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Fig. 8. TNF
induces activation of caspase 8. (A) Time course of activation of caspase 8 in LNCaP cells infected with Ad-Control (
) or Ad-Cx43 (
) after treatment with 10 ng/ml TNF for variable lengths of time. Caspase 8 activity was significantly greater in the Ad-Cx43-infected cells as compared to those infected with control virus at 30 minutes, 1 hour and 3 hours (P<0.01). (B) Bar graph showing the effects of pre-treatment with TNFR neutralizing antibodies (10 µg/ml) upon the TNF-induced caspase 8 activation in LNCaP cells infected with Ad-Control (black bars) or Ad-Cx43 (gray bars). Ab-R1 and Ab-R2 indicate antibodies directed against TNFR1 and TNFR2, respectively. Ad-Control-infected LNCaP cells showed a small increase in caspase 8 activity that was abolished by pretreatment with anti-TNFR1 antibodies (P=0.0183 for Ad-Control + TNF vs Ad-Control alone; P=0.0381 for Ad-Control + TNF vs Ad-Control + TNF + Ab-R1; and not significant for Ad-Control + TNF + Ab-R1 vs Ad-Control alone). Ad-Cx43-infected cells showed a much larger increase in caspase 8 activity that was abolished by pretreatment with anti-TNFR1 antibodies or anti-TNFR1 plus anti-TNFR2 antibodies (P<0.0001 for Ad-Cx43 + TNF vs Ad-Cx43 alone or Ad-Cx43 + TNF +Ab-R1 or Ad-Cx43 + TNF +Ab-R1 +Ab-R2; Ad-Cx43 + TNF +Ab-R1 or Ad-Cx43 + TNF +Ab-R1 +Ab-R2 did not significantly differ from Ad-Cx43 alone). Pretreatment of Ad-Cx43-infected cells with anti-TNFR2 antibodies partially antagonized the TNF-induced activation of caspase 8, since it significantly reduced the caspase 8 activity as compared to Ad-Cx43-infected cells treated with TNF (P=0.0009), but levels were still much higher than those in untreated Cx43-expressing cells (P<0.0001).
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