CANCER GENOMICS & PROTEOMICS 7: 87-92 (2010) Inhibition of c-Jun N-terminal Kinase by SP600125: A cDNA Microarray Analysis PIERRE CHAMPELOVIER1, MICHELE EL ATIFI-BOREL2, JEAN PAUL ISSARTEL2,4, JEAN BOUTONNAT1,3, FRANÇOIS BERGER2,3 and DANIEL SEIGNEURIN1,3 1Laboratoire de Cytologie, Département d’Anatomie et de Cytologie Pathologiques, CHU de Grenoble, Hôpital A. Michallon, BP217, 38043 Grenoble, France; 2Plate-forme Transcriptome et Protéome Cliniques, INSERM U836 - Université Joseph Fourier - CHU, Grenoble, Institut des Neurosciences (Equipe 7) 38043 Grenoble, France; 3Université Joseph Fourier, Bâtiment Jean Roget, Faculté de Médecine, Grenoble, F-38700, France; 4CNRS, France Abstract. Background: In a previous investigation, we extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N- showed that the janus kinase (JNK) inhibitor SP600125 terminal kinase (JNK), and P38 MAPK. In mammals, the induced several phenotypic and genomic changes in JNK family contains four members: JNK1, JNK2, and TYK2, leukemia cells. However, the molecular mechanisms that which are ubiquitously expressed, and JNK3, which is sustain these changes remain unknown. The purpose of the predominantly expressed in the brain, testis, and heart. JNK present study was to examine gene expression changes in members act through signal transducer and activator of THP-1 leukemia cells treated with SP600125. Materials and transcription factors (STATs), activation mediated by Methods: Gene expression levels were investigated using phosphorylation upon cytokine stimulation. Subsequently, Affymetrix hybridization technology and quantitative reverse pSTATs dimerize and translocate to the nucleus to induce transcriptase polymerase chain reaction. Results: Affymetrix target gene transcription including c-JUN, c-KIT, c-MYC, technology showed that the expression of 1,038 genes with a BCL-xL, BCL-2 and p21/WAF1 (for review see 1). In biological process description well known in gene ontology leukemia, constitutive activation of STAT can be due to was modulated. Fifteen genes were related to kinases or overexpression of either the cytokine or cytokine receptor phosphatases, 20 genes were involved in the cell cycle expression, but also as a consequence of excessive JNK regulation, and 23 genes were involved in apoptosis. A activity (2-4). In this context, inhibition of the JNK activity network of 15 correlated genes was obtained showing a can be a therapeutic target for acute myeloid leukemia (AML) primordial role for the myelocytomatosis viral oncogene treatment (5). Recent publications showed that JNK inhibition homolog (MYC). Conclusion: These findings show that using the putative JNK-specific inhibitor SP600125 induced SP600125 exhibits cytostatic and cytolytic activities through G2/M cell cycle arrest and apoptosis and caused an MYC gene modulation. endoreplication process in leukemia cells (6-7). Since proliferation, endoreplication and cell survival are processes In normal hematopoietic development, cytokines control cell regulated by numerous genes, Affymetrix microarray analysis growth and differentiation through two major kinase signaling may be particularly well suited to address the question of the pathways that involve mitogen-activated protein kinase effect of SP600125 on leukemia cells at the molecular level. (MAPK) and phosphatidylinositol 3 kinase (PI3K). Three Using transcriptomics, we recently successfully shed light on mammalian MAPK subgroups have been identified: the tumor necrosis factor (TNF)α and transforming growth factor (TGF)β-induced tumoral progression in bladder carcinoma and the resistance phenomenon of UM384 cells to phorbol ester-induced differentiation (8-10). Correspondence to: Dr. Pierre Champelovier, Laboratoire de Recently, using four myeloid cell lines, we have shown Cytologie, Département d’Anatomie et de Cytologie Pathologiques, that SP600125 is able to arrest cells in G2 phase and to Centre Hospitalier Universitaire de Grenoble, BP 217X, 38043 induce endoreplicative and apoptotic processes (7). In the Grenoble cedex 09, France. Tel: +33 476765489, Fax: +33 476765992, e-mail: [email protected] present study, we focused our experiment on the molecular mechanisms induced by SP600125 using numerous genes, Key Words: Cell cycle regulation, apoptosis, endoreplication, microarray analysis may be particularly well suited for this microarray. investigation. 1109-6535/2010 $2.00+.40 87 CANCER GENOMICS & PROTEOMICS 7: 87-92 (2010) Materials and Methods of 1,038 genes that exhibited significant relative changes in their expression level (more than a twofold increase or Maintenance, culture of human leukemia cell line, drugs and decrease) were identified. Out of these 1,038 genes, 15 that reagents. Human leukemia-derived cell line THP-1, generously encoded for kinases or phosphatases were associated with provided by Pr. F. Laporte (Laboratoire de Biochimie, Grenoble), was either the PI3K cascade (PTEN induced putative kinase 1 maintained in RPMI-1640 medium with 10% (v/v) inactivated fetal (PINK1), phosphoinositide-3-kinase, catalytic alpha subunit calf serum (FCS) (Gibco BRL, Eragny, France), antibiotics (penicillin 100 IU/ml−1), streptomycin (100 μg/ml−1), and L-glutamine (2 mM) (p110) (PIK3CA), phosphoinositide-3-kinase, catalytic beta (Roche, Meylan, France). SP600125 was obtained from Sigma (St. polypeptide (PIK3CB), ribosomal protein S6 kinase, 70 kDa Quentin Fallavier, France) and dissolved in DMSO (Sigma). In (RPS6KB2), TIP41, TOR signaling pathway regulator-like induced cultures, cells were seeded at 0.3×106 cells ml−1 for 24 h (TIPRL) or the MAPK cascade mitogen-activated protein with either SP600125 (30 μM) or DMSO (0.1%) (control vehicle). kinase (MAPK) kinase 4 (MAP2K4), MAPK kinase 5 (MAP2K5), MAPK interacting serine/threonine kinase 1 Molecular analysis. Affymetrix analysis: Total RNAs were isolated (MKNK1), V-raf murine sarcoma virus oncogene homology from cells with the mirVana™ isolation kit (Ambion, Austin, TX, USA). The quantity and the quality of extracted RNA were checked B1 (BRAF). Moreover, 20 genes, known to modify the cell using RNA LabChips run on a 2100 BioAnalyser (Agilent cycle (G0/G1 and G2/M transition, mitosis and cytokinesis Technologies, Palo Alto, CA, USA). For microarray analysis, 5 μg regulation), and 23 genes that belong to an of RNA were amplified with the One-Cycle Eukaryotic Target apoptosis/survival-related group were modulated (Table I). Labeling Assay (Affymetrix, Santa Clara, CA, USA) and then Following this, using BioNetworks, we focused our ® hybridized on GeneChip Human Genome U133 Plus 2.0 according investigation on the relationships between all these genes to Affymetrix specifications. The expression values, reported in according to their co-citation in reports from the literature. arbitrary units, were processed and validated using the MAS5 algorithm and then all the individual genes in the treated cells This analysis revealed that 9 out of the 20 cell cycle-related compared with those in the control cells. genes were in close relationships (co-citation of 2 different Reverse transcriptase-quantitative polymerase chain reaction: To genes was scored a minimum of 10 times for all pairs of validate results from the hybridization assays, changes in gene genes) whereas 10 of the 23 apoptosis-related genes had a expression for v-myc viral oncogene homolog (MYC), v-fos viral high score of co-citation in reports (high co-citation score oncogene homolog (FOS), jun D proto-oncogene (JUND), cell genes are indicated by asterisks in Table I). Moreover, using division cycle 25 homolog C (CDC25C), collagen type IV alpha 1 the Pathway Studio network, we showed connectivity (COL4A1), and cytochrome P450 (CYP1B1) were analyzed by reverse transcriptase-quantitative PCR (RT-QPCR) analysis (7). between 15 genes constituting a network pinpointing the Ribosomal protein large subunit 27 (RPL27) and beta actin (ACTB) central role for v-myc viral oncogene homolog (MYC) in the gene products were used as references. PCR primers for each gene regulation of both cell cycle and apoptosis (Figure 1). were designed (http://www.rocheappliedscience.com/sis/rtpcr/upl/ index.jsp?id=UP030000). In RT-QPCR analysis, 2 μg of each total Validation of microarray analysis. RT-QPCR analysis was RNA were transcribed into cDNA using Promega Reverse used to validate the microarray results. The mRNA levels Transcription reagents with random (dN6) primer (8). PCR was then from eight genes were quantified using RT-QPCR and performed according to the SYBR Green methodology on a Light Cycler™ (Roche Diagnostics GmbH, Germany). The specificity of compared to the results obtained using Affymetrix DNA PCR products was monitored by melting curve analysis. Results chips. The results obtained by both approaches for MYC, were normalized to an exogenous standard used in our previously FOS, JUND, CDC25C, COL4A1, CYP1B1, RPL27 and described microarray experiments (8). ACTB were not statistically different (Table II). Gene connectivity. The BioNetworks expression analysis tool Discussion (PubGene) was used to determine the relationships between the differentially expressed genes found in the literature (http:// www.pubgene.org/tools/GeneSearch/GeneSearch.cgi) and Pathway Affymetrix technology was used to investigate the effects of Studio software (Ariadne, Genomics Inc, Rockville, MD, USA) SP600125 on the THP-1 cells and we showed that 1,038 genes was used to analyze the functional connectivity between the with a known biological process