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INTRODUCTION |
Retroviral insertional mutagenesis in BXH-2 recombinant inbred
mice induces a high incidence of acute myeloid leukemia
(AML),1 and the proviral
integration sites in the leukemias provide powerful genetic tags for
disease gene identification (2, 3). During the past several years, a
number of disease genes have been identified in BXH-2 AMLs by proviral
tagging. They include a tumor suppressor gene, Neurofibromatosis
type 1 (Nf1); a gene with homology to the
lymphoid-restricted type II membrane protein Jaw1, Mrv integration site
1 (Mrvi1); a gene encoding a hematopoietic cell growth and differentiation factor, myeloblastosis oncogene (Myb); three
homeobox genes, homeobox A7 (Hoxa7), homeobox A9
(Hoxa9), and myeloid ecotropic viral integration site 1 (Meis1); a zinc finger protein gene, ecotropic viral
integration site 9 (Evi9); and a novel cytokine receptor (Evi27) (4-12). Importantly, two of these genes,
NF1 and HOXA9, are known human myeloid leukemia
disease genes, validating the usefulness of this approach for human
disease gene identification (9, 13).
Mutations in the human NF1 gene are responsible for the
cancer predisposition syndrome neurofibromatosis type 1 (13).
NF1 encodes neurofibromin, a GTPase-activating protein (GAP)
that is active on the four true Ras proteins (Ha-Ras, K-RasA, K-RasB, and N-Ras) as well as R-Ras (14-16). In BXH2 AMLs, about 15% of the
AMLs have proviral insertions in the Nf1 gene (4, 5). These
proviral insertions inhibit neurofibromin expression, and no wild type
neurofibromin is expressed in these AMLs (5). Loss of neurofibromin in
myeloid cells is associated with increased and prolonged Ras activation
after cytokine stimulation in chronic myeloid leukemia and in primary
cells (17). These results contribute to a large body of evidence
implicating aberrant regulation of Ras signaling in myeloid leukemia.
The other 85% of BXH-2 AMLs do not have proviral insertions in the
Nf1 gene (4, 5). This has led to the suggestion that these
AMLs have insertions in or near other genes involved in Ras regulation
and that these insertions activate Ras in the absence of inactivating
insertions at Nf1. One class of likely targets are the
guanine nucleotide exchange factors (GEF) that catalyze the exchange of
GTP for GDP on Ras and consequently turn on Ras. One such GEF is RasGRP
(18, 19). RasGRP is one of three guanine exchange factors that have
calcium-binding "EF hands" in addition to diacylglycerol
(DAG)-binding domains (18-20). It has been speculated that this class
of GEFs couple cell surface receptors that signal through
Ca2+ and DAG to the Ras pathway (18, 20).
In the studies described here we report the recurrent proviral
activation of the calcium- and diacylglycerol-binding guanine nucleotide exchange factor 1 (CalDAG-GEF I) gene in BXH-2 AML. Like
RasGRP, CalDAG-GEF I has EF hands and DAG domains. Previous studies,
however, have shown that CalDAG-GEF I is not a Ras GEF, rather it
appears to be a Rap1 GEF (1). We also show that forced overexpression
of CalDAG-GEF I results in transformation of cultured fibroblasts and
implicate Rap1 signaling in producing these effects. This is the first
evidence implicating Rap1 signaling in myeloid leukemia in human or mouse.
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EXPERIMENTAL PROCEDURES |
Molecular Cloning of CalDAG-GEF I Proviral
Insertions--
Proviral insertions in the CalDAG-GEF I
first intron were discovered as part of a large scale cloning effort
that utilized a long template, inverse PCR method, described in detail
elsewhere (6). In brief, 5 µg of genomic DNA from individual BXH-2
AMLs was digested with SacII overnight; the enzyme was
inactivated by heating at 65 °C for 10 min, and the DNA fragments
were ligated in 500-µl reactions, using 5 units of T4 DNA ligase
(Stratagene) at 4 °C overnight, to produce circular
provirus/cellular DNA templates for PCR amplification. The ligated
material was precipitated in ethanol and resuspended in 20 µl of
Tris-EDTA (pH 8.0). Two µl of this precipitated material was used as
template in a primary PCR in a 50-µl reaction volume containing 20 nmol of each dNTP, 10 pmol each forward and reverse primer, 1× buffer
2, and 2.5 units of enzyme mix in the ExpandTM Long
Template PCR System (Roche Molecular Biochemicals). Amplification was
performed with a Omnigene Hybaid thermocycler programmed as follows:
92 °C for 2 min; 10 cycles of 92 °C for 10 s, 63 °C for 30 s, 68 °C for 10 min; 20 cycles of 92 °C for 10 s,
63 °C for 30 s, 68 °C for 10 min with 20-s auto-extension.
The amount of primary PCR product was semi-quantified by 1% agarose
gel electrophoresis and 0.01 to 1 µl of the primary PCR product was
used as template in a secondary PCR under the same conditions except
that the secondary primers were used. The secondary PCR product was
separated on a 1% agarose gel, purified using the Geneclean II kit
(Bio 101), and directly cloned using CloneAmp® pAMP1 System (Life
Technologies, Inc.) according to supplied protocol. These particular
clones were obtained using primer pairs designed to amplify proviruses located 3' of genomic SacII sites. The primers used in the
primary PCRs are as follows: 5' ECO F1 (5'-GGCTGCCATGCACGATGACCTT-3') and 5' ECO R4 (5'-CGGCCAGTACTGCAACTGACCAT-3'). For the secondary PCR
the primer pair was 5' ECO F2-dUMP (5'-GAGGCCACCTCCACTTCTGAGAT-3') and
U3REVD-dUMP (5'-CTCTGTCGCCATCTCCGTCAGA-3'. The cloned PCR products were
sequenced using the PRISMTM BigDyeTM Cycle
Sequencing Kit (PerkinElmer Life Sciences) on an ABI model 373A DNA
Sequencer (Applied Biosystems). SP6 and T7 sequencing primers were
purchased from Life Technologies, Inc.
Southern and Northern Blot Analysis--
Isolation and analysis
of BXH2 genomic DNA and poly(A)+ RNA, by Southern and
Northern blotting, was performed using random primed
[32P]dCTP-labeled probe (Roche Molecular Biochemicals) as
described before (21).
Western Blot Analysis--
Stably or transiently transfected
cells were lysed in RIPA buffer (10 mM Tris-HCl (pH 8.0),
150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS) at a concentration of 10 × 106 cells per ml at 4 °C for 10 min. The lysates were
clarified by centrifugation for 15 min at 14,000 rpm to remove
insoluble material. The supernatants were frozen in liquid nitrogen and
stored at
70 °C. The concentration of protein in the supernatants
was determined using the Bradford assay. Fifty µg of protein was
loaded onto SDS-polyacrylamide gels, run, and transferred to
nitrocellulose as described (5). The blots were hybridized with
anti-tubulin or anti-V5 antibody (Invitrogen) according to
manufacturer's instructions and visualized with the ECL
chemiluminescence kit (Amersham Pharmacia Biotech).
Expression Constructs, Electroporation, and Retroviral
Vectors--
The CalDAG-GEF Ia and CalDAG-GEF Ib
open reading frames were cloned from a mouse bone marrow cDNA
library using the HIFI polymerase chain reaction kit from Roche
Molecular Biochemicals. Primer sequences used to amplify full-length
CalDAG-GEF Ia are forward 5'-ACCGGCAGCCATGACGA-3' and
reverse 5'-GTGGATGTCAAACACTCCGTCCTC-3'. PCR products were gel-purified
and cloned into the pcDNA3.1/V5/His-TOPO vector from Invitrogen.
This vector added a V5 epitope tag to the C-terminal end of the
CalDAG-GEF Ia protein. Primer sequences used to amplify full-length
CalDAG-GEF Ib are forward 5'-CTCGAGCCGTGGGAGGCTCTGAGA-3' and reverse
5'-TCTAGAAGCACATATGGTGTAGCATGCG-3'. These primers added a
XhoI site and a XbaI site to the 5' and 3' ends
of the PCR product, respectively. PCR products were gel-purified,
digested with XhoI and XbaI, and then cloned into
the XhoI and XbaI sites of the
pcDNA3.1/Myc-His(+) from Invitrogen. This vector added a Myc
epitope tag to the C-terminal end of the CalDAG-GEF Ib protein. Both
the CalDAG-GEF Ia and CalDAG-GEF Ib expression constructs were
sequenced for errors. Both expression constructs were used in an
in vitro transcription/translation experiment (Promega) to
confirm that they produced an ~70-kDa V5-tagged protein for CalDAG-GEF Ia and 19-kDa Myc-tagged protein for CalDAG-GEF
Ib. The CalDAG-GEF Ia insert was subcloned into the MSCV2.1 retroviral expression vector and electroporated into the PT67 amphotropic packaging cell line (CLONTECH). The
electroporated PT67 cells were selected in 400 µg/ml G418 in 10%
fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM),
and penicillin/streptomycin (P/S). Resistant colonies were pooled and
replated at 1-2 × 106 cells/ml. Viral supernatants
were harvested 24 h after cells reached confluence and stored at
70 °C.
Cell Culture Assays--
The following cell types were
transduced with MSCV-CalDAG-GEF Ia or MSCV2.1 empty vector virus and
selected in G418: Rat2, Swiss 3T3, FDCP1, and 32Dcl3Gr. All lines were
obtained from the ATCC, apart from 32Dcl3Gr, which was obtained from D. Askew (University of Cincinnati). Rat2 and Swiss 3T3 were grown in 10%
FBS/DMEM supplemented with P/S and selected in 400 µg/ml G418. FDCP1
and 32Dcl3Gr were grown in 20% WEHI-3 cell line conditioned medium in
10% FBS/DMEM supplemented with 10% NCTC-109, P/S, Hepes buffer (pH
7.4), 10 units/ml insulin, glutamine, sodium pyruvate, nonessential amino acids, and 10
5 M
-mercaptoethanol. All media and supplements were from Life Technologies, Inc., except insulin and
-mercaptoethanol (Sigma). FDCP1 and 32Dcl3Gr cells were selected in 1 mg/ml G418. Retroviral transduction was done by overnight infection of 10 × 106 (FDCP1 and 32Dcl3Gr) or 3 × 106 cells
(Rat2 and Swiss 3T3) in 10 ml of a 50:50 mix of high titer retroviral
pool and regular growth medium supplemented with 16 µg/ml Polybrene
(Sigma). Twenty four hours after infection, the cells were switched to
selective media. Pooled G418-resistant populations were analyzed for
phenotypes after mock-infected parallel cultures in G418 had cleared.
Rat2 cells were tested for colony formation by plating at 4 × 102 cells in 2 ml of 5 or 0.5% FBS/DMEM/P/S medium in
6-well plates. After 10 days (5% FBS) or 24 days (0.5% FBS) in
culture, the colonies were fixed and stained with methylene blue and
counted. Rat2 cells were also plated in soft agar colony forming assays
as described (22). Ten-centimeter plates of G418-resistant Swiss 3T3
transductants were examined for foci of morphologically transformed
cells after coming to confluence. These cells were trypsinized,
counted, and replated at 5 × 105 cells/10-cm plate in
10 ml of 10% FBS/DMEM or 5% calf serum (CS)/DMEM. Secondary foci
(from 10% FBS/DMEM cultures) and total cells per plate (from 10%
FBS/DMEM and 5% CS/DMEM cultures) were counted 14 days after the cells
were replated. For growth rates and saturation density, FDCP1
transductants were plated at 2 × 105 cells in 2 ml of
growth medium with 0, 10, or 50 ng/ml phorbol 12-myristate 13-acetate
(PMA, from Sigma) and counted every day for 4 days. For apoptosis
assays, transduced 32Dcl3Gr cells were washed three times in 1× PBS
and plated at a density of 1 × 106 cells per ml in
normal growth medium (previously described) lacking WEHI-3-conditioned
medium, the IL-3 source. PMA was also included in a subset of plates at
a concentration of 1 µM. Cells were scored at the
indicated time points for total cell count by trypan blue dye exclusion
and viability.
Measurement of Ras and Rap1 Activation States--
The
activation states of Ras and Rap1 were measured as described previously
in coupled enzymatic assays that measure the total amount of GTP and
GTP plus GDP bound to Ras or Rap1 (23-25). Briefly, cells were
harvested rapidly and snap-frozen in liquid nitrogen. For measuring Ras
activation, the frozen cells were extracted in a Hepes-based buffer
containing 1% Triton X-100, and after removing nuclei and subcellular
organelles from the extracts by brief centrifugation, the extracts were
split in half, one-half receiving the pan-Ras rat monoclonal antibody
Y13-259 (the experimental sample) and the other half receiving rat IgG
(the control sample). After shaking for 1 h at 4 °C, Ras was
collected on protein G-agarose beads to which goat anti-rat antibody
was bound. The immunoprecipitated Ras was washed four times in lysis
buffer, and GTP and GDP bound to Ras were eluted by heating. The
samples were split in half, and GTP was measured in one-half by
conversion to ATP using ADP, and the enzyme nucleoside diphosphate
kinase and the resulting ATP was measured by the firefly luciferase
system that is sensitive to 1 fmol. The control sample was subtracted
from the experimental sample, and the amount of GTP was determined from
standard curves prepared with each set of samples. The sum of GTP plus
GDP was measured in the other half of the sample by converting GDP to GTP using phosphoenolpyruvate and pyruvate kinase, and then GTP, which
now represents the sum of GTP plus GDP, was measured as above. Rap1
activation was measured similarly, but since there are no good
immunoprecipitating antibodies for Rap1, the Rap-binding domain of Ral
GDS fused to glutathione S-transferase was used to isolate
Rap GTP from one-half of the sample on glutathione-Sepharose beads
(25). The GTP was then eluted from Rap and measured as described above.
To measure the sum of GTP and GDP bound to Rap in the other half of the
sample, extracts were incubated in the absence of magnesium with 10 µM GTP to convert Rap-GDP to Rap-GTP. Excess free GTP was
removed by a temperature-dependent phase extraction since,
as part of the procedure for measuring Rap1 activation, cells were
extracted in 0.92% Triton X-114, 0.08% Triton X-45; an aqueous phase
containing the free GTP and a detergent phase containing the Rap1 were
generated by warming the samples to 15 °C for 2 min. Rap-GTP, which
now represented the sum of Rap-GTP plus Rap-GDP, was then isolated by
binding to the Rap binding domain glutathione S-transferase
fusion protein, and the GTP was eluted and measured as described above.
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RESULTS |
Proviral Integration at CalDAG-GEF I in BXH-2 AML Leads to
Increased CalDAG-GEF I Expression--
Proviral insertions within the
first intron of the CalDAG-GEF I gene were identified as
part of a large screen to discover proviral insertions that are located
near CpG islands (6). Sequence analysis of two of the clones showed
that they were located in the first intron of the mouse homolog of the
human CDC25-like gene (Fig.
