The role of bivalent cations and choline in ATP induced apoptosis via P2Z purinoceptor was investigated in human leukemic lymphocytes. In vitro exposure of leukemic lymphocytes with P2Z rec...The role of bivalent cations and choline in ATP induced apoptosis via P2Z purinoceptor was investigated in human leukemic lymphocytes. In vitro exposure of leukemic lymphocytes with P2Z receptors to 1 mmol/L ATP or 0 1 mmol/L benzoylbenzoic ATP(BzATP) for 8 h in the presence of choline, 1 mmol/L Mg 2+ or other bivalent cations, and ATP induced DNA breaks, associated with apoptosis were quantified by TdT assay. We observed that (1) Extracellular Mg 2+ or Ca 2+ stimulated ATP induced DNA fragmentation in a dose dependent manner, and the compatible evidence was provided by the inhibition of ATP induced DNA fragmentation in the present of EGTA or EDTA; (2) ATP induced DNA fragmentation was completely inhibited by 1 mmol/L Zn 2+ ;(3)ATP induced DNA breaks were not affected by Ba 2+ , Sr 2+ , Co 2+ when they were substituted for extracellular Mg 2+ or Ca 2+ ;(4)Choline, an inhibitor of phospholipase D(PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); (5)ATP induced DNA fragmentation was completely obliterated when the temperature was lower than 10℃. These results suggest that the endonuclease and PLD may be involved in ATP induced apoptosis in human lymphocytes via P2Z receptor.展开更多
The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors.These...The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors.These methods used were electron microscopy(EM), detection of internucleosomal DNA fragmentation by agarose gel electrophoresis, autoradiographic analysis of DNA fragmentation, in situ labeling of DNA strand breaks with fluorescein dUTP and exogenous terminal deoxynucleotidyl transferase (TUNEL), quantitation of 3 ends of DNA breaks by labeling with α 32 PdCTP(TdT assay), and quantitation of apoptotic cells with fluorescein annexin V using flow cytometry(FCA).We found EM and detection of DNA ladder pattern by agarose gel electrophoresis to be specific,but lacking in sensitivity. The combination of autoradiography and gel electrophoresis gave an increase in sensitivity of at least 50 fold although, of all the methods, the TdT assay was shown to be most sensitive. The four methods for quantifying apoptosis EM, FCA, TUNEL and TdT assay proved to be reliable and gave statistically similar results on apoptotic lymphocytes. These observations indicate it is essential to combine specific, sensitive and quantitative techniques in detecting apoptosis.展开更多
文摘The role of bivalent cations and choline in ATP induced apoptosis via P2Z purinoceptor was investigated in human leukemic lymphocytes. In vitro exposure of leukemic lymphocytes with P2Z receptors to 1 mmol/L ATP or 0 1 mmol/L benzoylbenzoic ATP(BzATP) for 8 h in the presence of choline, 1 mmol/L Mg 2+ or other bivalent cations, and ATP induced DNA breaks, associated with apoptosis were quantified by TdT assay. We observed that (1) Extracellular Mg 2+ or Ca 2+ stimulated ATP induced DNA fragmentation in a dose dependent manner, and the compatible evidence was provided by the inhibition of ATP induced DNA fragmentation in the present of EGTA or EDTA; (2) ATP induced DNA fragmentation was completely inhibited by 1 mmol/L Zn 2+ ;(3)ATP induced DNA breaks were not affected by Ba 2+ , Sr 2+ , Co 2+ when they were substituted for extracellular Mg 2+ or Ca 2+ ;(4)Choline, an inhibitor of phospholipase D(PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); (5)ATP induced DNA fragmentation was completely obliterated when the temperature was lower than 10℃. These results suggest that the endonuclease and PLD may be involved in ATP induced apoptosis in human lymphocytes via P2Z receptor.
文摘The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors.These methods used were electron microscopy(EM), detection of internucleosomal DNA fragmentation by agarose gel electrophoresis, autoradiographic analysis of DNA fragmentation, in situ labeling of DNA strand breaks with fluorescein dUTP and exogenous terminal deoxynucleotidyl transferase (TUNEL), quantitation of 3 ends of DNA breaks by labeling with α 32 PdCTP(TdT assay), and quantitation of apoptotic cells with fluorescein annexin V using flow cytometry(FCA).We found EM and detection of DNA ladder pattern by agarose gel electrophoresis to be specific,but lacking in sensitivity. The combination of autoradiography and gel electrophoresis gave an increase in sensitivity of at least 50 fold although, of all the methods, the TdT assay was shown to be most sensitive. The four methods for quantifying apoptosis EM, FCA, TUNEL and TdT assay proved to be reliable and gave statistically similar results on apoptotic lymphocytes. These observations indicate it is essential to combine specific, sensitive and quantitative techniques in detecting apoptosis.
