This paper represents the first direct demonstration of an oncogenic effect of REL protein expression in a human B-lymphoid cell system. That is, we show that overexpression of an activated REL mutant, RELΔTAD1, increases the oncogenic properties of the human B-cell lymphoma BJAB cell line, as measured by increased soft agar colony-forming ability, tumor formation in immunocompromised mice, and adhesion. Moreover, the mRNA expression profile of BJAB cells overexpressing RELΔTAD1 is substantially altered; in particular, there is increased expression of many NF-κB target genes whose expression is associated with the more aggressive ABC subtype of diffuse large B-cell lymphoma. Furthermore, many of the up-regulated genes in BJAB-RELΔTAD1 cells can be classified as genes implicated in immunological diseases (), suggesting that BJAB-RELΔTAD1 cells have a phenotype that is more similar to aggressive DLBCL than is the GCB-like phenotype of control BJAB cells. As such, the cell system we describe here may provide an in vitro model system for understanding DLBCL transition from a low-grade (GCB-like) to a high-grade (ABC-like) oncogenic state.
Although v-Rel, c-Rel and their derivatives have been shown to be oncogenic in avian and mouse systems (Gilmore, 1999
; Gilmore et al., 2004
), there has been controversy about whether REL is a true oncoprotein for human B-lymphoid cells (Shaffer et al., 2002
; Houldsworth et al., 2004
). For example, the REL
gene is amplified in a high percentage of GCB-type DLBCLs, but these cells do not have particularly high levels of nuclear κB site-binding activity (Davis et al
., 2001). Moreover, the lack of oncogenic activity by overexpressed REL in mouse B-lymphoid cells in vitro
or in vivo
has cast doubt on whether REL acts as an oncoprotein in human B-cell malignancies, which are the sole human cancer cell type wherein the REL
gene has been found to undergo amplification and mutation (Gilmore et al., 2004
). The results we present herein strongly suggest that REL can exert an oncogenic effect in human B-lymphoma cells, and indicate that REL or certain REL target genes may be suitable therapeutic targets for some human B-cell lymphomas.
There are several likely explanations for the susceptibility of BJAB cells to the transforming activity of RELΔTAD1. First, BJAB cells express relatively low levels of endogenous REL protein () as compared to several other human B-lymphoma cell lines. Thus, in BJAB cells it is possible to achieve a higher ratio of RELΔTAD1 protein to endogenous REL, and this relatively high level of RELΔTAD1 may be required for its transforming effect in human B cells. Second, BJAB cells have a GCB mRNA profile (Ngo et al., 2006
), which is correlated with a better clinical outcome in DLBCL patients (Rosenwald et al., 2002
; Shipp et al., 2002
), suggesting that BJAB cells are not as “transformed” as some other human B-cell lines. Third, in soft agar and tumor-forming assays similar to those we have conducted here, BJAB cells have been shown to be susceptible to oncogenic effects of other factors, including the Epstein-Barr virus (EBV) LMP1 protein (Enberg et al., 1983
; Wennborg et al., 1987
), EBV small RNAs (Yamamoto et al., 2000
) and the AP12-MALT1 fusion protein from MALT lymphomas (Ho et al., 2005
). Interestingly, LMP1 and AP12-MALT1 are both inducers of NF-κB (Hammarskjold et al., 1992
; Lucas et al
., 2007) and both can increase the resistance of BJAB cells to inducers of apoptosis (Stoffel et al., 2004
; Ho et al., 2005
; Lucas et al
., 2007). In addition, LMP1 can induce expression of BCL2 and IRF4, which are required for apoptosis resistance (Henderson et al., 1991
; Finke et al., 1992
; Snow et al., 2006
), enhanced adhesion (Mainou and Raab-Traub, 2006
), and cell motility (Mainou and Raab-Traub, 2006
). Moreover, MALT1
chromosomal gains are also associated with ABC-subtype gene expression, including high levels of BCL2 expression and poorer prognosis (Dierlamm et al., 2008
Many of the upregulated genes in BJAB-RELΔTAD1 cells are connected with processes that involve the plasma membrane, i.e., cell-to-cell communication, the extracellular matrix, adhesion, and membrane binding (see ). These genes include VCAM1, CD44, CD40, ITGAX
, and many chemokines and chemokine receptors including CCL22, CCR7, CXCR4
. Additionally, BJAB-RELΔTAD1 cells are more adherent to a culture dish than control BJAB-MSCV cells (). This is consistent with the large cohort of increased cDNAs in RELΔTAD1 cells that are classified as related to adhesion (). NF-κB signaling is also known to be downstream of many adhesion-related signaling pathways (Perez et al., 1994
; Lee et al., 1999
; Zarnegar et al., 2004
). Furthermore, CD40 and VCAM1 mRNA and protein expression are upregulated in the BJAB-RELΔTAD1 cells. While CD40
mRNA was only modestly increased (1.4-fold) in BJAB-RELΔTAD1 cells, this did translate into similarly increased CD40 protein levels (). CD40 has been shown to be important in B-cell aggregation (Lee et al., 1999
), and both VCAM1 and CD40 play roles in adhesion (Springer and Vonderheide, 1992
; Lee et al., 1999
). Taken together, these results suggest that overexpression of RELΔTAD1 in BJAB cells causes upregulation of many adhesion-associated genes, which results in a phenotype of the cells being more adherent, which may contribute to their enhanced ability to form colonies in soft agar and tumors in SCID mice.
, genes whose expression is up-regulated in BJAB-RELΔTAD1 cells, are markers for ABC DLBCL, whereas CD10
is down-regulated in both ABC DLBCLs and BJAB-RELΔTAD1 cells (Alizadeh et al., 2000
; Wright et al., 2003
). The increased expression of BCL2
in ABC DLBCLs correlates with a poorer clinical prognosis (Iqbal et al., 2006
). The transcription factor IRF4 can synergize with v-Rel in the transformation of chicken fibroblasts and knockdown of IRF4 expression reduces the soft agar colony-forming ability of v-Rel-transformed cells (Hrdlicková et al
., 2001). Of note, multiple myelomas are dependent on IRF4 for growth, whereas the growth of GCB-DLCBL does not require IRF4 (Shaffer et al., 2008
). Taken together, these results are consistent with BCL2 and IRF4 playing a role in the enhanced transformed phenotype that we describe for BJAB-RELΔTAD1 cells.
We also found that many other ABC-defining genes (including several not known to be NF-κB targets) are significantly upregulated in BJAB-RELΔTAD1 cells. These ABC genes include MARCKS, BATF, BMI1, LITAF
and others (see Tables and S2
). Some of these ABC-type upregulated genes may reflect an overall shift in gene expression, induced indirectly by NF-κB/REL. In addition, some GCB-subtype genes are significantly down-regulated in BJAB-RELΔTAD1 cells (Tables and S3
). These genes are, for the most part, non-NF-κB targets, suggesting that these decreases in GCB-type gene expression are also indirectly affected by RELΔTAD1.
Approximately 4% of total NF-κB targets (www.nf-kb.org
) were up-regulated in BJAB-RELΔTAD1 cells as compared to 59% of ABC-specific NF-κB targets (). The selective increase in expression of only a small number of NF-κB target genes in BJAB-RELΔTAD1 cells suggests that the BJAB cells have been transformed to a more aggressive form of DLBCL by RELΔTAD1 through activation of a minor subset of NF-κB/REL targets. These ABC-specific NF-κB target genes may be poised for activation by RELΔTAD1 in B-lymphoma cells, possibly due to their chromosomal state or to cooperation of RELΔTAD1 with other B cell-specific transcription factors.
There are 40 genes whose expression is reduced by at least ten-fold in BJAB-RELΔTAD1 cells (Table S1
). The reduced expression of CD10 mRNA and protein in BJAB-RELΔTAD1 cells () is consistent with the enhanced transformed properties of these cells, given that reduced CD10 expression correlates with a poorer prognosis in the clinic (van Imhoff et al., 2006
). Gupta et al. (2008)
have shown that expression of two B-cell proteins BLNK and BCAP are down-regulated directly by Rel in v-Rel-transformed avian cells. In our study, the level of only BLNK was significantly reduced in BJAB-RELΔTAD1 cells. Such results raise the possibility that down-regulation of gene expression is important for REL-induced effects on B-cell oncogenesis, and that some of these genes are specifically repressed by RELΔTAD1.
BJAB-RELΔTAD1 cells show a reduced induction of caspase-3 activity following treatment with 1 μg/ml doxorubicin, although the ability of doxorubicin to decrease viability is unchanged in BJAB-RELΔTAD1 cells (). These data are consistent with previous results showing that CD40 ligand, an inducer of NF-κB, can reduce the ability of this concentration of doxorubicin to induce caspase activity in BJAB cells without affecting its ability to induce apoptosis (Voorzanger-Rousselot et al., 1998
). These results indicate that doxorubicin induces apoptosis in BJAB cells through a caspase-independent mechanism, which is not blocked by increased Rel/NF-κB activity.
The majority of the nuclear κB site-binding activity in RELΔTAD1-BJAB cells contains REL protein, whereas in control BJAB-MSCV cells, only a small fraction of the binding activity is supershifted by REL antiserum (). In addition, there are increased nuclear levels of NF-κB p50 in BJAB-RELΔTAD1 cells, presumably because RELΔTAD1 and p50 readily interact (Figure S1
). Taken together, these data suggest that a shift in the composition of nuclear NF-κB/REL dimers occurs upon overexpression of RELΔTAD1.
Only a small number of the genes upregulated by more than ten-fold in BJAB-RELΔTAD1 cells are ABC-defining (5 genes) or known NF-κB targets (8 genes) (). As such, some of these genes may be novel ABC DLBCL markers or NF-κB/REL targets. In addition, there are 14 ABC-defining genes that are significantly up-regulated in BJAB-RELΔTAD1 cells that have yet to be classified as NF-κB/REL targets (Tables and S2
). Future work will be directed at determining which genes are direct RELΔTAD1 targets and which contribute to the phenotypic changes that occur in RELΔTAD1 “transformed” BJAB cells.