1A) also called
CalDAG-GEF I (1). Northern blot analysis of one of the
leukemias (number 22) showed that CalDAG-GEF I expression
was elevated compared with other BXH-2 leukemias (numbers 13, 29, 106, 117, and 132) that did not contain viral integrations at
CalDAG-GEF I (Fig. 1B). These results show that viral integration at CalDAG-GEF I leads to increased
CalDAG-GEF I expression.

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Fig. 1.
A, proviral insertions in the first
intron of the CalDAG-GEF I gene. Exons are shown as
black boxes, and the position and orientation of the
proviruses at CalDAG-GEF I are indicated by an
arrow. B, Northern blot analysis of BXH-2 AMLs
with proviral insertions at CalDAG-GEF I. BXH-2
poly(A)+ RNAs are shown hybridized with a CalDAG-GEF
Ib cDNA probe. The size (in kilobase pairs (kb)) and position
of migration of RNA markers are indicated to the left. Tumor
22 has a proviral insertion in the first intron of the CalDAG-GEF
I gene. Below the blot are shown the Gapdh-normalized
CalDAG-GEF I transcript levels as determined using
PhosphorImager analysis.
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Two CalDAG-GEF I-hybridizing transcripts were detected in
all BXH-2 leukemias tested. This was surprising because the shorter transcript has not been reported in humans (1). Interestingly, both
transcripts were up-regulated by viral integration at CalDAG-GEF I (Fig. 1B). This prompted us to examine normal mouse
tissues for the shorter CalDAG-GEF I transcript. As shown in
Fig. 2, both CalDAG-GEF I
transcripts are expressed in normal mouse tissues. Expression was
highest in brain, heart, and lung followed by spleen, liver and kidney.
Expression was not detected in skeletal muscle. Both transcripts are
also expressed in the embryo with the highest expression found between
15 and 17 days of development. Subsequently, we confirmed that the
shorter transcript is also expressed in humans (data not shown). The
shorter transcript hybridized to probes composed of CalDAG-GEF
I exons 1-5 but not with probes from farther downstream (data not
shown). In fact, the size of the shorter transcript is approximately
what would be expected for an mRNA that was polyadenylated shortly
after the fifth exon. Quantitation by PhosphorImager analysis of normal
tissue Northern blots, hybridized with a probe containing exons 1-5,
showed that the long and short transcripts are usually present at a
roughly 1:1 ratio, although in some tissues more long form is expressed (Fig. 2).

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Fig. 2.
Expression of CalDAG-GEF I
mRNA in adult tissues and the embryo.
Poly(A)+ RNA, isolated from day 7, 11, 15, and 17 normal
mouse embryos or normal adult heart (Hr), brain
(Br), spleen (Sp), lung (Lg), liver
(Li), skeletal muscle (Sm), kidney
(Kd), and testis (Ts), was hybridized with a
CalDAG-GEF Ib cDNA probe. CalDAG-GEF Ia and
CalDAG-GEF Ib transcripts are indicated.
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Alternate Polyadenylation Predicts the Expression of a Full-length
(CalDAG-GEF Ia) and a C-terminal Truncated (CalDAG-GEF Ib)
Protein--
Sequence analysis of the human CalDAG-GEF I
genomic locus revealed a putative alternate polyadenylation site that
was located just after the splice donor site within intron 5 (Fig.
3). This polyadenylation site is also
present in the mouse. Examination of EST data bases revealed a number
of mouse and human ESTs representing cDNA clones in which
polyadenylation apparently occurred at this site rather than at the end
of the last CalDAG-GEF I exon. These cDNA clones contain
CalDAG-GEF I sequences from exon 5, which then read into the
fifth intron and are polyadenylated after a consensus ATAATA site.
Polyadenylation at this site would produce a transcript of ~0.6
kilobase pairs.

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Fig. 3.
Alternate polyadenylation of the
CalDAG-GEF I transcript and predicted primary sequence
of CalDAG-GEF Ia and CalDAG-GEF Ib isoforms. The predicted protein
domain structures for CalDAG-GEF Ia and CalDAG-GEF Ib are shown
above. Shown below are the nucleotide sequences
surrounding the splice donor site at the end of the mouse and human
fifth exon. Exon 5 sequences are shown in bold. The
consensus polyadenylation site (AATAAA) present in both mouse and human
intron 5 is shown in bold at the end of each sequence. The
amino acid sequence of the C terminus of CalDAG-GEF Ib, encoded by
intron 5 sequences, is shown for both mouse and human. Significant
homology exists between intron 5 encoded amino acid sequence between
human and mouse.
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The short CalDAG-GEF Ib transcript is predicted to encode a
truncated protein. CalDAG-GEF Ib would include the Ras exchanger motif
(REM) domain, amino acid sequences between the REM domain and CDC25
domain (i.e. the catalytic core domain similar to that from
the yeast CDC25 gene), and 17 (mouse) or 20 (human)
amino acids encoded by intron 5 (Fig. 3). Comparison between the human and mouse CalDAG-GEF I genomic sequences showed conservation
in the region surrounding the alternate polyadenylation site in the fifth intron and indeed the whole fifth intron (72% identity). In
contrast, other intron sequences showed much less conservation (e.g. fourth intron, 51% identity). These data indicate
that the CalDAG-GEF I locus produces two transcripts through
alternate polyadenylation, which encode a full-length protein,
CalDAG-GEF Ia, and a truncated protein, CalDAG-GEF Ib.
