(Received for publication, August 8, 1995; and in revised form, September 7, 1995)
From the
The chimeric gene E2A-PBX1 is formed by the t(1;19) chromosomal
translocation exclusively associated with pediatric pre-B cell acute
lymphoblastic leukemia (pre-B ALL). The resultant fusion protein from
this chimeric gene contains the DNA-binding homeodomain of Pbx1. The
first and only functional Pbx1 binding site has been localized in
bovine CYP17 to a sequence (CRS1) that participates in cAMP-dependent
transcription of this gene encoding the steroid hydroxylase,
17-hydroxylase cytochrome P450. Because Pbx1 is not expressed in
pre-B cells, it may be possible that the E2a-Pbx1 fusion protein
expressed in pre-B cells having this translocation will activate, in
response to cAMP, transcription of genes not normally expressed in
these cells leading to arrest of differentiation at the pre-B cell
stage. We have now shown that reporter genes comprising CRS1 are
activated transcriptionally by protein kinase A (PKA) in the pre-B cell
line 697, which endogenously expresses the fusion protein, and that
overexpression of E2A-Pbx1 in additional cell lines enhances
transcription of reporter genes in a PKA-dependent fashion. Thus, it
seems plausible that arrest in the pre-B stage leading to pre-B ALL
includes cAMP-dependent activation of E2A-Pbx1.
Acute lymphoblastic leukemia (ALL) ()is predominantly
a disease of children and young adults. Approximately 1800 new cases
are diagnosed each year in the United States in individuals under 15
years of age. A chromosomal translocation t(1;19)(q23;p13.3) is
observed in 25% of the cases of pediatric pre-B cell
ALL(1, 2) . This translocation producing the E2A-PBX1
chimeric gene results in synthesis of transcripts which encode the
N-terminal half of the E2A gene product and the C-terminal 75% of
Pbx1(3, 4) . The E2A gene product belongs to the
helix-loop-helix family of transcription factors and dimerizes with
itself or with other family members in order to bind
DNA(5, 6) . Dimerization and DNA binding functions of
E2A are closely linked in a domain located in the C-terminal half of
the protein. The N-terminal half of E2A contains two transactivation
domains that can confer a strong transactivation function upon
heterologous DNA-binding domains (7) . In the E2A-Pbx1 fusion
protein, the dimerization and DNA-binding domains of E2A have been
replaced by the majority of the Pbx1 sequence including the DNA-binding
homeodomain.
PBX is a homeobox gene family consisting of at least
three members, PBX1, PBX2, and PBX3(8) . Alternative splicing
of the PBX1 transcript generates two different Pbx1 proteins, Pbx1a and
Pbx1b, which vary in their C-terminal sequence. In the same way as
Pbx1, alternative splicing of E2A-Pbx1 transcripts leads to the
production of two different E2A-Pbx1 fusion proteins, E2A-Pbx1a and
E2A-Pbx1b(3, 4) . These fusion proteins containing the
transactivation domain of E2A and the DNA-binding homeodomain of Pbx1
have been investigated intensively and shown to be capable of
transformation in fibroblast(9) , myeloid (10) , and T
lineage cells(11) . Intriguingly, no B lineage tumors have been
observed in transgenic mice expressing E2A-Pbx1(11) . E2A-Pbx1
as well as Pbx family members have been reported to bind the Pbx1
recognition sequence, designated PRS, which was identified by the
polymerase chain reaction-mediated random DNA binding site selection (12, 13) . Independently, we discovered the Pbx1
binding site in a cAMP-regulatory sequence, designated CRS1, which
enhances the expression of 17-hydroxylase cytochrome P450
(P450c17) encoded by the CYP17 gene(14, 15) . Both
Pbx1a and Pbx1b are involved in the cAMP-dependent regulation of
CYP17(15) . Although E2A-Pbx1 is reported to enhance reporter
gene transcription through PRS(12, 13, 16) ,
its role as a transcription factor in leukemogenesis remains unknown.
Because E2A gene products are required for B cell formation(17, 18) , and E2A-Pbx1 is under the control of the E2A promoter, E2A-Pbx1 is expected to be expressed and activate target genes at a certain stage of B cell differentiation leading to pre-B ALL. Since E2A-Pbx1 retains a putative protein kinase A (PKA) recognition site derived from Pbx1, and Pbx1 is involved in cAMP-dependent transcription of CYP17, the possibility arose that E2A-Pbx1 is activated by a cAMP signaling pathway. In the B cell developmental process, an increase of intracellular cAMP level plays an important role in both differentiation and apoptotic cell death(19, 20) , since induced expression of interleukin-2 receptor was observed in 70Z/3 pre-B cells in the presence of both cAMP and interleukin-1. Therefore, if E2A-Pbx1 responds to cAMP to activate transcription of target genes, this event may be involved in pre-B cell leukemogenesis.
In this study, we have investigated whether the fusion protein E2A-Pbx1a transactivates transcription in a cAMP-dependent manner. Utilizing cotransfection of expression constructs of E2A-Pbx1a and the PKA catalytic subunit (PKA-C) with a luciferase reporter construct containing the CRS1 sequence in different cell lines, we demonstrate that the transactivation of the fusion protein is PKA-dependent but not tissue-specific.
Luciferase assays were performed using a kit (Promega) following the manufacturer's instructions. Light production from luciferase reaction mixtures was measured by a liquid scintillation counter (Wallac 1409, Pharmacia Biotech Inc.).
Figure 1: Immunodetection of Pbx1 in nuclear extracts from Y1, S194, and 697 cells. After SDS-polyacrylamide (10%) gel electrophoresis and transfer onto a polyvinylidene difluoride membrane, anti-Pbx1 and anti-CREB (A) and anti-E47 (B) antibodies were used in immunoblot analysis. Pbx1a, Pbx1b, E2A-Pbx1a, and 43-kDa CREB are indicated.
Figure 2:
Gel shift analysis of nuclear extracts
from S194 and 697 cells with and without anti-Pbx1 antibody. Nuclear
extracts from S194 (12.5 µg/lane) and 697 cells (5 µg/lane)
were incubated with P-labeled CRS1 and the indicated
amount of anti-Pbx1 antibody or control serum (1 µl) for 5 min on
ice. U and M indicate protein-DNA complexes observed
in S194 nuclear extracts, and S indicates complexes
supershifted by the antibody. U` indicates protein-DNA
complexes specific for 697 cells, and S` indicates complexes
supershifted.
Figure 3:
Activation of E2A-Pbx1 by co-expression of
PKA in lymphocytes. The reporter gene plasmid 4 CRS1-Luc (16
µg) was co-transfected with the PKA-C expression vector PKAp (2
µg) by electroporation into 697 pre-B cells (A). The total
amount of plasmid was adjusted with RSVpBK vector to 18 µg in each
transfection. As a mock transfection, the luciferase plasmid containing
no cis-activating sequence (Vacant Luc) was used. For
S194 lymphocytes (B), 4
CRS1-Luc (5 µg) was
co-transfected with the E2A-Pbx1a expression vector E2A-PBX RSV (3
µg) and PKAp (1 µg) by the DEAE-dextran method. The total
amount of transfected plasmids was adjusted to 9 µg with the RSVpBK
plasmid (RSV) containing no cDNA insert in a series of CRS1-Luc
transfection experiments. The 4
CRE-Luc plasmid (5 µg) was
co-transfected with the CREB expression vector CREB RSV (3 µg) and
PKAp (3 µg) to adjust the total amount of plasmids in this series
to 11 µg as described above. The luciferase activity of 4
CRS1-Luc without E2A-Pbx1a or PKA-C expression was set as 1 relative
activity.
The S194 B cells show very low CRS1-mediated
PKA-dependent transcription (Fig. 3B). However, when
S194 cells were co-transfected with PKA-C and E2A-Pbx1a expression
plasmids (PKAp and E2A-PBX RSV, respectively) and 4 CRS1-Luc (Fig. 3B), a low basal level of luciferase activity
without the expression of PKA-C but a strong activity with the
expression of PKA-C (approximately 70-fold enhancement) was observed.
This enhancement by the fusion protein was 2-2.5-fold greater
than that by Pbx1a (Pbx RSV) in response to PKA-C. As control
experiments, co-transfection with PKAp and CREB RSV (a CREB expression
plasmid) and 4
CRE-Luc has been performed. Because of the
presence of endogenous CREB in S194 cells (Fig. 1), PKA-C weakly
enhanced the luciferase activity through 4
CRE without
co-transfection of CREB RSV. When CREB RSV was transfected, the
co-transfection of PKAp greatly enhanced the luciferase activity. The
level of the enhancement by PKA-C with CREB was approximately the same
as that with E2A-Pbx1a, suggesting that the transactivation by
E2A-Pbx1a in response to PKA is as strong as that observed with CREB.
To examine whether the transactivation of E2A-Pbx1a is cell-specific, similar co-transfection experiments have been performed utilizing Y1 (mouse adrenal tumor), Jeg3 (human choriocarcinoma), and HepG2 cells (human hepatocarcinoma). Because Y1 cells express Pbx1b and Pbx1a (Fig. 1A), luciferase activities were enhanced without co-transfection of E2A-PBX RSV by either forskolin treatment, a stimulator of intracellular cAMP, or cotransfection of PKAp. Y1 cells transfected with E2A-PBX RSV showed a basal activity of the reporter gene slightly higher than that of control plasmid-transfected cells (Fig. 4A). Either forskolin treatment or PKAp co-transfection greatly enhanced the activity. Activation levels in different cell lines vary, perhaps due to experimental variation, however, the transactivation by E2A-Pbx1a was greatly enhanced by the co-expression of PKA-C in both Jeg3 and HepG2 (Fig. 4, B and C). These results indicate that the cAMP-dependent transactivation of E2A-Pbx1 is not tissue-dependent. Thus, E2A-Pbx1 appears to require no complex formation with tissue-specific factors to transactivate its target genes.
Figure 4:
Activation of E2A-Pbx1a by forskolin
treatment or co-expression of PKA in cell lines other than lymphoid
cells. The 4 CRS1-Luc plasmid (10 µg for Y1 and Jeg3 and 5
µg for HepG2 cells) was co-transfected with and without the
E2A-Pbx1a expression vector, E2A-PBX RSV (5 µg for Y1 and Jeg3 and
3 µg for HepG2), and the PKA-C expression vector, PKAp (2 µg
for Y1, Jeg3, and HepG2). The total amount of plasmids transfected was
adjusted to 17 µg for Y1 and Jeg3 and 10 µg for HepG2 with the
RSVpBK plasmid (RSV) containing no cDNA insert. The luciferase activity
of CRS1-Luc without any treatment or co-expression was set as 1
relative activity in each cell line.
In the regulatory region of the
CYP17 gene, the first example of a native target gene for Pbx1, the
Pbx-binding site of CRS1 (TTGATGGAC) is placed in the opposite
orientation to PRS (ATCAATCAA)(12, 13, 16) .
To compare activation by both the difference in orientation and DNA
sequence, we constructed the 4 PRS-Luc plasmid containing the
consensus PRS sequence in the same orientation as used by other groups (12, 13, 16) in the luciferase reporter
plasmid. In S194 cells, 4
PRS-Luc also showed little
enhancement of basal transcription upon expression of E2A-Pbx1a but
showed a dramatic enhancement with the co-expression of E2A-Pbx1a and
PKA-C as did 4
CRS1-Luc (Fig. 5). This indicates that
the orientation and sequence variation between CRS1 and PRS are not
critical for the cis-activation of reporter genes. We also
examined the promoter dependence of the reporter gene activation by
replacing the SV40 minimal promoter in 4
CRS-Luc with
-globin and CYP17 minimal promoters. Although the absolute level
of luciferase activities varied depending on minimal promoters, the
pattern of activation by PKA-C showed no difference (data not shown).
These results indicate that E2A-Pbx1a is able to transactivate its
target genes upon stimulation by cAMP via PKA and also suggest that the
activation of E2A-Pbx1a by PKA in the B cell lineage may trigger gene
transcription leading to the development of pre-B ALL.
Figure 5:
Comparison of 4 CRS1 and 4
PRS as cis-activating sequence. The reporter gene vectors
containing 4
CRS1 (4xCRS1-Luc) and 4
PRS (4xPRS-Luc) as cis-activating sequence were
co-transfected with E2A-PBX RSV and PKAp into S194 cells as described
in Fig. 3.
In this study we have shown that 697 pre-B ALL cells, which express E2A-Pbx1, have a gel shift pattern clearly distinct from cells which express members of the PBX gene family, yet they enhance transcription mediated by the Pbx binding site in a cAMP-dependent fashion. Not only does endogenous E2A-Pbx1 have this effect in pre-B ALL cells, but co-expression of E2A-Pbx1 shows this effect in several different cell types.
Extradenticle, a Drosophila homologue
of Pbx proteins, interacts with homeotic selector (HOM) proteins to
modulate the developmental
specificity(28, 29, 30) . In addition,
E2A-Pbx1 and Pbx1a have also been shown to interact in vitro with Hox proteins, mammalian counterparts of Drosophila HOM proteins(31) . Lu et al.(32) have
reported that E2A-Pbx1 and Pbx1 form complexes with Hox proteins
leading to repression of transactivation by co-transfection analysis
using the PRS sequence. Intriguingly, they have also reported that Pbx1
does not form complexes with Hox proteins when the CRS1 sequence is
used as a Pbx binding site(32) . In fact, the gel shift pattern
observed with 697 cells in this study showed no slower migrating
complexes that have been reported previously for E2A-Pbx1Hox
protein complexes in in vitro studies(31, 32) . This suggests that it is not
necessary for E2A-Pbx1a to form complexes with Hox proteins to bind to
its target genes, at least in 697 pre-B ALL cells. Furthermore, the
PKA-dependent transactivation of E2A-Pbx1a has been shown in all cell
lines examined ( Fig. 3and Fig. 4), suggesting that
E2A-Pbx1a does not require tissue-specific complex formation for
cAMP-dependent transactivation. Although our data do not eliminate the
possibility that E2A-Pbx1 undergoes complex formation with ubiquitous
factors, E2A-Pbx1 may be capable of transactivating its target genes
without complex formation with other transcription factors.
The E2A breakpoints in the t(1;19) translocation occur in the 3.5-kilobase intron 13. The t(17;19) translocation also has breakpoints within E2A intron 13 fused to intron 3 of hepatic leukemia factor (Hlf) gene(33, 34) . This translocation results in synthesis of an E2A-Hlf fusion protein comprising the identical portion of the E2A gene product in the E2A-Pbx1 fusion protein. Whereas the t(1;19) translocation is exclusively associated with pre-B ALL, the t(17;19) translocation causes pro-B ALL. This suggests that target genes of Pbx1 and their regulation by the fusion protein play an important role in development of pre-B ALL. Because Pbx1 is activated by cAMP-dependent protein kinase A (PKA) and E2A-Pbx1 retains a putative PKA recognition site in its Pbx1-portion, it was important to determine whether E2A-Pbx1 is activated by a cAMP signaling pathway, as a first step in understanding its role in pre-B ALL.
We have observed dramatic
cAMP-dependent transactivation of E2A-Pbx1a, but not basal activation.
However, other groups have observed basal activation of the reporter
gene by co-transfection with the E2A-Pbx1 expression
vector(12, 13, 16) . To clarify this
inconsistency, we constructed the reporter gene plasmid, 4
PRS-Luc, containing four copies of the same PRS sequence, ATCAATCAA, as
described(13) , and used it instead of 4
CRS1 whose
Pbx1 binding sequence, TTGATTGGA, is similar but in the opposite
orientation to that of PRS(15) . Upon the co-transfection of 4
PRS-Luc with the E2A-Pbx1a expression vector on S194 cells, we
again observed no remarkable enhancement of luciferase activity without
PKA-C co-expression but a great activation with PKA-C. The difference
among the SV40,
-globin, and CYP17 minimal promoters was also
examined. Although the variation of absolute light emission levels in
luciferase assay was observed, no significant difference in the
transactivation pattern depending on promoters was found (data not
shown). The inconsistency over basal transcription with previous
reports (12, 13, 16) was not resolved in this
study. Because the transactivation level of E2A-Pbx1a induced by PKA is
in a comparable range with the SV40 enhancer and the CRE/CREB
system-positive controls in all cell types examined, we conclude that
this PKA-dependent transactivation of E2A-Pbx1a is biologically
significant.
Although the detailed cellular mechanisms are not known, the cAMP signaling pathway is important for differentiation and apoptosis of lymphocytes(18, 19, 35, 36) . The expression of the E2A-Pbx1 fusion protein is exclusively associated with a subgroup of childhood leukemia that exhibit arrest at the stage of pre-B cell differentiation(1, 2) . The present study therefore suggests that a cAMP signaling pathway required for proper differentiation and/or apoptotic cell death may activate the target genes of E2A-Pbx1 resulting in triggering the malignant transformation and proliferation of pre-B cells containing the t(1;19) translocation.