Apoptotic events induced by the phosphatase inhibitor okadaic acid in normal human lung fibroblasts.
We have studied different biochemical indicators of apoptosis in okadaic acid-treated normal human lung fibroblasts (NHLF). Apoptosis was identified by fluorimetric microplate measurements of DNA content, caspase-3 activation and changes in mitochondrial and plasma membrane after 1-48-h treatments with 1-1000 nM okadaic acid. Cells exposed to okadaic acid showed activation of caspase-3, decreased DNA content (<50% of controls at >100 nM okadaic acid after 12 h of incubation) and translocation of phosphatidylserine to the outer leaflet of the plasma membrane, as indicated by the increase in Merocyanine 540 fluorescence after 4 h of incubation with more than 250 nM okadaic acid. Decreased mitochondrial membrane potential (53-98% of controls) was observed with MitoTracker Red CMXRos in all cases, which indicated an active role of mitochondria during the early phase of apoptosis. However, reactive oxygen species were significantly reduced in okadaic acid-treated fibroblasts (50-70% of controls at 1000 nM after 3 h of incubation), which indicates that ROS cannot be considered as a hallmark of apoptosis in okadaic acid-treated cells. These results provide evidence of apoptotic events induced by okadaic acid in NHLF, which can be detected by means of sensitive and reliable fluorimetric microplate assays.
Leira
F
F
ANFACO-CECOPESCA, Campus Universitario de Vigo, 36310, Vigo, Spain. fleira@anfaco.cesga.es
Vieites
J M
JM
Vieytes
M R
MR
Botana
L M
LM
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay; D005470:Fluorometry; D006801:Humans; D008168:Lung / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013997:Time Factors
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D011933:Reagent Kits, Diagnostic; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases