anisomycin and Cell-Transformation--Neoplastic

We have previously shown that transcription of immediate-early c-fos protooncogene is becoming strongly repressed in rat embryo fibroblasts transformed by oncogenes E1A and cHa-ras, so that serum only slightly stimulated c-fos transcription in these cells in contrast to high level of c-fos activation in non-transformed REF52 cells. Here we showed that stress-inducing agent anisomycin was able to override the c-fos repression and to induce c-fos transcription in E1A + ras transformants. In vitro kinase assay data demonstrated that anisomycin increased phosphorylation of transactivation domain of Elk-1 transcription factor--a key regulator of inducible c-fos transcription. Importantly, this activation was mediated through up-regulation of MEK/ERK but not stress-kinase cascades JNK or p38. The activating effect of anisomycin on c-fos transcription could be abrogated by a prior treatment with N-acetyl-L-cysteine. This indicates that anisomycin potentiates generation of reactive oxygen species (ROS), which, in turn, can modulate the activity of MAP kinase-specific phosphatases (MKPs). As anisomycin did not cause acetylation of nucleosome core histones, the present work focuses on the molecular mechanisms mediating the HDAC-independent induction of IEG c-fos by anisomycin in E1A + cHa-ras-transformed fibroblasts.

Topics: Adenovirus E1A Proteins; Animals; Anisomycin; Cell Line, Transformed; Cell Transformation, Neoplastic; Enzyme Activation; ets-Domain Protein Elk-1; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, fos; Genes, ras; Immediate-Early Proteins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-fos; Rats; Transcriptional Activation

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

Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1 trans-activating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expressing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1 trans-activating activity are not absolute requirements for ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the protease-dependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype. These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. In ras-transformed NIH 3T3 fibroblasts, the uPA- and CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.

Topics: 3T3 Cells; Animals; Anisomycin; Basement Membrane; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Cathepsin L; Cathepsins; Cell Transformation, Neoplastic; Cysteine Endopeptidases; Endopeptidases; Enzyme Activation; Genes, ras; Mice; Mutation; Neoplasm Invasiveness; Phenotype; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-jun; RNA, Messenger; Urokinase-Type Plasminogen Activator