RESUMO
Normal cell proliferation is controlled by a balance between signals that promote or halt cell proliferation. Micro RNAs are emerging as key elements in providing fine signal balance in different physiological situations. Here we report that STAT5 signaling induces the miRNAs miR-19 and miR-155, which potentially antagonize the tumor suppressor axis composed by the STAT5 target gene SOCS1 (suppressor of cytokine signaling-1) and its downstream effector p53. MiRNA sponges against miR-19 or miR-155 inhibit the functions of these miRNAs and potentiate the induction of SOCS1 and p53 in mouse leukemia cells and in human myeloma cells. Adding a catalytic RNA motif of the hammerhead type within miRNA sponges against miR-155 leads to decreased miR-155 levels and increased their ability of inhibiting cell growth and cell migration in myeloma cells. The results indicate that antagonizing miRNA activity can reactivate tumor suppressor pathways downstream cytokine stimulation in tumor cells.
Assuntos
Leucemia/metabolismo , MicroRNAs/metabolismo , Mieloma Múltiplo/metabolismo , RNA Catalítico/biossíntese , RNA Neoplásico/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Leucemia/genética , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Mieloma Múltiplo/genética , Células RAW 264.7 , RNA Catalítico/genética , RNA Neoplásico/antagonistas & inibidores , RNA Neoplásico/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The expression of the forkhead transcription factor checkpoint suppressor 1 (CHES1), also known as FOXN3, is reduced in many types of cancers. We show here that CHES1 decreases protein synthesis and cell proliferation in tumor cell lines but not in normal fibroblasts. Conversely, short hairpin RNA-mediated depletion of CHES1 increases tumor cell proliferation. Growth suppression depends on the CHES1 forkhead DNA-binding domain and correlates with the nuclear localization of CHES1. CHES1 represses the expression of multiple genes, including the kinases PIM2 and DYRK3, which regulate protein biosynthesis, and a number of genes in cilium biogenesis. CHES1 binds directly to the promoter of PIM2, and in cells expressing CHES1 the levels of PIM2 are reduced, as well as the phosphorylation of the PIM2 target 4EBP1. Overexpression of PIM2 or eIF4E partially reverses the antiproliferative effect of CHES1, indicating that PIM2 and protein biosynthesis are important targets of the antiproliferative effect of CHES1. In several human hematopoietic cancers, CHES1 and PIM2 expressions are inversely correlated, suggesting that repression of PIM2 by CHES1 is clinically relevant.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proliferação de Células , Biossíntese de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Fatores de Transcrição Forkhead , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
An efficient method for digestion and extraction of proteolytic peptides from silver-stained proteins was applied to the characterization of nuclear proteins from the small cell lung cancer H82 (ATCC HTB 175) cell line previously separated by high-resolution large format two-dimensional gel electrophoresis. From 68 spots, evenly distributed on the gel area and representing a wide range of spot intensities, 63 (92%) were successfully identified by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionozation-mass spectrometry (ESI-MS). In five cases where the identification was not possible, the presence of an intense background apparently due to the leakage of polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post-translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reported in sequence databases was also detected.