[Bcl-xl blocks tumor necrosis factor alpha-induced caspase 8 activation and apoptosis].
OBJECTIVE
To explore the effect of Bcl-xl on tumor necrosis factor-alpha (TNFalpha)-induced apoptosis signal pathway and apoptosis.


METHODS
A dominant negative mutant of ikB (pmi kappaB) and Green Fluorescent Protein (GFP) expression plasmid pEGFP-C1, pmi kappab and pEGFP-C1 and Bcl-xl expression construct pBcl-xl/HA, were co-transfected into HeLa cells. Expression plasmid pBcl-xl/HA was introduced into p65-/-MEF cells in which nuclear factor-kappaB (NF-kappaB)/p65 was deficient, to establish cell line p65-/-Bcl-xl expressing Bcl-xl by selection with puromycin. These cells were treated with TNFalpha at a concentration of 10 ng/ml, and apoptotic cell death was examined microscopically with trypan blue staining. The proteins were abstracted from treated cells, and caspase 8 activation and cleavage of poly (ADP-ribose) polymerase (PARP) were examined by western blot using a specific antibody that recognized cleaved caspase 8 and cleaved PARP, respectively.


RESULTS
HeLa cells transfected with pmi kappaB, TNFalpha showed significant cell death as they became rounded, shrank, and detached. However in HeLa cells co-transfected with pBcl-xl and pmi kappaB, no cell death was observed after treatment with TNFalpha. In p65-/- MEF cells; cell death was observed at 4 hours after treatment with TNFalpha, and cell death reached 90% at 12 hours after the treatment. However, in p65-/-Bcl-xl/HA cells expressing Bcl-xl, no cell death was seen even when treated with TNFa for 24 hours. Meanwhile, in pmikB/HeLa cells transfected with pmi kappaB, TNFalpha induced caspase 8 activation and PARP cleavage, but in the HeLa cells co-transfected with pBcl-xl and pmi kappaB, no activated caspase 8 and cleaved PARP were observed after treatment with TNFalpha.


CONCLUSIONS
In the experimental system in which NF-kB was inhibited, Bcl-xl blocked TNFalpha-induced apoptosis signal pathway and apoptosis. These results bring to light that further studies of the pathogenesis and therapy of TNFa-related diseases are needed.
Yang
Lin
L
Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China. linyang992hotmail.com
Deng
Yi-Bin
YB
Wu
Xiang-Wei
XW
D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D051020:bcl-X Protein / Q000494:pharmacology*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D051020:bcl-X Protein; D053181:Caspase 8