lithium-chloride and Cell-Transformation--Neoplastic

c-Myc oncogene is an important regulator of cell cycle and apoptosis, and its dysregulated expression is associated with many malignancies. Myc is instrumental in directly or indirectly regulating the progression through the G1 phase and G1/S transition, and transformation by Myc results in perturbed cell cycle. Also contributory to the control of G1 is the Ras effector pathway Raf/MEK/ERK MAP kinase. Together with GSK3, ERK plays an important role in the critical hierarchical phosphorylation of S62/T58 controlling Myc protein levels. Therefore, our main aim was to examine the levels of MAPK in Myc transformed cells in light of the roles of ERK in cell cycle and control of Myc protein levels. We found that active forms of ERK were barely detectable in v-Myc (MC29) transformed cells. Furthermore, we could only detect reduced levels of activated ERK in c-Myc transformed cells compared to the non-transformed primary chick embryo fibroblast cells. The addition of LiCl inhibited GSK3 and successfully restored the levels of ERK in v-Myc and c-Myc transformed cells to those found in non-transformed cells. In addition, LiCl stabilised Myc protein in the non-transformed and c-Myc transformed cells but not in v-Myc transformed cells. These results can provide an important insight into the role of MAPK in the mechanism of Myc induced transformation and carcinogenesis.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Transformed; Cell Transformation, Neoplastic; Chick Embryo; Culture Media, Serum-Free; Fibroblasts; Genes, myc; Glycogen Synthase Kinase 3; Half-Life; Lithium Chloride; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Oncogene Protein p55(v-myc); Phosphorylation

Beta-catenin plays a key role in the Wnt signaling cascade. The levels of beta-catenin within a cell are regulated via phosphorylation of the N terminus of beta-catenin by GSK-3beta. The phosphorylation leads to ubiquitination and subsequent degradation of the protein. Thus far three serines (S33, 37, 45) and one threonine (T41) are considered to be the substrates for GSK-3beta phosphorylation. Indeed, these amino acids are regularly mutated in tumors, resulting in beta-catenin molecules with enhanced transcriptional activity. Aligning N-terminal sequences of beta-catenin homologues of different species revealed two other highly conserved serines (S23, 29), which have also been found mutated in tumors. We show that these serines are modified in the same fashion as that of the known regulatory residues. During embryogenesis, the phosphorylation status of S23 and S29 appears to be actively regulated. Nevertheless, constructs harboring the mutations found in tumors fail to show enhanced transcriptional activity or transforming properties.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; beta Catenin; Calcium-Calmodulin-Dependent Protein Kinases; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Embryo, Mammalian; Eukaryotic Cells; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Humans; Lithium Chloride; Mice; Mutation; Neoplasms; Phosphorylation; Protein Structure, Tertiary; Serine; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured

This research was to observe the effects of lithium chloride (LiCl) and Harringtonine (HT) on the proliferation and differentiation of HL-60 leukemia cells. The results obtained by liquid suspension culture, semi-solid colony culture and 3H-TdR incorporation into HL-60 cells indicated that different concentrations of LiCl (5-20 mmol/L) and HT (10(-8)-10(-5)mol/L) exerted the inhibitory effects in a dose-dependent manner on HL-60 cell proliferation respectively. When LiCl (10 mmol/L) and HT (10(-7) mol/L) were added together in the liquid culture or semi-solid culture of HL-60 cells, they showed much greater inhibitory effect than that by each agent separately. It was discovered that there was induction of the differentiation of HL-60 cells by lithium and HT and the induction of HL-60 cells differentiation by HT was markedly enhanced by the addition of low concentration of lithium. This work also showed that by treating HL-60 cells with lithium and HT, the expression of the c-myc proto-oncogene was markedly decreased as measured by RT/PCR-mRNA (P < 0.01). These findings provide some evidence of the mechanismcausing leukemic change and of the potential use of lithium and HT in the treatment of leukemia and in vitro purging of leukemic cells for autologous bone marrow transplantation.

Topics: Antineoplastic Agents; Cell Division; Cell Transformation, Neoplastic; Drug Synergism; Genes, myc; Harringtonines; HL-60 Cells; Humans; Lithium Chloride; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc

The metabolism of phosphatidylinositol (PtdIns) was studied in a mink lung epithelial cell line and its subclones transformed by feline sarcoma viruses containing either the v-fms or v-fes oncogenes. The transformed cell lines had a higher rate of PtdIns turnover but did not have elevated levels of phosphorylated PtdIns species or PtdIns kinase activity. Significantly higher specific activities of a guanine nucleotide-activated PtdIns-4,5-diphosphate phospholipase C were detected in both transformed cell lines (F3CL7(v-fes), 55 pmol/min/mg of protein and G2M(v-fms), 18 pmol/min/mg of protein) as compared to the nontransformed parental cell line (CCL64, 2 pmol/min/mg of protein). The guanine nucleotide-stimulated phospholipase C activity was specific for PtdIns-4,5-diphosphate, and the water-soluble hydrolysis product was inositol 1,4,5-triphosphate. Both GTP and nonhydrolyzable GTP analogs activated the phospholipase C, whereas ATP was weakly effective and GDP was inactive. The phospholipase C activity was maximally active in the presence of 9 mM sodium cholate, had a sharp pH optimum of pH 6.5, and was not activated by calcium although hydrolysis was inhibited by high concentrations of EDTA. These data point to enhanced production of diacylglycerol and inositol 1,4,5-triphosphate second messengers in transformed cells due to the activation of guanine nucleotide-dependent PtdIns-4,5-diphosphate-specific phospholipase C and suggest that the generation of aberrant hormonally independent signals is associated with cell transformation by oncogenes encoding tyrosine-specific protein kinases.

Topics: 1-Phosphatidylinositol 4-Kinase; Animals; Calcium; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chlorides; Epithelium; Guanine Nucleotides; Inositol; Kinetics; Lithium; Lithium Chloride; Lung; Mink; Oncogenes; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphotransferases; Retroviridae; Sarcoma Viruses, Feline; Type C Phospholipases