5 mM MgCl2 and 1 U of Taq DNA polymerase (Gibco). In preliminary experiments, the optimal numbers of PCR cycles were found to be 23 for c-jun, 26 for ATF3, and 26 for b2-microglobulin (internal standard) cDNAs. These numbers yielded PCR products within the linear exponential range. Each cycle consisted of 30 s at 948C, followed by 30 s at 608C and 1 min at 728C. The sequence of the primers were presented elsewhere (Yu et al., 1996a). Since c-jun is an intronless gene (Hattori et al., 1988) very small amounts of DNA present in the RNA preparations could be a source of false-positive signals in PCR. Preliminary PCR was carried out with the omission of the reverse transcription step, i.e., DNase-treated RNA was used directly as the template. No detectable signals were visualized with non-reverse transcribed RNAs after 30 cycles (data not shown). PCR products were ampli®ed on a OmniGene Temperature Cycler (Hybaid Ltd, Woodridge, NJ, USA) in separate tubes (Yu et al., 1996a, Komarov et al.,1996), mixed and resolved in 7.5% PAGE. The gels were dried and exposed to Kodak X-ray ®lm for 2 h at 7708C. Immunocytochemical detection of tubulin MCF-7 cells were grown on glass coverslips for 36 h, treated with either taxol, colchicine, or doxorubicin for indicated times (see Results), washed with solution A (PBS containing 0.1 mM CaCl2 and 1 mM MgCl2, pH 7.2), ®xed with buered 3% paraformaldehyde (10 min) and permeabilized with 0.5% Triton X-100 (1 min). Non-speci®c binding was blocked by incubating in solution A containing 5% normal goat serum, 1% bovine serum albumin, and 0.5% Tween-20. Cells were then incubated with mouse anti-rat b-tubulin monoclonal antibody (TUB2.1; Sigma) diluted 1 : 200 in solution A for 1 h, washed, and then incubated with FITC-conjugated rabbit anti-mouse IgG (Chemicon, Temecula, CA, USA; diluted 1 : 500). Cells were examined using a Zeiss Axioscop microscope equipped with a 100 W mercury epi¯uorescence illuminator and ®lter sets for ¯uorescein and for propidium iodide (PI). Cells were photographed using a 1006 oil immersion lens with either Kodak Tri-X or Pro 400MC color ®lm (400 ASA). Cell cycle analysis Exponentially growing MCF-7 cells (106 per treatment) were treated with colchicine or taxol, washed with ice-cold PBS, and resuspended in staining solution (0.1% sodium citrate, 0.3% Nonidet P-40, 100 mg/ml RNase A and 50 mg/ ml propidium iodide, PI). The cell lysates were vortexed at high speed for 1 min and incubated on ice for 45 min. DNA content (PI ¯uorescence) was measured using an EPICS ELITE ESP ¯ow cytometer (Coulter Corp., Hialeah, FL, USA) equipped with an argon laser for 488 nm excitation. The PI ¯uorescence was collected after passage through the standard optics and a 610 nm bandpass ®lter. Cell cycle analyses were performed using the MULTICYCLE Av Program (Phoenix Flow Systems, San Diego, CA, USA). Abbreviations ATF, activation transcription factor; BAPTA/AM, 1,2-bis(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-

383


384

(acetoxymethyl)-ester; ERK, extracellular signal-regulated protein kinase; GST, glutathione S-transferase; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MBA, microtubule binding agent; NAC, N-acetylL-cysteine; ROI, reactive oxygen intermediates

Acknowledgements The authors are grateful to Drs V Gelfand and T Ignatova for critical discussion of the manuscript. This work was supported by National Institutes of Health grants R37CA40333 (to IBR), R01-AI38649 (to T-HT) and R01ES06887 (to ANTK). AAS is a Fellow of the Oncology Research Faculty Development Program of the National Cancer Institute.

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