- Email: [email protected]
Nemo-like kinase induces apoptosis in DLD-1 human colon cancer cells
BBRC Biochemical and Biophysical Research Communications 308 (2003) 227–233 www.elsevier.com/locate/ybbrc
Nemo-like kinase induces apoptosis in DLD-1 human colon cancer cells Jun Yasuda,a,* Akira Tsuchiya,b Tesshi Yamada,b Michiie Sakamoto,b,1 Takao Sekiya,c and Setsuo Hirohashib a
Cancer Transcriptome Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan b Pathology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan c Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-1252, Japan Received 18 June 2003
Abstract Deregulation of Wnt/b-catenin signaling is thought to play a critical role in human carcinogenesis. Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase that suppresses b-catenin/T-cell factor (TCF) complex transcriptional activity through phosphorylation of TCF. Since NLK may be a tumor suppressor as a negative regulator of Wnt/b-catenin pathway, we established tetracycline-inducible NLK and its kinase-negative mutant expression in DLD-1 human colon cancer cells to analyze the effect of NLK on cell growth and viability. The induction of wild-type NLK in DLD-1 cells caused suppression of cell growth whereas the kinase-negative mutant did not. Flow cytometry indicated that NLK expression increased the number of apoptotic cells but did not induce obvious cell cycle arrest. Apoptosis induction by wild-type NLK was confirmed using TUNEL assays. Our results suggest that overexpression of NLK may have targets other than TCF for induction of apoptosis in human colon carcinoma cells. Ó 2003 Elsevier Inc. All rights reserved. Keywords: NLK; b-Catenin; TCF; Colorectal carcinoma; Apoptosis
Many of the genes mutated in tumor cells play pivotal roles in the regulation of cell differentiation during embryogenesis [1–4]. Wnt/Wingless signaling is an evolutionarily conserved developmental signal transduction pathway and is known to be involved in human carcinogenesis [5,6]. Somatic genetic alterations in most of the components of Wnt/Wingless pathway, such as APC, b-catenin, and Axin, have been found in many human cancers [1,7]. This signaling pathway is thought to suppress degradation of the b-catenin protein, an essential component of cell–cell junctions in epithelial tissues. Stabilized b-catenin protein accumulates in the cell nucleus and forms complex with and activates TCF/ LEF transcription factors. Studies of transgenic mice also indicated that overexpression of b-catenin in intestinal epithelium caused intestinal tumorigenesis [8,9]. *
Corresponding author. Fax: +81-3-3542-0688. E-mail address: [email protected] (J. Yasuda). 1 Present address: Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01343-3
Among the target genes of the b-catenin/TCF complex, several cell growth related genes have been identified (reviewed in [10]) including cyclin D1 [11,12], c-myc [13], MDR1 [14], and PPAR-d [15]. Nemo-like kinase (NLK) was originally identified by the screening of cDNAs coding for protein kinases [16] and was recently characterized as a suppressor of the Wnt signaling pathway in mammalian cells [17–19]. NLK is an evolutionarily conserved serine/threonine kinase and belongs to the proline directed protein kinase superfamily which consists of mitogen activated protein kinases (MAPKs) and cyclin-dependent protein kinases (CDKs) [16]. A member of the MAPK kinase kinase superfamily, TAK1, is identified as a potential activator for NLK [17,20,21]. A recent study showed that Wnt5aCa2þ signaling, which is known as a negative regulator of Wnt [22], can be an upstream activator of TAK1NLK for suppression of canonical Wnt/b-catenin signaling in Xenopus system [18]. Genetic studies of the homologs of NLK in model organisms show that the kinase plays a suppressive role in Wnt/b-catenin signaling. A mutant allele of Nmo, a Drosophila homolog
228
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
of NLK, was originally found to cause a defect of epithelial planar polarity in the fly’s eyes [23]. Another group independently identified a more severe Nmo mutant allele by the screening of Notch modifier mutants and the Nmo mutant restored the wing phenotype of the overexpression of fruit fly b-catenin, Armadillo, indicating that Nmo acts as a suppressor of Wnt signaling in fly wing development [24]. The Caenorhabditis elegans NLK homolog, LIT-1, has been identified as a critical regulator of embryonic cell fate determination accompanying anterior–posterior cell division [25]. In the development of C. elegans, Wnt signaling pathway is also known to determine cell polarity and differentiation [26,27]. Genetic and biochemical analyses showed that LIT-1 could phosphorylate a worm TCF homolog, POP-1, and regulate its subcellular distribution [20,25,28]. In mammals, NLK can suppress transcriptional activity of the b-catenin/T-cell factor (TCF) complex through phosphorylation of TCF in vivo and in vitro [17]. Therefore, NLK/Nmo/LIT-1 works as a downstream regulator of Wnt/Wingless signaling in a variety of organisms. Based on the evolutionary conservation of the function of NLK in Wnt/wingless signaling, it seems very likely that NLK suppresses cell growth stimulated by deregulated b-catenin/TCF complex transcriptional activity in human carcinoma cells. In a recent study, inhibition of the b-catenin/TCF complex by dominant-negative TCF caused suppression of cell growth and G1 arrest of human colorectal carcinoma cell lines including DLD-1 [29]. Similarly, our previous study showed that induction of dominant-negative TCF in DLD-1 cells decreased colony growth rate in soft agar [30]. Based on these studies, it is obvious that transcriptional activity of the b-catenin/ TCF complex is critical to the cell growth of colon cancer cell lines including DLD-1. Therefore, we hypothesized that overexpression of NLK may induce growth arrest in DLD-1 cells mimicking the effect of dominant-negative TCF. However, it is not known whether NLK actually affects cell growth and viability. To address the above issue, we established tetracycline-inducible NLK and its kinase-negative mutants in the DLD-1 cell line. We found that wild-type NLK suppressed cell growth and anchorage independence of the DLD-1 cells. Flow cytometry indicated that NLK expression increased apoptotic cell rate but did not induce cell cycle arrest. Moreover, induction of NLK in these cells increased the rate of apoptotic cells in TUNEL assay.
Materials and methods Plasmids. The plasmids pCMV-Flag-NLK and pCMV-Flag-NLK K155M were kindly provided by Dr. Kunihiro Matsumoto. These plasmids were used as templates of PCR to construct the following expression vector. The plasmids pTRE2PURO-HA-NLK and its
kinase-negative variant pTRE2PURO-HA-NLK K155M were constructed by the insertion of PCR-derived cDNA fragments of NLK in the BamHI and NotI sites of the pTRE2PURO vector (Clontech). pGEXhLEF-1 was constructed by cloning of the RT-PCR product of human lymphoid enhancer factor-1 (LEF-1) cDNA [31]. The pGEXhLEF-1 was introduced in the BL21 competent cells (Stratagene) and the GST-LEF-1 fusion proteins were induced following standard protocols. pRL-tk was purchased from Promega. pTOPFLASH was purchased from Upstate biosystems. The detailed construction strategy of the plasmids is available upon request. Cell culture, transfection, and luciferase assays. DLD-1 tet-on cells [30] were cultured with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1 mg/ml of penicillin/streptomycin (Invitrogen), and 0.4 mg/ml G-418 (Invitrogen) in 10 cm dishes. Seventy per cent confluent cells were subjected to transfection with Lipofectamine 2000 following the manufacturer’s instructions with modifications. pTRE2 Puro HA-NLK and the corresponding mutant plasmids were stably transfected in the presence of 2 lg/ml of puromycin and 0.4 mg/ml G-418. After 2 weeks of selection with G-418 and puromycin, independent clones were isolated by the limiting dilution method or single colony pickup with micropipette tips under a phase contrast microscope. For transient transfection studies, cells were cultured with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 mg/ml of penicillin/streptomycin (Invitrogen) in 10 cm dishes. Cells at 80% confluence were subjected to transfection with Lipofectamine 2000 following the manufacturer’s instructions with modifications. The Dual-luciferase assay system (Promega) was used for analysis of luciferase activity. As much as 1.2 ng of pRL-tk and 0.1 lg of luciferase reporter plasmids were mixed with Lipofectamine 2000 and added to a well of a 24-well plate. After 24 h of transfection, the cells were lysed with passive lysis buffer (Promega) following the manufacturer’s instructions. The induction of NLKs was done 24 h prior to the transfection of reporter genes. The experiments were performed at least twice in triplicate. Luminescence was assayed with a Lumat LB9507 luminomator (EG&G Berthold). The firefly luciferase activity was adjusted with Renilla luciferase activity for each sample. Western blotting and immunoprecipitation-protein kinase assay. Antibodies to Hemagglutinin (HA) epitope tag (mouse: 12CA5, rat: 3F10, Roche), b-catenin (C19220: Transduction Laboratories), and atubulin (Amersham) were used in this study. Cells in 10 cm dishes were lysed in 800 ll of Triton X-lysis buffer for in vitro kinase assays [32]. The Triton X-lysis buffer consisted of 137 mM NaCl, 20 mM Tris–Cl (pH 7.4), 1% of Triton X-100, 25 mM b-glycerophosphate, 2 mM EDTA (pH 8.0), 2 mM sodium pyrophosphate, and 10% glycerol, supplemented with 0.1 mM sodium orthovanadate and complete protease inhibitor (Roche). The lysate was centrifuged at 15,000g for 15 min at 4 °C and the supernatant was subjected to further analysis. Western blotting was performed following the instructions provided by the antibody suppliers. The lysate was subjected to SDS– PAGE and the proteins were transferred onto Immobilon membrane (Millipore) by the use of an electroblotting apparatus (Bio-Rad Transblot semi-dry electrotransfer cell). Immune complexes were detected by enhanced chemiluminescence (Perkin–Elmer). The in vitro kinase reaction was performed as described previously [32]. Briefly, immunoprecipitation was performed with 1 lg of mouse monoclonal antibody for HA tag (12CA5) bound to Dynabeads protein G (Dynal). The lysate with 24 h induction with doxycyclin was mixed with the antibody bound to beads and incubated for 12–16 h at 4 °C with rotation. The immune complex bound to the magnetic beads was washed four times with the same lysis buffer and subjected to the kinase reaction. One microgram of GST-LEF1 fusion protein was added to the immune complex with 20 ll of kinase reaction buffer (25 mM Hepes, pH 7.4, 25 mM b-glycerophosphate, 25 mM MgCl2 , 2 mM DTT, 1 lCi [c-32 P]ATP, 50 lM ATP, and 100 lM sodium orthovanadate). The reaction was performed at 30 °C for 10 min. The reaction was stopped by the addition of SDS–PAGE loading buffer
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233 and the samples were subjected to SDS–PAGE analysis. The phosphorylated proteins were visualized by autoradiography. Cell growth and colony formation assay. Cell growth was estimated with the use of Premix WST-1 cell proliferation assay system (TAKARA), which analyzes succinate-tetrazolium reductase activity. The number of viable cells can be analyzed by measuring the optical density (OD) at 455 nm of the reaction samples as there is a linear correlation between OD and the number of viable cells [33]. The cells were trypsinized and plated at 1.8 104 cells/well in the 24-well plates. The next day, the medium was replaced with the fresh DMEM with 10% FBS with or without 1 lg/ml of doxycyclin for 1–7 days. During the cell culturing, the medium was replaced with fresh one every other day. At the analysis, the medium was replaced with fresh DMEM with 10% FBS with or without 1 lg/ml of doxycyclin containing 3% of WST reagent and the cells were incubated at 37 °C for 1 h with 5% of CO2 for succinate-tetrazolium reductase reaction. The OD of the samples was analyzed with Bio-Rad Model 3550 UV microplate reader at 455 nm. After 7 days of cell culturing and analysis, the cells were lysed and subjected to immunoblotting analysis to determine the expression of the transgene. The amount of proteins loaded on the SDS–PAGE was adjusted as 5 lg/lane. Soft-agar colony formation assay was performed as described [30]. In brief, 1 ml of 0.6% agarose in culture medium was placed on the bottom of the wells in 6-well plates and trypsinized cells (1.5 104 / well) were inoculated in 0.3% agarose in culture medium. The wells were covered with 1 ml of culture medium. The cells were incubated with or without doxycyclin for 3 weeks at 37 °C. The colonies were stained with 0.05% crystal violet for visualization. Flow cytometry. DLD-1 tet-on cell lines (3 105 cells/dish at the beginning) were incubated after 4 days with or without doxycyclin. The floating cells were collected by centrifugation and the adherent cells were collected after trypsinization with 0.25% Trypsin/EDTA solution (Invitrogen). The cell pellets from the floating and the adherent cells were mixed and fixed in 70% ethanol at 4 °C overnight. Fixed cells were incubated with PBS containing 1 mg/ml RNase A at 37 °C for 5 min and stained with 10 lg/ml propidium iodide (Sigma) at room temperature for 20 min. The stained samples were analyzed with a Becton Dickinson FACScalibur flow cytometer using CellQuest software (Becton Dickinson). Each sample was counted up to 20,000 events for analysis in duplicate three times. TUNEL assays. For the detection of apoptotic cells, TUNEL assays were performed with an in situ cell death detection kit (Roche) following the manufacturer’s instructions. The cells were incubated with or without doxycyclin for 5 days on L -polylysine coated glass coverslips (Iwaki, Japan). The cells were then washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 min, and placed in 100% of methanol at )20 °C for 10 min. The nuclei were visualized with DAPI in Vectashield (Vector). The Axiovert fluorescence microscopy system (Zeiss) was used for the generation of fluorescent images. The TUNEL positive and negative cells were counted manually with the aid of Metaphor software (Universal Imaging). An area of DAPI staining of half the average or larger in size was counted as a cell. Apparent mitotic cells showing chromatids were not counted.
Results and discussion NLK induction causes cell growth suppression in DLD-1 cells Fig. 1A shows schematic diagrams of the structures of HA-NLK and its kinase-negative mutants used in this study. A recent study indicated that wild-type NLK may have a shorter isoform, which lacks 71 amino acid residues in N-terminal compared with the conventional form
229
Fig. 1. Induction of NLK with doxycyclin in DLD-1 tet-on cells. (A) Schematic diagrams of the NLK constructs in this study. The Nterminal single-amino acid stretches are shown. The methionine codon for candidate alternative translation initiation for shorter form (see text) is located at just downstream of the alanine stretch (72nd amino acid). (B) Effect of induced NLK on b-catenin/TCF complex transcriptional activity in DLD-1 cells. The vertical axis shows the relative value of normalized fire fly luciferase activity in the cell lysates. The luciferase activity of the cell lysate without doxycyclin used as 100% for each cell line. The horizontal axis shows the cell line and doxycyclin status in the media indicated with + (added) or ) (not added). Error bars indicate the standard error of measurement (SEM). (C) In vitro kinase assay of HA-NLKs induced in DLD-1 cells. Upper panel is an autoradiogram of phosphoproteins in a SDS–PAGE gel. Lower panel is the Western blotting with anti-HA antibody to indicate the amount of proteins loaded in the immunoprecipitation reaction.
[34]. The missing part includes stretches of single-amino acids consisting of histidines and alanines shown in Fig. 1A. Despite the fact that even the histidine stretches in the N-terminal sequences are conserved between fish and mammals [35], it may be possible that the shorter isoform has different functions from the longer, wellcharacterized, isoform which is used in the present study. Four independent clones of the DLD-1 tet-on HANLK cell line were established, two clones for wild-type and two clones for kinase-negative mutants HA-NLK K155M. We performed two distinct assays to confirm whether the induced kinases would work as theoretically expected: suppression of the b-catenin/TCF complex and phosphorylation of TCF/LEF (Figs. 1B and C, respectively). A luciferase assay was performed to test the former using a reporter plasmid (pTOPFLASH [36]) and an in vitro kinase assay was done with GST-LEF1 fusion protein [18] to examine the latter. Induction of wild-type NLK suppressed the reporter gene expression from pTOPFLASH in the DLD-1 cells while mutant
230
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
NLK induction did not cause suppression of the reporter gene expression (Fig. 1B). Immunoprecipitationprotein kinase assay indicated that the wild-type NLK induced in DLD-1 cells could phosphorylate recombinant GST-LEF1 fusion proteins and itself (Fig. 1C). These results were compatible with previous observations [17–19]. As shown here and previous studies, NLK is constitutively active when overexpressed in cultured mammalian cells [16,17]. The WST assay revealed that cell growth was suppressed 3 days after the induction of wild-type NLK by doxycyclin (Fig. 2A). Without induction of the transgenes by doxycyclin, the wild-type NLK cells grew as fast as the parental cells (Fig. 2A). Since the NLK kinase-negative transformants and the parental cells showed similar growth potential regardless of the presence or absence of doxycyclin in the culture medium (Figs. 2A and B), the toxicity of doxycyclin on DLD-1 NLK cells seemed to be negligible. Growth suppression by the induction of NLK was also confirmed by the softagar colony formation assay (Figs. 2C and D).
NLK could induce apoptotic cell death in DLD-1 cells The cause of growth suppression of the DLD-1 cells by wild-type NLK could be either cell cycle arrest or increased rate of apoptosis or both. To examine the cell cycle state and apoptotic cell death in these cell lines, we analyzed the DNA content of the DLD-1 NLK (and its mutant) cells with flow cytometry. Flow cytometric analysis indicated that the ratio of apoptotic cells, measured as sub-G1 populations, was about threefold higher in DLD-1 HA-NLK cells with induction (19.4%) compared with those without induction (6.71%) and 4to 5-fold higher than in DLD-1 HA-NLK KM cells with or without induction (3.28% and 4.10%, respectively). It was also obvious that the relative populations of cells in other phases of the cell cycle were not changed so dramatically (Fig. 3B), even though the G2/M phase cells decreased more than cells in other phases in wild-type NLK-induced cells (Fig. 3B). As mentioned earlier, a recent study by van de Wetering et al. [29] showed that p53 independent induction of p21/WAF1 was strongly
Fig. 2. Growth suppression of DLD-1 tet-on cells by induction of wild-type NLK. (A) WST assay of the DLD-1 NLK cell lines: the name of the clones is indicated in each panel. Open and closed symbols indicate the cell lines without doxycyclin and with doxycyclin, respectively. The graphs show the average of optical density (OD) of the samples over the period of the experiments. The bars show the standard error of measurement (SEM). (B) Clonal variation of the cell growth estimated with the WST assay system. The vertical axis of the graph indicates the average OD at 455 nm with WST reagent of the samples on the seventh day of culturing with or without doxycyclin. Open and closed bars indicate the average OD of the WST reagent for the cell lines without doxycyclin and with doxycyclin, respectively. The Western blots of HA-NLK and a-tubulin of the corresponding cells are shown below the graph. (C) Colony formation assay of DLD-1 tet-on NLK clone 6 with or without doxycyclin. Representative well for each is shown. The cells (1.0 103 cells/well in 6-well plate) were incubated for 3 weeks with or without doxycyclin in the medium in soft agar. (D) The graph indicates the average numbers of colonies in three wells with SEM.
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
231
Fig. 3. Flow cytometric analyses of DLD-1 tet-on NLK cell lines. (A) Histograms of flow cytometrical analysis of DNA contents of DLD-1 tet-on NLK cell lines with (right) or without (left) doxycyclin. The numbers in the panels indicate the percentage of cells with a sub-G1 DNA content. (B) State of cell cycle of DLD-1 tet-on NLK cell lines. The percentage was based on the number of events counted as G0/G1, S, and G2/M but not sub-G1.
correlated with cell cycle arrest of DLD-1 cells by induced dominant-negative TCF. We analyzed the p21/ WAF1 expression by Western blot but did not observe the change of expression level of p21 by NLK in DLD-1 cells (data not shown). These two findings, lack of cell cycle arrest and lack of p21 induction in wild-type NLKinduced DLD-1 cells, are compatible both with each other and with the study by van de Wetering et al. [29]. Nuclear condensation and fragmentation, typical characteristics for apoptotic cells, were observed in numerous floating cells stained with DAPI after 7 days of induction of wild-type NLK in DLD-1 cells (data not shown). We performed TUNEL assays to confirm the induction of apoptosis in wild-type NLK-induced DLD1 cells (Figs. 4A and B). The induction of NLK caused
increased numbers of TUNEL positive cells in DLD-1 cells. Fluorescent microscopy revealed that substantial numbers of the cells were positive in TUNEL assays (labeled with FITC) and many of the TUNEL positive cells exhibited nuclear fragmentation (Fig. 4A). The ratio of TUNEL positive cells in the population increased 3.7-fold in the NLK-induced DLD-1 cells (Fig. 4B). The loss of anchorage independence by the induction of wild-type NLK shown in Figs. 2C and D might be related to this increase of apoptosis tendency. Suppression of the b-catenin/TCF complex by induction of dominant-negative TCF in DLD-1 cells resulted in growth suppression of varying extent without apoptosis [29,30]. The difference between previous studies and the present study suggests that NLK may
Fig. 4. Induction of NLK causes apoptosis of the DLD-1 tet-on cell lines. (A) The TUNEL assays were performed with in situ cell death detection kit and the TUNEL positive cells were visualized with FITC. Arrowhead indicates the TUNEL positive cells and those cells showing nuclear condensation and fragmentation. (B) Percentage of TUNEL positive cells. The DLD-1 tet-on NLK clone 6 cells were counted based on DAPI staining in 200 visual field. The average percentage of TUNEL positive cells in visual fields was indicated with SEM. Total counted doxycyclin free cells, n ¼ 1083 and doxycyclin positive cells, n ¼ 903.
232
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
Fig. 5. Induction of wild-type or mutant NLK does not affect the amount of b-catenin in DLD-1 cells. The samples are the same as in Fig. 2B. Western blotting with anti-a-tubulin antibody was done for control of loading.
have functions other than the suppression of the bcatenin/TCF complex. Alternatively, the difference in the mode of suppression of the b-catenin/TCF complex may explain the difference of outcome between dominant-negative TCF and NLK. It has been suggested that NLK could regulate the affinity of the b-catenin/TCF complex to the target DNA sequences [17]. If the phosphorylated TCF by NLK specifically lost its affinity to target DNA sequences for anti-apoptotic signals, the NLK-overexpressed cells would not show cell cycle arrest but commit apoptosis. We speculated that degradation of b-catenin might cause apoptosis [37] in DLD-1 cells with wild-type NLK induction. Western blotting analysis of b-catenin revealed that this was not the case. We induced NLK and its mutant expression in the DLD-1 tet-on NLK lines and found that the amount of b-catenin was not reduced in the presence or absence of induction of NLK and its mutants (Fig. 5). We concluded that the apoptosis of DLD-1 cells induced by NLK was not related to degradation of b-catenin. Mechanism of apoptosis induction by NLK is still elusive The present study showed that induction of wild-type NLK in a human colorectal carcinoma cell line (DLD-1) caused suppression of cell growth, loss of anchorage independence, and induction of apoptosis in a kinasedependent manner. Notably, this apoptosis induction is p53 independent because both alleles of the p53 gene were inactivated in DLD-1 cells [38]. Given the fact that most of the Wnt signaling mediators play roles in carcinogenesis, NLK may also be involved in human carcinogenesis caused by the deregulation of Wnt/b-catenin signaling. Our results showed that NLK affects cell growth by stimulating p53 independent apoptosis pathway as well as suppressing the b-catenin/TCF complex, which mediates a strong tumorigenic signaling pathway. Since reduction of apoptosis is one of the critical traits in tumorigenesis [39], NLK may be a potential tumor suppressor because
of its ability to induce apoptosis. So far, somatic genetic alterations of the NLK gene have not, however, been observed in human colon and breast cancers [40]. This may mean that the NLK gene is not an “authentic” tumor suppressor gene as such. Further investigation is required into the relationship between NLK expression and carcinogenesis. In terms of the mechanism of apoptosis induction by NLK, there is a possibility that NLK may suppress b-catenin/TCF complex and consequential survival signaling transduced by the complex. Several studies show that the stabilized b-catenin is involved in anti-apoptotic signaling in human cells [37,41,42]. The present study is consistent with these observations as NLK is a suppressor of Wnt/b-catenin signaling. Alternatively, it is possible that TCF/LEF-1 may not be the sole target of NLK for apoptosis induction. Studies of model organisms suggest that NLK and its homologs might have regulatory functions in other signaling pathways [24,43,44]. The search for other downstream targets of NLK may provide clues that will further clarify the role of NLK in apoptosis.
Acknowledgments We thank Hitoshi Ichikawa, Issay Kitabayashi, and Panayiotis Loukopoulos for their critical comments on the manuscript, our colleagues for providing essential reagents, and Kunihiro Matsumoto for the NLK plasmids. This work was supported in part by a Grant-in Aid for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan and a Grantin-Aid from the Ministry of Education, Sciences, Sports and Culture of Japan.
References [1] M. Peifer, P. Polakis, Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus, Science 287 (2000) 1606–1609. [2] M.M. Shen, A.F. Schier, The EGF-CFC gene family in vertebrate development, Trends Genet. 16 (2000) 303–309. [3] R. Derynck, R.J. Akhurst, A. Balmain, TGF-beta signaling in tumor suppression and cancer progression, Nat. Genet. 29 (2001) 117–129. [4] J. Taipale, P.A. Beachy, The Hedgehog and Wnt signalling pathways in cancer, Nature 411 (2001) 349–354. [5] M. Bienz, H. Clevers, Linking colorectal cancer to Wnt signaling, Cell 103 (2000) 311–320. [6] N.S. Fearnhead, M.P. Britton, W.F. Bodmer, The ABC of APC, Hum. Mol. Genet. 10 (2001) 721–733. [7] P. Polakis, The oncogenic activation of beta-catenin, Curr. Opin. Genet. Dev. 9 (1999) 15–21. [8] N. Harada, Y. Tamai, T. Ishikawa, B. Sauer, K. Takaku, M. Oshima, M.M. Taketo, Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene, Embo J. 18 (1999) 5931–5942. [9] B. Romagnolo, D. Berrebi, S. Saadi-Keddoucci, A. Porteu, A.L. Pichard, M. Peuchmaur, A. Vandewalle, A. Kahn, C. Perret, Intestinal dysplasia and adenoma in transgenic mice after over-
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
[10] [11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
expression of an activated beta-catenin, Cancer Res. 59 (1999) 3875–3879. R. Nusse, WNT targets. Repression and activation, Trends Genet. 15 (1999) 1–3. M. Shtutman, J. Zhurinsky, I. Simcha, C. Albanese, M. D’Amico, R. Pestell, A. Ben-Ze’ev, The cyclin D1 gene is a target of the betacatenin/LEF-1 pathway, Proc. Natl. Acad. Sci. USA 96 (1999) 5522–5527. O. Tetsu, F. McCormick, Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells, Nature 398 (1999) 422–426. T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin, B. Vogelstein, K.W. Kinzler, Identification of c-MYC as a target of the APC pathway, Science 281 (1998) 1509– 1512. T. Yamada, A.S. Takaoka, Y. Naishiro, R. Hayashi, K. Maruyama, C. Maesawa, A. Ochiai, S. Hirohashi, Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis, Cancer Res. 60 (2000) 4761–4766. T.C. He, T.A. Chan, B. Vogelstein, K.W. Kinzler, PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs, Cell 99 (1999) 335–345. B.K. Brott, B.A. Pinsky, R.L. Erikson, Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus, Proc. Natl. Acad. Sci. USA 95 (1998) 963–968. T. Ishitani, J. Ninomiya-Tsuji, S. Nagai, M. Nishita, M. Meneghini, N. Barker, M. Waterman, B. Bowerman, H. Clevers, H. Shibuya, K. Matsumoto, The TAK1-NLK-MAPK-related pathway antagonizes signalling between beta-catenin and transcription factor TCF, Nature 399 (1999) 798–802. T. Ishitani, S. Kishida, J. Hyodo-Miura, N. Ueno, J. Yasuda, M. Waterman, H. Shibuya, R.T. Moon, J. Ninomiya-Tsuji, K. Matsumoto, The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling, Mol. Cell Biol. 23 (2003) 131–139. T. Ishitani, J. Ninomiya-Tsuji, K. Matsumoto, Regulation of lymphoid enhancer factor 1/T-cell factor by mitogen-activated protein kinase-related Nemo-like kinase-dependent phosphorylation in Wnt/beta-catenin signaling, Mol. Cell. Biol. 23 (2003) 1379–1389. M.D. Meneghini, T. Ishitani, J.C. Carter, N. Hisamoto, J. Ninomiya-Tsuji, C.J. Thorpe, D.R. Hamill, K. Matsumoto, B. Bowerman, MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans, Nature 399 (1999) 793–797. T.H. Shin, J. Yasuda, C.E. Rocheleau, R. Lin, M. Soto, Y. Bei, R.J. Davis, C.C. Mello, MOM-4, a MAP kinase kinase kinaserelated protein, activates WRM-1/LIT-1 kinase to transduce anterior/posterior polarity signals in C. elegans, Mol. Cell 4 (1999) 275–280. M. Kuhl, L.C. Sheldahl, M. Park, J.R. Miller, R.T. Moon, The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape, Trends Genet. 16 (2000) 279–283. K.W. Choi, S. Benzer, Rotation of photoreceptor clusters in the developing Drosophila eye requires the nemo gene, Cell 78 (1994) 125–136. E.M. Verheyen, I. Mirkovic, S.J. MacLean, C. Langmann, B.C. Andrews, C. MacKinnon, The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development, Mech. Dev. 101 (2001) 119–132. T. Kaletta, H. Schnabel, R. Schnabel, Binary specification of the embryonic lineage in Caenorhabditis elegans, Nature 390 (1997) 294–298. C.E. Rocheleau, W.D. Downs, R. Lin, C. Wittmann, Y. Bei, Y.H. Cha, M. Ali, J.R. Priess, C.C. Mello, Wnt signaling and an APCrelated gene specify endoderm in early C. elegans embryos, Cell 90 (1997) 707–716.
233
[27] C.J. Thorpe, A. Schlesinger, J.C. Carter, B. Bowerman, Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm, Cell 90 (1997) 695–705. [28] C.E. Rocheleau, J. Yasuda, T.H. Shin, R. Lin, H. Sawa, H. Okano, J.R. Priess, R.J. Davis, C.C. Mello, WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans, Cell 97 (1999) 717–726. [29] M. van de Wetering, E. Sancho, C. Verweij, W. de Lau, I. Oving, A. Hurlstone, K. van der Horn, E. Batlle, D. Coudreuse, A.P. Haramis, M. Tjon-Pon-Fong, P. Moerer, M. van den Born, G. Soete, S. Pals, M. Eilers, R. Medema, H. Clevers, The betacatenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells, Cell 111 (2002) 241–250. [30] Y. Naishiro, T. Yamada, A.S. Takaoka, R. Hayashi, F. Hasegawa, K. Imai, S. Hirohashi, Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of beta-catenin/Tcell factor 4-mediated gene transactivation, Cancer Res. 61 (2001) 2751–2758. [31] K. Hovanes, T.W. Li, M.L. Waterman, The human LEF-1 gene contains a promoter preferentially active in lymphocytes and encodes multiple isoforms derived from alternative splicing, Nucleic Acids Res. 28 (2000) 1994–2003. [32] J. Yasuda, A.J. Whitmarsh, J. Cavanagh, M. Sharma, R.J. Davis, The JIP group of mitogen-activated protein kinase scaffold proteins, Mol. Cell. Biol. 19 (1999) 7245–7254. [33] D.R. Cook, R.L. Stiller, J.N. Weakly, S. Chakravorti, B.W. Brandom, R.M. Welch, In vitro metabolism of mivacurium chloride (BW B1090U) and succinylcholine, Anesth. Analg. 68 (1989) 452–456. [34] M. Kortenjann, C. Wehrle, M.C. Nehls, T. Boehm, Only one nemo-like kinase gene homologue in invertebrate and mammalian genomes, Gene 278 (2001) 161–165. [35] H. Kehrer-Sawatzki, E. Moschgath, C. Maier, E. Legius, G. Elgar, W. Krone, Characterization of the Fugu rubripes NLK and FN5 genes flanking the NF1 (Neurofibromatosis type 1) gene in the 50 direction and mapping of the human counterparts, Gene 251 (2000) 63–71. [36] V. Korinek, N. Barker, P.J. Morin, D. van Wichen, R. de Weger, K.W. Kinzler, B. Vogelstein, H. Clevers, Constitutive transcriptional activation by a beta-catenin–Tcf complex in APC)/) colon carcinoma, Science 275 (1997) 1784–1787. [37] I.M. Shih, J. Yu, T.C. He, B. Vogelstein, K.W. Kinzler, The betacatenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression, Cancer Res. 60 (2000) 1671–1676. [38] S. Casares, Y. Ionov, H.Y. Ge, E. Stanbridge, M. Perucho, The microsatellite mutator phenotype of colon cancer cells is often recessive, Oncogene 11 (1995) 2303–2310. [39] D.R. Green, G.I. Evan, A matter of life and death, Cancer Cell 1 (2002) 19–30. [40] H. Harada, S. Yoshida, Y. Nobe, Y. Ezura, T. Atake, T. Koguchi, M. Emi, Genomic structure of the human NLK (nemo-like kinase) gene and analysis of its promoter region, Gene 285 (2002) 175–182. [41] S. Chen, D.C. Guttridge, Z. You, Z. Zhang, A. Fribley, M.W. Mayo, J. Kitajewski, C.Y. Wang, Wnt-1 signaling inhibits apoptosis by activating beta-catenin/T cell factor-mediated transcription, J. Cell Biol. 152 (2001) 87–96. [42] Y. Ueda, M. Hijikata, S. Takagi, R. Takada, S. Takada, T. Chiba, K. Shimotohno, Wnt/beta-catenin signaling suppresses apoptosis in low serum medium and induces morphologic change in rodent fibroblasts, Int. J. Cancer 99 (2002) 681–688. [43] J. Hyodo-Miura, S. Urushiyama, S. Nagai, M. Nishita, N. Ueno, H. Shibuya, Involvement of NLK and Sox11 in neural induction in Xenopus development, Genes Cells 7 (2002) 487–496. [44] M. Kortenjann, M. Nehls, A.J. Smith, R. Carsetti, J. Schuler, G. Kohler, T. Boehm, Abnormal bone marrow stroma in mice deficient for nemo-like kinase, Nlk, Eur. J. Immunol. 31 (2001) 3580–3587.
Nemo-like kinase induces apoptosis in DLD-1 human colon cancer cells Jun Yasuda,a,* Akira Tsuchiya,b Tesshi Yamada,b Michiie Sakamoto,b,1 Takao Sekiya,c and Setsuo Hirohashib a
Cancer Transcriptome Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan b Pathology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan c Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-1252, Japan Received 18 June 2003
Abstract Deregulation of Wnt/b-catenin signaling is thought to play a critical role in human carcinogenesis. Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase that suppresses b-catenin/T-cell factor (TCF) complex transcriptional activity through phosphorylation of TCF. Since NLK may be a tumor suppressor as a negative regulator of Wnt/b-catenin pathway, we established tetracycline-inducible NLK and its kinase-negative mutant expression in DLD-1 human colon cancer cells to analyze the effect of NLK on cell growth and viability. The induction of wild-type NLK in DLD-1 cells caused suppression of cell growth whereas the kinase-negative mutant did not. Flow cytometry indicated that NLK expression increased the number of apoptotic cells but did not induce obvious cell cycle arrest. Apoptosis induction by wild-type NLK was confirmed using TUNEL assays. Our results suggest that overexpression of NLK may have targets other than TCF for induction of apoptosis in human colon carcinoma cells. Ó 2003 Elsevier Inc. All rights reserved. Keywords: NLK; b-Catenin; TCF; Colorectal carcinoma; Apoptosis
Many of the genes mutated in tumor cells play pivotal roles in the regulation of cell differentiation during embryogenesis [1–4]. Wnt/Wingless signaling is an evolutionarily conserved developmental signal transduction pathway and is known to be involved in human carcinogenesis [5,6]. Somatic genetic alterations in most of the components of Wnt/Wingless pathway, such as APC, b-catenin, and Axin, have been found in many human cancers [1,7]. This signaling pathway is thought to suppress degradation of the b-catenin protein, an essential component of cell–cell junctions in epithelial tissues. Stabilized b-catenin protein accumulates in the cell nucleus and forms complex with and activates TCF/ LEF transcription factors. Studies of transgenic mice also indicated that overexpression of b-catenin in intestinal epithelium caused intestinal tumorigenesis [8,9]. *
Corresponding author. Fax: +81-3-3542-0688. E-mail address: [email protected] (J. Yasuda). 1 Present address: Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01343-3
Among the target genes of the b-catenin/TCF complex, several cell growth related genes have been identified (reviewed in [10]) including cyclin D1 [11,12], c-myc [13], MDR1 [14], and PPAR-d [15]. Nemo-like kinase (NLK) was originally identified by the screening of cDNAs coding for protein kinases [16] and was recently characterized as a suppressor of the Wnt signaling pathway in mammalian cells [17–19]. NLK is an evolutionarily conserved serine/threonine kinase and belongs to the proline directed protein kinase superfamily which consists of mitogen activated protein kinases (MAPKs) and cyclin-dependent protein kinases (CDKs) [16]. A member of the MAPK kinase kinase superfamily, TAK1, is identified as a potential activator for NLK [17,20,21]. A recent study showed that Wnt5aCa2þ signaling, which is known as a negative regulator of Wnt [22], can be an upstream activator of TAK1NLK for suppression of canonical Wnt/b-catenin signaling in Xenopus system [18]. Genetic studies of the homologs of NLK in model organisms show that the kinase plays a suppressive role in Wnt/b-catenin signaling. A mutant allele of Nmo, a Drosophila homolog
228
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
of NLK, was originally found to cause a defect of epithelial planar polarity in the fly’s eyes [23]. Another group independently identified a more severe Nmo mutant allele by the screening of Notch modifier mutants and the Nmo mutant restored the wing phenotype of the overexpression of fruit fly b-catenin, Armadillo, indicating that Nmo acts as a suppressor of Wnt signaling in fly wing development [24]. The Caenorhabditis elegans NLK homolog, LIT-1, has been identified as a critical regulator of embryonic cell fate determination accompanying anterior–posterior cell division [25]. In the development of C. elegans, Wnt signaling pathway is also known to determine cell polarity and differentiation [26,27]. Genetic and biochemical analyses showed that LIT-1 could phosphorylate a worm TCF homolog, POP-1, and regulate its subcellular distribution [20,25,28]. In mammals, NLK can suppress transcriptional activity of the b-catenin/T-cell factor (TCF) complex through phosphorylation of TCF in vivo and in vitro [17]. Therefore, NLK/Nmo/LIT-1 works as a downstream regulator of Wnt/Wingless signaling in a variety of organisms. Based on the evolutionary conservation of the function of NLK in Wnt/wingless signaling, it seems very likely that NLK suppresses cell growth stimulated by deregulated b-catenin/TCF complex transcriptional activity in human carcinoma cells. In a recent study, inhibition of the b-catenin/TCF complex by dominant-negative TCF caused suppression of cell growth and G1 arrest of human colorectal carcinoma cell lines including DLD-1 [29]. Similarly, our previous study showed that induction of dominant-negative TCF in DLD-1 cells decreased colony growth rate in soft agar [30]. Based on these studies, it is obvious that transcriptional activity of the b-catenin/ TCF complex is critical to the cell growth of colon cancer cell lines including DLD-1. Therefore, we hypothesized that overexpression of NLK may induce growth arrest in DLD-1 cells mimicking the effect of dominant-negative TCF. However, it is not known whether NLK actually affects cell growth and viability. To address the above issue, we established tetracycline-inducible NLK and its kinase-negative mutants in the DLD-1 cell line. We found that wild-type NLK suppressed cell growth and anchorage independence of the DLD-1 cells. Flow cytometry indicated that NLK expression increased apoptotic cell rate but did not induce cell cycle arrest. Moreover, induction of NLK in these cells increased the rate of apoptotic cells in TUNEL assay.
Materials and methods Plasmids. The plasmids pCMV-Flag-NLK and pCMV-Flag-NLK K155M were kindly provided by Dr. Kunihiro Matsumoto. These plasmids were used as templates of PCR to construct the following expression vector. The plasmids pTRE2PURO-HA-NLK and its
kinase-negative variant pTRE2PURO-HA-NLK K155M were constructed by the insertion of PCR-derived cDNA fragments of NLK in the BamHI and NotI sites of the pTRE2PURO vector (Clontech). pGEXhLEF-1 was constructed by cloning of the RT-PCR product of human lymphoid enhancer factor-1 (LEF-1) cDNA [31]. The pGEXhLEF-1 was introduced in the BL21 competent cells (Stratagene) and the GST-LEF-1 fusion proteins were induced following standard protocols. pRL-tk was purchased from Promega. pTOPFLASH was purchased from Upstate biosystems. The detailed construction strategy of the plasmids is available upon request. Cell culture, transfection, and luciferase assays. DLD-1 tet-on cells [30] were cultured with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1 mg/ml of penicillin/streptomycin (Invitrogen), and 0.4 mg/ml G-418 (Invitrogen) in 10 cm dishes. Seventy per cent confluent cells were subjected to transfection with Lipofectamine 2000 following the manufacturer’s instructions with modifications. pTRE2 Puro HA-NLK and the corresponding mutant plasmids were stably transfected in the presence of 2 lg/ml of puromycin and 0.4 mg/ml G-418. After 2 weeks of selection with G-418 and puromycin, independent clones were isolated by the limiting dilution method or single colony pickup with micropipette tips under a phase contrast microscope. For transient transfection studies, cells were cultured with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 mg/ml of penicillin/streptomycin (Invitrogen) in 10 cm dishes. Cells at 80% confluence were subjected to transfection with Lipofectamine 2000 following the manufacturer’s instructions with modifications. The Dual-luciferase assay system (Promega) was used for analysis of luciferase activity. As much as 1.2 ng of pRL-tk and 0.1 lg of luciferase reporter plasmids were mixed with Lipofectamine 2000 and added to a well of a 24-well plate. After 24 h of transfection, the cells were lysed with passive lysis buffer (Promega) following the manufacturer’s instructions. The induction of NLKs was done 24 h prior to the transfection of reporter genes. The experiments were performed at least twice in triplicate. Luminescence was assayed with a Lumat LB9507 luminomator (EG&G Berthold). The firefly luciferase activity was adjusted with Renilla luciferase activity for each sample. Western blotting and immunoprecipitation-protein kinase assay. Antibodies to Hemagglutinin (HA) epitope tag (mouse: 12CA5, rat: 3F10, Roche), b-catenin (C19220: Transduction Laboratories), and atubulin (Amersham) were used in this study. Cells in 10 cm dishes were lysed in 800 ll of Triton X-lysis buffer for in vitro kinase assays [32]. The Triton X-lysis buffer consisted of 137 mM NaCl, 20 mM Tris–Cl (pH 7.4), 1% of Triton X-100, 25 mM b-glycerophosphate, 2 mM EDTA (pH 8.0), 2 mM sodium pyrophosphate, and 10% glycerol, supplemented with 0.1 mM sodium orthovanadate and complete protease inhibitor (Roche). The lysate was centrifuged at 15,000g for 15 min at 4 °C and the supernatant was subjected to further analysis. Western blotting was performed following the instructions provided by the antibody suppliers. The lysate was subjected to SDS– PAGE and the proteins were transferred onto Immobilon membrane (Millipore) by the use of an electroblotting apparatus (Bio-Rad Transblot semi-dry electrotransfer cell). Immune complexes were detected by enhanced chemiluminescence (Perkin–Elmer). The in vitro kinase reaction was performed as described previously [32]. Briefly, immunoprecipitation was performed with 1 lg of mouse monoclonal antibody for HA tag (12CA5) bound to Dynabeads protein G (Dynal). The lysate with 24 h induction with doxycyclin was mixed with the antibody bound to beads and incubated for 12–16 h at 4 °C with rotation. The immune complex bound to the magnetic beads was washed four times with the same lysis buffer and subjected to the kinase reaction. One microgram of GST-LEF1 fusion protein was added to the immune complex with 20 ll of kinase reaction buffer (25 mM Hepes, pH 7.4, 25 mM b-glycerophosphate, 25 mM MgCl2 , 2 mM DTT, 1 lCi [c-32 P]ATP, 50 lM ATP, and 100 lM sodium orthovanadate). The reaction was performed at 30 °C for 10 min. The reaction was stopped by the addition of SDS–PAGE loading buffer
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233 and the samples were subjected to SDS–PAGE analysis. The phosphorylated proteins were visualized by autoradiography. Cell growth and colony formation assay. Cell growth was estimated with the use of Premix WST-1 cell proliferation assay system (TAKARA), which analyzes succinate-tetrazolium reductase activity. The number of viable cells can be analyzed by measuring the optical density (OD) at 455 nm of the reaction samples as there is a linear correlation between OD and the number of viable cells [33]. The cells were trypsinized and plated at 1.8 104 cells/well in the 24-well plates. The next day, the medium was replaced with the fresh DMEM with 10% FBS with or without 1 lg/ml of doxycyclin for 1–7 days. During the cell culturing, the medium was replaced with fresh one every other day. At the analysis, the medium was replaced with fresh DMEM with 10% FBS with or without 1 lg/ml of doxycyclin containing 3% of WST reagent and the cells were incubated at 37 °C for 1 h with 5% of CO2 for succinate-tetrazolium reductase reaction. The OD of the samples was analyzed with Bio-Rad Model 3550 UV microplate reader at 455 nm. After 7 days of cell culturing and analysis, the cells were lysed and subjected to immunoblotting analysis to determine the expression of the transgene. The amount of proteins loaded on the SDS–PAGE was adjusted as 5 lg/lane. Soft-agar colony formation assay was performed as described [30]. In brief, 1 ml of 0.6% agarose in culture medium was placed on the bottom of the wells in 6-well plates and trypsinized cells (1.5 104 / well) were inoculated in 0.3% agarose in culture medium. The wells were covered with 1 ml of culture medium. The cells were incubated with or without doxycyclin for 3 weeks at 37 °C. The colonies were stained with 0.05% crystal violet for visualization. Flow cytometry. DLD-1 tet-on cell lines (3 105 cells/dish at the beginning) were incubated after 4 days with or without doxycyclin. The floating cells were collected by centrifugation and the adherent cells were collected after trypsinization with 0.25% Trypsin/EDTA solution (Invitrogen). The cell pellets from the floating and the adherent cells were mixed and fixed in 70% ethanol at 4 °C overnight. Fixed cells were incubated with PBS containing 1 mg/ml RNase A at 37 °C for 5 min and stained with 10 lg/ml propidium iodide (Sigma) at room temperature for 20 min. The stained samples were analyzed with a Becton Dickinson FACScalibur flow cytometer using CellQuest software (Becton Dickinson). Each sample was counted up to 20,000 events for analysis in duplicate three times. TUNEL assays. For the detection of apoptotic cells, TUNEL assays were performed with an in situ cell death detection kit (Roche) following the manufacturer’s instructions. The cells were incubated with or without doxycyclin for 5 days on L -polylysine coated glass coverslips (Iwaki, Japan). The cells were then washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 min, and placed in 100% of methanol at )20 °C for 10 min. The nuclei were visualized with DAPI in Vectashield (Vector). The Axiovert fluorescence microscopy system (Zeiss) was used for the generation of fluorescent images. The TUNEL positive and negative cells were counted manually with the aid of Metaphor software (Universal Imaging). An area of DAPI staining of half the average or larger in size was counted as a cell. Apparent mitotic cells showing chromatids were not counted.
Results and discussion NLK induction causes cell growth suppression in DLD-1 cells Fig. 1A shows schematic diagrams of the structures of HA-NLK and its kinase-negative mutants used in this study. A recent study indicated that wild-type NLK may have a shorter isoform, which lacks 71 amino acid residues in N-terminal compared with the conventional form
229
Fig. 1. Induction of NLK with doxycyclin in DLD-1 tet-on cells. (A) Schematic diagrams of the NLK constructs in this study. The Nterminal single-amino acid stretches are shown. The methionine codon for candidate alternative translation initiation for shorter form (see text) is located at just downstream of the alanine stretch (72nd amino acid). (B) Effect of induced NLK on b-catenin/TCF complex transcriptional activity in DLD-1 cells. The vertical axis shows the relative value of normalized fire fly luciferase activity in the cell lysates. The luciferase activity of the cell lysate without doxycyclin used as 100% for each cell line. The horizontal axis shows the cell line and doxycyclin status in the media indicated with + (added) or ) (not added). Error bars indicate the standard error of measurement (SEM). (C) In vitro kinase assay of HA-NLKs induced in DLD-1 cells. Upper panel is an autoradiogram of phosphoproteins in a SDS–PAGE gel. Lower panel is the Western blotting with anti-HA antibody to indicate the amount of proteins loaded in the immunoprecipitation reaction.
[34]. The missing part includes stretches of single-amino acids consisting of histidines and alanines shown in Fig. 1A. Despite the fact that even the histidine stretches in the N-terminal sequences are conserved between fish and mammals [35], it may be possible that the shorter isoform has different functions from the longer, wellcharacterized, isoform which is used in the present study. Four independent clones of the DLD-1 tet-on HANLK cell line were established, two clones for wild-type and two clones for kinase-negative mutants HA-NLK K155M. We performed two distinct assays to confirm whether the induced kinases would work as theoretically expected: suppression of the b-catenin/TCF complex and phosphorylation of TCF/LEF (Figs. 1B and C, respectively). A luciferase assay was performed to test the former using a reporter plasmid (pTOPFLASH [36]) and an in vitro kinase assay was done with GST-LEF1 fusion protein [18] to examine the latter. Induction of wild-type NLK suppressed the reporter gene expression from pTOPFLASH in the DLD-1 cells while mutant
230
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
NLK induction did not cause suppression of the reporter gene expression (Fig. 1B). Immunoprecipitationprotein kinase assay indicated that the wild-type NLK induced in DLD-1 cells could phosphorylate recombinant GST-LEF1 fusion proteins and itself (Fig. 1C). These results were compatible with previous observations [17–19]. As shown here and previous studies, NLK is constitutively active when overexpressed in cultured mammalian cells [16,17]. The WST assay revealed that cell growth was suppressed 3 days after the induction of wild-type NLK by doxycyclin (Fig. 2A). Without induction of the transgenes by doxycyclin, the wild-type NLK cells grew as fast as the parental cells (Fig. 2A). Since the NLK kinase-negative transformants and the parental cells showed similar growth potential regardless of the presence or absence of doxycyclin in the culture medium (Figs. 2A and B), the toxicity of doxycyclin on DLD-1 NLK cells seemed to be negligible. Growth suppression by the induction of NLK was also confirmed by the softagar colony formation assay (Figs. 2C and D).
NLK could induce apoptotic cell death in DLD-1 cells The cause of growth suppression of the DLD-1 cells by wild-type NLK could be either cell cycle arrest or increased rate of apoptosis or both. To examine the cell cycle state and apoptotic cell death in these cell lines, we analyzed the DNA content of the DLD-1 NLK (and its mutant) cells with flow cytometry. Flow cytometric analysis indicated that the ratio of apoptotic cells, measured as sub-G1 populations, was about threefold higher in DLD-1 HA-NLK cells with induction (19.4%) compared with those without induction (6.71%) and 4to 5-fold higher than in DLD-1 HA-NLK KM cells with or without induction (3.28% and 4.10%, respectively). It was also obvious that the relative populations of cells in other phases of the cell cycle were not changed so dramatically (Fig. 3B), even though the G2/M phase cells decreased more than cells in other phases in wild-type NLK-induced cells (Fig. 3B). As mentioned earlier, a recent study by van de Wetering et al. [29] showed that p53 independent induction of p21/WAF1 was strongly
Fig. 2. Growth suppression of DLD-1 tet-on cells by induction of wild-type NLK. (A) WST assay of the DLD-1 NLK cell lines: the name of the clones is indicated in each panel. Open and closed symbols indicate the cell lines without doxycyclin and with doxycyclin, respectively. The graphs show the average of optical density (OD) of the samples over the period of the experiments. The bars show the standard error of measurement (SEM). (B) Clonal variation of the cell growth estimated with the WST assay system. The vertical axis of the graph indicates the average OD at 455 nm with WST reagent of the samples on the seventh day of culturing with or without doxycyclin. Open and closed bars indicate the average OD of the WST reagent for the cell lines without doxycyclin and with doxycyclin, respectively. The Western blots of HA-NLK and a-tubulin of the corresponding cells are shown below the graph. (C) Colony formation assay of DLD-1 tet-on NLK clone 6 with or without doxycyclin. Representative well for each is shown. The cells (1.0 103 cells/well in 6-well plate) were incubated for 3 weeks with or without doxycyclin in the medium in soft agar. (D) The graph indicates the average numbers of colonies in three wells with SEM.
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
231
Fig. 3. Flow cytometric analyses of DLD-1 tet-on NLK cell lines. (A) Histograms of flow cytometrical analysis of DNA contents of DLD-1 tet-on NLK cell lines with (right) or without (left) doxycyclin. The numbers in the panels indicate the percentage of cells with a sub-G1 DNA content. (B) State of cell cycle of DLD-1 tet-on NLK cell lines. The percentage was based on the number of events counted as G0/G1, S, and G2/M but not sub-G1.
correlated with cell cycle arrest of DLD-1 cells by induced dominant-negative TCF. We analyzed the p21/ WAF1 expression by Western blot but did not observe the change of expression level of p21 by NLK in DLD-1 cells (data not shown). These two findings, lack of cell cycle arrest and lack of p21 induction in wild-type NLKinduced DLD-1 cells, are compatible both with each other and with the study by van de Wetering et al. [29]. Nuclear condensation and fragmentation, typical characteristics for apoptotic cells, were observed in numerous floating cells stained with DAPI after 7 days of induction of wild-type NLK in DLD-1 cells (data not shown). We performed TUNEL assays to confirm the induction of apoptosis in wild-type NLK-induced DLD1 cells (Figs. 4A and B). The induction of NLK caused
increased numbers of TUNEL positive cells in DLD-1 cells. Fluorescent microscopy revealed that substantial numbers of the cells were positive in TUNEL assays (labeled with FITC) and many of the TUNEL positive cells exhibited nuclear fragmentation (Fig. 4A). The ratio of TUNEL positive cells in the population increased 3.7-fold in the NLK-induced DLD-1 cells (Fig. 4B). The loss of anchorage independence by the induction of wild-type NLK shown in Figs. 2C and D might be related to this increase of apoptosis tendency. Suppression of the b-catenin/TCF complex by induction of dominant-negative TCF in DLD-1 cells resulted in growth suppression of varying extent without apoptosis [29,30]. The difference between previous studies and the present study suggests that NLK may
Fig. 4. Induction of NLK causes apoptosis of the DLD-1 tet-on cell lines. (A) The TUNEL assays were performed with in situ cell death detection kit and the TUNEL positive cells were visualized with FITC. Arrowhead indicates the TUNEL positive cells and those cells showing nuclear condensation and fragmentation. (B) Percentage of TUNEL positive cells. The DLD-1 tet-on NLK clone 6 cells were counted based on DAPI staining in 200 visual field. The average percentage of TUNEL positive cells in visual fields was indicated with SEM. Total counted doxycyclin free cells, n ¼ 1083 and doxycyclin positive cells, n ¼ 903.
232
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
Fig. 5. Induction of wild-type or mutant NLK does not affect the amount of b-catenin in DLD-1 cells. The samples are the same as in Fig. 2B. Western blotting with anti-a-tubulin antibody was done for control of loading.
have functions other than the suppression of the bcatenin/TCF complex. Alternatively, the difference in the mode of suppression of the b-catenin/TCF complex may explain the difference of outcome between dominant-negative TCF and NLK. It has been suggested that NLK could regulate the affinity of the b-catenin/TCF complex to the target DNA sequences [17]. If the phosphorylated TCF by NLK specifically lost its affinity to target DNA sequences for anti-apoptotic signals, the NLK-overexpressed cells would not show cell cycle arrest but commit apoptosis. We speculated that degradation of b-catenin might cause apoptosis [37] in DLD-1 cells with wild-type NLK induction. Western blotting analysis of b-catenin revealed that this was not the case. We induced NLK and its mutant expression in the DLD-1 tet-on NLK lines and found that the amount of b-catenin was not reduced in the presence or absence of induction of NLK and its mutants (Fig. 5). We concluded that the apoptosis of DLD-1 cells induced by NLK was not related to degradation of b-catenin. Mechanism of apoptosis induction by NLK is still elusive The present study showed that induction of wild-type NLK in a human colorectal carcinoma cell line (DLD-1) caused suppression of cell growth, loss of anchorage independence, and induction of apoptosis in a kinasedependent manner. Notably, this apoptosis induction is p53 independent because both alleles of the p53 gene were inactivated in DLD-1 cells [38]. Given the fact that most of the Wnt signaling mediators play roles in carcinogenesis, NLK may also be involved in human carcinogenesis caused by the deregulation of Wnt/b-catenin signaling. Our results showed that NLK affects cell growth by stimulating p53 independent apoptosis pathway as well as suppressing the b-catenin/TCF complex, which mediates a strong tumorigenic signaling pathway. Since reduction of apoptosis is one of the critical traits in tumorigenesis [39], NLK may be a potential tumor suppressor because
of its ability to induce apoptosis. So far, somatic genetic alterations of the NLK gene have not, however, been observed in human colon and breast cancers [40]. This may mean that the NLK gene is not an “authentic” tumor suppressor gene as such. Further investigation is required into the relationship between NLK expression and carcinogenesis. In terms of the mechanism of apoptosis induction by NLK, there is a possibility that NLK may suppress b-catenin/TCF complex and consequential survival signaling transduced by the complex. Several studies show that the stabilized b-catenin is involved in anti-apoptotic signaling in human cells [37,41,42]. The present study is consistent with these observations as NLK is a suppressor of Wnt/b-catenin signaling. Alternatively, it is possible that TCF/LEF-1 may not be the sole target of NLK for apoptosis induction. Studies of model organisms suggest that NLK and its homologs might have regulatory functions in other signaling pathways [24,43,44]. The search for other downstream targets of NLK may provide clues that will further clarify the role of NLK in apoptosis.
Acknowledgments We thank Hitoshi Ichikawa, Issay Kitabayashi, and Panayiotis Loukopoulos for their critical comments on the manuscript, our colleagues for providing essential reagents, and Kunihiro Matsumoto for the NLK plasmids. This work was supported in part by a Grant-in Aid for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan and a Grantin-Aid from the Ministry of Education, Sciences, Sports and Culture of Japan.
References [1] M. Peifer, P. Polakis, Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus, Science 287 (2000) 1606–1609. [2] M.M. Shen, A.F. Schier, The EGF-CFC gene family in vertebrate development, Trends Genet. 16 (2000) 303–309. [3] R. Derynck, R.J. Akhurst, A. Balmain, TGF-beta signaling in tumor suppression and cancer progression, Nat. Genet. 29 (2001) 117–129. [4] J. Taipale, P.A. Beachy, The Hedgehog and Wnt signalling pathways in cancer, Nature 411 (2001) 349–354. [5] M. Bienz, H. Clevers, Linking colorectal cancer to Wnt signaling, Cell 103 (2000) 311–320. [6] N.S. Fearnhead, M.P. Britton, W.F. Bodmer, The ABC of APC, Hum. Mol. Genet. 10 (2001) 721–733. [7] P. Polakis, The oncogenic activation of beta-catenin, Curr. Opin. Genet. Dev. 9 (1999) 15–21. [8] N. Harada, Y. Tamai, T. Ishikawa, B. Sauer, K. Takaku, M. Oshima, M.M. Taketo, Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene, Embo J. 18 (1999) 5931–5942. [9] B. Romagnolo, D. Berrebi, S. Saadi-Keddoucci, A. Porteu, A.L. Pichard, M. Peuchmaur, A. Vandewalle, A. Kahn, C. Perret, Intestinal dysplasia and adenoma in transgenic mice after over-
J. Yasuda et al. / Biochemical and Biophysical Research Communications 308 (2003) 227–233
[10] [11]
[12] [13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
expression of an activated beta-catenin, Cancer Res. 59 (1999) 3875–3879. R. Nusse, WNT targets. Repression and activation, Trends Genet. 15 (1999) 1–3. M. Shtutman, J. Zhurinsky, I. Simcha, C. Albanese, M. D’Amico, R. Pestell, A. Ben-Ze’ev, The cyclin D1 gene is a target of the betacatenin/LEF-1 pathway, Proc. Natl. Acad. Sci. USA 96 (1999) 5522–5527. O. Tetsu, F. McCormick, Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells, Nature 398 (1999) 422–426. T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin, B. Vogelstein, K.W. Kinzler, Identification of c-MYC as a target of the APC pathway, Science 281 (1998) 1509– 1512. T. Yamada, A.S. Takaoka, Y. Naishiro, R. Hayashi, K. Maruyama, C. Maesawa, A. Ochiai, S. Hirohashi, Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis, Cancer Res. 60 (2000) 4761–4766. T.C. He, T.A. Chan, B. Vogelstein, K.W. Kinzler, PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs, Cell 99 (1999) 335–345. B.K. Brott, B.A. Pinsky, R.L. Erikson, Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus, Proc. Natl. Acad. Sci. USA 95 (1998) 963–968. T. Ishitani, J. Ninomiya-Tsuji, S. Nagai, M. Nishita, M. Meneghini, N. Barker, M. Waterman, B. Bowerman, H. Clevers, H. Shibuya, K. Matsumoto, The TAK1-NLK-MAPK-related pathway antagonizes signalling between beta-catenin and transcription factor TCF, Nature 399 (1999) 798–802. T. Ishitani, S. Kishida, J. Hyodo-Miura, N. Ueno, J. Yasuda, M. Waterman, H. Shibuya, R.T. Moon, J. Ninomiya-Tsuji, K. Matsumoto, The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling, Mol. Cell Biol. 23 (2003) 131–139. T. Ishitani, J. Ninomiya-Tsuji, K. Matsumoto, Regulation of lymphoid enhancer factor 1/T-cell factor by mitogen-activated protein kinase-related Nemo-like kinase-dependent phosphorylation in Wnt/beta-catenin signaling, Mol. Cell. Biol. 23 (2003) 1379–1389. M.D. Meneghini, T. Ishitani, J.C. Carter, N. Hisamoto, J. Ninomiya-Tsuji, C.J. Thorpe, D.R. Hamill, K. Matsumoto, B. Bowerman, MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans, Nature 399 (1999) 793–797. T.H. Shin, J. Yasuda, C.E. Rocheleau, R. Lin, M. Soto, Y. Bei, R.J. Davis, C.C. Mello, MOM-4, a MAP kinase kinase kinaserelated protein, activates WRM-1/LIT-1 kinase to transduce anterior/posterior polarity signals in C. elegans, Mol. Cell 4 (1999) 275–280. M. Kuhl, L.C. Sheldahl, M. Park, J.R. Miller, R.T. Moon, The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape, Trends Genet. 16 (2000) 279–283. K.W. Choi, S. Benzer, Rotation of photoreceptor clusters in the developing Drosophila eye requires the nemo gene, Cell 78 (1994) 125–136. E.M. Verheyen, I. Mirkovic, S.J. MacLean, C. Langmann, B.C. Andrews, C. MacKinnon, The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development, Mech. Dev. 101 (2001) 119–132. T. Kaletta, H. Schnabel, R. Schnabel, Binary specification of the embryonic lineage in Caenorhabditis elegans, Nature 390 (1997) 294–298. C.E. Rocheleau, W.D. Downs, R. Lin, C. Wittmann, Y. Bei, Y.H. Cha, M. Ali, J.R. Priess, C.C. Mello, Wnt signaling and an APCrelated gene specify endoderm in early C. elegans embryos, Cell 90 (1997) 707–716.
233
[27] C.J. Thorpe, A. Schlesinger, J.C. Carter, B. Bowerman, Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm, Cell 90 (1997) 695–705. [28] C.E. Rocheleau, J. Yasuda, T.H. Shin, R. Lin, H. Sawa, H. Okano, J.R. Priess, R.J. Davis, C.C. Mello, WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans, Cell 97 (1999) 717–726. [29] M. van de Wetering, E. Sancho, C. Verweij, W. de Lau, I. Oving, A. Hurlstone, K. van der Horn, E. Batlle, D. Coudreuse, A.P. Haramis, M. Tjon-Pon-Fong, P. Moerer, M. van den Born, G. Soete, S. Pals, M. Eilers, R. Medema, H. Clevers, The betacatenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells, Cell 111 (2002) 241–250. [30] Y. Naishiro, T. Yamada, A.S. Takaoka, R. Hayashi, F. Hasegawa, K. Imai, S. Hirohashi, Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of beta-catenin/Tcell factor 4-mediated gene transactivation, Cancer Res. 61 (2001) 2751–2758. [31] K. Hovanes, T.W. Li, M.L. Waterman, The human LEF-1 gene contains a promoter preferentially active in lymphocytes and encodes multiple isoforms derived from alternative splicing, Nucleic Acids Res. 28 (2000) 1994–2003. [32] J. Yasuda, A.J. Whitmarsh, J. Cavanagh, M. Sharma, R.J. Davis, The JIP group of mitogen-activated protein kinase scaffold proteins, Mol. Cell. Biol. 19 (1999) 7245–7254. [33] D.R. Cook, R.L. Stiller, J.N. Weakly, S. Chakravorti, B.W. Brandom, R.M. Welch, In vitro metabolism of mivacurium chloride (BW B1090U) and succinylcholine, Anesth. Analg. 68 (1989) 452–456. [34] M. Kortenjann, C. Wehrle, M.C. Nehls, T. Boehm, Only one nemo-like kinase gene homologue in invertebrate and mammalian genomes, Gene 278 (2001) 161–165. [35] H. Kehrer-Sawatzki, E. Moschgath, C. Maier, E. Legius, G. Elgar, W. Krone, Characterization of the Fugu rubripes NLK and FN5 genes flanking the NF1 (Neurofibromatosis type 1) gene in the 50 direction and mapping of the human counterparts, Gene 251 (2000) 63–71. [36] V. Korinek, N. Barker, P.J. Morin, D. van Wichen, R. de Weger, K.W. Kinzler, B. Vogelstein, H. Clevers, Constitutive transcriptional activation by a beta-catenin–Tcf complex in APC)/) colon carcinoma, Science 275 (1997) 1784–1787. [37] I.M. Shih, J. Yu, T.C. He, B. Vogelstein, K.W. Kinzler, The betacatenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression, Cancer Res. 60 (2000) 1671–1676. [38] S. Casares, Y. Ionov, H.Y. Ge, E. Stanbridge, M. Perucho, The microsatellite mutator phenotype of colon cancer cells is often recessive, Oncogene 11 (1995) 2303–2310. [39] D.R. Green, G.I. Evan, A matter of life and death, Cancer Cell 1 (2002) 19–30. [40] H. Harada, S. Yoshida, Y. Nobe, Y. Ezura, T. Atake, T. Koguchi, M. Emi, Genomic structure of the human NLK (nemo-like kinase) gene and analysis of its promoter region, Gene 285 (2002) 175–182. [41] S. Chen, D.C. Guttridge, Z. You, Z. Zhang, A. Fribley, M.W. Mayo, J. Kitajewski, C.Y. Wang, Wnt-1 signaling inhibits apoptosis by activating beta-catenin/T cell factor-mediated transcription, J. Cell Biol. 152 (2001) 87–96. [42] Y. Ueda, M. Hijikata, S. Takagi, R. Takada, S. Takada, T. Chiba, K. Shimotohno, Wnt/beta-catenin signaling suppresses apoptosis in low serum medium and induces morphologic change in rodent fibroblasts, Int. J. Cancer 99 (2002) 681–688. [43] J. Hyodo-Miura, S. Urushiyama, S. Nagai, M. Nishita, N. Ueno, H. Shibuya, Involvement of NLK and Sox11 in neural induction in Xenopus development, Genes Cells 7 (2002) 487–496. [44] M. Kortenjann, M. Nehls, A.J. Smith, R. Carsetti, J. Schuler, G. Kohler, T. Boehm, Abnormal bone marrow stroma in mice deficient for nemo-like kinase, Nlk, Eur. J. Immunol. 31 (2001) 3580–3587.