Abstract
Apoptosis can be induced by various stimuli including DNA-damaging anticancer drugs and chemical agents such as the protein kinase inhibitor staurosporine. To investigate the expression of apoptosis and cell cycle regulating proteins the lymphoma cell lines JURKAT (non-EBV transformed T-ALL) and EHEB (EBV-transformed B-CLL) were incubated with staurosporine. FACS analyses were performed with double staining of Annexin PE-V/7AAD to determine the rates of staurosporine induced apoptosis and for detection of active Caspase-3 after 24 hours and 48 hours. Similar rates of apoptosis which were achieved with lower concentrations of staurosporine in JURKAT (0.125μM-0.5μM vs. 0.5μM-2μM in EHEB). Expression changes after staurosporine treatment were examined for the following proteins: procaspase-8, procaspase-9, Apaf-1, active caspase-3, PARP, CDK4, CDK2, Survivin, p21, p27, BCL-2, BAX, Cyclin-D1/D2/D3, Rb, cIAP2, XIAP, and Akt1 by Western blotting. Cleavage of procaspase-8 and procaspase-9 was observed in both cell lines upon treatment. In JURKAT, subsequent activation of caspase-3 could be detected by Western Blotting as well as by FACS. In contrast, no active caspase-3 was detected in treated EHEB cells by Western blotting and only moderate activation was observed by FACS, although PARP-cleavage was clearly detected in both cell lines by Western blotting. Apoptotic regulators were differentially regulated when comparing treated JURKAT and EHEB cells. Treatment of JURKAT cells led to an up-regulation of BCL-2 and down-regulation of Akt1 and BAX, but not to expression changes of XIAP and Apaf-1. In contrast, XIAP and Apaf-1 were down-regulated in EHEB upon treatment, whereas no change in protein levels was observed for BCL-2 and BAX. Furthermore, differences between the two cell lines in response to staurosporine treatment were observed for the cell cycle proteins p27, p21, CDK4, Cyclin-D1/D2/D3 and Rb. Down-regulation of p27, p21 and Cyclin-D1 and up-regulation of Cyclin-D3 was only seen in treated EHEB cells. In the opposite, JURKAT showed up-regulation of Cyclin-D1 and down-regulation of Cyclin-D3 and CDK4 upon treatment. Interestingly, in EHEB Cyclin-D2 was initially down-regulated (after 24 hours) followed by an up-regulation later on. Both cell lines responded with cleavage of Rb upon treatment. Levels of cIAP2, Survivin and CDK2 were not altered in either cell line. In summary, characteristic responses to staurosporine treatment were detected in EHEB and JURKAT. In both cell lines apoptosis induction resulted in a cleavage of Rb despite opposite effects on Cyclin-D1 and Cyclin-D3 expression. The most striking difference in response to staurosporine incubation was a PARP cleavage in EHEB cells without significant activation of caspase-3 or alteration in BCL-2 expression in combination with a higher resistance to apoptosis induction by staurosporine when compared to JURKAT. A previous study indicated that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and an elevated expression of BCL-2 that is not altered by etoposide treatment. Therefore, the distinct protein expression response of EHEB to staurosporine treatment might be in part a result of its immortalization by EBV transformation. Further analyses are in progress to elucidate the response of lymphoma cell lines to fludarabine and etoposide.
Oxidative Stress Activates the Transcription Factors FoxO 1a and FoxO 3a in the Hippocampus of Rats Exposed to Low Doses of Ozone