Effects of CalDAG-GEF Ia Overexpression in Fibroblasts and Myeloid
Cells--
Both CalDAG-GEF I transcripts were cloned into
mammalian expression vectors through PCR amplification of lymph node
cDNA. Primers were designed for both transcripts to allow the PCR
fragments to be cloned in-frame into an expression vector and to add an epitope tag to the 3' end of each cDNA. This resulted in a
Cdgla clone tagged with a V5 epitope and a CalDAG-GEF
Ib clone tagged with a Myc epitope. The cytomegalovirus promoter
was used to drive both constructs. To determine whether the expression
vectors functioned as expected, we transiently transfected each vector
into HeLa cells and then followed their expression using antibodies to
the epitope tag. Surprisingly, we were unable to detect CalDAG-GEF Ib
protein expression by Western analysis. This contrasts with in
vitro transcription/translation studies where we were able to
detect Cgd1b epitope-tagged protein (data not shown). We have also
failed to generate stable CalDAG-GEF Ib-expressing transfectants. These
results suggest that the CalDAG-GEF Ib protein is toxic, unstable, or
produced at low levels.
To test for possible transforming effects of the CalDAG-GEF Ia protein,
an MSCV2.1-based expression vector for CalDAG-GEF Ia was constructed
(MSCV-CalDAG-GEF Ia) and tested for its ability to express V5
epitope-tagged CalDAG-GEF Ia protein in FDCP1, Rat2, and 32Dcl3Gr
cells. All MSCV-CalDAG-GEF Ia vector transduced cells expressed readily
detectable protein (Fig. 4). The Rat2
fibroblast cell line was subsequently transduced with CalDAG-GEF
Ia-expressing virus or empty vector virus pools and selected in G418.
Stable Rat2 transductants were obtained, and pools of greater than 50 colonies of each were assayed for growth in soft agar and growth rates
in 0.5 or 5% FBS, with or without added PMA, a diacylglycerol analog,
were measured. Neither population contained cells capable of forming
large colonies in soft agar (data not shown). The number of colonies
formed in liquid culture in 0.5% FBS/DMEM was significantly higher for
MSCV-CalDAG-GEF Ia transductants than for the empty vector
transductants both with and without added PMA (Fig.
5A). At higher serum
concentrations, both populations gave rise to a similar number of
colonies.

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Fig. 4.
Expression of epitope-tagged CalDAG-GEF Ia in
fibroblast and myeloblast cell lines. Total cell lysates from
MSCV2.1 empty vector (M) or MSCV-CalDAG-GEF Ia vector
(C)-transduced FDCP1, Rat2, and 32Dcl3Gr cells were
hybridized with an anti-V5-specific monoclonal antibody. All
MSCV-CalDAG-GEF Ia vector-transduced cells expressed readily detectable
protein.
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Fig. 5.
Activity of CalDAG-GEF Ia in Rat2 and Swiss
3T3 cells. A, colony formation at low serum in Rat2
cells. The number of colonies present after seeding 4 × 102 cells per plate of MSCV2.1-transduced Rat 2 cells
(white bars) or MSCV-CalDAG-GEF Ia-transduced Rat2 cells
(black bars) is indicated. Error bars indicate
standard deviations. B, transformed foci in CalDAG-GEF
Ia-transduced Swiss 3T3 cells. Photomicrograph of parental Swiss
3T3 cells, MSCV2.1-transduced Swiss 3T3 cells (MSCV2.1), and of four
different transformed foci of MSCV-CalDAG-GEF Ia-transduced Swiss 3T3
cells (MSCV-CalDAG-GEF Ia). C, primary and secondary foci
and saturation density in Swiss 3T3. The number of Swiss 3T3 cells per
10-cm dish at confluency is shown for MSCV2.1-transduced cells
(white bars) and for MSCV-CalDAG-GEF Ia-transduced Swiss 3T3
cells (black bars). The experiment was performed in 10% FBS
or 5% CS. In addition, the average number of transformed foci of
parental Swiss 3T3, MSCV2.1-transduced, or MSCV-CalDAG-GEF
Ia-transduced Swiss 3T3 cells is shown. Primary foci are the number
that appeared after G418 selection and growth to confluency. Secondary
foci are the number that appeared after replating and growth to
confluency. Error bars indicate standard deviations.
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In contrast to Rat2 cells, Swiss 3T3 cells transduced with
MSCV-CalDAG-GEF Ia formed multiple, transformed foci upon reaching confluence after G418 selection (Fig. 5B). These foci
appeared as areas of more spindle-shaped cells, growing at high density and in multiple layers. The size of these foci increased with time in
culture. Upon replating, many secondary foci were present in cultures
transduced with the MSCV-CalDAG-GEF Ia vector. These transformed foci
were more apparent in 10% FBS than in 5% calf serum (CS) medium. The
CalDAG-GEF Ia-transduced Swiss 3T3 cells also reached a
higher saturation density than did empty vector transductants (Fig.
