myelin-basic-protein and Carcinoma--Hepatocellular

To test the effects of the 21.5 kDa human brain myelin basic protein (MBP) on the proliferation and apoptosis of HepG-2.. pSVCEP-MBP-CAT plasmid containing the full-length 21.5 kDa MBP cDNA was transfected into human hepatoma carcinoma cells (HepG-2). The pSVCEP-CAT vector transfected HepG-2 cells served as negative control. RT-PCR and Western blot were performed to confirm the effectiveness of the transfection. MTT measures were used to determine the proliferative curve of cells. H2O2 was then added to induce cell apoptosis. DNA ladder, immunohistochemistry assay, comet electrophoresis and TUNEL (Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) were used to detect the apoptosis and relevant protein expressions.. The 21.5 KDa MBP cDNA was transfected into HepG-2 cells successfully. MTT measures of pSVCEP-MBP-CAT transfected group showed increased proliferation and anti-apoptosis. The control group displayed with more typical DNA ladders and much higher level of Caspase-3 than the MBP group. Comet and TUNEL assays revealed that the control group cells had significant DNA damages and serious apoptosis, whereas the MBP group showed slight changes.. The 21.5 kDa MBP promotes the proliferation of HepG-2 and blocks apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Hep G2 Cells; Humans; Liver Neoplasms; Myelin Basic Protein; Nerve Tissue Proteins; Transcription Factors; Transfection

IRS-1 has been found to relay the signals from the receptors for insulin, insulin-like growth factor-1, growth hormone, and many cytokines for the downstream effects in the various cell types tested. For interleukin 4 signaling, most studies were performed on hematopoietic cells and cell lines transfected with rat liver IRS-1 cDNA. In a liver cell lineage, IRS-1 expression has been found to be increased in hepatoma cells and hepatocytes in regenerating liver. To elucidate the possible function and the signal transduction pathway for interleukin 4, in comparison with insulin, in liver cells, we used the Hep 3B hepatoma cell line as a model system. Following insulin and interleukin 4 stimulation, rapid tyrosyl phosphorylation of IRS-1 occurred. Interleukin 4, but not insulin, stimulated the tyrosine phosphorylation of JAK1 and, to a lesser extent, JAK2. In contrast to the other cell types, the association of IRS-1 and Grb2 through the SH2 of Grb2 was demonstrated after IL-4 and insulin stimulation of the Hep3B hepatoma cells. Both insulin and interleukin 4 stimulated tyrosine phosphorylation and the enzyme activity of Erk1 kinase. Our results indicate that interleukin 4 and insulin might modulate hepatic cell growth and differentiation through many different or common pathways for the activation of JAK kinases and the usage of IRS-1 as a docking protein. The binding of IRS-1 with Grb2 after IL-4 as well as insulin stimulation may lead to MAP kinase activation, probably through the Grb2/sos/p21ras pathway.

Topics: Adaptor Proteins, Signal Transducing; Antigens, CD; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Hepatocellular; Enzyme Activation; GRB2 Adaptor Protein; Humans; Insulin; Insulin Receptor Substrate Proteins; Interleukin-4; Janus Kinase 1; Janus Kinase 2; Liver; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myelin Basic Protein; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins; Receptors, Interleukin; Receptors, Interleukin-4; Signal Transduction; src Homology Domains; Tumor Cells, Cultured; Tyrosine