CANCER GENOMICS & PROTEOMICS 7: 87-92 (2010)

Inhibition of c-Jun N-terminal 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 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 with a BCL-xL, BCL-2 and p21/WAF1 (for review see 1). In biological process description well known in leukemia, constitutive activation of STAT can be due to was modulated. Fifteen genes were related to 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 -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 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 description in gene ontology modulated genes. were modulated. Among them, 20 genes were related to the cell cycle and proliferation process and 23 genes to the Results apoptotic process. It was noted that 15 genes were related to kinases or phosphatases, including three genes of the MAPK Transcriptomic analysis. To identify genes responsive to family (mitogen-activated protein kinase kinase 4, mitogen- SP600125 treatment in THP-1 cells, we carried out RNA activated protein kinase kinase 5, MAPK interacting microarray analysis of cells that had been growing for 24 h serine/threonine kinase 1) and five genes of the PI3K pathway in the presence of either DMSO (control vehicle) or (PTEN-induced putative kinase 1, phosphoinositide-3-kinase, SP600125 (30 μM). Among the 17,900 genes analyzed, a set catalytic alpha subunit (p110), phosphoinositide-3-kinase,

88 Champelovier et al: Microarray Analysis of SP600125 Inhibition of JNK

Table I. 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 symbol Gene description Fold change

Kinases and phosphatases ADK Adenylate kinase 0.5 BRAF V-raf murine sarcoma virus oncogene homology B1 0.5 CDK6 Cyclin-dependent kinase 6 0.4 DMPK Dystrophia myotonica protein kinase 2.2 INPP4A Inositol polyphosphate-4-phosphatase type I, 107 kDa 0.4 MAP2K4 Mitogen-activated protein kinase (MAPK) kinase 4 2 MAP2K5 Mitogen-activated protein kinase (MAPK) kinase 5 2 MKNK1 MAPK-interacting serine/threonine kinase 1 2.1 PDK1 Pyruvate dehydrogenase kinase, isozyme 1 2.1 PIK3CA Phosphoinositide-3-kinase, catalytic alpha subunit (p110) 2 PIK3CB Phosphoinositide-3-kinase, catalytic beta polypeptide 0.5 PINK1 PTEN-induced putative kinase 1 2.4 PPP1R15A Protein phosphatase 1, regulatory subunit 15A 2 RPS6KB2 Ribosomal protein S6 kinase, 70 kDa 0.4 TIPRL TIP41, TOR signaling pathway regulator-like 0.5

Cell cycle and proliferation ANAPC7 Anaphase promoting complex, subunit 7 0.4 BRAF* V-raf murine sarcoma virus oncogene homology B1 0.5 CCL3L1 G0/G1 switch regulatoring protein 19.1 0.5 CCNG2 Cyclin G2 2 CDC25A* Cell division cycle 25 homolog A 0.5 CDC25C* Cell division cycle 25 homolog C 2.6 CDK6* Cyclin-dependent kinase 6 0.4 FOS* v-fos viral oncogene homolog 2.9 JUND JUN D proto-oncogene 0.5 MINA* Myc-induced nuclear antigen 0.5 MYC* v-myc viral oncogene homolog 0.5 NAP1L5 Nucleosome assembly protein 1-like 6 0.4 NBS1* Nibrin 0.5 PAK1IP1 p21/cdc2/Rac 1-activated kinase 1-interacting protein 1 0.4 PPP1R15A Protein phosphatase 1, regulatory subunit 15A 2 RET* RET proto-oncogene 2.1 RPS6KB2 Ribosomal protein S6 kinase, 70 kDa 0.4 SCIN Scinderin 0.3 SHC1 SHC transforming protein 1 0.5 TIPRL TIP41, TOR signaling pathway regulator-like 0.5

