l-744832 and Cell-Transformation--Neoplastic

Interactions between the Chk1 inhibitor UCN-01 and the farnesyltransferase inhibitor L744832 were examined in human leukemia cells. Combined exposure of U937 cells to subtoxic concentrations of UCN-01 and L744832 resulted in a dramatic increase in mitochondrial dysfunction, apoptosis, and loss of clonogenicity. Similar interactions were noted in other leukemia cells (HL-60, Raji, Jurkat) and primary acute myeloid leukemia (AML) blasts. Coadministration of L744832 blocked UCN-01-mediated phosphorylation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), leading to down-regulation of phospho-cyclic adenosine monophosphate responsive element-binding protein (phospho-CREB) and -p90(RSK) and activation of p34(cdc2) and stress-activated protein kinase/ERK kinase/c-Jun N-terminal kinase (SEK/JNK). Combined treatment also resulted in pronounced reductions in levels of phospho-Akt, -glycogen synthase kinase-3 (-GSK-3), -p70(S6K), -mammalian target of rapamycin (-mTOR), -forkhead transcription factor (-FKHR), -caspase-9, and -Bad. Ectopic expression of Bcl-2 or Bcl-xL but not dominant-negative caspase-8 blocked UCN-01/L744832-mediated mitochondrial dysfunction and apoptosis but did not prevent activation of p34(cdc2) and JNK or inactivation of MEK/ERK and Akt. Enforced expression of myristoylated Akt but not constitutively active MEK significantly attenuated UCN-01/L744832-induced apoptosis. However, dual transfection with Akt and MEK resulted in further protection from UCN-01/L744832-mediated lethality. Finally, down-regulation of JNK1 by siRNA significantly reduced the lethality of the UCN-01/L744832 regimen. Together, these findings suggest that farnesyltransferase inhibitors interrupt the cytoprotective Akt and MAPK pathways while reciprocally activating SAPK/JNK in leukemia cells exposed to UCN-01 and, in so doing, dramatically increase mitochondria-dependent apoptosis.

Topics: Acute Disease; Alkyl and Aryl Transferases; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspases; Cell Transformation, Neoplastic; Checkpoint Kinase 1; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Farnesyltranstransferase; HL-60 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukemia, Myeloid; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Potentials; Methionine; Mitochondria; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Staurosporine; U937 Cells

Recent genetic investigations have established that RhoB gain-of-function is sufficient to mediate the antitransforming effects of farnesyltransferase inhibitors (FTIs) in H-Ras-transformed fibroblast systems. In this study, we addressed the breadth and mechanism of RhoB action in epithelial cells transformed by oncoproteins which are themselves insensitive to FTI inactivation. Rat intestinal epithelial (RIE) cells transformed by activated K-Ras or Rac1 were highly sensitive to FTI-induced actin reorganization and growth inhibition, despite the inability of FTI to block prenylation of either K-Ras or Rac1. Ectopic expression of the geranylgeranylated RhoB isoform elicited in cells by FTI treatment phenocopied these effects. Analysis of RhoB effector domain mutants pointed to a role for PRK, a Rho effector kinase implicated in the physiological function of RhoB in intracellular receptor trafficking, and these findings were supported further by experiments in a fibroblast system. We propose that FTIs recruit the antioncogenic RhoB protein in the guise of RhoB-GG to interfere with signaling by pro-oncogenic Rho proteins, possibly by sequestering common exchange factors or effectors such as PRK that are important for cell transformation.

Topics: Actin Cytoskeleton; Actins; Alkyl and Aryl Transferases; Animals; Bacterial Proteins; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; DNA-Binding Proteins; Enzyme Inhibitors; Epithelial Cells; Escherichia coli Proteins; Farnesyltranstransferase; Fibroblasts; Genes, ras; Intestinal Mucosa; Methionine; Models, Biological; Protein Isoforms; Protein Kinase C; Protein Prenylation; Protein Processing, Post-Translational; rac1 GTP-Binding Protein; Rats; Signal Transduction

Neoplastic transformation sensitizes many cells to apoptosis. This phenomenon may underlie the therapeutic benefit of many anticancer drugs, but its molecular basis is poorly understood. We have used a selective and potent farnesyltransferase inhibitor (FTI) to probe a mechanism of apoptosis that is peculiarly linked to neoplastic transformation. While nontoxic to untransformed mouse cells, FTI triggers a massive RhoB-dependent, p53-independent apoptosis in mouse cells that are neoplastically transformed. Here we offer evidence that the BAR adapter-encoding tumor suppressor gene Bin1 is required for this transformation-selective death program. Targeted deletion of Bin1 in primary mouse embyro fibroblasts (MEFs) transformed by E1A+Ras did not affect FTI-induced reversion, actin fiber formation, or growth inhibition, but it abolished FTI-induced apoptosis. The previously defined requirement for RhoB in these effects suggests that Bin1 adapter proteins act downstream or in parallel to RhoB in cell death signaling. The death defect in Bin1 null cells was significant insofar as it abolished FTI efficacy in tumor xenograft assays. p53 deletion did not phenocopy the effects of Bin1 deletion. However, MEFs transformed by SV40 large T antigen+Ras were also resistant to apoptosis by FTI, consistent with other evidence that large T inhibits Bin1-dependent cell death by a p53-independent mechanism. Taken together, the results define a function for Bin1 in apoptosis that is conditional on transformation stress. This study advances understanding of the functions of BAR adapter proteins, which are poorly understood, by revealing genetic interactions with an Rho small GTPase that functions in stress signaling. The frequent losses of Bin1 expression that occur in human breast and prostate cancers may promote tumor progression and limit susceptibility to FTI or other therapeutic agents that exploit the heightened sensitivity of neoplastic cells to apoptosis.

Topics: Actins; Adaptor Proteins, Signal Transducing; Adenovirus E1A Proteins; Alkyl and Aryl Transferases; Animals; Antigens, Polyomavirus Transforming; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Inhibitors; Farnesyltranstransferase; Fibroblasts; Gene Deletion; Membrane Proteins; Methionine; Mice; Mice, SCID; Nerve Tissue Proteins; Nuclear Proteins; ras Proteins; rhoB GTP-Binding Protein; Stress, Physiological; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are approximately 50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-kappaB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation.

Topics: 3T3 Cells; Animals; Cell Transformation, Neoplastic; Contact Inhibition; Culture Media, Serum-Free; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Female; Gene Expression Regulation; Genes, jun; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Methionine; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; NF-kappa B; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Phenotype; Phosphatidylinositol 3-Kinases; Protein Prenylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; ras Proteins; Receptor Protein-Tyrosine Kinases; Receptor, EphB4; Receptors, Eph Family; rhoA GTP-Binding Protein; Serum Response Factor; Signal Transduction; Tumor Stem Cell Assay