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Activation of MST/Krs and c-Jun N-terminal Kinases by Different Signaling Pathways during Cytotrienin A-induced Apoptosis

2000; Elsevier BV; Volume: 275; Issue: 12 Linguagem: Inglês

10.1074/jbc.275.12.8766

1083-351X

Masahiko Watabe, Hideaki Kakeya, Rie Onose, Hiroyuki Osada,

Cancer-related Molecular Pathways

We found that antitumor drugs such as cytotrienin A, camptothecin, taxol, and 5-fluorouracil induced the activation of a 36-kDa protein kinase (p36 myelin basic protein (MBP) kinase) during apoptosis in human promyelocytic leukemia HL-60 cells. This p36 MBP kinase, which phosphorylates MBP in an in-gel kinase assay, results from the caspase-3-mediated proteolytic cleavage of MST/Krs protein, a mammalian Ste20-like serine/threonine kinase. Herein the correlation between cytotrienin A-induced apoptosis and the activation of MST/Krs proteins was examined in human tumor cell lines, including leukemia-, lung-, epidermoid-, cervix-, stomach-, and brain-derived cell lines. In cytotrienin A-sensitive cell lines, we observed a strong activation of p36 MBP kinase by cleavage of the C-terminal regulatory domain of full-length MST/Krs proteins by caspase-3. When the kinase-inactive mutant form of MST/Krs protein was overexpressed in cytotrienin A-sensitive HL-60 cells, the cytotrienin A-induced apoptosis was partially inhibited. Because cytotrienin A also activated c-Jun N-terminal kinase, we examined the effect of the expression of dominant negative c-Jun on cytotrienin A-induced apoptosis. The expression of dominant negative c-Jun also partially inhibited cytotrienin A-induced apoptosis. Furthermore, coexpression of kinase-inactive MST/Krs protein and dominant negative c-Jun completely suppressed cytotrienin A-induced apoptosis. These findings suggest that the proteolytic activation of MST/Krs and c-Jun N-terminal kinase activation are involved in cytotrienin A-induced apoptosis in human tumor cell lines. We found that antitumor drugs such as cytotrienin A, camptothecin, taxol, and 5-fluorouracil induced the activation of a 36-kDa protein kinase (p36 myelin basic protein (MBP) kinase) during apoptosis in human promyelocytic leukemia HL-60 cells. This p36 MBP kinase, which phosphorylates MBP in an in-gel kinase assay, results from the caspase-3-mediated proteolytic cleavage of MST/Krs protein, a mammalian Ste20-like serine/threonine kinase. Herein the correlation between cytotrienin A-induced apoptosis and the activation of MST/Krs proteins was examined in human tumor cell lines, including leukemia-, lung-, epidermoid-, cervix-, stomach-, and brain-derived cell lines. In cytotrienin A-sensitive cell lines, we observed a strong activation of p36 MBP kinase by cleavage of the C-terminal regulatory domain of full-length MST/Krs proteins by caspase-3. When the kinase-inactive mutant form of MST/Krs protein was overexpressed in cytotrienin A-sensitive HL-60 cells, the cytotrienin A-induced apoptosis was partially inhibited. Because cytotrienin A also activated c-Jun N-terminal kinase, we examined the effect of the expression of dominant negative c-Jun on cytotrienin A-induced apoptosis. The expression of dominant negative c-Jun also partially inhibited cytotrienin A-induced apoptosis. Furthermore, coexpression of kinase-inactive MST/Krs protein and dominant negative c-Jun completely suppressed cytotrienin A-induced apoptosis. These findings suggest that the proteolytic activation of MST/Krs and c-Jun N-terminal kinase activation are involved in cytotrienin A-induced apoptosis in human tumor cell lines. c-Jun N-terminal kinase myelin basic protein poly(ADP-ribose) polymerase reactive oxygen species 3-acetyl-4,5-dimethyl-5-octyloxy-3-pyrrolin-2-one reactive oxygen species benzoyloxycarbonyl-Asp-CH2O(CO)-2,6-dichlorobenzene Apoptosis is induced by a wide variety of cellular stresses, including DNA damage, UV radiation, ionizing radiation, and oxidative stress (1.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4577) Google Scholar, 2.Arends M.J. Wyllie A.H. Int. Rev. Exp. Pathol. 1991; 32: 223-254Crossref PubMed Scopus (1405) Google Scholar). It is morphologically distinct from necrosis in many of its characteristic changes as follows: DNA fragmentation, chromatin condensation, membrane blebbing, and cell shrinkage. Antitumor agents also induce apoptosis in some cancer cells both in vitro andin vivo, indicating that apoptosis plays a very important role in cancer chemotherapy (3.Kaufmann S.H. Cancer Res. 1989; 49: 5870-5878PubMed Google Scholar, 4.Meyn R.E. Stephens L.C. Hunter N.R. Milas L. Anti-Cancer Drugs. 1995; 6: 443-450Crossref PubMed Scopus (113) Google Scholar). However, the biochemical mechanism of apoptosis induction by antitumor agents is not yet fully understood. Previous studies indicate that the apoptotic process is triggered by the activation of a caspase cascade (1.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4577) Google Scholar, 5.Nicholson D.W. Thornbery N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar). Caspases are a family of cysteine proteases expressed ubiquitously in multicellular organisms as latent pro-enzyme forms. Activation of pro-caspases is an obligatory step in the execution of apoptosis, and this activation takes place by proteolytic cleavage. In the nematode Caenorhabditis elegans, three apoptosis-related genes have been cloned,ced-3, ced-4, and ced-9 (6.Hengartner M.O. Horvitz H.R. Curr. Opin. Genet. & Dev. 1994; 4: 581-586Crossref PubMed Scopus (337) Google Scholar). The former two genes are required for execution of the death program, whereas ced-9 prevents cell death. Mammalian homologues of Ced-3 and Ced-9 proteins have been identified as the interleukin-1β-converting enzyme cysteine protease (termed caspase-1) and Bcl-2, respectively (7.Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2271) Google Scholar, 8.Hengartner M.O. Horvitz H.R. Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1055) Google Scholar). The Ced-4 protein is homologous to the human protein Apaf-1, which participates in the activation of caspase-3 (9.Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2764) Google Scholar, 10.Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6316) Google Scholar). Caspase-3, an important effector caspase, is responsible for the cleavage of crucial substrates such as structural proteins, signaling proteins, and transcription-regulating proteins involved in the apoptotic process (1.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4577) Google Scholar, 5.Nicholson D.W. Thornbery N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2201) Google Scholar). In particular, the fact that caspases regulate the activity of several protein kinases indicates that protein phosphorylation/dephosphorylation mechanisms may play an important role in the initiation and progression of apoptosis. However, the role of individual targets in the common apoptotic signaling pathway triggered by anticancer agents is still uncertain. Cytotrienin A (Fig. 1), a novel ansamycin antitumor agent, was isolated from Streptomyces sp. as an apoptosis inducer (11.Kakeya H. Zhang H.-P. Kobinata K. Onose R. Onozawa C. Kudo T. Osada H. J. Antibiot. (Tokyo). 1997; 50: 370-372Crossref PubMed Scopus (52) Google Scholar, 12.Zhang H.-P. Kakeya H. Osada H. Tetrahedron Lett. 1997; 38: 1789-1792Crossref Scopus (44) Google Scholar). Recently, we have found that JNK1 and a 36-kDa kinase (termed p36 MBP kinase) are activated during the cytotrienin A-induced apoptosis in human promyelocytic leukemia HL-60 cells (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). This p36 MBP kinase is an active proteolytic product of the MST2/Krs1 and MST1/Krs2 protein kinases and were originally cloned by virtue of their homology to the budding yeast Ste20 kinase (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). Two other groups (14.Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (328) Google Scholar,15.Lee K.-K. Murakawa M. Nishida E. Tsubuki S. Kawashima S.-I. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar) have also reported that MST1/Krs2 is cleaved by a caspase-3-like activity during Fas-induced apoptosis. Notably, Graves et al. (14.Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (328) Google Scholar) extensively examined the involvement of MST1/Krs2 together with mitogen-activated protein kinase cascade during apoptosis. It is possible that most apoptotic signals including the Fas-mediated signal and the cytotrienin-induced signal activate several pathways for apoptosis in a target cell. Unlike these apoptosis inducers, MT-21 (3-acetyl-4,5-dimethyl-5-octyloxy-3-pyrrolin-2-one), our synthetic apoptosis inducer, activates the signal from caspase-3, MST/Krs to JNK in a rather simple manner (16.Kakeya H. Onozawa C. Sato M. Arai K. Osada H. J. Med. Chem. 1997; 40: 391-394Crossref PubMed Scopus (86) Google Scholar, 17.Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar). In the present study, we have investigated the caspase-mediated proteolytic activation of MST/Krs proteins in cytotrienin A-sensitive and -resistant human tumor cell lines. Cytotrienin A was isolated fromStreptomyces sp. RK95-74, as described previously (11.Kakeya H. Zhang H.-P. Kobinata K. Onose R. Onozawa C. Kudo T. Osada H. J. Antibiot. (Tokyo). 1997; 50: 370-372Crossref PubMed Scopus (52) Google Scholar, 12.Zhang H.-P. Kakeya H. Osada H. Tetrahedron Lett. 1997; 38: 1789-1792Crossref Scopus (44) Google Scholar). MBP was purchased from Sigma. Anti-Krs1 antibody, anti-Krs2 antibody, and anti-PARP antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Bcl-2 and Bax were purchased from Dako (Glostrup, Denmark) and MBL (Nagoya, Japan), respectively, and anti-caspase-3 antibody and [γ-32P]ATP were from Transduction Laboratories (Lexington, KY) and ICN Biochemicals Inc. (Costa Mesa, CA), respectively. Camptothecin, etoposide, mitomycin C, doxorubicin, bleomycin, staurosporine, actinomycin D, colcemid, taxol, and 5-fluorouracil were purchased from Wako Pure Chemicals, Ltd. (Osaka, Japan). Cloning of human full-length MST/krs1 andMST/krs2 cDNA and constructions of expression vectors of kinase-inactive MST/krs1 and MST/krs2 were performed as described previously (17.Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar). HL-60 (human promyelocytic leukemia cells), U937 (human monoblastoid leukemia cells), K562 (human chronic myelogenous leukemia cells), Jurkat (human acute T-cell leukemia cells), and MKN74 (human stomach adenocarcinoma cells) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. WI-38 (human normal lung fibroblast cells), A431 (human epidermoid carcinoma cells), HeLa (human cervix epithelioid carcinoma cells), and SH-SY5Y (human neuroblastoma cells) were maintained in Dulbecco's modified Eagle's medium including 10% fetal bovine serum. A549 (human lung carcinoma cells) cells were maintained in Eagle's minimum essential medium supplemented with 10% fetal bovine serum and non-essential amino acids. SMS-KCN (human neuroblastoma cells) were maintained in RPMI 1640 with 10% fetal bovine serum and OPI medium supplement (Sigma). All cells were maintained in a 5% CO2 humidified atmosphere at 37 °C. U937, WI-38, and HeLa cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). A549 and MKN74 cells were purchased from Health Science Research Resources Bank (Osaka, Japan). Jurkat cells were supplied by Dr. M. Ishizuka (Institute of Chemotherapy, Shizuoka, Japan) and SH-SY5Y and SMS-KCN by Dr. H. Matsui (St. Marianna University, Tokyo, Japan) and Dr. A. Nakagawara (Chiba Cancer Res. Inst., Chiba, Japan), respectively. U937 cells transfected with dominant negative c-Jun were kindly provided by Dr. Nakaya (Showa University, Tokyo, Japan). Exponentially growing cells were exposed to various concentrations of antitumor agents. After incubation for 24 h at 37 °C, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt and 1-methoxy-5-methylphenazinium methyl sulfate were added into the culture medium and incubated for a further 2–3 h. Then the cell viability was determined by the measurement ofA 405. DNA fragmentation assays were carried out as described previously (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 17.Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar). The DNA ladders were visualized by UV illumination after ethidium bromide staining. Preparation of whole-cell extracts and Western blotting analyses were performed as described previously (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). Bands were visualized by a Western blot Chemiluminescence Reagent (Pierce). Cells treated/untreated with various drugs were lysed in lysis buffer (20 mm Tris-HCl (pH 7.5), 5 mm EGTA, 0.5% Triton X-100, 50 mmβ-glycerophosphate, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 6 mmdithiothreitol, and 2% aprotinin), and the protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad). To assay the total cell lysate, equal amounts of protein (50–100 μg) were electrophoresed in 10% SDS-polyacrylamide gels containing 0.5 mg/ml MBP as a substrate. Following electrophoresis, SDS was removed from the gel; the protein was renatured, and a kinase assay was carried out by incubating the gel in buffer (40 mm HEPES (pH 7.5), 10 mm MgCl2, 2 mm dithiothreitol, and 0.1 mm EGTA) containing 20 μm[γ-32P]ATP. Gels were washed and dried, and the incorporated radioactivity was analyzed by autoradiography. JNK activity in whole-cell extracts (0.1 mg) was measured by incubation with 10 μg of glutathione S-transferase-c-Jun-(1–79) conjugated to glutathione-agarose beads (prepared according to the manufacturer's instructions, Amersham Pharmacia Biotech) for 1 h at 4 °C. The agarose beads were collected by microcentrifugation and washed 5 times with HEPES binding buffer (20 mm HEPES (pH 8.0), 20 mm MgCl2, 0.1 mm EDTA, 50 mm NaCl, 0.05% Triton X-100). The final wash was performed in kinase buffer (20 mm HEPES (pH 8.0), 20 mmMgCl2, 20 mm β-glycerophosphate, 0.1 mm sodium vanadate, 2 mm dithiothreitol). The kinase reaction was initiated by resuspending the pelleted beads in 30 μl of kinase buffer containing [γ-32P]ATP (20 μm, 0.5 μCi/reaction) for 10 min at 30 °C. The reaction was terminated by addition of 1 ml of the ice-cold HEPES binding buffer. The beads were pelleted, resuspended in SDS-PAGE loading buffer, and boiled for 5 min. Proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel followed by autoradiography. HL-60 and U937 cells were transfected in 60-mm dishes with 2 μg of the appropriate plasmids using 10 μl of FuGENETM 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. We previously reported that MST/Krs proteins consisting of MST1/Krs2 and MST2/Krs1 were activated during cytotrienin A-induced apoptosis (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). To characterize the relationship between the antitumor agent-induced apoptosis and the activation of MST/Krs proteins, we examined the effects of antitumor agents including cytotrienin A, camptothecin, etoposide, mitomycin C, doxorubicin, bleomycin, staurosporine, actinomycin D, colcemid, taxol, and 5-fluorouracil on proteolytic activation of MST/Krs proteins in HL-60 cells. As shown in Fig. 2 A, a DNA ladder pattern typical of internucleosomal fragmentation, which is considered to be an early event in apoptosis, was detected upon treatment with all the antitumor agents tested here. In addition, the activation of p36 MBP kinase upon proteolysis of MST2/Krs1 and MST1/Krs2 was induced by these antitumor agents (Fig. 2 B). In the case of colcemid treatment, the induction of DNA fragmentation was slightly weaker than other agents, and the activation of p36 MBP kinase also was weak. These results suggest that the proteolytic activation of Krs proteins may be closely related to the apoptotic program induced by antitumor agents, despite the differences in their modes of action. We examined 10 human tumor cell lines in addition to one human normal cell line for cytotrienin A sensitivity as shown in TableI. Exponentially growing cultures of human tumor cell lines were exposed to various concentrations of cytotrienin A, and the effects of cytotrienin A on cell viability were evaluated by a colorimetric method. The sensitivity to cytotrienin A-induced apoptosis in T-cell leukemia, Jurkat cells, was approximately the same as the promyelocytic leukemia HL-60 cell line, which is extremely sensitive to cytotrienin A. In monoblastoid leukemia U937 and myelogenous leukemia K562 cells, cytotrienin A showed apoptosis-inducing activity with an IC50 of 50 ng/ml, whereas in stomach adenocarcinoma MKN74 cells, it showed weaker activity with an IC50 of 1000 ng/ml. In contrast, six other cell lines, including human normal lung fibroblast WI-38 cells, were found to be largely resistant to cytotrienin A treatment.Table IEffects of cytotrienin A on cell growth in human cancer cell linesCell lineOriginIC50ng/mlHL-60Promyelocytic leukemia7U937Monoblastoid leukemia50K562Myelogenous leukemia50JurkatT-cell leukemia9WI-38Normal lung fibroblast>3000A549Lung carcinoma>3000A431Epidermoid carcinoma>3000HeLaCervix epithelioid carcinoma>3000MKN74Stomach adenocarcinoma1000SH-SY5YNeuroblastoma2000SMS-KCNNeuroblastoma2000Each cell line was cultured with various concentrations of cytotrienin A. Cell viability was measured following a 24-h incubation. Open table in a new tab Each cell line was cultured with various concentrations of cytotrienin A. Cell viability was measured following a 24-h incubation. To investigate whether the expression of MST/Krs proteins is correlated with cytotrienin A sensitivity, we examined MST/Krs protein levels by Western blotting using specific antibodies (Fig. 3 A). The MST/Krs proteins were abundantly expressed in the cytotrienin A-sensitive cell lines HL-60, U937, K562, and Jurkat as shown in TableI. However, cytotrienin A-resistant cell lines such as WI-38 and A549 expressed lower levels of MST/Krs proteins. Next, we examined whether the proteolytic activation of MST/Krs proteins by cytotrienin A is correlated with cytotrienin A sensitivity. Proteolytic activation and p36 MBP kinase activity were detected by an in-gel kinase assay using MBP as a substrate. As shown in Fig. 3 B, the activation of MST/Krs proteins by cytotrienin A was easily detected in the cytotrienin A-sensitive cell lines such as HL-60, U937, and Jurkat. However, no activation of MST/Krs proteins by cytotrienin A was detected in cytotrienin A-resistant cell lines such as WI-38, A549, A431, HeLa, MKN74, SH-SY5Y, and SMS-KCN. Although MKN74 cells expressed high levels of MST/Krs, p36 MBP kinase activation was not observed, and MKN74 cells showed low cytotrienin A sensitivity. The sole exception to this correlation is K562 cells. K562 cells are cytotrienin A-sensitive, but no activation of p36 MBP kinase was detected upon cytotrienin A treatment. K562 cells have the Philadelphia chromosome creating a Bcr-Abl fusion that is known to inhibit apoptosis induced by some antitumor agents (18.Amarante-Mendes G.P. McGahon A.J. Nishioka W.K. Afar D.EH Witte N.O. Green D.R. Oncogene. 1996; 16: 1383-1390Crossref Scopus (200) Google Scholar). It is possible that Bcr-Abl acts to inhibit the proteolytic activation of MST/Krs proteins in K562 cells but does not prevent cell death. We went on to examine the protein expression level of three other apoptosis-related molecules: caspase-3, Bcl-2, and Bax. HL-60, U937, SH-SY5Y, and SMC-KCN cells were found to express a high level of caspase-3, Bcl-2, and Bax, whereas K562, Jurkat, and MKN74 cells express a high level of caspase-3 with a low level of Bcl-2 or/and Bax. Taken together, we found a rough correlation between the sensitivity to cytotrienin A-induced apoptosis and expression of MST/Krs proteins but not the expression of caspase-3, Bcl-2, or Bax. We previously reported that activation of p36 MBP kinase required caspase activation via production of reactive oxygen species (ROS) (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). To investigate the role of caspase-3 activation and ROS production in cytotrienin A-sensitive and -resistant cells, we examined the effects of benzoyloxycarbonyl-Asp-CH2O(CO)-2,6-dichlorobenzene (Z-Asp-CH2-DCB), a synthetic inhibitor of caspases, orN-acetylcysteine, a radical scavenger, on the activation of p36 MBP kinase in cytotrienin A-sensitive (HL-60 and Jurkat cells) and -resistant cells (WI-38 cells). As expected the activation of p36 MBP kinase and the cleavage of full-length MST/Krs proteins induced by cytotrienin A were inhibited by Z-Asp-CH2-DCB orN-acetylcysteine (Fig. 4,A and B) in both HL-60 and Jurkat cells but not in WI-38 cells. Furthermore, to confirm the activation of caspase-3, we analyzed the proteolytic digestion of poly(ADP-ribose) polymerase (PARP), a known substrate of caspase-3. The 28-kDa proteolytic fragment of PARP was detected by immunoblotting with an anti-PARP antibody in response to cytotrienin A-treatment in HL-60 and Jurkat cells but not in WI-38 cells. This cleavage was also inhibited by Z-Asp-CH2-DCB or N-acetylcysteine (Fig.4 B). We previously reported that cytotrienin A also activates JNK and that this activation was dependent on the production of ROS but not on caspase activation (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). These results suggest that ROS play a key role in the caspase-mediated activation of p36 MBP kinase and in the activation of JNK during cytotrienin A-induced apoptosis. To demonstrate a role for the MST/Krs proteins in cytotrienin A-induced apoptosis, we constructed expression vectors for kinase-inactive MST2/Krs1 or MST1/Krs2. Following the method of Creasy et al. (19.Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), kinase-inactive forms of MST2/Krs1 and MST1/Krs2 were constructed by mutating Lys-56 of MST2/Krs1 or Lys-59 of MST1/Krs2 to Arg, disrupting ATP binding. We expected that kinase-negative MST/Krs proteins would compete with endogenous MST/Krs proteins for substrates and function as dominant negative forms of the proteins. When these kinase-inactive MST/Krs proteins were transiently expressed in HL-60 cells, the activation of p36 MBP kinase by cytotrienin A was almost completely inhibited (data not shown). We then went on to examine the effect of kinase-inactive MST/Krs proteins on the induction of apoptosis by cytotrienin A. As shown in Fig.5 A, DNA fragmentation was partially inhibited by the transient expression of either inactive MST2/Krs1 or MST1/Krs2. The kinase-inactive MST/Krs proteins were abundantly expressed, and almost completely inhibited activation of p36 MBP kinase, but these mutant proteins were unable to completely inhibit cytotrienin A-induced DNA fragmentation. This result showed that other MST/Krs-independent pathways were capable of mediating cytotrienin A-induced cell death. We previously reported that cytotrienin A induced the activation of JNK during apoptosis in HL-60 cells, a cytotrienin A-sensitive cell line (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). To confirm whether the JNK signal was also important for cytotrienin A-induced apoptosis, we examined the effect of expressing dominant negative c-Jun. Dominant negative c-Jun, lacking the transactivation domain, is capable of dimerization with members of the c-Jun and c-Fos family and binding to DNA. However, these dimers do not possess transactivation activity. We examined the apoptosis-induced DNA fragmentation in U937 cells stably expressing dominant negative c-Jun upon cytotrienin A treatment. As shown in Fig. 5 B, DNA fragmentation induced by cytotrienin A was partially inhibited by the expression of dominant negative c-Jun. We confirmed that the expression of dominant negative c-Jun had no effect on cytotrienin A-induced activation of p36 MBP kinase or JNK activity (Fig. 5, C andD). Furthermore, in order to examine the effects of coexpression of kinase-inactive MST/Krs proteins along with dominant negative c-Jun on cytotrienin A-induced apoptosis, we transiently expressed kinase-inactive MST/Krs proteins in U937 cells that are stably expressing dominant negative c-Jun. As shown in Fig.5 B, DNA fragmentation induced by cytotrienin A was almost completely inhibited by the coexpression of kinase-inactive MST/Krs proteins and dominant negative c-Jun. This treatment blocked cytotrienin A-induced activation of p36 MBP kinase, whereas cytotrienin A-induced activation of JNK was not inhibited (Fig. 5, C andD). Therefore, the kinase-inactive MST/Krs proteins could not inhibit cytotrienin A-induced JNK activation. These results suggest that cytotrienin A induces apoptosis by activating the MST/Krs proteins and JNK via two different signaling pathways. In our previous studies (11.Kakeya H. Zhang H.-P. Kobinata K. Onose R. Onozawa C. Kudo T. Osada H. J. Antibiot. (Tokyo). 1997; 50: 370-372Crossref PubMed Scopus (52) Google Scholar, 12.Zhang H.-P. Kakeya H. Osada H. Tetrahedron Lett. 1997; 38: 1789-1792Crossref Scopus (44) Google Scholar), we identified a novel anticancer drug, named cytotrienin A, which is an ansamycin with a unique 1-aminocyclopropane-1-carboxylic acid in the molecule. Moreover, cytotrienin A induces apoptosis in human leukemia HL-60 cells via activation of p36 MBP kinase, an active proteolytic fragment of MST/Krs proteins (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). The MST/Krs proteins are Ste20-related protein kinases and are classified in the germinal center kinase subfamily (20.Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (266) Google Scholar). The germinal center kinase subfamily of p21-activated protein kinase-like kinases, which play regulatory roles in diverse cellular phenomena such as morphogenesis, stress-response, and proliferation, comprises at least 9 distinct members (germinal center kinase, HPK1, NIK, MST1/Krs2, MST2/Krs1, KHS, SOK1, Sps1p, and Nrk1p) (20.Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (266) Google Scholar). Very little is known about the regulation and function of these kinases, including the MST/Krs proteins. All of the antitumor agents that were used in the present study induced the activation of the MST/Krs proteins and DNA fragmentation in HL-60 cells (Fig. 2). As MST/Krs proteins might be a general component of apoptosis induction by antitumor agents, we investigated the relationship between the expression and proteolytic activation of MST/Krs proteins in response to anticancer agents, including cytotrienin A, in various human tumor cell lines. In HL-60 and Jurkat cells, which are highly sensitive to cytotrienin A, robust expression and proteolytic activation of MST/Krs proteins by caspase-3 were observed (Table I and Figs. 3 and 4). In contrast, cytotrienin A-resistant cell lines such as WI-38, A549, A431, HeLa, SH-SY5Y, and SMS-KCN expressed a very low amount of MST/Krs proteins, and there was no detectable proteolytic activation of MST/Krs proteins (Table I and Figs. 3 and 4). These effects of cytotrienin A are not dependent on the doubling time of each tumor cell line (data not shown). Although MKN74 cells expressed high levels of MST2/Krs1 and showed resistance to cytotrienin A treatment, there was no detectable proteolytic activation of MST/Krs by caspases (Fig. 3 B). K562 cells are also cytotrienin A-sensitive, but no activation of p36 MBP kinase was induced by cytotrienin A-treatment. K562 cells possess a Philadelphia chromosome with a translocation between chromosomes 9 and 22 that generates the Bcr-Abl tyrosine kinase by the fusion of Bcr sequences upstream of the second exon of c-Abl. Bcr-Abl is known to inhibit chemotherapeutic drug-induced apoptosis (18.Amarante-Mendes G.P. McGahon A.J. Nishioka W.K. Afar D.EH Witte N.O. Green D.R. Oncogene. 1996; 16: 1383-1390Crossref Scopus (200) Google Scholar). Considering this, it may be possible that Bcr-Abl acts as an inhibitor of the signal that activates MST/Krs proteins and that there is a different signal for cytotrienin A-induced apoptosis not affected by Bcr-Abl. These results suggest that the proteolytic activation of MST/Krs proteins rather than expression level might correlate with apoptosis. There appears to be no correlation between the expression level of crucial components of the apoptotic program such as caspase-3, Bcl-2, and Bax (1.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4577) Google Scholar, 21.Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1392) Google Scholar, 22.Chresta C.M. Masters J.R.W. Hickman J.A. Cancer Res. 1996; 56: 1834-1841PubMed Google Scholar) and the cytotrienin A sensitivity of the tested human tumor cell lines. To date, we have only examined the expression and activation of MST/Krs proteins in a limited number of cell lines in response to a small number of antitumor agents. A more systematic analysis of the expression and the activation of MST/Krs proteins should be helpful in studies aimed at finding new antitumor agents and testing the sensitivity of antitumor agents in human tumor cells. MST/Krs proteins consist of two family members, MST2/Krs1 and MST1/Krs2. Both proteins undergo caspase-mediated cleavage during apoptosis. MST2/Krs1 and MST/Krs2 possess consensus recognition sites for caspase-3, 319DELD322S and323DEMD326S, respectively (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar, 14.Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (328) Google Scholar, 15.Lee K.-K. Murakawa M. Nishida E. Tsubuki S. Kawashima S.-I. Sakamaki K. Yonehara S. Oncogene. 1998; 16: 3029-3037Crossref PubMed Scopus (120) Google Scholar, 17.Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar, 19.Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar,23.Creasy C.L. Chernoff J. J. Biol. Chem. 1995; 270: 21695-21700Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 24.Taylor L.K. Wang H.-C.R. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10099-10104Crossref PubMed Scopus (141) Google Scholar, 25.Creasy C.L. Chernoff J. Gene (Amst.). 1995; 167: 303-306Crossref PubMed Scopus (119) Google Scholar). As shown in Fig. 4, A and B, a synthetic inhibitor of caspase-3, Z-Asp-CH2-DCB, blocked proteolytic activation of both MST2/Krs1 and MST1/Krs2 proteins in cytotrienin A-sensitive human tumor cell lines, indicating that caspase-3 activation is necessary for the activation of p36 MBP kinase. In addition, we have shown that in both HL-60 and Jurkat cells, ROS induced by treatment with cytotrienin A play an important role in the caspase-mediated activation of p36 MBP kinase (Fig. 4, A andB). These results indicate that MST/Krs proteins are responsive to oxidative stress. Apoptosis induced by MT-21, a synthetic apoptosis inducer, is also blocked by N-acetylcysteine, a radical scavenger (17.Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar), supporting our present studies. SOK-1, another germinal center kinase family member, is directly activated via autophosphorylation triggered by ROS, but the kinase is not activated by growth factors, alkylating agents, cytokines, or environmental stresses such as heat shock and osmotic stress (26.Pombo C.M. Bonventre J.V. Molnar A. Kyriakis J. Force T. EMBO J. 1996; 15: 4537-4546Crossref PubMed Scopus (137) Google Scholar). Although MST/Krs proteins are also activated by ROS, the mechanism of activation of MST/Krs proteins is apparently different from that of SOK-1. Therefore, MST/Krs activation may be an important event in response to oxidative stress in human tumor cell lines. To demonstrate a role for MST/Krs in cytotrienin A-induced apoptosis, we constructed expression vectors for kinase-inactive MST/Krs proteins by mutating a conserved Lys residue in an ATP binding pocket as Creasyet al. (19.Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) reported previously. We expected that the overexpression of kinase-inactive MST/Krs proteins could compete with endogenous MST/Krs proteins and function in a dominant negative fashion. As a result, the overexpression of kinase-inactive MST/Krs proteins may inhibit cytotrienin A-induced apoptosis. Indeed, the overexpression of the kinase-inactive MST/Krs proteins partially suppressed cytotrienin A-induced apoptosis (Fig. 5 A). On the other hand, the overexpression of the dominant negative c-Jun also partially suppressed cytotrienin A-induced apoptosis (Fig.5 B). We have previously shown that overexpression of kinase-inactive MST/Krs proteins inhibited apoptosis induced by treatment with MT-21 and that MST/Krs proteins also act upstream of JNK during MT-21-induced apoptosis (17.Watabe M. Kakeya H. Osada H. Oncogene. 1999; 18: 5211-5220Crossref PubMed Scopus (48) Google Scholar). If both MST/Krs and JNK were located on the same signal cascade during cytotrienin A-induced apoptosis, the cotransfection of kinase-inactive MST/Krs and dominant negative c-Jun would also show strong inhibition of DNA fragmentation (Fig. 5 B). However, this linear model is contradicted by the following data: the activation of MST/Krs (p36) by cytotrienin A treatment was almost completely suppressed by the overexpression of kinase-inactive MST/Krs (Fig. 5 C), but the activation of JNK was not suppressed by the overexpression of kinase-inactive MST/Krs (Fig. 5 D). In addition, we previously reported that a caspase inhibitor, Z-Asp, suppressed MST/Krs activation but not JNK activation (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). Therefore, it is more likely that JNK activation by cytotrienin A is independent of the caspase-MST/Krs signal and that two different signals from MST/Krs and JNK are required for a full induction of apoptosis by cytotrienin A. We previously reported that radical scavengers such as N-acetylcysteine and reduced glutathione suppressed cytotrienin A-induced activation of JNK and MST/Krs induced by cytotrienin A (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar). Therefore, two different signaling pathways may be divided downstream of ROS and upstream of the caspase cascade. Considering these results, both the activation of JNK and the caspase-mediated cleavage of MST/Krs proteins appear to be important for the induction of cell death by antitumor agents (Fig.6). This schematic model incorporates the previous reports that MST/Krs proteins function upstream of JNK (13.Kakeya H. Onose R. Osada H. Cancer Res. 1998; 58: 4888-4894PubMed Google Scholar,14.Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (328) Google Scholar). It suggests that there are several signaling pathways for JNK activation in response to stimulants. We are grateful to Dr. K. Nakaya for the dominant negative c-Jun expressing U937 cells and to Eric C. Griffith for critical reading of the manuscript.