h-89 and Cell-Transformation--Neoplastic

Altered oxidative stress has long been observed in cancer cells, and this biochemical property of cancer cells represents a specific vulnerability that can be exploited for therapeutic benefit. The major role of an elevated oxidative stress for the efficacy of molecular targeted drugs is under investigation. Menadione is considered an attractive model for the study of oxidative stress, which can induce apoptosis in human leukemia HL-60 cell lines. Prostaglandin E(2) (PGE(2)) via its receptors not only promotes cell survival but also reverses apoptosis and promotes cancer progression. Here, we present evidence for the biological role of PGE(2) as a protective agent of oxidative stress-induced apoptosis in monocytic cells. Pretreatment of HL-60 cells with PGE(2) markedly ameliorated the menadione-induced apoptosis and inhibited the degradation of PARP and lamin B. The EP(2) receptor antagonist AH6809 abrogated the inhibitory effect of PGE(2), suggesting the role of the EP(2)/cAMP system. The PKA inhibitor H89 also reversed apoptosis and decreased the PKA activity that was elevated 10-fold by PGE(2). The treatment of HL-60 cells with NAC or zinc chloride showed a similar protective effect as with PGE(2) on menadione-treated cells. Furthermore, PGE(2) activated the Ras/Raf/MEK pathway, which in turn initiated ERK activation, and ultimately protected menadione-induced apoptosis. These results imply that PGE(2) via cell survival pathways may protect oxidative stress-induced apoptosis in monocytic cells. This study warrants further pre-clinical investigation as well as application towards leukemia clinics.

Topics: Apoptosis; Cell Survival; Cell Transformation, Neoplastic; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Isoquinolines; Lamin Type B; Leukemia; Lipid Peroxidation; Oxidative Stress; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Receptors, Prostaglandin E, EP2 Subtype; Sulfonamides; Vitamin K 3; Xanthones

Activated by the RAS-MAPK signaling pathway, MSK1 is recruited to immediate-early gene (IEG) regulatory regions, where it phosphorylates histone H3 at Ser-10 or Ser-28. Chromatin remodelers and modifiers are then recruited by 14-3-3 proteins, readers of phosphoserine marks, leading to the occupancy of IEG promoters by the initiation-engaged form of RNA polymerase II and the onset of transcription. In this study, we show that this mechanism of IEG induction, initially elucidated in parental 10T1/2 murine fibroblast cells, applies to metastatic Hras1-transformed Ciras-3 cells. As the RAS-MAPK pathway is constitutively activated in Ciras-3 cells, MSK1 activity and phosphorylated H3 steady-state levels are elevated. We found that steady-state levels of the IEG products AP-1 and COX-2 were also elevated in Ciras-3 cells. When MSK1 activity was inhibited or MSK1 expression was knocked down in Ciras-3 cells, the induction of IEG expression and the steady-state levels of COX-2, FRA-1, and JUN were greatly reduced. Furthermore, MSK1 knockdown Ciras-3 cells lost their malignant phenotype, as reflected by the absence of anchorage-independent growth.

Topics: 14-3-3 Proteins; Animals; Cell Line; Cell Transformation, Neoplastic; Chromatin Assembly and Disassembly; Cyclooxygenase 2; Fibroblasts; Genes, Immediate-Early; Histones; Isoquinolines; Mice; Oncogene Protein p21(ras); Phenotype; Phorbol Esters; Phosphorylation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Regulatory Sequences, Nucleic Acid; Ribosomal Protein S6 Kinases, 90-kDa; Sulfonamides; Transcriptional Activation

Mitogen- and stress-activated kinase 1 (MSK1) belongs to a family of dual protein kinases that are activated by either extracellular signal-regulated kinase or p38 mitogen-activated protein kinases in response to stress or mitogenic extracellular stimuli. The physiologic role of MSK1 in malignant transformation and cancer development is not well understood. Here, we report that MSK1 is involved in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced neoplastic transformation of JB6 Cl41 cells. H89, a potent inhibitor of MSK1, strongly suppressed TPA-induced or EGF-induced cell transformation. When cells overexpressing wild-type MSK1 were treated with TPA or EGF, colony formation increased substantially compared with untreated cells or cells that did not overexpress MSK1. In contrast, MSK1 COOH terminal or NH(2) terminal dead dominant negative mutants dramatically suppressed cell transformation. Introduction of small interfering RNA-MSK1 into JB6 Cl41 cells resulted in suppressed TPA-induced or EGF-induced cell transformation. In addition, cell proliferation was inhibited in MSK1 knockdown cells compared with MSK1 wild-type cells. In wild-type MSK1-overexpressing cells, activator protein (AP-1) activation increased after TPA or EGF stimulation, whereas AP-1 activation decreased in both MSK1 dominant-negative mutants and in MSK1 knockdown cells. Moreover, TPA-induced or EGF-induced phosphorylation of histone H3 at Ser(10) was increased in wild-type cells but the induced phosphorylation was abolished in MSK1 dominant-negative mutant or MSK1 knockdown cells. Thus, MSK1 is required for tumor promoter-induced cell transformation through its phosphorylation of histone H3 at Ser(10) and AP-1 activation.

Topics: Animals; Cell Growth Processes; Cell Transformation, Neoplastic; Enzyme Activation; Epidermal Growth Factor; G1 Phase; Histones; Isoquinolines; Mice; Phosphorylation; Protein Kinase Inhibitors; Ribosomal Protein S6 Kinases, 90-kDa; RNA, Small Interfering; Skin; Sulfonamides; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

Previous reports showed that activation of the thromboxane receptor (TP) induced some types of cells to proliferate. We report here that TPalpha activates beta-catenin/T-cell factor (Tcf)/lymphoid enhancer factor (Lef) pathway through phosphorylation of glycogen synthase kinase (GSK)-3. TP agonist [1S-alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) induced both alpha and beta forms of GSK-3 phosphorylation in human embryonic kidney (HEK)293 cells stably overexpressing TPalpha (HEK293-TPalpha). N-[2-(4-Bromocinnamylamino)ethyl]-5-isoquinoline (H89), a protein kinase A (PKA) inhibitor, totally blocked the phosphorylation of GSK-3, whereas wortmannin, a phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, partially attenuated it, suggesting that PKA as well as PI-3 kinase/Akt pathway were involved in TP-induced phosphorylation of GSK-3. I-BOP consistently stimulated an approximately 8-fold increase over basal Tcf/Lef reporter gene activity in HEK293-TPalpha cells. Furthermore, I-BOP-induced Tcf/Lef reporter gene activity was totally inhibited by H89 and partially inhibited by wortmannin. I-BOP also induced overexpression of Tcf/Lef downstream target gene cyclin D1. Blockade of the beta-catenin expression by small interfering RNA approach attenuated I-BOP-induced expression of cyclin D1, indicating that the induction was mediated by beta-catenin/Tcf/Lef pathway. Finally, I-BOP resulted in the morphology change, such as cell rounding and aggregation, in HEK293-TPalpha cells after 1-h incubation. However, HEK293-TPalpha cells were not able to revert back to normal shape even 24 h after the removal of the agonist, suggesting that the prolonged activation of the Tcf/Lef promoter induced downstream gene expression leading to cell permanent morphology change that was related to cell transformation. Together, our results showed for the first time TP agonist-induced phosphorylation of GSK-3 and activation of Tcf/Lef signaling leading to cell proliferation and transformation.

Topics: Androstadienes; Binding, Competitive; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Fatty Acids, Unsaturated; Genes, Reporter; Glycogen Synthase Kinase 3; Humans; Isoquinolines; Phosphorylation; Radioligand Assay; Receptors, Thromboxane; Signal Transduction; Sulfonamides; TCF Transcription Factors; Transfection; Wortmannin