Effect of antisense oligodeoxynucleotide directed to NF-κB-RelA on Bcl-x_L mRNA in extended drug resistance leukemia cell line HL-60/E6

Abstract

To explore the effect of NF-κB on bcl-x gene transcription in extended drug resistance leukemia cell line HL-60/E6, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF-κB-RelA in HL-60/E6 cells. FCM analysis and RT-PCR were used to detect the efficiency of liposome-mediated ODN transfection and the change of bcl-X_L. mRNA levels after 5 μmol/L phosphorothioate (PS)-derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL-60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL-60/E6 cells, but in the cytoplasm of HL-60 cells, the efficiency of liposomemediated ODN transfection was significantly higher than that of null ODN (P<0. 01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL-60/E6 cells to 5 μmol/L AS-PS-ODN directed to RelA led to a maximal 40 % decline of bcl-x_L mRNA levels within 8 h. The inhibition rate of bcl-X_L. mRNA was (15±1. 79) %, (28±2. 34) %, (40±3.47) %, (20±1.54) % in 4 h,6h,8h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF-κB was involved in regulating bcl-x transcription. It was suggested that NF-κB was an important factor for drug resistance in leukemia cells.

Keywords

cell line / HL-60/E6 / RelA / antisense oligodeoxynucleotide / drug resistance / bcl-X_L

References

[1] ChenF, CastranovaV, ShiX, et al.. New insights into the role of nuclear factor-κB, a ubiquitous transcription factor in the initiation of diseases. Clin Chem, 1999, 45: 7-7

[2] ReedJ C. Double identity for proteins of the Bcl-2 family. Nature, 1997, 387: 773-773

[3] LeeH H, DadgostarH, ChengQ, et al.. NF-κB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci USA, 1999, 96: 9136-9136

[4] KawasakiA M, CasperM D, FreierS M, et al.. Uniformly modified 2′-deoxy-2′-fluorophosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets. J Med Chem, 1993, 36: 831-831

[5] ReuningU, WilhelmO, NishiguchiT, et al.. Inhibition of NF-κB-RelA expression by antisense oligodeoxynucleotides suppression synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1. Nucleic Acids Res, 1995, 23: 3887-3887