Poly(ADP-ribose) polymerase activation and changes in Bax protein expression associated with extracellular ATP-mediated apoptosis in human embryonic kidney 293-P2X7 cells.
Extracellular ATP is a potent signaling factor that modulates a variety of cellular functions through the activation of P2 purinergic receptors. Extracellular ATP at higher concentrations exerts cytostatic as well as cytotoxic effects in a variety of cell systems, the mechanism of which is not fully understood. In this study, we used cultured human embryonic kidney (HEK) cells stably transfected with human P2X(7) receptors (HEK-P2X(7)) to investigate the mechanism of ATP-induced cell death. The cytotoxic effects of ATP in HEK-P2X(7) cells were dose- and time-dependent, whereas ADP, AMP, and UTP had no effect. ATP treatment induced a significant increase in apoptotic HEK-P2X(7) cells as ascertained by the terminal deoxynucleotidyl transferase dUTP nick-end labeling technique and flow cytometry. An ATP-induced decrease in the pro-apoptotic bax gene expression was detected by apoptosis-related cDNA microarray analysis, which correlated with a decrease of Bax protein expression. Western blot analysis revealed that ATP treatment resulted in the processing of pro-caspase 3 to its active form and cleavage of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). Both ATP-induced molecular alterations in HEK-P2X(7) cells (i.e., decrease of Bax expression and increase of PARP cleavage) were blocked by the purinergic P2X(7) receptor antagonist oxidized ATP. In conclusion, we demonstrated the importance of the P2X(7) receptor in ATP induced cell death of HEK-P2X(7) cells, which seems to be independent of bax expression; however, the activation of caspases is required.
Wen
Long T
LT
Microchemical Core Facility, Department of Biology, San Diego State University, San Diego, California 92182-1030, USA. lwen@sciences.sdsu.edu
Caldwell
Charles C
CC
Knowles
Aileen F
AF
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D018048:Receptors, Purinergic P2 / Q000502:physiology; D058486:Receptors, Purinergic P2X7; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C546468:P2RX7 protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D051028:bcl-2-Associated X Protein; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases