calyculin-a and Lymphoma--Large-B-Cell--Diffuse

Signal transduction pathways involved in apoptotic cell death are poorly understood, although recent studies have implicated sphingomyelin hydrolysis and generation of the second messenger, ceramide. Previous work in this laboratory demonstrated that a serine protease termed AP24 was activated by TNF or UV light and induced DNA fragmentation in isolated nuclei. This study extended these findings to examine the role of these enzymes in apoptosis of the U937 cell line and the mechanism of resistance of its variant, U9-TR. Although this subclone was selected by growth in TNF, it was unexpectedly found to resist apoptosis induced by UV light, but was still sensitive to anti-Fas-induced DNA fragmentation. Here we show that in contrast to normal U937 cells, UV light and TNF both failed to activate neutral or acidic sphingomyelinase or AP24 in the U9-TR variant. However, anti-Fas activated both neutral and acidic sphingomyelinase in the variant comparable to that seen in parental U937. The U9-TR variant could be sensitized to TNF or UV light activation of both sphingomyelinase and DNA fragmentation by the protein phosphatase inhibitors okadaic acid and calyculin A. Furthermore, exogenous bacterial-derived sphingomyelinase caused U9-TR activation of AP24 and DNA fragmentation comparable to that in the parental U937. Exposure of permeabilized U937 cells to ceramide caused internucleosomal DNA cleavage that was blocked by an inhibitor of AP24. Taken altogether, these findings demonstrate that TNF or UV light activate sphingomyelinase that leads to the generation of ceramide resulting in activation of AP24 and DNA fragmentation in sensitive cells. A selective defect in signals leading to sphingomyelinase activation can confer resistance to apoptosis even though the variant is still sensitive to downstream apoptotic signals such as nuclear DNA fragmentation by activated exogenous AP24.

Topics: Apoptosis; DNA; Enzyme Activation; Ethers, Cyclic; Humans; Lymphoma, Large B-Cell, Diffuse; Marine Toxins; Okadaic Acid; Oxazoles; Serine Endopeptidases; Sphingomyelin Phosphodiesterase; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Ultraviolet Rays

This study examined the role of protein phosphorylation in TNF induction of apoptosis in several tumor cell lines by testing the effects of agents that either stimulate or inhibit protein phosphorylation. The serine-threonine phosphatase inhibitors, okadaic acid (OKA) and calyculin A (CLA), synergistically augmented TNF-induced apoptosis in several TNF-sensitive tumor cell lines including the U937 histiocytic lymphoma, the BT-20 mammary carcinoma, and the LNCap prostatic tumor cell line. Furthermore, the phosphatase inhibitors completely reversed the TNF resistance of a variant (U9-TR) derived from U937. CLA also inhibited phosphatase activity in cell-free extracts from both U937 and U9-TR at the same concentrations (0.4-2.0 nM) that it synergized with TNF. In contrast, TNF treatment of U937 cells did not result in inhibition of phosphatase activity mediated by protein phosphatase 1 (PP1) and PP2A in cell extracts. Since the phosphatase inhibitors are known to increase the overall levels of protein phosphorylation in cells, this suggested that TNF may act by stimulating protein kinase (PK) activity. This hypothesis was supported by the results of testing a panel of relatively specific protein kinase inhibitors. TNF activation of DNA fragmentation was blocked by a potent inhibitor of myosin light chain kinase (MLCK) but was unaffected by inhibitors of cAMP or cGMP-dependent PKs. We postulate that a defect in the activation of MLCK or possibly some other as yet unknown PK may be responsible for the TNF resistance of U9-TR. Furthermore, this resistance may be circumvented by promoting protein phosphorylation with the serine-threonine-dependent phosphatase inhibitors.

Topics: Alkaloids; Apoptosis; Breast Neoplasms; Carbazoles; DNA; Drug Synergism; Ethers, Cyclic; Humans; Indoles; Lymphoma, Large B-Cell, Diffuse; Male; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Prostatic Neoplasms; Protein Kinase Inhibitors; Recombinant Proteins; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha