ABSTRACT
<p><b>BACKGROUND</b>The aim of this study was to explore whether the inhibition of nuclear factor-kappaB (NF-kappaB) activation by mutant IkappaBalpha (S32, 36-->A) can enhance TNF-alpha-induced apoptosis of leukemia cells and to investigate the possible mechanism.</p><p><b>METHODS</b>The mutant IkappaBalpha gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IkappaBalpha were obtained by the limiting dilution method. TNF-alpha-induced NF-kappaB 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-alpha. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM).</p><p><b>RESULTS</b>Mutant IkappaBalpha protein was confirmed to exist by Western blot. The results of EMSA showed that NF-kappaB activation by TNF-alpha in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IkappaBalpha repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-alpha, but changed very little in transfected HL-60 cells. The inhibition of NF-kappaB activation by mutant IkappaBalpha enhanced TNF-alpha-induced apoptosis. The cytotoxic effects of TNF-alpha were amplified in a time- and dose-dependent manner.</p><p><b>CONCLUSIONS</b>NF-kappaB activation plays an important role in the resistance to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB by mutant IkappaBalpha could provide a new approach that may enhance the anti-leukemia effects of TNF-alpha or even of other cytotoxic agents.</p>
Subject(s)
Humans , Apoptosis , Gene Expression Regulation, Leukemic , HL-60 Cells , I-kappa B Proteins , Physiology , NF-KappaB Inhibitor alpha , NF-kappa B , Proto-Oncogene Proteins c-bcl-2 , Genetics , Tumor Necrosis Factor-alpha , Pharmacology , bcl-X ProteinABSTRACT
In order to explore the effect of nuclear factor-kappa B (NF-kappaB) on bcl-x gene transcrtiption in drug-resistant leukemia cell line HL-60/E6, first of all, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin, and then bcl-x(L) mRNA levels were detected by RT-PCR after exposing HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA at different times. Morever, indirect immuno-fluorescence and FCM were used to demonstrate the location of NF-kappaB-RelA in HL-60/E6 cells and the efficiency of liposome-mediated ODN transfection. The results showed that RelA kept active and located at the nuclei of HL-60/E6 cells, and the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P < 0.01 in 4, 6 and 12 h). Exposure of HL-60/E6 cells to 5 micro mol/L AS-PS-ODN-RelA led to a maximal 40% decline of bcl-x(L) mRNA levels. No significant change of bcl-x(L) mRNA level occurred in control group. It was concluded that NF-kappaB was involved in regulating bcl-x transcription. Suppressing NF-kappaB-RelA by antisense technology may down-regulate level of bcl-x(L) mRNA.