thapsigargin and Leukemia-Lymphoma--Adult-T-Cell

The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.

Topics: Calcium; Calcium Channels; Depression, Chemical; Enzyme Inhibitors; Extracellular Space; Genistein; Humans; Intracellular Fluid; Ionomycin; Ionophores; Isoflavones; Leukemia-Lymphoma, Adult T-Cell; Muromonab-CD3; Neoplasm Proteins; Phenols; Phosphatidylserines; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Terpenes; Thapsigargin; Tumor Cells, Cultured

Thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, depletes intracellular Ca2+ stores and induces a sustained Ca2+ influx without altering phosphatidyl inositol levels. TG plus phorbol myristate acetate (PMA) but not TG alone induced IL-2 in Jurkat T cells, suggesting that TG had no effect on protein kinase C (PKC). However, TG induced increases in IL-2R alpha protein as well as IL-2R alpha mRNA in Jurkat T cells in a dose-dependent manner. A similar increase in IL-2R alpha by TG was also observed in human peripheral T cells. Further, like PMA, TG markedly induced NF kappa B in Jurkat T cells. However, TG and PMA exhibited a synergistic action on IL-2R alpha expression, suggesting that TG and PMA induce IL-2R alpha through distinct pathways. PMA- but not TG-induced IL-2R alpha is inhibited by the PKC inhibitor H7, whereas TG- but not PMA-induced IL-2R alpha was inhibited by cholera toxin, forskolin and 1,9-dideoxy forskolin. In toto, these results suggest that TG induces IL-2R alpha in human T cells through a PKC-independent pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Base Sequence; Calcimycin; Calcium; Calcium-Transporting ATPases; Cholera Toxin; Colforsin; Drug Synergism; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Humans; Hydroquinones; Isoquinolines; Leukemia-Lymphoma, Adult T-Cell; Molecular Sequence Data; Neoplasm Proteins; NF-kappa B; Piperazines; Protein Kinase C; Receptors, Interleukin-2; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Staurosporine; T-Lymphocytes; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

The regulation by protein kinase C (PKC) of TCR-mediated changes in phosphoinositide metabolism and intracellular calcium ([Ca2+]i) was investigated in HPB-ALL T cells. Low concentrations (< 1 microgram/ml) of the anti-CD3 OKT3 mAb triggered large calcium signals but not detectable increase in D-myo-inositol 1,4,5-trisophate (IP3) production. CD3-CD4 coligation amplified the calcium signal twofold, compared with CD3 cross-linking alone, but this protocol also did not stimulate IP3 production. At higher OKT3 concentrations (> 2.5 micrograms/ml), IP3 production was detected but was not inhibited by activating PKC with phorbol ester. In contrast, PKC activation caused a marked inhibition (53 to 64%) of the CD3- or CD3-CD4-triggered calcium signals, but had only a small inhibitory effect (20 to 30%) on the release of intracellular Ca2+. PKC activation also inhibited by 47% calcium signals triggered by thapsigargin, an inhibition that was completely reversed by addition of the specific PKC inhibitor RO 31-8220 (1 microM). Addition of 1 microM RO 31-8220 caused a twofold stimulation of CD3-induced calcium signals. This effect was not mediated at the level of Ca2+ influx, because RO 31-8220 did not significantly increase thapsigargin-triggered calcium signals. However, RO 31-8220 did slightly increase the CD3-induced release of intracellular Ca2+, suggesting that amplification of Ca2+ influx may be secondary to increased release of Ca2+ from intracellular stores. Our results indicate that PKC regulates TCR-mediated changes in [Ca2+]i in HPB-ALL T cells by two distinct mechanisms. First, PKC activation causes a marked inhibition of Ca2+ influx by a mechanism independent of changes in IP3 production, possibly involving inhibition of ion channels. Second, PKC activity causes a small inhibition of intracellular Ca2+ release, most likely by promoting Ca2+ sequestration.

Topics: Calcium; Calcium-Transporting ATPases; Enzyme Activation; Humans; Indoles; Inositol 1,4,5-Trisphosphate; Leukemia-Lymphoma, Adult T-Cell; Microtubule Proteins; Phorbol 12,13-Dibutyrate; Phosphoproteins; Phosphorylation; Protein Kinase C; Receptors, Antigen, T-Cell; Signal Transduction; Stathmin; T-Lymphocytes; Terpenes; Thapsigargin; Tumor Cells, Cultured