Abstract
Background: In a previous investigation, we showed that the janus kinase (JNK) inhibitor SP600125 induced several phenotypic and genomic changes in leukemia cells. However, the molecular mechanisms that sustain these changes remain unknown. The purpose of the present study was to examine gene expression changes in THP-1 leukemia cells treated with SP600125. Materials and Methods: Gene expression levels were investigated using Affymetrix hybridization technology and quantitative reverse transcriptase polymerase chain reaction. Results: Affymetrix technology showed that the expression of 1,038 genes with a biological process description well known in gene ontology was modulated. Fifteen genes were related to kinases or phosphatases, 20 genes were involved in the cell cycle regulation, and 23 genes were involved in apoptosis. A network of 15 correlated genes was obtained showing a primordial role for the myelocytomatosis viral oncogene homolog (MYC). Conclusion: These findings show that SP600125 exhibits cytostatic and cytolytic activities through MYC gene modulation.
In normal hematopoietic development, cytokines control cell growth and differentiation through two major kinase signaling pathways that involve mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K). Three mammalian MAPK subgroups have been identified: extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and P38 MAPK. In mammals, the JNK family contains four members: JNK1, JNK2, and TYK2, which are ubiquitously expressed, and JNK3, which is predominantly expressed in the brain, testis, and heart. JNK members act through signal transducer and activator of transcription factors (STATs), activation mediated by phosphorylation upon cytokine stimulation. Subsequently, pSTATs dimerize and translocate to the nucleus to induce target gene transcription including c-JUN, c-KIT, c-MYC, BCL-xL, BCL-2 and p21/WAF1 (for review see 1). In leukemia, constitutive activation of STAT can be due to overexpression of either the cytokine or cytokine receptor expression, but also as a consequence of excessive JNK activity (2-4). In this context, inhibition of the JNK activity can be a therapeutic target for acute myeloid leukemia (AML) treatment (5). Recent publications showed that JNK inhibition using the putative JNK-specific inhibitor SP600125 induced G2/M cell cycle arrest and apoptosis and caused an endoreplication process in leukemia cells (6-7). Since proliferation, endoreplication and cell survival are processes regulated by numerous genes, Affymetrix microarray analysis may be particularly well suited to address the question of the effect of SP600125 on leukemia cells at the molecular level. Using transcriptomics, we recently successfully shed light on 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).
Recently, using four myeloid cell lines, we have shown that SP600125 is able to arrest cells in G2 phase and to induce endoreplicative and apoptotic processes (7). In the present study, we focused our experiment on the molecular mechanisms induced by SP600125 using numerous genes, microarray analysis may be particularly well suited for this investigation.
Materials and Methods
Maintenance, culture of human leukemia cell line, drugs and reagents. Human leukemia-derived cell line THP-1, generously provided by Pr. F. Laporte (Laboratoire de Biochimie, Grenoble), was maintained in RPMI-1640 medium with 10% (v/v) inactivated fetal calf serum (FCS) (Gibco BRL, Eragny, France), antibiotics (penicillin 100 IU/ml–1), streptomycin (100 μg/ml–1), and L-glutamine (2 mM) (Roche, Meylan, France). SP600125 was obtained from Sigma (St. Quentin Fallavier, France) and dissolved in DMSO (Sigma). In induced cultures, cells were seeded at 0.3×106 cells ml–1 for 24 h with either SP600125 (30 μM) or DMSO (0.1%) (control vehicle).
Molecular analysis. Affymetrix analysis: Total RNAs were isolated from cells with the mirVana™ isolation kit (Ambion, Austin, TX, USA). The quantity and the quality of extracted RNA were checked using RNA LabChips run on a 2100 BioAnalyser (Agilent Technologies, Palo Alto, CA, USA). For microarray analysis, 5 μg of RNA were amplified with the One-Cycle Eukaryotic Target Labeling Assay (Affymetrix, Santa Clara, CA, USA) and then hybridized on GeneChip® Human Genome U133 Plus 2.0 according to Affymetrix specifications. The expression values, reported in arbitrary units, were processed and validated using the MAS5 algorithm and then all the individual genes in the treated cells compared with those in the control cells.
Reverse transcriptase-quantitative polymerase chain reaction: To validate results from the hybridization assays, changes in gene expression for v-myc viral oncogene homolog (MYC), v-fos viral oncogene homolog (FOS), jun D proto-oncogene (JUND), cell division cycle 25 homolog C (CDC25C), collagen type IV alpha 1 (COL4A1), and cytochrome P450 (CYP1B1) were analyzed by reverse transcriptase-quantitative PCR (RT-QPCR) analysis (7). Ribosomal protein large subunit 27 (RPL27) and beta actin (ACTB) gene products were used as references. PCR primers for each gene were designed (http://www.rocheappliedscience.com/sis/rtpcr/upl/index.jsp?id=UP030000). In RT-QPCR analysis, 2 μg of each total RNA were transcribed into cDNA using Promega Reverse Transcription reagents with random (dN6) primer (8). PCR was then performed according to the SYBR Green methodology on a Light Cycler™ (Roche Diagnostics GmbH, Germany). The specificity of PCR products was monitored by melting curve analysis. Results were normalized to an exogenous standard used in our previously described microarray experiments (8).
Gene connectivity. The BioNetworks expression analysis tool (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 Studio software (Ariadne, Genomics Inc, Rockville, MD, USA) was used to analyze the functional connectivity between the modulated genes.
Results
Transcriptomic analysis. To identify genes responsive to SP600125 treatment in THP-1 cells, we carried out RNA microarray analysis of cells that had been growing for 24 h in the presence of either DMSO (control vehicle) or SP600125 (30 μM). Among the 17,900 genes analyzed, a set of 1,038 genes that exhibited significant relative changes in their expression level (more than a twofold increase or decrease) were identified. Out of these 1,038 genes, 15 that encoded for kinases or phosphatases were associated with either the PI3K cascade (PTEN induced putative kinase 1 (PINK1), phosphoinositide-3-kinase, catalytic alpha subunit (p110) (PIK3CA), phosphoinositide-3-kinase, catalytic beta polypeptide (PIK3CB), ribosomal protein S6 kinase, 70 kDa (RPS6KB2), TIP41, TOR signaling pathway regulator-like (TIPRL) or the MAPK cascade mitogen-activated protein kinase (MAPK) kinase 4 (MAP2K4), MAPK kinase 5 (MAP2K5), MAPK interacting serine/threonine kinase 1 (MKNK1), V-raf murine sarcoma virus oncogene homology B1 (BRAF). Moreover, 20 genes, known to modify the cell cycle (G0/G1 and G2/M transition, mitosis and cytokinesis regulation), and 23 genes that belong to an apoptosis/survival-related group were modulated (Table I). Following this, using BioNetworks, we focused our investigation on the relationships between all these genes according to their co-citation in reports from the literature. This analysis revealed that 9 out of the 20 cell cycle-related genes were in close relationships (co-citation of 2 different genes was scored a minimum of 10 times for all pairs of genes) whereas 10 of the 23 apoptosis-related genes had a high score of co-citation in reports (high co-citation score genes are indicated by asterisks in Table I). Moreover, using the Pathway Studio network, we showed connectivity between 15 genes constituting a network pinpointing the central role for v-myc viral oncogene homolog (MYC) in the regulation of both cell cycle and apoptosis (Figure 1).
Validation of microarray analysis. RT-QPCR analysis was used to validate the microarray results. The mRNA levels from eight genes were quantified using RT-QPCR and compared to the results obtained using Affymetrix DNA chips. The results obtained by both approaches for MYC, FOS, JUND, CDC25C, COL4A1, CYP1B1, RPL27 and ACTB were not statistically different (Table II).
Discussion
Affymetrix technology was used to investigate the effects of SP600125 on the THP-1 cells and we showed that 1,038 genes with a known biological process description in gene ontology were modulated. Among them, 20 genes were related to the cell cycle and proliferation process and 23 genes to the apoptotic process. It was noted that 15 genes were related to kinases or phosphatases, including three genes of the MAPK family (mitogen-activated protein kinase kinase 4, mitogen-activated protein kinase kinase 5, MAPK interacting serine/threonine kinase 1) and five genes of the PI3K pathway (PTEN-induced putative kinase 1, phosphoinositide-3-kinase, catalytic alpha subunit (p110), phosphoinositide-3-kinase, catalytic beta polypeptide, ribosomal protein S6 kinase, 70 kDa, TIP41, TOR signaling pathway regulator-like). SP600125 was also shown to change the expression of several genes coding for protein phosphatases including protein phosphatase 1 regulatory subunit 15A (PPP1R15A) and inositol polyphosphate 4 phosphatase type 1 107kDa (INPP4A) (Table I). All these results confirm that SP600125 is not a specific inhibitor of the JNK pathway (11). Our data confirm that PIK3CA and PIK3CB regulate several mechanisms controlling cell proliferation (12) and that JNK modulates PIK3CB expression (13). SP600125 increases the expression of the mitogen-activated protein kinase kinase 5 (MAP2K5) gene known to stimulate cell proliferation. Moreover, the fact that SP600125 also up-regulates PIK3CA present at the G0/G1 transition and S phase entry (14, 15) could suggest that in our experiment, cell proliferation is not stopped but only mitosis aborted, inducing an endoreplicative process.
SP600125 up- and down-regulated genes clustered by biological activity. Genes were classified using biological activity as described in the literature using either PubMed or PubGene. Due to the fact that some genes act in several processes, they can appear in several classes: e.g. RET and FOS acting in the regulation of both the cell cycle and apoptotic processes. *Genes taking part in the network obtained with BioNetworks (PubGene).
Gene network. The gene network was realized from the transcriptomic data analyzed using Pathway Studio software. Rectangles: apoptosis-associated genes; ovals: cell cycle-associated genes.
Transcriptomic analysis also showed a decrease in the expression of cell cycle-associated genes such as cyclin G2 (CCNG2) and cyclin-dependent kinase 6 (CDK6) and an increase in the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A) (7), all of which are genes involved in the cell cycle arrest-initiating mechanisms (16, 17). However, in spite of a substantial cell cycle arrest in G2, transcriptomic data revealed an increase in the expression of cell division cycle 25 homolog C (CDC25C) recently associated with the regulation of events preceding cell division such as spindle formation, chromatin condensation, and fragmentation of the nuclear envelope. This suggest that SP600125 probably does not affect the entry into mitosis (G2/M transition). SP600125 decreases the expression of nibrin (NBS1) recently associated with the control of temozolomide-induced G2 arrest and cytotoxicity, suggesting that SP600125 did not act like temozolomide to block the cells in the G2 phase of the cell cycle (18).
Validation of the microarray analysis. THP-1 cells were grown for 24 hours without inducer (control) or in the presence of SP600125 (30 μM). mRNAs used in the RT-QPCR were from 3 independent extractions. Numbers±SD indicated the fold change between the control and the SP600125-induced culture.
SP600125 also increases the expression of MYC-induced nuclear antigen (MINA) and decreases the expression of MYC, suggesting that MINA could possess MYC-independent functions regulating cell cycle process (19). Moreover, after 48 h of treatment, SP600125 induced an endoreplicative process. Transcriptomic analysis shows that SP600125 modulates genes recently associated with either aberrant mitosis and endoreplication including CDKN1A (20) or with the final steps of mitosis including mitotic spindle formation and cytokinesis (TIP41, TOR signaling pathway regulator-like (TIPRL), scinderin (SCIN) and anaphase promoting complex, subunit 7 (ANAPC7)) (21-23). Therefore, the loss of cytokinesis without arrest of DNA synthesis is consistant with the observed endoreplicative process.
Moreover, SP600125 modulates cytochrome c (CYCS), CYP1B1, superoxide dismutase 2, mitochondrial (SOD2), phospholipase A2 (CPLA2), glutathione peroxidase 3 (GPX3), tumor protein p53 inducible nuclear protein 1 (TP53INP1), TNF (ligand) superfamily, member 10 (CD253/TRAIL) (TNFSF10), and TNF (ligand) superfamily, member 13 (CD256/APRIL) (TNFSF13). All these genes, which have been associated with either reactive oxygen species (ROS) or arachidonic acid/ceramide pathways, or mitochondrial dysfunction, may be able to induce apoptosis (24-27). The removal of ROS by the free radical scavenger N-acetyl-cysteine inhibits SP600125-mediated apoptosis from 30% to 20% (data not shown), confirming the action of ROS on the apoptotic process.
In conclusion, SP600125 modulated the PI3K and the ERK pathways, confirming that the effect of this inhibitor is not restricted to the JNK pathway. Moreover, the MYC gene seems to be the key factor in the regulation of cell growth, survival, and endoreplication processes induced by SP600125 in THP-1 cells. Based on its cytostatic and cytolytic activities, SP600125 could be put forward as a novel potential drug for the treatment of leukemia cells, either used alone as a therapeutic agent, or in association with other antitumor agents.
Acknowledgments
This study was supported, in part, by the Association Grenobloise d’Etude de la Cellule Cancéreuse (AGECC). We express our thanks to Linda Northrup (Ph.D., ELS) for her help in English language editing.
- Received December 3, 2009.
- Revision received February 19, 2010.
- Accepted February 22, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
