fti-277 and Cell-Transformation--Neoplastic

The v-Abl tyrosine kinase activates several signaling pathways during transformation of bone marrow cells in mice. Because the SH2-containing inositol 5'-phosphatase (SHIP) and Downstream of tyrosine kinase 1 (Dok1) have been shown to interact with Abl, the effect of SHIP and Dok1 deficiency on v-Abl transformation was investigated. Bone marrow cells from either Dok1- or SHIP-deficient mice are more susceptible to transformation by v-Abl. v-Abl-transformed preB cells from these knockout mice show Abl kinase-dependent hyperproliferation and moderate resistance to apoptosis. Elevated activation of Ras, Raf-1, and Erk, but not of Akt, was observed in either SHIP(-/-) or Dok1(-/-) v-Abl-transformed cells. This activation is sensitive to treatment with STI571. Furthermore, treatment of these cells with either a farnesyltransferase inhibitor or a MEK1/2 inhibitor abrogates the increased proliferation of SHIP(-/-) or Dok1(-/-) cells in a dose-dependent manner. Complementation of SHIP(-/-) or Dok1(-/-) cells abrogates their hyperproliferation and intracellular Erk activation. These data indicate that both SHIP and Dok1 functionally regulate the activation of Ras-Erk pathway by v-Abl and affect the mitogenic activity of v-Abl transformed bone marrow cells.

Topics: Animals; Apoptosis; Benzamides; Bone Marrow Cells; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Farnesyltranstransferase; Gene Expression Regulation, Leukemic; Imatinib Mesylate; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Methionine; Mice; Oncogene Proteins v-abl; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Phosphoproteins; Phosphoric Monoester Hydrolases; Piperazines; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Pyrimidines; ras Proteins; RNA-Binding Proteins; Signal Transduction

Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) beta. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFalpha expression, or TGFbeta sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFbeta resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFbeta sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.

Topics: Alkyl and Aryl Transferases; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Enzyme Activation; Farnesyltranstransferase; Gene Expression Regulation, Neoplastic; Genes, Dominant; Intestinal Mucosa; Methionine; Mice; Mutation; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Rats; rhoA GTP-Binding Protein; Transfection; Transforming Growth Factor beta; Tumor Stem Cell Assay; 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

Several small GTPases of the Ras superfamily have been shown to antagonize TGFbeta signaling in human tumor cell lines. Some of these GTPases are post-translationally modified by farnesylation, a lipid modification catalyzed by farnesyltransferase and required for the proteins to attach to membranes and to function. In this study, we investigated the effect of the farnesyltransferase inhibitor FTI-277 on TGFbeta-regulated cell growth and transcription. Treatment of the human pancreatic tumor cell line, Panc-1, with FTI-277 enhanced the ability of TGFbeta to inhibit both anchorage-dependent and -independent tumor cell growth. FTI-277 also enhanced the ability of TGFbeta to induce transcription, as measured by p3TP-lux reporter activity and collagen synthesis. The enhancement of TGFbeta responses by FTI-277 correlated with the stimulation of transcription and protein expression of type II TGFbeta receptor (TbetaRII). Consequently, FTI-277-treated cells exhibited a higher level of TGFbeta binding to its receptor. Thus, inhibition of protein farnesylation stimulates TbetaRII expression, which leads to increased TGFbeta receptor binding and signaling as well as inhibition of tumor cell growth and transformation.

Topics: 3T3 Cells; Alkyl and Aryl Transferases; Animals; Cell Division; Cell Transformation, Neoplastic; Drug Synergism; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Methionine; Mice; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function. Since we recently showed that the signaling pathways that mediate Ras transformation of RIE-1 rat intestinal epithelial cells are distinct from those that cause Ras transformation of fibroblasts, we utilized three approaches to determine if Src transformation of RIE-1 cells is dependent on Ras. First, although both Ras and Src cause upregulation of an epidermal growth factor (EGF) receptor-dependent autocrine growth loop, only Ras transformation required this activity. Second, whereas both Src and Ras caused upregulation of the p42 and p44 mitogen-activated protein kinases (MAPKs), only Ras transformation was blocked by the inhibition of MAPK activation by treatment with the PD 98059 MEK inhibitor. Third, treatment with the farnesyltransferase inhibitor FTI-277 blocked Ras, but not Src, transformation. Taken together, these observations suggest that Src transformation of RIE-1 cells is not dependent on Ras. Finally, we determined that Ras activation of Raf-independent pathways alone is sufficient to cause growth transformation of RIE-1 cells. Thus, both Ras and Src cause transformation of RIE-1 cells via pathways distinct from those required to cause transformation of NIH3T3 cells.

Topics: Alkyl and Aryl Transferases; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Inhibitors; Epithelial Cells; ErbB Receptors; Farnesyltranstransferase; Genes, ras; Genes, src; Methionine; Mutation; Proto-Oncogene Proteins c-raf; Rats; Up-Regulation

Many tumor cells have a greater resistance to ionizing radiation than their normal counterparts, suggesting that the development of drugs that can reduce that radioresistance would potentiate the efficacy of radiation therapy. Because activated H-ras expression has been shown to markedly increase radiation resistance in some transformed cells, the inactivation of H-ras would then be predicted to radiosensitize these tumor cells, while leaving normal cells unaffected. H-ras depends for activity upon farnesylation, which can be blocked by farnesylation inhibitors, including the compound FTI-277. In keeping with this prediction, inhibition of H-ras processing using FTI-277 resulted in higher levels of apoptosis after irradiation and increased radiosensitivity in H-ras-transformed rat embryo cells but did not affect control cells. These experiments suggest that farnesylation inhibitors may prove clinically useful as radiosensitizers of tumors that depend on ras function.

Topics: Alkyl and Aryl Transferases; Animals; Apoptosis; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Embryo, Mammalian; Enzyme Inhibitors; Farnesyltranstransferase; Fibroblasts; Genes, myc; Genes, ras; Kinetics; Methionine; Radiation-Sensitizing Agents; Rats; Transfection; Transferases; Urinary Bladder Neoplasms

In order to assess the relative contributions of farnesylated and/or geranylgeranylated proteins on cell cycle progression from G1 to S phase we designed potent and selective farnesyltransferase (FTI-277) and geranylgeranyltransferase-I (GGTI-298) inhibitors. Flow cytometry studies showed that treatment of NIH3T3 cells with GGTI-298 or lovastatin, which inhibits both protein farnesylation and geranylgeranylation, arrested cells in G0/G1 whereas cells treated with FTI-277 progressed normally through the cell cycle. [3H]thymidine incorporation studies showed that mevalonate and geranylgeraniol, but not farnesol, released the lovastatin G1 block. Furthermore, mevalonate release of the lovastatin G1 block was inhibited by GGTI-298 but not by FTI-277. These results demonstrate that geranylgeranylated proteins are required for cells to proceed from G1 to S phase, and that farnesylated proteins do not play an essential role in the G1 to S phase transition

Topics: Actins; Alkyl and Aryl Transferases; Animals; Benzamides; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeleton; Diterpenes; DNA; Enzyme Inhibitors; Farnesol; Fibroblasts; G1 Phase; Genes, ras; GTP-Binding Proteins; Lovastatin; Methionine; Mevalonic Acid; Mice; Protein Prenylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; rap GTP-Binding Proteins; ras Proteins; Resting Phase, Cell Cycle; S Phase; Transferases

Ras-induced malignant transformation requires Ras farnesylation, a lipid posttranslational modification catalyzed by farnesyltransferase (FTase). Inhibitors of this enzyme have been shown to block Ras-dependent transformation, but the mechanism by which this occurs remains largely unknown. We have designed FTI-276, a peptide mimetic of the COOH-terminal Cys-Val-Ile-Met of K-Ras4B that inhibited potently FTase in vitro (IC50 = 500 pM) and was highly selective for FTase over geranylgeranyltransferase I (GGTase I) (IC50 = 50 nM). FTI-277, the methyl ester derivative of FTI-276, was extremely potent (IC50 = 100 nM) at inhibiting H-Ras, but not the geranylgeranylated Rap1A processing in whole cells. Treatment of H-Ras oncogene-transformed NIH 3T3 cells with FTI-277 blocked recruitment to the plasma membrane and subsequent activation of the serine/threonine kinase c-Raf-1 in cells transformed by farnesylated Ras (H-RasF), but not geranylgeranylated, Ras (H-RasGG). FTI-277 induced accumulation of cytoplasmic non-farnesylated H-Ras that was able to bind Raf and form cytoplasmic Ras/Raf complexes in which Raf kinase was not activated. Furthermore, FTI-277 blocked constitutive activation of mitogen-activated protein kinase (MAPK) in H-RasF, but not H-RasGG, or Raf-transformed cells. FTI-277 also inhibited oncogenic K-Ras4B processing and constitutive activation of MAPK, but the concentrations required were 100-fold higher than those needed for H-Ras inhibition. The results demonstrate that FTI-277 blocks Ras oncogenic signaling by accumulating inactive Ras/Raf complexes in the cytoplasm, hence preventing constitutive activation of the MAPK cascade.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Transformation, Neoplastic; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Methionine; Molecular Sequence Data; Oligopeptides; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; ras Proteins; Signal Transduction; Transferases