anisomycin and Rhabdomyosarcoma

We have previously suggested that PKCalpha has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCalpha mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCalpha, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.

Topics: Anisomycin; Cell Differentiation; Cell Transformation, Neoplastic; Child; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Muscle Fibers, Skeletal; Muscle Neoplasms; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Kinase C-alpha; Rhabdomyosarcoma; Stem Cells; Tissue Plasminogen Activator; Tumor Cells, Cultured

Expression of the various components of the plasminogen activation system is under tight regulation by hormones, cytokines, and growth factors under physiologic conditions. Like early-response genes, these components are modulated by inhibitors of protein synthesis in some cell lines. To clarify the specific expression and regulation of mRNAs for urokinase (uPA), its receptor (uPAR), and type-1 plasminogen activator inhibitor (PAI-1), I analyzed RNA from four human cancer cell lines by RNA blotting after treatment with cycloheximide, anisomycin, emetine or puromycin. These inhibitors, all of which induced translational arrest, induced a very diverse response in the various transcripts, suggesting that the inhibitors mediate their effects through different molecular mechanisms. Dose-response analysis showed that, in A549 cells, anisomycin strongly induced uPAR and PAI-1 mRNA at concentrations that did not cause complete inhibition of protein synthesis, whereas cycloheximide induced these transcripts in a dose-dependent manner only at concentrations sufficient to inhibit total protein synthesis by >90%. Puromycin induced the 3.4-kb transcript of PAI-1 mRNA in A549 and RD cells, whereas it decreased the expression of both the 3.4-kb and 2.4-kb PAI-1 transcripts in HT-1080 cells. Different time patterns of induction for uPA, uPAR and PAI-1 mRNA suggest that even in the same cell type, inhibitors of protein synthesis mediate their effects on various genes through different mechanisms. Thus, induction of uPA, uPAR, and PAI-1 transcripts by inhibitors of protein synthesis was dependent on the gene, the cell line, and the type of inhibitor, and inhibition of protein synthesis per se was not sufficient for induction of these transcripts.

Topics: Anisomycin; Cell Line; Cycloheximide; Dose-Response Relationship, Drug; Emetine; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Lung Neoplasms; Protein Synthesis Inhibitors; Puromycin; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Rhabdomyosarcoma; RNA, Messenger; RNA, Neoplasm; Time Factors; Transcription, Genetic; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator