okadaic-acid and Multiple-Myeloma

To understand the roles of reactive oxygen intermediates (ROI) in Fas-mediated apoptosis of myeloma cells, the effects of antioxidants were tested. Fas-mediated apoptosis was further increased in the presence of antioxidants such as N-acetyl-L-cysteine and glutathione, but it was decreased when hydrogen peroxide was added. The intracellular ROI level was significantly decreased in myeloma cells treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A). To clarify the direct roles of PP2A in myeloma cell growth, the PP2A transfected cell lines, sense- or antisense-PP2A transfectants, were established. Spontaneous cell growth of antisense-PP2A transfectants was reduced compared with that of vector transfectants. The intracellular ROI level was significantly decreased in antisense-PP2A transfectants but increased in sense-PP2A transfectants compared with vector controls. In addition, anti-apoptotic factors such as bcl-2 and IL-6 were reduced in antisense-PP2A transfectants. Taken together, these results indicate that PP2A is an essential factor for survival and growth of myeloma cells via regulation of intracellular ROI and anti-apoptotic factors.

Topics: 3T3 Cells; Acetylcysteine; Animals; Antioxidants; Apoptosis; Cell Division; Enzyme Inhibitors; Gene Expression; Glutathione; Humans; Interleukin-6; Intracellular Fluid; Mice; Multiple Myeloma; NF-kappa B; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Transfection; Tumor Cells, Cultured

Okadaic acid (OA) is a potent inhibitor of PP1 and PP2A serine/threonine phosphatases and an inhibitor of phosphatidylinositol 3'-kinase (PI 3-kinase) recruitment/ activation. Here we report that PI 3-kinase associates with a serine kinase activated by OA. Whole cell phosphorylation studies showed that PI 3-kinase associates with a wortmannin insensitive 76 kDa serine phosphoprotein (pp76) distinct from the p85 subunit of PI 3-kinase. Serine kinase assays demonstrated that pp76 phosphorylation was dependent upon a wortmannin insensitive serine kinase contained within PI 3-kinase/pp76 complexes and that this kinase had different cation requirements than PI 3-kinase serine kinase. Treatment of whole cells with OA lead to a wortmannin-independent 7.6-fold increase in pp76 serine phosphorylation and to a 7-fold rise in pp76 kinase activity. Together, these findings indicate that pp76 is a PI 3-kinase associated phosphoprotein and suggest that pp76 may be a novel PI 3-kinase associated serine kinase that is activated by OA.

Topics: Androstadienes; Blotting, Western; Enzyme Inhibitors; Humans; Multiple Myeloma; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Phosphoserine; Precipitin Tests; Protein Serine-Threonine Kinases; Tumor Cells, Cultured; Wortmannin

Recently, there has been an increasing interest in the expression pattern and biological significance of the CD45 molecule in myeloma cells. In this study, we have further defined the phenotypic pattern of CD45 expression on myeloma cells. Using a panel of myeloma cell lines, we showed that CD45 showed a remarkably heterogeneous pattern of expression. Whereas some cell lines were CD45(+) and others were CD45(-), the U-266 cell line, although predominantly CD45(-), still had a considerable subpopulation of CD45(+) cells. Among the myeloma cell lines examined, there was a direct correlation between interleukin-6 (IL-6) dependency and CD45 positivity. Moreover, we showed that IL-6 stimulation led to the induction of expression of CD45 and cellular proliferation. Using independent experimental approaches, we could show that the IL-6-induced effects were closely linked to CD45 expression. First, sorting out CD45(+) and CD45(-) subsets of U-266 cell line followed by IL-6 stimulation, only the CD45(+) cells showed a proliferative advantage after IL-6 stimulation. Second, IL-6 stimulation of sorted CD45(-) cells was gradually followed by phenotypic conversion to CD45(+) cells that started after 2 days as judged by the detection of CD45 mRNA by reverse transcription polymerase chain reaction (RT-PCR) and immunophenotypic analysis by flow cytometry. Withdrawal of IL-6 from the medium led to gradual loss of CD45 expression in CD45(+) flow-sorted U-266 cells. Third, the use of vanadate, a potent inhibitor of protein tyrosine phosphatase (PTP), abrogated the IL-6-induced proliferation in the CD45(+) myeloma cells. On the other hand, cellular proliferation induced by IL-6 was not affected by the serine-threonine phosphatase inhibitor okadaic acid. Our data show that the expression pattern of CD45 in myeloma cell lines is heterogeneous and show for the first time that CD45 expression can be induced by IL-6 stimulation. Finally, these data shed some light on the biological role of CD45 in myeloma by determining the proliferative population among myeloma cells.

Topics: Antigens, Neoplasm; Cell Division; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Leukocyte Common Antigens; Multiple Myeloma; Neoplasm Proteins; Neoplastic Stem Cells; Okadaic Acid; Phosphoprotein Phosphatases; Protein Tyrosine Phosphatases; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured; Vanadates

Deregulation of IL-6 production is one of the major causes for human multiple myeloma. Exogenous IL-6 stimulated the proliferation of fresh human myeloma cells and the myeloma cell line, U266, which produced IL-6 spontaneously. Anti-IL-6 antibody and IL-6 antisense oligonucleotide suppressed the IL-6 stimulated myeloma cell proliferation, indicating that IL-6 induced the myeloma cell proliferation via an autocrine loop. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, inhibited the U266 cell proliferation at a concentration of less than 1 ng/ml. At this concentration, okadaic acid suppressed the IL-6-induced IL-6 gene expression of myeloma cells. It seems that the okadaic acid blocked the myeloma cell proliferation by reducing IL-6 synthesis in myeloma cells. In addition, IL-6 itself also regulated IL-6 receptor expression. Analysis by FACScan and RT-PCR showed that anti-IL-6 antibody treatment up-regulated IL-6 receptor expression. Interestingly, the presence of okadaic acid induced the up-regulation of IL-6 receptor expression as well as the down-regulation of IL-6-induced gp130 phosphorylation in the myeloma cells. Taken together, these data suggest that protein phosphatase 1 and 2A are involved in IL-6-mediated autocrine growth of human myeloma cells by modulating IL-6 signaling and IL-6 receptor expression in myeloma cells.

Topics: Animals; Antigens, CD; Autoreceptors; Base Sequence; Cell Division; Enzyme Inhibitors; Ethers, Cyclic; Gene Expression Regulation, Neoplastic; Humans; Hybridomas; Interleukin-6; Mice; Multiple Myeloma; Neoplasm Proteins; Okadaic Acid; Oligonucleotides, Antisense; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Processing, Post-Translational; Receptors, Interleukin; Receptors, Interleukin-6; Signal Transduction; Sulfonamides; Tumor Cells, Cultured; Up-Regulation