Rearranged NF-kappa B2 gene in an adult T-cell leukemia cell line

Abstract

Adult T-cell leukemia (ATL) is an aggressive type of leukemia, originating from T-cells infected with human T-cell leukemia virus type 1. Accumulating evidence suggests the aberrant activation of NF-kappaB to be a causative factor mediating the abnormal proliferation of leukemic cells, thus resulting in the development of ATL. A rearranged NF-kappa B2/p100 gene was isolated from an ATL-derived cell line, which was generated by a chromosomal translocation. The isolated NF-kappa B2 mutant is fused with the with no (lysine) deficient protein kinase 1 gene, coding for a 58 kDa protein that retains the DNA binding Rel homology domain, but it lacks the entire ankyrin repeat inhibitory domain, thus suggesting its constitutive activation. This rearranged NF-kappa B2 gene product (p58) was localized in the nucleus, and formed a complex with NF-kappaB p65 or RelB. Moreover, a T-cell line expressing p58 increased the amount of an NF-kappa B2-inducible gene, NF-kappa B2/p100 by itself. These results suggest that such NF-kappa B2 gene rearrangement may therefore be a factor in the constitutive activation of NF-kappaB in ATL, and thereby playing a role in the ATL pathogenesis.

Figure 1

Expression of NF‐κB2 in human T‐cell lines. Cell lysates prepared from indicated human T‐cell lines were characterized by a Western blot analysis using anti‐NF‐κB2/p100, anti‐Tax, or anti‐tubulin antibody. The positions of NF‐κB2/p100 and p52 are indicated. The three adult T‐cell leukemia (ATL)‐derived cell lines KK1, KOB, and ST1 are IL‐2‐dependent cell lines, while the others including TL‐OmI and MT‐1 are IL‐2‐independent lines. HTLV‐1, human T‐cell leukemia virus type 1.

Figure 2

Structure of tumor‐associated NF‐κB2 mutants. The positions of the Rel homology domain (RHD) and ankyrin repeat are shown. The RC685, LB40 and p85 (HUT78) are previously characterized tumor‐associated NF‐κB2 mutants.^{( 19} , ^{21 )} WNK1, with no K (lysine) lysine deficient protein kinase 1.

Figure 3

Genomic and cDNA structures of NF‐κB2/p58 in TL‐OmI. (a) Horizontal and vertical arrows indicate the positions of the primer sequences to amplify genomic NF‐κB2 DNA and the chromosomal breakpoints, respectively. (b) Genomic DNA was extracted from TL‐OmI and Jurkat cells, and NF‐κB2 gene was amplified by polymerase chain reaction. The specific amplification from TL‐OmI is indicated by an arrow. (c) The expected genomic DNA and cDNA structures of NF‐κB2/p58 in TL‐OmI are shown. The nucleotide and amino acid sequences encompassing the boundary region between p100 and WNK1 in p58 are shown.

Figure 4

Subcellular localization of NF‐κB2/p58 and p100 in 293T cells. 293T cells were transfected with either the pEFneo‐p58, pEFneo‐p100 or pEFneo plasmid. The cells were then stained with anti‐p100 (red) and with Hoechst 33258 (blue) for nuclear staining. The stained cells were examined using fluorescent light microscopy.

Figure 5

NF‐κB2/p58 activates the endogenous NF‐κB2/p100 expression in a T‐cell line. (a, b) The cell lysates were prepared from TL‐OmI (lane 1), and CTLL‐2 infected with either p58‐virus (lane 2), p52‐virus (lane 3), control virus (lane 4) or Tax‐virus (lane 5). NF‐κB2 proteins in cell lysates were analyzed by a Western blot analysis using an anti‐p100 antibody. The CTLL‐2 cells expressing p58 or p52 in (a) and (b) were independently established. (c) The CTLL‐2 cells characterized above were cultured in the absence of IL‐2, and viable cell numbers were counted using the trypan blue exclusion assay under microscopy.

Figure 6

The interaction of NF‐κB2/p58 with other NF‐κB subunits. The cell lysates were prepared from 293T cells transfected either with pCMV‐HA‐p100, pCMV‐HA‐p52, or pCMV‐HA‐p58, and they were immunoprecipitated with anti‐hemagglutinin(HA) antibody. The total cell lysates (Input) and immunoprecipitates (HA IP) were then characterized by a Western blot analysis by using either anti‐RelB, anti‐p65, or anti‐HA antibody.

Figure 7

Transcriptional activity of NF‐κB2/p58 in a T‐cell line. (a) Cell lysates were prepared from 293T cells transfected with either the pEFneo‐p58, pEFneo‐p100 or pEFneo plasmid (2 µg), and the amount of NF‐κB2 protein in each lysate was measured by a Western blot analysis using an antip100 antibody. The arrows indicate the p58 and p52 recognized by the antibody. (b, c) Jurkat cells were transfected with either the pEFneo‐p58, pEFneo‐p100 or pEFneo plasmid (0.1 µg) together with the luciferase plasmid (0.5 µg) regulated by the NF‐κB element (kB‐Luc) and the β‐galactosidase plasmid (0.1 µg). The pSG‐p65 plasmid (0.1 µg) was cotransfected with increasing amount of pEFneo‐p100 or pEFneo‐p58 (0.05, 0.1, and 0.2 µg) into Jurkat cells as indicated in (c). Cell lysates were prepared from transfected cells, and the luciferase and β‐galactosidase activities were determined. The luciferase activity normalized by the β‐galactosidase activity was shown as the average with standard deviations. Three independent experiments were carried out to confirm reproducibility.