Overexpression of an activated REL mutant enhances the transformed state of the human B-lymphoma BJAB cell line and alters its gene expression profile

Abstract

The human REL proto-oncogene encodes a transcription factor in the nuclear factor (NF)-kappaB family. Overexpression of REL is acutely transforming in chicken lymphoid cells, but has not been shown to transform any mammalian lymphoid cell type. In this report, we show that overexpression of a highly transforming mutant of REL (RELDeltaTAD1) increases the oncogenic properties of the human B-cell lymphoma BJAB cell line, as shown by increased colony formation in soft agar, tumor formation in SCID (severe combined immunodeficient) mice, and adhesion. BJAB-RELDeltaTAD1 cells also show decreased activation of caspase in response to doxorubicin. BJAB-RELDeltaTAD1 cells have increased levels of active nuclear REL protein as determined by immunofluorescence, subcellular fractionation and electrophoretic mobility shift assay. Overexpression of RELDeltaTAD1 in BJAB cells has transformed the gene expression profile of BJAB cells from that of a germinal center B-cell subtype of diffuse large B-cell lymphoma (DLBCL) (GCB-DLBCL) to that of an activated B-cell subtype (ABC-DLBCL), as evidenced by increased expression of many ABC-defining mRNAs. Upregulated genes in BJAB-RELDeltaTAD1 cells include several NF-kappaB targets that encode proteins previously implicated in B-cell development or oncogenesis, including BCL2, IRF4, CD40 and VCAM1. The cell system we describe here may be valuable for further characterizing the molecular details of REL-induced lymphoma in humans.

Figure 1

Overexpression of RELΔTAD1 increases the soft agar colony-forming ability of BJAB cells. (a) Structure of MSCV retroviral vectors used in these studies. (b) Anti-REL Western blotting of cells stably transduced with MSCV or MSCV-RELΔTAD1 (RELΔTAD1). Endogenous REL and introduced RELΔTAD1 are indicated. (c) Relative soft agar colony formation of BJAB-MSCV cells (1.0) and BJAB-RELΔTAD1 cells. Values are the averages of four assays performed in triplicate; error bars indicate standard error. (d) Comparison of the proliferation of BJAB-MSCV cells and BJAB-RELΔTAD1 cells. Cells were plated at 10⁵ cells/well and were counted each day following plating. (e) BJAB-MSCV cells (MSCV) and BJAB-RELΔTAD1 cells (ΔTAD1) were treated with 1 μg/ml doxorubicin (DOX) for the indicated times and caspase-3 activity was measured or PARP cleavage was monitored by Western blotting (bottom panel). For each cell type, caspase activity is relative to the activity seen with untreated cells at the same time point (1.0). Cell viability was measured after treatment with 1.0 μg/ml of doxorubicin at the indicated time points (right panel). Values are the averages of four (caspase-3 activity) or three experiments (cell viability), each performed with triplicate samples.

Figure 2

BJAB-RELΔTAD1 cells have increased nuclear REL protein activity as compared to BJAB-MSCV cells. BJAB-RELΔTAD1 cells and BJAB-MSCV cells were compared by subcellular fractionation (a, c), indirect immunofluorescence using an anti-REL primary antiserum (b), and by EMSA analysis of nuclear extracts (d). In (a) and (c), nuclear (N) and cytoplasmic fractions (C) were subjected to Western blotting using equal proportions of each fraction for analysis of REL, p50, RelA, and 14-3-3 and CD40 proteins (as cytoplasmic controls) or RNA polymerase II (as a nuclear control). In (b), the indicated BJAB cells were stained with an anti-REL antibody and viewed by confocal microscopy. The left panel contains BJAB-RELΔTAD1 cells; the middle and right panels show BJAB-MSCV cells. The left and middle panels were imaged using the same exposure time (Exp), while the right panel was imaged using a longer exposure time to detect the low level of endogenous REL in BJAB-MSCV cells. In (d), an EMSA was performed on equalized amounts (5 μg) of nuclear extracts using a κB-site probe from the human MHC1 enhancer. Where indicated, competitions were performed using an excess of cold probe or samples were supershifted (SS) using anti-REL antiserum. The relevant complexes are indicated.

Figure 3

Analysis of mRNA and protein from select genes in BJAB-MSCV and BJAB-RELΔTAD1 cells. (a) Heat map of NF-κB-specific ABC-target gene expression in BJAB-RELΔTAD1 cells as compared to BJAB-MSCV cells. The map was created using the matrix2png program (Pavlidis and Noble, 2003). The expression scale is shown below the map. (b) RT-PCR of the indicated mRNAs: BCL2, IRF4, CCR7, CD10, VCAM1, REL (as a positive control), and GAPDH (as a normalization control). Water control (-); BJAB-MSCV (MSCV); BJAB-RELΔTAD1 (RELΔTAD1). (c) Western blotting for REL, VCAM1, BCL2, CD40, CD10 and β-actin (as a normalization control) of extracts from BJAB-MSCV cells (MSCV) and BJAB-RELΔTAD1 cells (RELΔTAD1).

Figure 4

BJAB-RELΔTAD1 cells show increased adherence to culture dishes. (a) BJAB-MSCV and BJAB-RELΔTAD1 cells (1×10⁶) were grown in petri dishes for 36 h and imaged at 200 × magnification (top panel); dishes were then washed with PBS and cells in the same field were imaged again (“Washed” panels). The arrows point to a clump of BJAB-RELΔTAD1 cells that adhere to the culture dish. (b) The percentage of attached cells was determined by measuring the total protein content of floating cells isolated directly from the media and from cells that remained attached to the culture dish. The assay was performed with triplicate plates; error bars represent standard error.

Figure 5

Expression of REL in several human B-lymphoma cell lines. (a) The following human B-lymphoma cell lines were used: BJAB (EBV-negative Burkitt-like lymphoma), SUDHL-4 (DLBCL), RC-K8 (DLBCL), IB4 (umbilical cordblood B-cell lymphoblastoid line infected with EBV), Daudi (EBV-positive Burkitt’s lymphoma), and BL41 (Burkitt’s lymphoma). Lysates were prepared from actively growing cells, and 20 μg of total protein was analyzed by anti-REL Western blotting (top). At the bottom is shown a Coomassie blue-stained gel of equalized total protein extracts. Rel. Amt. indicates the relative amount of REL in each cell type, as compared to BJAB cells (1.0), determined by scanning of the film in the top panel. (b) Anti-REL Western blotting of control BJAB cells, BJAB-RELΔTAD1 cells, control Daudi cells, and a Daudi-RELΔTAD1 cell line. (c) Relative soft agar colony forming ability of control vs Daudi-RELΔTAD1 cells. Assays were performed as in Figure 1c, values are the averages of 5 experiments performed with triplicate plates, and were normalized to the number of colonies obtained with control Daudi cells (1.0).