Vol. 63, Issue 3, 706-713, March 2003

Poly(ADP-ribose) Polymerase Activation and Changes in Bax Protein Expression Associated with Extracellular ATP-Mediated Apoptosis in Human Embryonic Kidney 293-P2X₇ Cells

Long T. Wen, Charles C. Caldwell, and Aileen F. Knowles

Microchemical Core Facility, Department of Biology (L.T.W.) and Department of Chemistry (A.F.K.), San Diego State University, San Diego, California; and Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland (C.C.C.)

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 P2X₇ receptors (HEK-P2X₇) to investigate the mechanism of ATP-induced cell death. The cytotoxic effects of ATP in HEK-P2X₇ cells were dose- and time-dependent, whereas ADP, AMP, and UTP had no effect. ATP treatment induced a significant increase in apoptotic HEK-P2X₇ 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-P2X₇ cells (i.e., decrease of Bax expression and increase of PARP cleavage) were blocked by the purinergic P2X₇ receptor antagonist oxidized ATP. In conclusion, we demonstrated the importance of the P2X₇ receptor in ATP induced cell death of HEK-P2X₇ cells, which seems to be independent of bax expression; however, the activation of caspases is required.