PD98059 attenuates hydrogen peroxide-induced cell death through inhibition of Jun N-Terminal Kinase in HT29 cells.
We have investigated the effects of hydrogen peroxide (H(2)O(2)), a potent naturally occurring oxidant on cell signaling and viability in the pluripotent HT29 intestinal cell line. There was a dose-dependent reduction in cell viability upon exposure to H(2)O(2) as measured by the XTT assay. Features of apoptosis were indicated by the findings of PARP and caspase 3 cleavage, as well as changes in cell morphology using phase contrast and nuclear fragmentation using fluorescence microscopy. There was a dose-dependent increase in the activation of p45-JNK, p42/p44-ERK, and p38-HOG. Surprisingly, oxidant-induced cell injury could be attenuated by preincubation with PD98059 to 50% of untreated control cells (P = 0.002). This and UO126, another MEK inhibitor were ably to reproducibly inhibit p45-JNK activation induced by hydrogen peroxide. Transfection with kinase-inactive constructs of JNK and ERK revealed that the improvement in cell viability was due to inhibition of JNK and not ERK. Transient transfections with AP-1 and NF-kappaB luciferase reporter constructs did not reveal any transcriptional activation due to hydrogen peroxide exposure however, in both cases the basal levels of transcriptional activity were suppressed in the presence of PD98059. It is concluded that JNK mediates H(2)O(2)-induced cellular injury in the HT29 cell line, and additionally, we report for the first time that JNK activation can be inhibited by both PD98059 and UO126 at conventional doses used to inhibit MEK.
Salh
B S
BS
Jack Bell Research Centre, 2660 Oak Street, Vancouver, British Columbia, Canada V6H 3Z6. bsalh@interchange.ubc.ca
Martens
J
J
Hundal
R S
RS
Yoganathan
N
N
Charest
D
D
Mui
A
A
Gómez-Muñoz
A
A
D002070:Butadienes / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D014162:Transfection
D002070:Butadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009570:Nitriles; D016877:Oxidants; C113580:U 0126; D006861:Hydrogen Peroxide; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one