Apoptosis and survival BAG1 Bcl2-associated athanogene 0.4 BAX* Bcl2-associated X protein 0.5 BCL6* B-cell CLL/lymphoma 6 2 BCL11A* B-cell CLL/lymphoma 11A 0.5 BMP2* Bone morphogenic protein 2 0.5 CPLA2 Phospholipase A2 0.3 CYP1B1 Cytochrome P450 0.4 CYCS* Cytochrome c 0.5 DAPK3 Death-associated protein kinase 3 0.4 FOS* v-fos viral oncogene homolog 2.9 GPX3* Glutathione peroxidase 3 2.1 JUND JUN D proto-oncogene 0.5 MYC* v-myc viral oncogene homolog 0.5 PAK1IP1 p21/cdc2/Rac 1-activated kinase 1 interacting protein 1 0.4 PDE4B Phosphodiesterase 4B, cAMP-specific 2.3 PHCA Phytoceramidase alkalin 0.4 PIK3CA Phosphoinositide-3-kinase, catalytic alpha subunit 1 2 PIK3CB Phosphoinositide-3-kinase, catalytic beta polypeptide 0.5 RET* RET proto-oncogene 2.1 SOD2* Superoxide dismutase 2, mitochondrial 0.5 TNFSF10 TNF (ligand) superfamily, member 10 (CD253) 2 TNFSF13 TNF (ligand) superfamily, member 13 (CD256) 2.2 TP53INP1 Tumor protein p53-inducible nuclear protein 1 3.1

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Figure 1. 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.

catalytic beta polypeptide, ribosomal protein S6 kinase, Table II. Validation of the microarray analysis. THP-1 cells were grown 70 kDa, TIP41, TOR signaling pathway regulator-like). for 24 hours without inducer (control) or in the presence of SP600125 SP600125 was also shown to change the expression of several (30 μM). mRNAs used in the RT-QPCR were from 3 independent extractions. Numbers±SD indicated the fold change between the control genes coding for protein phosphatases including protein and the SP600125-induced culture. phosphatase 1 regulatory subunit 15A (PPP1R15A) and inositol polyphosphate 4 phosphatase type 1 107kDa (INPP4A) Genes Microarray RT-QPCR (Table I). All these results confirm that SP600125 is not a specific inhibitor of the JNK pathway (11). Our data confirm ACTB 1 1.2±0.2 CDC25C 3 7±2 that PIK3CA and PIK3CB regulate several mechanisms COL4A1 0.4 0.25±0.1 controlling cell proliferation (12) and that JNK modulates CYP1B1 0.4 0.2±0.1 PIK3CB expression (13). SP600125 increases the expression FOS 3 6±0.3 of the mitogen-activated protein kinase kinase 5 (MAP2K5) JUN 0.5 0.3±0.2 gene known to stimulate cell proliferation. Moreover, the fact MYC 0.5 0.4±0.2 RPL27 1.1 1.2±0.2 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. Transcriptomic analysis also showed a decrease in the and fragmentation of the nuclear envelope. This suggest that expression of cell cycle-associated genes such as cyclin G2 SP600125 probably does not affect the entry into mitosis (CCNG2) and cyclin-dependent kinase 6 (CDK6) and an (G2/M transition). SP600125 decreases the expression of increase in the expression of cyclin-dependent kinase nibrin (NBS1) recently associated with the control of inhibitor 1A (CDKN1A) (7), all of which are genes involved temozolomide-induced G2 arrest and cytotoxicity, suggesting in the cell cycle arrest-initiating mechanisms (16, 17). that SP600125 did not act like temozolomide to block the However, in spite of a substantial cell cycle arrest in G2, cells in the G2 phase of the cell cycle (18). transcriptomic data revealed an increase in the expression of SP600125 also increases the expression of MYC-induced cell division cycle 25 homolog C (CDC25C) recently nuclear antigen (MINA) and decreases the expression of MYC, associated with the regulation of events preceding cell suggesting that MINA could possess MYC-independent division such as spindle formation, chromatin condensation, functions regulating cell cycle process (19). Moreover, after

90 Champelovier et al: Microarray Analysis of SP600125 Inhibition of JNK

48 h of treatment, SP600125 induced an endoreplicative marrow phospho-STAT5 expression in non-CML chronic process. Transcriptomic analysis shows that SP600125 myeloproliferative disorders correlates with JAK2 V617F modulates genes recently associated with either aberrant mutation and provides evidence of in vivo JAK2 activation. Am J Surg Pathol 31: 233-239, 2007. mitosis and endoreplication including CDKN1A (20) or with 5 Zada AA, Singh SM, Reddy VA, Elsasser A, Meisel A, Haferlach the final steps of mitosis including mitotic spindle formation T, Tenen DG, hiddemann W and Behre G: Down regulation of and cytokinesis (TIP41, TOR signaling pathway regulator-like c-Jun expression and cell cycle regulatory molecules in acute (TIPRL), scinderin (SCIN) and anaphase promoting complex, myeloid leukemia cells upon CD44 ligation. Oncogene 2: 2296- subunit 7 (ANAPC7)) (21-23). Therefore, the loss of 2308, 2003. cytokinesis without arrest of DNA synthesis is consistant with 6 Amin HM, Medeiros LJ, Ma Y, feretzaki M, Das P, Leventaki V, the observed endoreplicative process. Rassidakis GZ, O’Connor SL, McDonnel TJ and Lai R: Moreover, SP600125 modulates cytochrome c (CYCS), Inhibition of JAK3 induces apoptosis and decreases anaplastic lymphoma kinase activity in anaplastic large cell lymphoma. CYP1B1 SOD2 , superoxide dismutase 2, mitochondrial ( ), Oncogene 22: 5399-5407, 2003. phospholipase A2 (CPLA2), glutathione peroxidase 3 (GPX3), 7 Champelovier P, El Atifi M, Pautre V, Rostaing B, Berger F and tumor protein p53 inducible nuclear protein 1 (TP53INP1), Seigneurin D: Specific inhibition of basal mitogen-activated TNF (ligand) superfamily, member 10 (CD253/TRAIL) protein kinase (MAPKs) and phosphatidylinositol-3 kinase (TNFSF10), and TNF (ligand) superfamily, member 13 (PI3K) activities in leukemia cells: a possible therapeutic role (CD256/APRIL) (TNFSF13). All these genes, which have for the kinase inhibitor. Exp Hematol 36: 28-36, 2008. been associated with either reactive oxygen species (ROS) or 8 Champelovier P, El Atifi M, Mantel F, Michallat S, Simon A, Rostaing B, Berger F and Seigneurin D: Effects of tumor necrosis arachidonic acid/ceramide pathways, or mitochondrial factor-α (TNF α) and interferon-γ (IFN γ) on gene expression dysfunction, may be able to induce apoptosis (24-27). The profiles in bladder carcinoma cells using oligonucleotide microarray removal of ROS by the free radical scavenger N-acetyl- analysis. Cancer Genomics Proteomics 1: 455-463, 2004. cysteine inhibits SP600125-mediated apoptosis from 30% to 9 Champelovier P, El Atifi M, Mantel F, Rostaing B, Simon A, 20% (data not shown), confirming the action of ROS on the Berger F and Seigneurin D: In vitro tumoral progression of human apoptotic process. bladder carcinoma: Role for TGFβ. Eur Urol 48: 846-851, 2005. In conclusion, SP600125 modulated the PI3K and the 10 Champelovier P, Pautre V, El Atifi M, Dupré I, Rostaing B, ERK pathways, confirming that the effect of this inhibitor is Michoud A, Berger F and Seigneurin D: Resistance to phorbol ester-induced differentiation in human myeloid leukemia cells: not restricted to the JNK pathway. Moreover, the MYC gene a hypothetic role for the mRNA stabilization process. Leukemia seems to be the key factor in the regulation of cell growth, Res 30: 1407-1416, 2006. survival, and endoreplication processes induced by 11 Bain J, McLauchlan H, Elliot M and Cohen P: The specificities SP600125 in THP-1 cells. Based on its cytostatic and of protein kinase inhibitors: an update. Biochem J 371: 199-204, cytolytic activities, SP600125 could be put forward as a 2003. novel potential drug for the treatment of leukemia cells, 12 Crowder RJ, Phommaly C, Tao Y, Hoog J, Luo J, Perou CM, either used alone as a therapeutic agent, or in association Parker JS, Miller MA, Huntsman DG, Lin L, Snider J, Davies SR, Olson JA Jr, Watson MA, Saporita A, Weber JD and Ellis with other antitumor agents. MJ: PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivative in estrogen receptor- Acknowledgements positive breast cancer. Cancer Res 69: 3955-3962, 2009. 13 Utsugi M, Dobashi K, Ono A, Ishizuka T, Matsuzaki S, Hisada This study was supported, in part, by the Association Grenobloise T, Shimizu Y, Kawata T, Aoki H, Kanide Y and Mori M: PI3K d’Etude de la Cellule Cancéreuse (AGECC). 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