Inhibition of NF-κb by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-XL expression
- Author:
- No author available
- Journal Title:
- CHINESE MEDICAL JOURNAL
- Issue:
- 7
- DOI:
- No doi available
- Key Word:
- mutant IκBα;nuclear fector-κB;TNF-α;cell line,HL-60;apoptosis;bcl-xL
Abstract： Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB) activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism.Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. The cytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner.Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the anti-leukemia effects of TNF-α or even of other cytotoxic agents.
- [1]Daniel J. Van Antwerp, Seamus J. Martin, Tal Kafri, Douglas R. Green, Inder M. Verma.Suppression of TNF-α-Induced Apoptosis by NF-κB[J].1996,274 (5288).
- [2]Nakamura H,Sumitomo M,Murai M,etc.An essential role for nuclear factor kappa B in preventing TNF-alpha-induced cell death in prostate cancer cells.[J].1999,161(2).
- [3]Wang, Cun-Yu,Mayo, Marty W..TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kappaB.[J].1996,274(5288).
- [4]Amer A. Beg, David Baltimore.An Essential Role for NF-κB in Preventing TNF-α-Induced Cell Death[J].1996,274 (5288).
- [5]A A, Beg,W C, Sha,R T, Bronson,etc.Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B.[J].1995,376(6536).
- [6]Albert S. Baldwin,Jr..THE NF-κB AND IκB PROTEINS: New Discoveries and Insights[J].1996,14.
- [7]Baldwin AS Jr,Liu R,Wang CY,etc.Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB.[J].1999,5(4).
- [8]Aggarwal BB,Haridas V,Manna SK.Bcl-x(L) suppresses TNF-mediated apoptosis and activation of nuclear factor-kappaB, activation protein-1, and c-Jun N-terminal kinase.[J].2000,20(8).
- [9]Ho H. Lee,Hajir Dadgostar,Qingwen Cheng,etc.NF-κB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes[J].1999,96(16).
- [10]Ghosh,S,May,MJ,Kopp,EB.NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses.[J].1998,16.
- [11]Chen C,Gelinas C,Edelstein LC.The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L).[J].2000,20(8).
- [12]Chen F.,Demers LM.,Castranova V.,etc.Involvement of 5 '-flanking kappa B-like sites within bcl-x gene insilica-induced Bcl-x expression[J].1999,274(50).
- [13]Cun-Yu Wang,Marty W. Mayo,Robert G. Korneluk,etc.NF-κB Antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to Suppress Caspase-8 Activation[J].1998,281 (5383).