Studies On Key Signal Events And Relationship In T. Cuspidata Undergoing Apoptosis Induced By Ce～(4+)

In order to well-understand key downstream signal events and entire signaltransduction pathway of Ce⁴⁺-induced apoptosis of cultured Taxus cuspidata cells, weinvestigated a series of downstream proteases activation associated with apoptosis.Based on those results, the mechanism of apoptosis induced by Ce⁴⁺ was furtheranalyzed and imagined. From the work above we gained some valuable results.(1) That Ce⁴⁺ could induce cell apoptosis was confirmed by the methods offluorescence microscope and apoptotic rate analysis. In gel assay of nucleaseactivity demonstrated the activation of three nuclease, T. cuspidata nuclease 1, 2,3 (TNU1, 2, 3), during the apoptotic progress. Molecular weights of TNU1, 2, 3were 34kD, 21kD and19kD respectively. Activities of TNU1, 2, 3 werestimulated by Ca²⁺ or Mg²⁺ and inhibited by Zn²⁺. Those had pH optima in theneutral range ( pH 6.8 ). TNU1 had a little activity in the alkaline range (pH 7.5) ,and activities of TNU 2, 3 were absolutely inhibited.(2) Caspase-3-like protease was an important signal molecule during the apoptosisinduced by Ce⁴⁺. A caspase-3 inhibitor-sensitivity studies further revealed that itcould markedly reduce apoptosis in T. cuspidata cells. A total of 15 protein spotschanged in response to caspase-3-like protease activation were identified bytwo-dimensional gel electrophoresis. Protein abundance of 12 of the 15 changedspots in Ce⁴⁺ -induced cells with 100 μM Ac-DEVD-CHO showed somesimilarity with that of the control. The change of other three spots differed fromthose12 protein spots. Some protein spots were analyzed by MALDI-TOF-MS.(3) Ce⁴⁺ could induce superoxide anions (O₂^-·) transient burst. Strikingly, westernblotting indicated that the activity of p-ERK was inhibited in the apoptosis of T.cuspidata cells. O₂^-· burst and inhibition of p-ERK were two interactional signalevents , and O₂^-· burst could in part modulate the inhibition of p-ERK. The burstof O₂^-· exerted its activity as an upstream of caspase-3-like and nuclease activation.Activities of TNU 2, 3 depended on caspase-3-like protease activation, but TNU 1could be independent of caspase-3-like.(4) Some analysis had proposed that other proteases instead of caspase-3-like proteasewere the critical role during the apoptosis in T. cuspidata cells. Our results alsoimplicated that other signal pathways independent of an O₂^-· burst possiblyparticipated in mediating caspase-3-like protease activation.