Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

MEK Inhibition Enhances Paclitaxel-induced Tumor Apoptosis

MEK Inhibition Enhances Paclitaxel-induced Tumor Apoptosis THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 50, Issue of December 15, pp. 38953–38956, 2000 Accelerated Publication © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. mitotic metaphase and anaphase. This prevents chromosome MEK Inhibition Enhances segregation, leading to tumor cell death. Paclitaxel-induced Tumor Combination therapy of paclitaxel and Herceptin, an anti- Her2-neu antibody, has produced impressive responses among Apoptosis* breast cancer patients (4), although this combination is obvi- ously limited to Her2-neu1 tumors. Combination therapy with Received for publication, September 28, 2000, and in revised other drugs, preferably via a rational molecular basis that is form, October 18, 2000 Published, JBC Papers in Press, October 18, 2000, widely applicable to many tumor types, is essential for im- DOI 10.1074/jbc.C000684200 proved cancer treatment. A combination of paclitaxel with re- agents that activate additional apoptotic signals, or inhibit ¶ ¶i Jeffrey P. MacKeigan‡§ , Timothy S. Collins‡ , survival signals, may provide a rational molecular basis for and Jenny P.-Y. Ting‡§** novel chemotherapeutic strategies. From the ‡Lineberger Comprehensive Cancer Center, A rational molecular target is the ERK mitogen-activated §Department of Microbiology and Immunology, iDepartment of Surgery, University of North Carolina, protein (MAP) kinase pathway that may serve as an opposing Chapel Hill, North Carolina 27599 force to Jun N-terminal kinase (JNK/SAPK). Previous reports have shown that JNK/SAPK leads to cell death, while MEK The anti-cancer drug paclitaxel (Taxol) alters micro- activation contributes to cell differentiation, proliferation, and tubule assembly and activates pro-apoptotic signaling survival (5, 6). Activated Raf-1, a serine-threonine kinase, ini- pathways. Previously, we and others found that pacli- tiates the signaling cascade through MEK, which in turn phos- taxel activates endogenous JNK in tumor cells, and the phorylates a second serine-threonine kinase ERK. ERK phos- activation of JNK contributes to tumor cell apoptosis. phorylates additional kinases and specific transcription Here we find that paclitaxel activates the prosurvival factors, such as Elk-1 and c-Fos, which are important in cell MEK/ERK pathway, which conversely may compromise proliferation. However, the link between Raf-1 and ERK acti- the efficacy of paclitaxel. Hence, a combination treat- vation and paclitaxel-induced cell death is not straightforward. ment of paclitaxel and MEK inhibitors was pursued to Several studies have shown that at a low concentration of the determine whether this treatment could lead to en- hanced apoptosis. The inhibition of MEK/ERK with a drug, paclitaxel-mediated apoptosis is attributed to activated pharmacologic inhibitor, U0126, together with pacli- Raf-1 (7–9). The role of the downstream ERK MAP kinase in taxel resulted in a dramatic enhancement of apoptosis paclitaxel-induced tumor apoptosis is also not entirely clear that is four times more than the additive value of the (10 –14). two drugs alone. Enhanced apoptosis was verified by In this report, we tested the combined effects of paclitaxel the terminal transferase-mediated dUTP nick end label- and inhibitors of MEK1/2 kinase on tumor cell apoptosis. The ing assay, by an enzyme-linked immunosorbent assay specificity of MEK1/2 inhibition was achieved by using two for histone-associated DNA fragments, and by flow cyto- different MEK inhibitors, and by the additional use of trans- metric analysis for DNA content. Specificity of the phar- dominant-negative mutants, which inhibit MEK/ERK activa- macologic inhibitor was confirmed by the use of (a)a tion. The reasons for selecting MEK1/2 as the target are: (i) second MEK/ERK inhibitor and (b) a transdominant- MEK is activated in many tumors (15, 16); (ii) small molecule- negative MEK. Enhanced apoptosis was verified in based MEK inhibitors are readily available, and a recent report breast, ovarian, and lung tumor cell lines, suggesting has described a novel MEK inhibitor that exhibited in vivo this effect is not cell type-specific. This is the first report efficacy in mice (17–19); and (iii) MEK is critical in transform- of enhanced apoptosis detected in the presence of pacli- ing cells, leading to tumor survival and proliferation (20, 21). In taxel and MEK inhibition and suggests a new anticancer the present study, we show that paclitaxel increases MEK1/2 strategy. activity. The combined treatment of paclitaxel plus MEK1/2 inhibition leads to enhanced cell death in breast, ovarian, and lung tumor lines. Paclitaxel is a promising frontline chemotherapy in the treatment of patients with ovarian, breast, and nonsmall cell EXPERIMENTAL PROCEDURES lung carcinomas (1, 2). Paclitaxel is isolated from the bark of JNK Kinase Assay—Following2hof paclitaxel (Sigma) treatment, the pacific yew (Taxus brevifolia) and functions by binding and cells were washed, harvested with lysis buffer, and centrifuged at 4 °C (10). Endogenous JNK was immunoprecipitated with anti-JNK anti- stabilizing microtubules (3). Binding of paclitaxel to microtu- body (Santa Cruz Biotechnology) and protein A-agarose beads for2hat bules blocks normal cell cycle progression during the merger of 4 °C. Immunoprecipitates were collected by centrifugation (2,500 rpm) at 4 °C. Immunoprecipitated JNK was mixed with 5 mg of glutathione * This work was supported by National Institutes of Health Grants 32 S-transferase-c-Jun and 10 mCi of [g- P]ATP and incubated for 30 min AI 41751 and AI45580 and by a grant from the Lineberger Comprehen- sive Cancer Center. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must The abbreviations used are: ERK, extracellular signal-regulated therefore be hereby marked “advertisement” in accordance with 18 kinase; MAP, mitogen-activated protein; JNK, c-Jun N-terminal ki- U.S.C. Section 1734 solely to indicate this fact. nase; SAPK, stress-activated protein kinase; MEK, MAP kinase kinase; ¶ These authors contributed equally to this study. dnMEK, dominant-negative MEK; PBS, phosphate-buffered saline; ** To whom correspondence should be addressed: Lineberger Com- PMSF, phenylmethylsulfonyl fluoride; PI, propidium iodide; TdT, ter- prehensive Cancer Center, Campus Box Number 7295, University of minal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-5538; Fax: 919- end-labeling; CMV, cytomegalovirus; PAGE, polyacrylamide gel elec- 966-8212; E-mail: [email protected]. trophoresis; ELISA, enzyme-linked immunosorbent assay. This paper is available on line at http://www.jbc.org 38953 This is an Open Access article under the CC BY license. 38954 MEK Inhibition Enhances Paclitaxel Apoptosis at 30 °C. The reactions were terminated with SDS sample buffer and resolved on a 10% SDS-PAGE gel. Immunoblot Analysis—H157 human lung carcinoma cells were se- rum-starved for 16 h and treated simultaneously with the indicated concentrations of paclitaxel with or without 10 mM U0126 (Promega). After 15 min of treatment, cells were lysed in 13 PBS, 1% Trition X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM Na VO ,10 mM leupeptin, and 10 mM pepstatin at 4 °C. Cell lysates were 3 4 separated with SDS-PAGE gels, transferred to nitrocellulose mem- branes, and subjected to immunoblot analysis with anti-ERK mono- clonal antibody for phosphorylated ERK1/2 (Santa Cruz Biotechnology). Cell Death ELISA—Manufacturer’s instructions were followed for PLUS the Cell Death Detection ELISA (Roche Molecular Biochemicals). Briefly, cells were plated at 5 3 10 cells/well in 96-well microtiter plates for 24 h. The cells were treated for 20 –24 h with the indicated doses of paclitaxel and U0126. Following lysis, the samples were cen- trifuged and 20 ml of the supernatant transferred to a streptavidin- coated microtiter plate as described (10). Anti-histone biotin and anti- DNA peroxidase antibodies were added to each well, and the plate was incubated at room temperature for 2 h. After three washes with incu- bation buffer, the peroxidase substrate was added to each well. Follow- FIG.1. Effects of paclitaxel and MEK inhibitor on MAP ki- ing a 15-min incubation, the plates were read at 405 nm in a microplate nases. A, paclitaxel-activated endogenous JNK. Human breast (BT474) reader. The data in this report are expressed as -fold increase in optical and lung (H358) carcinoma cell lines were treated with the indicated density as compared with control treated cells. concentrations of paclitaxel for 2 h, and JNK kinase activity was as- Cell Cycle Analysis—Adherent and detached cells were collected with sayed as described under “Experimental Procedures.” B, paclitaxel- trypsin and centrifuged at 200 3 g. Cells were resuspended at 2 3 10 activated endogenous ERK and activation is reversed by U0126. H157, cells/ml in PBS and fixed in ice-cold 70% ethanol for 2 h. Fixed cells a human lung carcinoma, and BT474 cells were serum-starved for 16 h were centrifuged at 200 3 g, and each sample resuspended in pro- and treated with the indicated concentrations of paclitaxel for 15 min. pidium iodide (PI) stain buffer (0.1% Triton X-100, 200 mg of DNase-free Cell lysates were subjected to immunoblot analysis with anti-ERK antibody for phosphorylated ERK1/2. The MEK inhibitor U0126 RNase A, 20 mg of PI) in PBS for 30 min. After staining, samples were blocked ERK activation by paclitaxel (lower panel). H157 cells were analyzed using a FACScan (Becton Dickinson) and ModFit LT (Verity serum-starved for 16 h and treated simultaneously with paclitaxel with Software). and without 10 mM U0126 for 15 min. TUNEL Assay—Cells were split at a density of 3 3 10 cells/well in a four-well chamber slide (Lab-Tek). Following a 36-h incubation, the The potential use of low dose chemotherapy is important, cells were treated with 10 nM paclitaxel in the presence or absence of 10 mM U0126 for 20 h. Following treatment, the cells were washed twice because lower dosages are more attainable during cancer ther- with PBS and fixed with 4% paraformaldehyde for 10 min. Cells were apy and likely to cause less toxicity in patients. We performed washed twice more with PBS and permeabilized with 0.2% Triton X-100 a dose-response analysis to assess the minimal concentration of for 5 min. After two more washes, each slide was covered with equili- paclitaxel, which when combined with U0126, causes enhanced bration buffer (Roche Molecular Biochemicals) for 10 more min. The cell death. Low doses of paclitaxel, starting at the 10 nM range, buffer was then aspirated, and the slides were incubated with TdT combined with U0126 cause enhanced cell death in both BT474 buffer at 37 °C for 1 h. The reaction was stopped with 23 SSC, and the slides were viewed with an immunofluorescence microscope. breast and H157 lung carcinoma cells (Fig. 2B). To control for pharmacologic specificity, two additional ex- RESULTS AND DISCUSSION periments were performed. First, a second MEK inhibitor The effect of paclitaxel on JNK and ERK activities is shown PD98059 was used and produced similar data (Fig. 2C), pro- in Fig. 1. Basal JNK activity was detected, and this activity viding additional evidence that the MEK enzyme is the target. was significantly enhanced by treatment with low, nanomolar However, pharmacologic approaches have their limitations, be- doses of paclitaxel in human lung and breast carcinoma cell cause the specificity of the drug can always be questioned. To lines (Fig. 1A). A basal level of ERK was also detected, and low provide further evidence for the effects of MEK inhibition, a doses of paclitaxel activated endogenous ERK1 and ERK2 (Fig. dominant-negative MEK (dnMEK) mutant was introduced into 1B). The MEK inhibitor, U0126, completely blocked ERK acti- H157 cells. Expression of dnMEK in the presence of low dose vation by paclitaxel. The activation of JNK in this scenario has (50 and 250 nM) paclitaxel enhanced apoptosis over the pCMV been previously found to contribute to apoptosis, while the role empty vector control (Fig. 2D). of paclitaxel-induced ERK has not been studied. In other sys- Table I summarizes enhanced apoptosis observed with pacli- tems, ERK generally plays a critical role in cell proliferation taxel and U0126. In H157 and OVCA194 cells, the combination and growth (22); thus, it was reasoned that ERK activation by treatment produced 4.0- and 2.5-fold enhancement of apoptosis paclitaxel might enhance cell proliferation and compromise the over the expected additive effect. This enhancement was efficacy of this drug. A logical approach is to use pharmacologic achieved with relative low dosages (1 mM paclitaxel, 10 mM blockers of MEK to inhibit paclitaxel-induced ERK activation U0126) of these two drugs. This trend was also observed with and its downstream effects. the breast carcinoma BT474 (not shown). To test this hypothesis, a combination of paclitaxel and a To examine the mechanism of U0126 and paclitaxel induced potent MEK1 inhibitor, U0126, was used to treat a variety of cell death, their effects on cell cycle progression was studied. human carcinoma lines, and cell death was measured by the The BT474 breast carcinoma cells were treated with paclitaxel cell death detection ELISA that detects DNA-histone fragmen- and/or U0126, and cell cycle progression was analyzed by in- tation. The combination of paclitaxel plus U0126 enhanced cell cubating the cells with propidium iodide, which allowed the death (Fig. 2A). The -fold increase in apoptosis was calculated analysis of DNA content by flow cytometry. U0126 arrested by comparing the ELISA optical density readings of treated BT474 cells in G , while 10 nM paclitaxel produced a dramatic samples, with the value of the untreated control as 1.0. In H157 G block (Fig. 3A). The percentage of control treated cells in cells, paclitaxel and U0126 combined caused four times more G -M was 13%, which increased to 75% after treatment with 10 cell death than paclitaxel alone, and eight times more cell nM paclitaxel for 24 h. Seventeen percentage of the cells under- death than U0126 alone. A similar trend was observed in went apoptosis in the presence of paclitaxel, while a negligible OVCA194 cells. increase in cell death was detected in the presence of U0126 MEK Inhibition Enhances Paclitaxel Apoptosis 38955 TABLE I Combination treatment with paclitaxel and MEK inhibitor U0126 H157 and OVCA194 cells were treated with the indicated concentra- tions of U0126 and paclitaxel for 24 h. Apoptosis was analyzed by cell death ELISA that measures DNA-histone release, and data are ex- pressed as absorbance [A 2 A ] 3 100. 405 nm 490 nm Combination treatment 10 mM 1 mM Fold increase U0126 paclitaxel a over additive Expected Observed H157 3.0 13.0 16.0 63.7 4.0 OVCA194 3.1 8.7 11.8 30.1 2.5 (Mean cell death of U0126) 1 (mean cell death of paclitaxel). (Observed combination)/(expected combination). To assess if the cell death observed above represents apo- ptosis, a TUNEL assay was performed with paclitaxel, U0126, or a combination of the two drugs. Singly, paclitaxel and U0126 caused little apoptosis (0.6 and 0.4%, see panels i–vi, Fig. 3B)as measured by the number of TUNEL-positive cells. When cells were treated with both, there was a dramatic increase in the number of TUNEL-positive cells to 11.1% (panels vii and viii, Fig. 3B). Phase-contrast photomicrographs of H157 cells re- vealed changes in morphology and cell membrane blebbing, which are characteristics of apoptosis (panel ix, Fig. 3B). These results further indicate that paclitaxel and U0126 enhance apoptosis. In the last 2 years, we and others have reported that pacli- taxel affects MAP kinases. The best documented is the activa- tion of JNK/SAPK by paclitaxel, which has been found in a variety of tumor cell lines (10 –13). JNK/SAPK activation is primarily a stress response, long proposed to be a determining factor in cell cycle arrest and apoptosis (23). Studies of hip- pocampal neuronal cells show that these cells do not undergo apoptosis when a JNK subgroup (jnk1, jnk2, or jnk3) is mu- tated. Very recently, the use of mice lacking functional JNK provides strong evidence that JNK is important in causing apoptosis (24 –26). Most relevant to this present study, JNK activation by paclitaxel directly contributes to apoptosis, as transdominant-negative JNK/SAPK significantly blocked pa- clitaxel-induced cell death (10 –13). Extensive research has identified potential mechanisms of paclitaxel-induced cell death, most prominent is the effect on BCL-2 family members and p53. Several reports indicate that paclitaxel causes the phosphorylation and inactivation of BCL-2 and its family members (7, 9, 27–29), while other stud- ies have found paclitaxel sensitivity varies with p53 status FIG.2. Analysis of paclitaxel and MEK inhibitor on carcinoma (30 –32). Additionally, a link between JNK and BCL-2 was cell death. A, paclitaxel and U0126 caused enhanced carcinoma cell found, where JNK mediated BCL-2 phosphorylation, and the death. H157 lung and OVCA194 ovarian carcinoma cells were treated inactivation of JNK inhibited paclitaxel-induced BCL-2 phos- with 1 mM paclitaxel, 10 mM MEK inhibitor U0126, or a combination of paclitaxel and MEK inhibitor. Twenty-four hours later, a cell death phorylation (33). This establishes the important roles of BCL-2 ELISA that measures cell death by DNA-histone release was performed and JNK family members in paclitaxel-induced apoptosis, al- as described under “Experimental Procedures.” B, low dose, nanomolar though other cell death and cell survival pathways are likely to range of paclitaxel and 10 mM U0126 caused enhanced killing. BT474 either enhance or intercede with this cytotoxicity. One of the breast carcinoma and H157 lung carcinoma cells were treated with the indicated concentrations of paclitaxel in the presence or absence of 10 findings described here is that paclitaxel also enhances the mM U0126, and -fold increase in cell death was measured by ELISA. C, activation of the MEK/ERK pathway, which is expected to a second MEK inhibitor, PD98059, and paclitaxel caused enhanced cell increase cell proliferation and survival, and may compromise death. H157 lung carcinoma cells were treated with either 10 mM U0126 the efficacy of paclitaxel in cancer treatment. or 50 mM PD98059 for 20 h and analyzed by the cell death ELISA. D, Based on a molecular approach, this report describes a novel dnMEK and paclitaxel caused enhanced cell death. H157 cells were transfected with 100 ng of pCMV vector control or dnMEK (34). After discovery that treatment with paclitaxel combined with the 24 h, cells were treated with the indicated amount of paclitaxel for 24 h inhibition of MEK1/2 lead to enhanced apoptosis of lung, ovar- and cell death assayed by ELISA. ian, and breast carcinoma cell lines. Two pharmacologic agents, paclitaxel and U0126, respectively, caused JNK activation that when compared with the control (6% compared with 4%). In promotes apoptosis, and MEK inhibition, which leads to cell contrast, the combination of paclitaxel and U0126 substan- cycle arrest. The two combined resulted in an impressive en- tially increased cell death as evidence by accumulation of a hancement of tumor cell killing. sub-G population that has ,2 N DNA (Fig. 3A) and represents In summary, these findings illustrate the power of molecular dead cells. These results further support the ELISA result that and rational drug targeting. Paclitaxel and MEK inhibitor com- low doses of paclitaxel and U0126 enhanced tumor cell death. bination therapy may allow the use of lower drug doses, likely 38956 MEK Inhibition Enhances Paclitaxel Apoptosis FIG.3. Paclitaxel and MEK inhibitor U0126 block cell cycle progression and cause enhanced cell death. A, BT474 breast carcinoma cells were treated for 24 h before staining with propidium iodide and analyzed by flow cytometry as described under “Experimental Procedures.” Histograms of control (Me SO-treated) cells exhibited normal cell cycle progression. Ten mM U0126 induced G growth arrest, 10 nM paclitaxel induced G growth arrest, and 10 nM paclitaxel plus 10 mM U0126 caused enhanced death by an accumulation of sub-G dead cells. The percentage 2 1 of cells in each phase of the cell cycle are shown below the histograms. B, paclitaxel and U0126 cause enhanced apoptosis. H157 cells were grown on coverslips and treated with 10 nM paclitaxel and/or 10 mM U0126 as indicated. After 16 h, the slides were incubated and TUNEL stained. The phase contrast photomicrographs (panels i, iii, v, and vii) and the corresponding immunofluorescence photomicrographs (panels ii, iv, vi, and viii) of cells undergoing apoptosis are shown. The number in the lower right of each panel represents the percentage of TUNEL-positive cells. 15. Sivaraman, V. S., Wang, H., Nuovo, G. J., and Malbon, C. C. (1997) J. Clin. leading to lowered toxicity and enhanced tumor killing in vivo. Invest. 99, 1478 –1483 The implications of these findings are broad for the potential 16. Mandell, J. W., Hussaini, I. M., Zecevic, M., Weber, M. J., and van den Berg, S. R. (1998) Am. J. Pathol. 153, 1411–1423 clinical usage of paclitaxel plus MEK inhibitors by: 1) improv- 17. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) ing the response rate and 2) expanding the usefulness of pacli- Proc. Natl. Acad. Sci. U. S. A. 92, 7686 –7689 taxel in the treatment of resistant tumors that affects a large 18. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, percentage of cancer patients. R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998) J. Biol. Chem. 273, 18623–18632 Acknowledgments—Channing Der kindly provided the MEK con- 19. Sebolt-Leopold, J. S., Dudley, D. T., Herrera, R., Van Becelaere, K., Wiland, A., structs. We thank Drs. Albert Baldwin, Lee Graves, Brian Martin, Gowan, R. C., Tecle, H., Barrett, S. D., Bridges, A., Przybranowski, S., Debra Taxman, Hank van Deventer, and Beverly Mitchell for helpful Leopold, W. R., and Saltiel, A. R. (1999) Nat. Med. 5, 810 – 816 comments and discussions. 20. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841– 852 21. Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., REFERENCES Fukasawa, K., vande Woude, G. F., and Ahn, N. G. (1994) Science 265, 966 –970 1. Ettinger, D. S. (1993) J. Natl. Cancer Inst. 15, 177–179 22. Karin, M., Liu, Z., and Zandi, E. (1997) Curr. Opin. Cell Biol. 9, 240 –246 2. Rowinsky, E. K., and Donehower, R. C. (1995) N. Engl. J. Med. 332, 23. Sanchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., 1004 –1014 Kyriakis, J. M., and Zon, L. I. (1994) Nature 372, 794 –798 3. Schiff, P. B., and Horwitz, S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 24. Yang, X., Khosravi-Far, R., Chang, H. Y., and Baltimore, D. (1997) Cell 89, 1561–1565 1067–1076 4. Baselga, J., Norton, L., Albanell, J., Kim, Y. M., and Mendelsohn, J. (1998) 25. Rincon, M., Whitmarsh, A., Yang, D. D., Weiss, L., Derijard, B., Jayaraj, P., Cancer Res. 58, 2825–2831 Davis, R. J., and Flavell, R. A. (1998) J. Exp. Med. 188, 1817–1830 5. Gupta, K., Kshirsagar, S., Li, W., Gui, L., Ramakrishnan, S., Gupta, P., Law, 26. Tournier, C., Hess, P., Yang, D. D., Xu, J., Turner, T. K., Nimnual, A., P. Y., and Hebbel, R. P. (1999) Exp. Cell Res. 247, 495–504 Bar-Sagi, D., Jones, S. N., Flavell, R. A., and Davis, R. J. (2000) Science 288, 6. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) 870 – 874 Science 270, 1326 –1331 27. Haldar, S., Jena, N., and Croce, C. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7. Blagosklonny, M. V., Giannakakou, P., El-Diery, W. S., Kingston, D. G. I., 4507– 4511 Higgs, P. I., Neckers, L. M., and Fojo, T. (1997) Cancer Res. 57, 130 –135 28. Haldar, S., Basu, A., and Croce, C. M. (1997) Cancer Res. 57, 229 –233 8. Torres, K., and Horwitz, S. B. (1998) Cancer Res. 58, 3620 –3626 29. Poruchynsky, M. S., Wang, E. E., Rudin, C. M., Blagosklonny, M. V., and Fojo, 9. Blagosklonny, V., Schulte, T., Nguyen, P., Trepel, J., and Neckers, L. M. (1996) T. (1998) Cancer Res. 58, 3331–3338 Cancer Res. 56, 1851–1854 30. Bhalla, K., Ibrado, A. M., Tourkina, E., Tang, C., Mahoney, M. E., and Huang, 10. Lee, L. F., Li, G., Templeton, D. J., and Ting, J. P. (1998) J. Biol. Chem. 273, 28253–28260 Y. (1993) Leukemia (Baltimore) 7, 563–568 31. Wahl, A. F., Donaldson, K. L., Fairchild, C., Lee, F. Y., Foster, S. A., Demers, 11. Wang, T.-Z., Wang, H.-S., Ichijo, H., Giannakakou, P., Foster, J. S., Fojo, T., and Wimalasena, J. (1998) J. Biol. Chem. 273, 4928 – 4936 G. W., and Galloway, D. A. (1996) Nat. Med. 2, 72–79 32. Lanni, J. S., Lowe, S. W., Licitra, E. J., Liu, J. O., and Jacks, T. (1997) Proc. 12. Yujiri, T., Sather, S., Fanger, G. R., and Johnson, G. L. (1998) Science 282, 1911–1914 Natl. Acad. Sci. U. S. A. 94, 9679 –9683 33. Yamamoto, K., Ichijo, H., and Korsmeyer, S. J. (1999) Mol. Cell. Biol. 19, 13. Amato, S. F., Swart, J. M., Berg, M., Wanebo, H. J., Mehta, S. R., and Chiles, T. C. (1998) Cancer Res. 58, 241–247 8469 – 8478 14. Huang, Y., Sheikh, M. S., Fornace, A. J., Jr., and Holbrook, N. J. (1999) 34. Seth, A., Gonzalez, F. A., Gupta, S., Raden, D. L., and Davis, R. J. (1992) Oncogene 18, 3431–3439 J. Biol. Chem. 267, 24796 –24804 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

MEK Inhibition Enhances Paclitaxel-induced Tumor Apoptosis

MacKeigan, Jeffrey P.; Collins, Timothy S.; Ting, Jenny P.-Y.
Journal of Biological Chemistry – Dec 1, 2000
Download PDF
4 pages

Loading next page...
 
/lp/unpaywall/mek-inhibition-enhances-paclitaxel-induced-tumor-apoptosis-Gs98qV3qXS

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.c000684200
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 50, Issue of December 15, pp. 38953–38956, 2000 Accelerated Publication © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. mitotic metaphase and anaphase. This prevents chromosome MEK Inhibition Enhances segregation, leading to tumor cell death. Paclitaxel-induced Tumor Combination therapy of paclitaxel and Herceptin, an anti- Her2-neu antibody, has produced impressive responses among Apoptosis* breast cancer patients (4), although this combination is obvi- ously limited to Her2-neu1 tumors. Combination therapy with Received for publication, September 28, 2000, and in revised other drugs, preferably via a rational molecular basis that is form, October 18, 2000 Published, JBC Papers in Press, October 18, 2000, widely applicable to many tumor types, is essential for im- DOI 10.1074/jbc.C000684200 proved cancer treatment. A combination of paclitaxel with re- agents that activate additional apoptotic signals, or inhibit ¶ ¶i Jeffrey P. MacKeigan‡§ , Timothy S. Collins‡ , survival signals, may provide a rational molecular basis for and Jenny P.-Y. Ting‡§** novel chemotherapeutic strategies. From the ‡Lineberger Comprehensive Cancer Center, A rational molecular target is the ERK mitogen-activated §Department of Microbiology and Immunology, iDepartment of Surgery, University of North Carolina, protein (MAP) kinase pathway that may serve as an opposing Chapel Hill, North Carolina 27599 force to Jun N-terminal kinase (JNK/SAPK). Previous reports have shown that JNK/SAPK leads to cell death, while MEK The anti-cancer drug paclitaxel (Taxol) alters micro- activation contributes to cell differentiation, proliferation, and tubule assembly and activates pro-apoptotic signaling survival (5, 6). Activated Raf-1, a serine-threonine kinase, ini- pathways. Previously, we and others found that pacli- tiates the signaling cascade through MEK, which in turn phos- taxel activates endogenous JNK in tumor cells, and the phorylates a second serine-threonine kinase ERK. ERK phos- activation of JNK contributes to tumor cell apoptosis. phorylates additional kinases and specific transcription Here we find that paclitaxel activates the prosurvival factors, such as Elk-1 and c-Fos, which are important in cell MEK/ERK pathway, which conversely may compromise proliferation. However, the link between Raf-1 and ERK acti- the efficacy of paclitaxel. Hence, a combination treat- vation and paclitaxel-induced cell death is not straightforward. ment of paclitaxel and MEK inhibitors was pursued to Several studies have shown that at a low concentration of the determine whether this treatment could lead to en- hanced apoptosis. The inhibition of MEK/ERK with a drug, paclitaxel-mediated apoptosis is attributed to activated pharmacologic inhibitor, U0126, together with pacli- Raf-1 (7–9). The role of the downstream ERK MAP kinase in taxel resulted in a dramatic enhancement of apoptosis paclitaxel-induced tumor apoptosis is also not entirely clear that is four times more than the additive value of the (10 –14). two drugs alone. Enhanced apoptosis was verified by In this report, we tested the combined effects of paclitaxel the terminal transferase-mediated dUTP nick end label- and inhibitors of MEK1/2 kinase on tumor cell apoptosis. The ing assay, by an enzyme-linked immunosorbent assay specificity of MEK1/2 inhibition was achieved by using two for histone-associated DNA fragments, and by flow cyto- different MEK inhibitors, and by the additional use of trans- metric analysis for DNA content. Specificity of the phar- dominant-negative mutants, which inhibit MEK/ERK activa- macologic inhibitor was confirmed by the use of (a)a tion. The reasons for selecting MEK1/2 as the target are: (i) second MEK/ERK inhibitor and (b) a transdominant- MEK is activated in many tumors (15, 16); (ii) small molecule- negative MEK. Enhanced apoptosis was verified in based MEK inhibitors are readily available, and a recent report breast, ovarian, and lung tumor cell lines, suggesting has described a novel MEK inhibitor that exhibited in vivo this effect is not cell type-specific. This is the first report efficacy in mice (17–19); and (iii) MEK is critical in transform- of enhanced apoptosis detected in the presence of pacli- ing cells, leading to tumor survival and proliferation (20, 21). In taxel and MEK inhibition and suggests a new anticancer the present study, we show that paclitaxel increases MEK1/2 strategy. activity. The combined treatment of paclitaxel plus MEK1/2 inhibition leads to enhanced cell death in breast, ovarian, and lung tumor lines. Paclitaxel is a promising frontline chemotherapy in the treatment of patients with ovarian, breast, and nonsmall cell EXPERIMENTAL PROCEDURES lung carcinomas (1, 2). Paclitaxel is isolated from the bark of JNK Kinase Assay—Following2hof paclitaxel (Sigma) treatment, the pacific yew (Taxus brevifolia) and functions by binding and cells were washed, harvested with lysis buffer, and centrifuged at 4 °C (10). Endogenous JNK was immunoprecipitated with anti-JNK anti- stabilizing microtubules (3). Binding of paclitaxel to microtu- body (Santa Cruz Biotechnology) and protein A-agarose beads for2hat bules blocks normal cell cycle progression during the merger of 4 °C. Immunoprecipitates were collected by centrifugation (2,500 rpm) at 4 °C. Immunoprecipitated JNK was mixed with 5 mg of glutathione * This work was supported by National Institutes of Health Grants 32 S-transferase-c-Jun and 10 mCi of [g- P]ATP and incubated for 30 min AI 41751 and AI45580 and by a grant from the Lineberger Comprehen- sive Cancer Center. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must The abbreviations used are: ERK, extracellular signal-regulated therefore be hereby marked “advertisement” in accordance with 18 kinase; MAP, mitogen-activated protein; JNK, c-Jun N-terminal ki- U.S.C. Section 1734 solely to indicate this fact. nase; SAPK, stress-activated protein kinase; MEK, MAP kinase kinase; ¶ These authors contributed equally to this study. dnMEK, dominant-negative MEK; PBS, phosphate-buffered saline; ** To whom correspondence should be addressed: Lineberger Com- PMSF, phenylmethylsulfonyl fluoride; PI, propidium iodide; TdT, ter- prehensive Cancer Center, Campus Box Number 7295, University of minal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-5538; Fax: 919- end-labeling; CMV, cytomegalovirus; PAGE, polyacrylamide gel elec- 966-8212; E-mail: [email protected]. trophoresis; ELISA, enzyme-linked immunosorbent assay. This paper is available on line at http://www.jbc.org 38953 This is an Open Access article under the CC BY license. 