5C).
The MSCV-CalDAG-GEF Ia and empty vector retroviruses were also used to
transduce cells from the myeloid cell lines 32Dcl3Gr and FDCP1 to test
the effects of CalDAG-GEF Ia overexpression on myeloid
cells. The growth and viability of 32Dcl3Gr and FDCP1 cells are
dependent on the presence of interleukin-3 (IL-3) in the culture
medium. If IL-3 is removed from the culture medium, these cells will
die by apoptosis. Previous studies have suggested that Rap1 plays a
role in regulating integrin-mediated cell adhesion to fibronectin
(26-28) and therefore fibronectin was included in some of these experiments.
The addition of PMA (50 ng/ml), in the absence of IL-3, caused a slight
delay in apoptosis in MSCV-CalDAG-GEF Ia-transduced but not control
empty vector-transduced 32Dcl3Gr cells, which was most apparent in
fibronectin-coated plates (data not shown). PMA and calcium
ionophore-treated MSCV-CalDAG-GEF Ia-transduced 32Dcl3Gr cells
displayed a substantial increase in adherence to fibronectin compared
with controls (Fig. 6A). FDCP1
cells expressing CalDAG-GEF Ia at high levels did not become
IL-3-independent but showed a subtle increase in growth rate and
saturation density compared with empty vector transductants (Fig.
6B). However, the difference in growth rate and saturation
density was evident only in the presence of 10 or 50 ng/ml PMA. PMA
activates the guanine nucleotide exchange activity of CalDAG-GEF I
(1).

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Fig. 6.
Activity of CalDAG-GEF Ia in FDCP1 and
32Dcl3Gr cells. A, appearance of adherent cells in PMA
plus calcium ionophore (white boxes; PMA + Ca) and IL-3 plus
PMA plus calcium ionophore (black boxes; IL-3 + PMA + Ca)-treated 32Dcl3Gr cells. The average number of cells per field
(at × magnification) remaining on the tissue culture dish 2 h after replating MSCV2.1-empty vector transductants or MSCV-CalDAG-GEF
Ia transductants and washing in PBS is shown. Standard deviations are
indicated. B, fold increase at saturation density of FDCP1
cells growing in IL-3. The maximal fold increases in cell number for
MSCV2.1-transduced FDCP1 cells (white boxes) or
MSCV-CalDAG-GEF Ia-transduced FDCP1 cells (black boxes) are
shown. Cells were initially plated in normal growth media with IL-3 at
0, 10, or 50 µg/ml PMA, and the cell number was determined every day
for 4 days. Error bars indicate standard deviations.
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Rap1, but Not Ras, Activation in Cells Expressing CalDAG-GEF Ia at
High Levels--
To determine whether enforced expression of
CalDAG-GEF Ia in cells resulted in increased endogenous Rap
activation in myeloid cells, we measured endogenous Rap-GDP and Rap-GTP
in empty vector or MSCV-CalDAG-GEF Ia transduced populations of
32Dcl3Gr cells. These cells were starved of serum and IL-3 for 10 h and stimulated with calcium ionophore and PMA to activate CalDAG-GEF
Ia for 15 min. CalDAG-GEF Ia-transduced cells showed an
increase in induced Rap-GTP levels at 15 min compared with control
cells (Fig. 7). The levels of Ras-GTP did
not vary significantly between CalDAG-GEF Ia and parental
32Dcl3Gr cells during this time (Fig. 7). This is consistent with other
reports showing that CalDAG-GEF I overexpression does not affect
Ras-GTP levels in transiently transected cells (1). Thus,
CalDAG-GEF Ia expression at high levels can influence the
level of endogenous Rap1 that becomes activated in response to
stimulation.

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Fig. 7.
Ras and Rap activation in CalDAG-GEF
Ia-transduced cells. The percentage of total Rap1
protein bound to GTP or total Ras protein bound to GTP is indicated for
32Dcl3Gr cells transduced with MSCV2.1-empty vector or MSCV-CalDAG-GEF
Ia viruses. These values are from cells at time 0 (white
bars) and 15 min after stimulation (black bars) of IL-3
and serum-starved cells with calcium ionophore and PMA. The average of
an experiment done in triplicate is shown.
|
|
As has been reported in HL60 cells (25), Rap1 protein in 32Dcl3Gr cells
shows a relatively high basal state of activation compared with Ras
(Fig. 7). This has been suggested to be due to the presence of a
threonine at position 61 in Rap1, which decreases its intrinsic GTPase
activity (25).
 |
DISCUSSION |
Proviral Activation of a Rap1 GEF Gene--
A screen of somatic
murine leukemia virus integration sites in BXH-2 AML has
identified a GEF gene, CalDAG-GEF I, that is a target for
murine leukemia virus integration in BXH2 AMLs. Proviral integration at
CalDAG-GEF I leads to increased CalDAG-GEF I
expression suggesting that CalDAG-GEF I is a proto-oncogene.
CalDAG-GEF I is the second GEF to be implicated as a myeloid
leukemia disease gene in humans or mouse. The NUP98 gene is
fused in frame, by translocation, to the broadly active GEF gene
smgGDS in some human myeloid leukemias, the result of which
may be an increase in GTPase activation levels (29).
CalDAG-GEF I is closely related to another GEF, RasGRP (1,
18, 19, 20). RasGRP has been shown to have GEF activity for Ha-Ras but
little activity for other Ras superfamily members such as Rap, Rho, or
Rac (1, 18, 19). In contrast, CalDAG-GEF I shows little GEF
activity for Ha-Ras but very high activity for Rap1 (1). More recently,
RasGRP, CalDAG-GEF I, and a newly identified GEF, CalDAG-GEF III, have
also been shown to have exchange activity for R-Ras and TC21 GTPases
(16, 30). Furthermore, another recent paper (31) suggests that
CalDAG-GEF I can cause exchange on N-Ras and K-Ras but not Ha-Ras
proteins in cells that are chronically stimulated with PMA or high
serum. Thus, activation of Rap1, Ha-Ras, N-Ras, R-Ras, and TC21 GTPases
could potentially mediate the oncogenic effects of CalDAG-GEF
I.
Small G Protein Signaling and Cancer--
There is abundant
genetic evidence from studies of primary cancers, cancer models, and
transformation systems for a central role for the so called "true"
Ras genes (HRAS, NRAS, and KRAS) in
cancer development (32, 33). Much less is known, however, about the
role of other small G proteins of the Ras-like subfamily in
oncogenesis. Rap1 was initially identified in a screen for genes that
can suppress the transformed phenotype of K-Ras-transformed fibroblasts
(34). This would suggest that Rap1 signals suppress rather than promote
oncogenesis. Later publications (35-37) showed that the Rap1 effector
domain, being virtually identical to that of Ras, can bind to many Ras
effectors without activating them. These data have led to the
hypothesis that Rap1 serves as a Ras antagonist by sequestering Ras
effectors. More recent work (38), however, has shown that Ras activity
is not inhibited by endogenous Rap1 activation, suggesting that Rap1
has distinct biological functions, apart from the inhibition of Ras.
Support for this hypothesis has come from studies of the
TSC2 tumor suppressor gene. TSC2 encodes tuberin,
which has Rap1 GAP activity (39). In addition, two common human
gliomas, astrocytoma and ependymoma, have been shown to have increased
Rap1 expression or reduced/absent tuberin expression in 50-60% of the
tumors examined (40). In Swiss 3T3 cells, Rap1-GTP can induce DNA
synthesis and a transformed morphology (41). Expression of wild type
Rap1 at high levels in Swiss 3T3 cells causes morphological
transformation and tumorigenicity in nude mice (42). This phenotype is
similar to our observations with CalDAG-GEF Ia, which causes
morphological transformation of Swiss 3T3 cells but not NIH 3T3 or Rat2 cells.
Our data suggest that signaling via CalDAG-GEF Ia in myeloid cells can
promote cellular proliferation and increase adherence. This is
consistent with other published data, which also suggest that Rap1
signaling regulates adherence. For example, overexpression of
the Rap1 GAP, SPA-1 in 32Dcl3 cells leads to a block in Rap1 activation and adherence during granulocyte-colony stimulating factor induced differentiation (28). Rap1 also seems to mediate adhesion induced by CD31 activation in lymphoid cells (27) and is a
major LFA1 activator, permitting adhesion to fibronectin (26). Our
results are consistent with a role for Rap1 activation in adherence to
fibronectin in myeloid cells. It could be imagined that adhesion to
fibronectin gives an AML clone a selective advantage, either by
suppression of apoptosis via the so-called "outside-in" integrin
signal or by permitting the clone to colonize extramedullary sites or
extravasate more readily.
Other data indirectly implicate Rap1 signaling in cell cycle control
and proliferation. Expression of the Rap1 GAP, tuberin, regulates the
abundance and subcellular distribution of p27 and cyclin D1 proteins in
fibroblasts (43). Our data suggest that overexpression of CalDAG-GEF Ia
can cause inappropriate cell division in Rat2 cells at low serum. This
may be analogous to the effects seen after tuberin depletion in other
fibroblast cell lines (43).
It is also possible that R-Ras and/or TC21 mediate the effects of
CalDAG-GEF Ia overexpression. Indeed, TC21 (43-45) and R-Ras (46, 47)
can cause malignant transformation of rodent fibroblasts. TC21 has been
shown to be overexpressed in breast carcinoma and activated by amino
acid substitution or insertional mutation in some breast and ovarian
cancer cell lines (48-50). Furthermore, activated R-Ras can suppress
apoptosis and stimulate adhesion in 32Dcl3 cells (51). Finally, recent
data suggest that CalDAG-GEF I is produced as both the form we have
identified and a longer form, referred to as RasGRP2, that is
myristoylated or palmitoylated and can act on N- or K-Ras (31).
Proviral insertion at this locus may therefore not only up-regulate
CalDAG-GEF Ia but also the longer form called RasGRP2. Both forms were
shown to be capable of GEF activity in cells for N-Ras or K-Ras.
However, the shorter nonfatty acid modified form of CalDAG-GEF I was
only capable of N- and K-Ras GEF activity after chronic PMA treatment
or growth in high serum, when it becomes localized to the plasma
membrane. However, it is not clear that the longer form of this
protein, called RasGRP2, is actually conserved in the mouse. We have
looked for mouse EST clones, analogous to the human RasGRP2 isoform, but could find none. Furthermore, the alternate exon that encodes most
of the additional N-terminal amino acids in RasGRP2 is not well conserved at the mouse CalDAG-GEF I locus (data not
shown). In addition, we have not ever detected increased Ras activation in CalDAG-GEF I overexpressing 32Dcl3 or FDCP1 cells. Nevertheless, it
is conceivable, and seems appealing, to consider that the combined activation of multiple small GTPases by CalDAG-GEF I is responsible for
its oncogenicity.
It is at present unclear which of the signaling pathways downstream of
Rap1, TC21, Ha-, N- or R-Ras activation can be linked to apoptosis
suppression, adherence, or hyperproliferation seen in CalDAG-GEF
Ia-overexpressing cells. Rap1 signaling has been shown to activate the
mitogen-activated protein kinase pathway independently of Ras signaling
via B-Raf (52, 53). In addition, Rap1-GTP, like Ras can bind to the Ral
guanine exchange factors RalGDS and Rgl1 (35, 36). The function of
Ral-GTP is not known, but Ral dominant-negative mutants block
R-Ras-induced adhesion to fibronectin in 32D cells (51). Ral is also
thought to be downstream of Ras signaling and forms a required
component of the Ras transformation response (54, 55). TC21 and R-Ras
have been shown to activate the stress-activated protein kinases, p38 and JNK, as well as phosphatidylinositol 3-kinase (44-47). Further work will be required to determine which of these pathways is important
for CalDAG-GEF I-induced leukemia. Furthermore, the CalDAG-GEF II (RasGRP1) Ras GEF gene is found at
a common site of proviral insertion and may be activated (6), and the
Ras GAP Nf1 gene is inactivated in BXH2 AML (4, 5).
Therefore, it will be important to determine whether and where Rap1 and
N-, K-, or Ha-Ras signalings overlap. Indeed, a lot of data have
accumulated showing that small G proteins can cooperate in cell growth
control (56) or must act in concert for cellular transformation to
occur (37, 51, 57-60).
Functional Role of a Truncated GEF--
CalDAG-GEF Ib, the
truncated form of CalDAG-GEF Ia, is unique in that no other GEFs
identified thus far have a truncated form. The simplest model to
explain the function of CalDAG-GEF Ib is that it acts as a
dominant-negative form of CalDAG-GEF Ia, serving to modulate its
activity. The only identified domain within CalDAG-GEF Ib is the REM
domain. A REM-like domain is found N-terminal to the core catalytic
CDC25-like domain of many GEF proteins. Although the function of the
REM domain is not entirely clear, an intact REM domain is required for
the transforming effects of RasGRP overexpression in fibroblasts (19).
The CDC25-like core catalytic domain of CDC25Mm will catalyze the
exchange of GDP for GTP on purified Ras protein, but the inclusion of
the REM domain increases the efficiency of this reaction (61).
The crystallization and structural determination of Ha-Ras protein
complexed with a fragment of the SOS protein lends some insight into
the role of the REM domain (62). The REM domain of SOS contains three
-helices. The first two of the helices interact with, and may
stabilize, a portion of the CDC25-like domain (62). The third
-helix
and the region of the protein between the REM domain and the CDC25-like
domain interact with neither the core catalytic CDC25-like domain nor
Ha-Ras. This portion of SOS may thus interact with other proteins. The
amino acid sequence of the region between the REM and CDC25-like
domains is not conserved between SOS and CalDAG-GEF I. The conservation of a short form of CalDAG-GEF I, CalDAG-GEF Ib, in both mouse and human
that includes the REM domain and most of the amino acid sequence
between the REM domain and the CDC25-like domain, implies that this
region of the protein may have an unidentified and unique regulatory
function. For example, CalDAG-GEF Ib may regulate CalDAG-GEF Ia
activity by interacting with and titrating a protein(s) that also binds
to and regulates CalDAG-GEF Ia and perhaps other GEFs.
CalDAG-GEF I in Other Malignancies--
CalDAG-GEF I
maps to human chromosome 11q13 (20). The 11q13 region has been
implicated in a variety of malignancies including breast
adenocarcinoma, bladder carcinoma, gastric cancer, B cell follicular
lymphoma, and oral squamous cell carcinoma (63, 64). Both
translocations and gene amplifications have been observed in these
cases. It is widely believed that activation of the cyclin D1 gene
(CYCD1) or fibroblast growth factor superfamily genes, such
as FGF and HIS, are the targets for genomic
alterations in this region (64). However, our identification of
CalDAG-GEF I as a proto-oncogene in BXH-2 AML suggests that
its activation may also be selected in some of these cases. Regarding
these tumors, it seems possible that selection is occurring due to
effects on multiple genes. Rap activity has been implicated in changes
in the adhesion and migratory capacity of cells in culture (26-28). Thus, it seems plausible that CalDAG-GEF I gene activation
could play a role in the metastatic behavior of these carcinomas.