9-(tetrahydro-2-furyl)-adenine and Leukemia--T-Cell

The expression by T lymphocytes (T cells) of more than one of the functionally distinct subtypes of prostaglandin E2 (PGE2) receptors (Rs), designated EP1, EP2, EP3, and EP4 Rs, is a principal determinant of specificity and diversity of the immune effects of PGE2. The cultured line of human leukemic T cells, termed HSB.2, co-expresses a total of 7282 +/- 1805 EP3, EP4, and EP2 Rs per cell with a Kd of 3.7 +/- 1.4 nM (mean +/- S.E., n = 9). The EP3/EP1 R-selective agonist sulprostone, EP3/EP2/EP4 R-selective agonists M&B 28767 and misoprostol, and EP2 R-selective agonist butaprost but not the EP1 R-selective antagonist SC-19220 competitively inhibited the binding of [3H]PGE2 to HSB.2 cells. Stimulation of increases in the intracellular concentration of cyclic AMP ([cAMP]i) by PGE2, misoprostol, and butaprost and of increases in the intracellular concentration of calcium ([Ca2+]i) by PGE2 and sulprostone demonstrated the respective involvement of EP2/EP4 Rs and EP3 Rs in transduction of biochemical signals. Matrix metalloproteinase (MMP)-9 was identified by zymography and Western blots as the principal MMP secreted by HSB.2 cells. The cytosolic level and secretion of MMP-9 were increased maximally after 24 h of incubation of HSB.2 cells with 10(-8)-10(-6) M PGE2, sulprostone, M&B 28767, and misoprostol but not with 10(-6) M PGF2alpha, PGD2, PGI2, or butaprost, suggesting a principal dependence on EP3 Rs. That stimulation of MMP-9 secretion by PGE2 was not diminished in Ca2+-free medium but was suppressed significantly and dose-dependently by thapsigargin, an inhibitor of endomembrane Ca2+-ATPase, suggested that MMP-9 expression by HSB.2 cells is mediated by increases in [Ca2+]i attributable to release of Ca2+ from intracellular stores. The lack of effect of dibutyryl cAMP, forskolin, and SQ 22536, an adenylyl cyclase inhibitor, on MMP-9 secretion by HSB.2 cells argued against any role for cAMP-dependent mechanisms linked to EP2/EP4 Rs. Cycloheximide and actinomycin D, which respectively inhibited protein and RNA synthesis, suppressed basal and PGE2 induction of MMP-9 production by HSB.2 cells. Northern analysis indicated that PGE2 and sulprostone time-dependently increased expression of MMP-9 mRNA. Thus, stimulation of MMP-9 in HSB.2 T cells by PGE2 is attributable to [Ca2+]i-dependent EP3 R-mediation of increases in message transcription.

Topics: Adenine; Alprostadil; Blotting, Northern; Bucladesine; Calcium; Cell Line; Colforsin; Collagenases; Cyclic AMP; Cycloheximide; Dactinomycin; Dinoprostone; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leukemia, T-Cell; Matrix Metalloproteinase 9; Misoprostol; Prostaglandins E, Synthetic; Receptors, Prostaglandin E; RNA, Messenger; T-Lymphocytes; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured