myelin-basic-protein and edelfosine

Guanine nucleotide regulatory proteins (G-proteins) represent an important transmembrane pathway whereby extra-cellular signals are transduced to intracellular signaling pathways. The mitogen-activated protein kinase (MAPK) cascade has been identified as a key factor in transducing numerous mitogenic stimuli. MAPK activity is regulated via numerous receptor types, including those linked to Gq/G11-proteins, which regulate phospholipase-C activity. We hypothesized that alterations in a Gq/G11-PLC pathway may contribute to the enhanced cellular mitogenesis characteristic of hepatocellular carcinoma (HCC), possibly via a MAPK-dependent pathway. By using an in vivo model of HCC we investigated changes in Gq/G11-protein expression in tumorigenic tissue versus adjacent, non-neoplastic liver. In addition we addressed the role of Gq/G11-proteins in the regulation of MAPK-linked mitogenesis by using rat hepatic tumorigenic cells (H4IIE) and isolated hepatocytes in culture. Western blot analysis showed significant increases in Gqalpha and G11alpha expression in tumorigenic liver versus normal liver specimens, an effect that was augmented in cultured H4IIE cells versus isolated cultured hepatocytes. Furthermore, phosphoinositol specific phospholipase-C (PLC) activity was significantly increased in HCC versus normal liver. A specific PLC inhibitor (Et-18-OCH3) caused a dose-dependent decrease in serum stimulated DNA synthesis in both cultured H4IIE cells and isolated rat hepatocytes, the H4IIE cell line showing greater sensitivity to Et-18-OCH3. In addition, serum-stimulated MAPK activity was significantly enhanced in H4IIE versus cultured hepatocytes. Moreover, treatment with Et-18-OCH3 significantly attenuated serum stimulated MAPK activity in both cultured hepatocytes and H4IIE cells. Furthermore, U73122 (Gqalpha-PLC specific uncoupler) and GP2A (Gqalpha specific inhibitor) mirrored the effects of those observed for Et-18-OCH3 whereas PD98059 (specific MEK inhibitor) completely abolished serum-stimulated DNA synthesis in tumorigenic H4IIE cells. We conclude that HCC is associated with enhanced Gq/G11-PLC expression/activity as compared with normal liver. Furthermore, a PLC-linked MAPK cascade plays a significant role in the progression of the enhanced mitogenesis characteristic of HCC.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; DNA; GTP-Binding Proteins; Immunoblotting; Liver Neoplasms, Experimental; Male; Mitogen-Activated Protein Kinase 1; Myelin Basic Protein; Phosphodiesterase Inhibitors; Phospholipid Ethers; Phosphorylation; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred ACI; Type C Phospholipases

Chronic relapsing experimental allergic encephalomyelitis (CR.EAE) was induced by immunizing Lewis rats with total guinea-pig spinal cord (GPSC) tissue emulsified in enriched complete Freund's adjuvant (CFA). The proliferative responses of draining inguinal and popliteal lymph node cells to GP.MBP, purified protein derivative (PPD) and concanavalin A (ConA) appeared significantly modulated according to the clinical state of the animals. Responses appeared significantly decreased in both lymphoid compartments during the recovery periods compared with that during relapses. Therapeutic treatment of CR.EAE with cyclosporin and different lysolecithin derivatives, such as ET-18-OCH3, SRI 62-843 and MLS 266-337, starting at the spontaneous remission of the first disease bout, could suppress the manifestation of further relapses. Whereas cyclosporin only delayed the onset of the disease relapse until discontinuation of treatment, all lysolecithins showed a curative effect in most animals. Plasma corticosterone levels measured at different time points in placebo, cyclosporin and MLS 266-377-treated rats showed a strong correlation with the clinical state of the animals. High corticosterone levels were detected during stages of acute paralysis, whereas a decrease to normal levels was noted during each recovery phase.

Topics: Animals; Autoimmune Diseases; Concanavalin A; Corticosterone; Cyclosporine; Dexamethasone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Furans; Guinea Pigs; Lymph Nodes; Lymphocyte Activation; Lysophosphatidylcholines; Multiple Sclerosis; Myelin Basic Protein; Phospholipid Ethers; Rats; Rats, Inbred Lew; Tuberculin

Alkyllysophospholipids are synthetic analogues of natural phospholipids possessing a high immunomodulating and antitumoral capacity. Experimental autoimmune encephalomyelitis is a model disease for multiple sclerosis which can be induced by injecting rats with myelin basic protein, MBP. The effect of one alkyllysophospholipid, ET-18-OCH3, on the course of experimental autoimmune encephalomyelitis was investigated. It was found that animals treated with ET-18-OCH3 showed only weak signs of disease. MBP specific T-cell lines were co-cultivated with ET-18-OCH3. The compound suppressed T-cell proliferation markedly, suggesting that this might be its mode of action in vivo. Since ET-18-OCH3 has only low toxicity in man, it could be of interest to perform further studies on its effects on autoimmune, demyelinating disease.

Topics: Adjuvants, Immunologic; Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Lymphocyte Activation; Myelin Basic Protein; Phospholipid Ethers; Rats; Rats, Inbred Lew; T-Lymphocytes

The substrate specificity determinants of protein kinase C are probed using synthetic peptides encompassing the major phosphorylation site serine 115 in bovine MBP. The results indicate that basic residues arginine 107 and 113 N-terminal to the phosphorylation site are essential for the substrate activity of the peptides. Substitutions of these basic residues by alanine cause a marked decrease in their substrate activity and the resulting peptide analogs become specific and rather potent inhibitors of protein kinase C. Leukemic cells are particularly abundant in protein kinase C and its substrate proteins, and the enzyme system has been shown to play a key role in cell growth. The agents that stimulate protein kinase C include tumor promoting phorbol esters (such as TPA) and mezerein, and the putative second messenger diacylglycerol. Many antineoplastic agents, on the other hand, inhibit the enzyme which include adriamycin, tamoxifen, alkyl-lysophospholipid, selenium, retinal and lipoidal amine CP-46, 665-1. Immunocytochemical studies of protein kinase C in leukemic cells indicate that it is localized in the plasma membrane, cytoplasm, nucleus and Golgi apparatus, and the subcellular distribution of the enzyme might be related to the phases of the cell cycle. TPA induces translocation of the enzyme to plasma membrane, suggesting an additional mode of action for the tumor promotor.

Topics: Animals; Antineoplastic Agents; Electrophoresis, Polyacrylamide Gel; Estrogen Antagonists; Humans; Kinetics; Leukemia, Experimental; Lysophosphatidylcholines; Microscopy, Electron; Myelin Basic Protein; Peptides; Phospholipid Ethers; Protein Kinase C; Rats; Substrate Specificity