38954 MEK Inhibition Enhances Paclitaxel Apoptosis at 30 °C. The reactions were terminated with SDS sample buffer and resolved on a 10% SDS-PAGE gel. Immunoblot Analysis—H157 human lung carcinoma cells were se- rum-starved for 16 h and treated simultaneously with the indicated concentrations of paclitaxel with or without 10 mM U0126 (Promega). After 15 min of treatment, cells were lysed in 13 PBS, 1% Trition X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM Na VO ,10 mM leupeptin, and 10 mM pepstatin at 4 °C. Cell lysates were 3 4 separated with SDS-PAGE gels, transferred to nitrocellulose mem- branes, and subjected to immunoblot analysis with anti-ERK mono- clonal antibody for phosphorylated ERK1/2 (Santa Cruz Biotechnology). Cell Death ELISA—Manufacturer’s instructions were followed for PLUS the Cell Death Detection ELISA (Roche Molecular Biochemicals). Briefly, cells were plated at 5 3 10 cells/well in 96-well microtiter plates for 24 h. The cells were treated for 20 –24 h with the indicated doses of paclitaxel and U0126. Following lysis, the samples were cen- trifuged and 20 ml of the supernatant transferred to a streptavidin- coated microtiter plate as described (10). Anti-histone biotin and anti- DNA peroxidase antibodies were added to each well, and the plate was incubated at room temperature for 2 h. After three washes with incu- bation buffer, the peroxidase substrate was added to each well. Follow- FIG.1. Effects of paclitaxel and MEK inhibitor on MAP ki- ing a 15-min incubation, the plates were read at 405 nm in a microplate nases. A, paclitaxel-activated endogenous JNK. Human breast (BT474) reader. The data in this report are expressed as -fold increase in optical and lung (H358) carcinoma cell lines were treated with the indicated density as compared with control treated cells. concentrations of paclitaxel for 2 h, and JNK kinase activity was as- Cell Cycle Analysis—Adherent and detached cells were collected with sayed as described under “Experimental Procedures.” B, paclitaxel- trypsin and centrifuged at 200 3 g. Cells were resuspended at 2 3 10 activated endogenous ERK and activation is reversed by U0126. H157, cells/ml in PBS and fixed in ice-cold 70% ethanol for 2 h. Fixed cells a human lung carcinoma, and BT474 cells were serum-starved for 16 h were centrifuged at 200 3 g, and each sample resuspended in pro- and treated with the indicated concentrations of paclitaxel for 15 min. pidium iodide (PI) stain buffer (0.1% Triton X-100, 200 mg of DNase-free Cell lysates were subjected to immunoblot analysis with anti-ERK antibody for phosphorylated ERK1/2. The MEK inhibitor U0126 RNase A, 20 mg of PI) in PBS for 30 min. After staining, samples were blocked ERK activation by paclitaxel (lower panel). H157 cells were analyzed using a FACScan (Becton Dickinson) and ModFit LT (Verity serum-starved for 16 h and treated simultaneously with paclitaxel with Software). and without 10 mM U0126 for 15 min. TUNEL Assay—Cells were split at a density of 3 3 10 cells/well in a four-well chamber slide (Lab-Tek). Following a 36-h incubation, the The potential use of low dose chemotherapy is important, cells were treated with 10 nM paclitaxel in the presence or absence of 10 mM U0126 for 20 h. Following treatment, the cells were washed twice because lower dosages are more attainable during cancer ther- with PBS and fixed with 4% paraformaldehyde for 10 min. Cells were apy and likely to cause less toxicity in patients. We performed washed twice more with PBS and permeabilized with 0.2% Triton X-100 a dose-response analysis to assess the minimal concentration of for 5 min. After two more washes, each slide was covered with equili- paclitaxel, which when combined with U0126, causes enhanced bration buffer (Roche Molecular Biochemicals) for 10 more min. The cell death. Low doses of paclitaxel, starting at the 10 nM range, buffer was then aspirated, and the slides were incubated with TdT combined with U0126 cause enhanced cell death in both BT474 buffer at 37 °C for 1 h. The reaction was stopped with 23 SSC, and the slides were viewed with an immunofluorescence microscope. breast and H157 lung carcinoma cells (Fig. 2B). To control for pharmacologic specificity, two additional ex- RESULTS AND DISCUSSION periments were performed. First, a second MEK inhibitor The effect of paclitaxel on JNK and ERK activities is shown PD98059 was used and produced similar data (Fig. 2C), pro- in Fig. 1. Basal JNK activity was detected, and this activity viding additional evidence that the MEK enzyme is the target. was significantly enhanced by treatment with low, nanomolar However, pharmacologic approaches have their limitations, be- doses of paclitaxel in human lung and breast carcinoma cell cause the specificity of the drug can always be questioned. To lines (Fig. 1A). A basal level of ERK was also detected, and low provide further evidence for the effects of MEK inhibition, a doses of paclitaxel activated endogenous ERK1 and ERK2 (Fig. dominant-negative MEK (dnMEK) mutant was introduced into 1B). The MEK inhibitor, U0126, completely blocked ERK acti- H157 cells. Expression of dnMEK in the presence of low dose vation by paclitaxel. The activation of JNK in this scenario has (50 and 250 nM) paclitaxel enhanced apoptosis over the pCMV been previously found to contribute to apoptosis, while the role empty vector control (Fig. 2D). of paclitaxel-induced ERK has not been studied. In other sys- Table I summarizes enhanced apoptosis observed with pacli- tems, ERK generally plays a critical role in cell proliferation taxel and U0126. In H157 and OVCA194 cells, the combination and growth (22); thus, it was reasoned that ERK activation by treatment produced 4.0- and 2.5-fold enhancement of apoptosis paclitaxel might enhance cell proliferation and compromise the over the expected additive effect. This enhancement was efficacy of this drug. A logical approach is to use pharmacologic achieved with relative low dosages (1 mM paclitaxel, 10 mM blockers of MEK to inhibit paclitaxel-induced ERK activation U0126) of these two drugs. This trend was also observed with and its downstream effects. the breast carcinoma BT474 (not shown). To test this hypothesis, a combination of paclitaxel and a To examine the mechanism of U0126 and paclitaxel induced potent MEK1 inhibitor, U0126, was used to treat a variety of cell death, their effects on cell cycle progression was studied. human carcinoma lines, and cell death was measured by the The BT474 breast carcinoma cells were treated with paclitaxel cell death detection ELISA that detects DNA-histone fragmen- and/or U0126, and cell cycle progression was analyzed by in- tation. The combination of paclitaxel plus U0126 enhanced cell cubating the cells with propidium iodide, which allowed the death (Fig. 2A). The -fold increase in apoptosis was calculated analysis of DNA content by flow cytometry. U0126 arrested by comparing the ELISA optical density readings of treated BT474 cells in G , while 10 nM paclitaxel produced a dramatic samples, with the value of the untreated control as 1.0. In H157 G block (Fig. 3A). The percentage of control treated cells in cells, paclitaxel and U0126 combined caused four times more G -M was 13%, which increased to 75% after treatment with 10 cell death than paclitaxel alone, and eight times more cell nM paclitaxel for 24 h. Seventeen percentage of the cells under- death than U0126 alone. A similar trend was observed in went apoptosis in the presence of paclitaxel, while a negligible OVCA194 cells. increase in cell death was detected in the presence of U0126 MEK Inhibition Enhances Paclitaxel Apoptosis 38955 TABLE I Combination treatment with paclitaxel and MEK inhibitor U0126 H157 and OVCA194 cells were treated with the indicated concentra- tions of U0126 and paclitaxel for 24 h. Apoptosis was analyzed by cell death ELISA that measures DNA-histone release, and data are ex- pressed as absorbance [A 2 A ] 3 100. 405 nm 490 nm Combination treatment 10 mM 1 mM Fold increase U0126 paclitaxel a over additive Expected Observed H157 3.0 13.0 16.0 63.7 4.0 OVCA194 3.1 8.7 11.8 30.1 2.5 (Mean cell death of U0126) 1 (mean cell death of paclitaxel). (Observed combination)/(expected combination). To assess if the cell death observed above represents apo- ptosis, a TUNEL assay was performed with paclitaxel, U0126, or a combination of the two drugs. Singly, paclitaxel and U0126 caused little apoptosis (0.6 and 0.4%, see panels i–vi, Fig. 3B)as measured by the number of TUNEL-positive cells. When cells were treated with both, there was a dramatic increase in the number of TUNEL-positive cells to 11.1% (panels vii and viii, Fig. 3B). Phase-contrast photomicrographs of H157 cells re- vealed changes in morphology and cell membrane blebbing, which are characteristics of apoptosis (panel ix, Fig. 3B). These results further indicate that paclitaxel and U0126 enhance apoptosis. In the last 2 years, we and others have reported that pacli- taxel affects MAP kinases. The best documented is the activa- tion of JNK/SAPK by paclitaxel, which has been found in a variety of tumor cell lines (10 –13). JNK/SAPK activation is primarily a stress response, long proposed to be a determining factor in cell cycle arrest and apoptosis (23). Studies of hip- pocampal neuronal cells show that these cells do not undergo apoptosis when a JNK subgroup (jnk1, jnk2, or jnk3) is mu- tated. Very recently, the use of mice lacking functional JNK provides strong evidence that JNK is important in causing apoptosis (24 –26). Most relevant to this present study, JNK activation by paclitaxel directly contributes to apoptosis, as transdominant-negative JNK/SAPK significantly blocked pa- clitaxel-induced cell death (10 –13). Extensive research has identified potential mechanisms of paclitaxel-induced cell death, most prominent is the effect on BCL-2 family members and p53. Several reports indicate that paclitaxel causes the phosphorylation and inactivation of BCL-2 and its family members (7, 9, 27–29), while other stud- ies have found paclitaxel sensitivity varies with p53 status FIG.2. Analysis of paclitaxel and MEK inhibitor on carcinoma (30 –32). Additionally, a link between JNK and BCL-2 was cell death. A, paclitaxel and U0126 caused enhanced carcinoma cell found, where JNK mediated BCL-2 phosphorylation, and the death. H157 lung and OVCA194 ovarian carcinoma cells were treated inactivation of JNK inhibited paclitaxel-induced BCL-2 phos- with 1 mM paclitaxel, 10 mM MEK inhibitor U0126, or a combination of paclitaxel and MEK inhibitor. Twenty-four hours later, a cell death phorylation (33). This establishes the important roles of BCL-2 ELISA that measures cell death by DNA-histone release was performed and JNK family members in paclitaxel-induced apoptosis, al- as described under “Experimental Procedures.” B, low dose, nanomolar though other cell death and cell survival pathways are likely to range of paclitaxel and 10 mM U0126 caused enhanced killing. BT474 either enhance or intercede with this cytotoxicity. One of the breast carcinoma and H157 lung carcinoma cells were treated with the indicated concentrations of paclitaxel in the presence or absence of 10 findings described here is that paclitaxel also enhances the mM U0126, and -fold increase in cell death was measured by ELISA. C, activation of the MEK/ERK pathway, which is expected to a second MEK inhibitor, PD98059, and paclitaxel caused enhanced cell increase cell proliferation and survival, and may compromise death. H157 lung carcinoma cells were treated with either 10 mM U0126 the efficacy of paclitaxel in cancer treatment. or 50 mM PD98059 for 20 h and analyzed by the cell death ELISA. D, Based on a molecular approach, this report describes a novel dnMEK and paclitaxel caused enhanced cell death. H157 cells were transfected with 100 ng of pCMV vector control or dnMEK (34). After discovery that treatment with paclitaxel combined with the 24 h, cells were treated with the indicated amount of paclitaxel for 24 h inhibition of MEK1/2 lead to enhanced apoptosis of lung, ovar- and cell death assayed by ELISA. ian, and breast carcinoma cell lines. Two pharmacologic agents, paclitaxel and U0126, respectively, caused JNK activation that when compared with the control (6% compared with 4%). In promotes apoptosis, and MEK inhibition, which leads to cell contrast, the combination of paclitaxel and U0126 substan- cycle arrest. The two combined resulted in an impressive en- tially increased cell death as evidence by accumulation of a hancement of tumor cell killing. sub-G population that has ,2 N DNA (Fig. 3A) and represents In summary, these findings illustrate the power of molecular dead cells. These results further support the ELISA result that and rational drug targeting. Paclitaxel and MEK inhibitor com- low doses of paclitaxel and U0126 enhanced tumor cell death. bination therapy may allow the use of lower drug doses, likely 38956 MEK Inhibition Enhances Paclitaxel Apoptosis FIG.3. Paclitaxel and MEK inhibitor U0126 block cell cycle progression and cause enhanced cell death. A, BT474 breast carcinoma cells were treated for 24 h before staining with propidium iodide and analyzed by flow cytometry as described under “Experimental Procedures.” Histograms of control (Me SO-treated) cells exhibited normal cell cycle progression. Ten mM U0126 induced G growth arrest, 10 nM paclitaxel induced G growth arrest, and 10 nM paclitaxel plus 10 mM U0126 caused enhanced death by an accumulation of sub-G dead cells. The percentage 2 1 of cells in each phase of the cell cycle are shown below the histograms. B, paclitaxel and U0126 cause enhanced apoptosis. H157 cells were grown on coverslips and treated with 10 nM paclitaxel and/or 10 mM U0126 as indicated. After 16 h, the slides were incubated and TUNEL stained. The phase contrast photomicrographs (panels i, iii, v, and vii) and the corresponding immunofluorescence photomicrographs (panels ii, iv, vi, and viii) of cells undergoing apoptosis are shown. The number in the lower right of each panel represents the percentage of TUNEL-positive cells. 15. Sivaraman, V. S., Wang, H., Nuovo, G. J., and Malbon, C. C. (1997) J. Clin. leading to lowered toxicity and enhanced tumor killing in vivo. Invest. 99, 1478 –1483 The implications of these findings are broad for the potential 16. Mandell, J. W., Hussaini, I. M., Zecevic, M., Weber, M. J., and van den Berg, S. R. (1998) Am. J. Pathol. 153, 1411–1423 clinical usage of paclitaxel plus MEK inhibitors by: 1) improv- 17. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) ing the response rate and 2) expanding the usefulness of pacli- Proc. Natl. Acad. Sci. U. S. A. 92, 7686 –7689 taxel in the treatment of resistant tumors that affects a large 18. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, percentage of cancer patients. R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998) J. Biol. Chem. 273, 18623–18632 Acknowledgments—Channing Der kindly provided the MEK con- 19. Sebolt-Leopold, J. S., Dudley, D. T., Herrera, R., Van Becelaere, K., Wiland, A., structs. We thank Drs. Albert Baldwin, Lee Graves, Brian Martin, Gowan, R. C., Tecle, H., Barrett, S. D., Bridges, A., Przybranowski, S., Debra Taxman, Hank van Deventer, and Beverly Mitchell for helpful Leopold, W. R., and Saltiel, A. R. (1999) Nat. Med. 5, 810 – 816 comments and discussions. 20. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841– 852 21. Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., REFERENCES Fukasawa, K., vande Woude, G. F., and Ahn, N. G. (1994) Science 265, 966 –970 1. Ettinger, D. S. (1993) J. Natl. Cancer Inst. 15, 177–179 22. Karin, M., Liu, Z., and Zandi, E. (1997) Curr. Opin. Cell Biol. 9, 240 –246 2. Rowinsky, E. K., and Donehower, R. C. (1995) N. Engl. J. Med. 332, 23. Sanchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., 1004 –1014 Kyriakis, J. M., and Zon, L. I. (1994) Nature 372, 794 –798 3. Schiff, P. B., and Horwitz, S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 24. Yang, X., Khosravi-Far, R., Chang, H. Y., and Baltimore, D. (1997) Cell 89, 1561–1565 1067–1076 4. Baselga, J., Norton, L., Albanell, J., Kim, Y. M., and Mendelsohn, J. (1998) 25. Rincon, M., Whitmarsh, A., Yang, D. D., Weiss, L., Derijard, B., Jayaraj, P., Cancer Res. 58, 2825–2831 Davis, R. J., and Flavell, R. A. (1998) J. Exp. Med. 188, 1817–1830 5. Gupta, K., Kshirsagar, S., Li, W., Gui, L., Ramakrishnan, S., Gupta, P., Law, 26. Tournier, C., Hess, P., Yang, D. D., Xu, J., Turner, T. K., Nimnual, A., P. Y., and Hebbel, R. P. (1999) Exp. Cell Res. 247, 495–504 Bar-Sagi, D., Jones, S. N., Flavell, R. A., and Davis, R. J. (2000) Science 288, 6. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) 870 – 874 Science 270, 1326 –1331 27. Haldar, S., Jena, N., and Croce, C. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7. Blagosklonny, M. V., Giannakakou, P., El-Diery, W. S., Kingston, D. G. I., 4507– 4511 Higgs, P. I., Neckers, L. M., and Fojo, T. (1997) Cancer Res. 57, 130 –135 28. Haldar, S., Basu, A., and Croce, C. M. (1997) Cancer Res. 57, 229 –233 8. Torres, K., and Horwitz, S. B. (1998) Cancer Res. 58, 3620 –3626 29. Poruchynsky, M. S., Wang, E. E., Rudin, C. M., Blagosklonny, M. V., and Fojo, 9. Blagosklonny, V., Schulte, T., Nguyen, P., Trepel, J., and Neckers, L. M. (1996) T. (1998) Cancer Res. 58, 3331–3338 Cancer Res. 56, 1851–1854 30. Bhalla, K., Ibrado, A. M., Tourkina, E., Tang, C., Mahoney, M. E., and Huang, 10. Lee, L. F., Li, G., Templeton, D. J., and Ting, J. P. (1998) J. Biol. Chem. 273, 28253–28260 Y. (1993) Leukemia (Baltimore) 7, 563–568 31. Wahl, A. F., Donaldson, K. L., Fairchild, C., Lee, F. Y., Foster, S. A., Demers, 11. Wang, T.-Z., Wang, H.-S., Ichijo, H., Giannakakou, P., Foster, J. S., Fojo, T., and Wimalasena, J. (1998) J. Biol. Chem. 273, 4928 – 4936 G. W., and Galloway, D. A. (1996) Nat. Med. 2, 72–79 32. Lanni, J. S., Lowe, S. W., Licitra, E. J., Liu, J. O., and Jacks, T. (1997) Proc. 12. Yujiri, T., Sather, S., Fanger, G. R., and Johnson, G. L. (1998) Science 282, 1911–1914 Natl. Acad. Sci. U. S. A. 94, 9679 –9683 33. Yamamoto, K., Ichijo, H., and Korsmeyer, S. J. (1999) Mol. Cell. Biol. 19, 13. Amato, S. F., Swart, J. M., Berg, M., Wanebo, H. J., Mehta, S. R., and Chiles, T. C. (1998) Cancer Res. 58, 241–247 8469 – 8478 14. Huang, Y., Sheikh, M. S., Fornace, A. J., Jr., and Holbrook, N. J. (1999) 34. Seth, A., Gonzalez, F. A., Gupta, S., Raden, D. L., and Davis, R. J. (1992) Oncogene 18, 3431–3439 J. Biol. Chem. 267, 24796 –24804

Journal

Journal of Biological Chemistry – Unpaywall

Published: Dec 1, 2000

There are no references for this article.