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Blood, 15 February 2002, Vol. 99, No. 4, pp. 1332-1340
NEOPLASIA
Mutations in the gene encoding the transcription factor
CCAAT/enhancer binding protein  in myelodysplastic syndromes and
acute myeloid leukemias
Adrian F. Gombart,
Wolf-K. Hofmann,
Seiji Kawano,
Seisho Takeuchi,
Utz Krug,
Scott H. Kwok,
Renee J. Larsen,
Hiroya Asou,
Carl W. Miller,
Dieter Hoelzer, and
H.
Phillip Koeffler
From Cedars-Sinai Medical Center, Burns and Allen
Research Institute, Division of Hematology/Oncology, University of
California-Los Angeles School of Medicine; Division of Hematology and
Clinical Laboratories, Keio University School of Medicine, Tokyo,
Japan; and Department of Hematology, University Hospital, Frankfurt am
Main, Germany.
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Abstract |
The CCAAT/enhancer binding protein  (C/EBP) protein is
essential for proper lung and liver function and granulocytic and adipose tissue differentation. It was hypothesized that
abnormalties in C/EBP function contribute to the development of
malignancies in a variety of tissues. To test this, genomic DNA from
408 patient samples and 5 cell lines representing 11 different cancers
was screened for mutations in the C/EBP gene. Two silent
polymorphisms termed P1 and P2 were present at frequencies of 13.5%
and 2.2%, respectively. Of the12 mutations detected in 10 patients,
silent changes were identified in one nonsmall cell lung cancer, one prostate cancer, and one acute myelogenous leukemia (AML) subtype M4.
The 9 remaining mutations were detected in 1 of 92 (1.1%) myelodysplastic syndrome (MDS) samples and 6 of 78 (7.7%) AML (AML-M2
and AML-M4) samples. Some mutations truncated the predicted protein
with loss of the DNA-binding (basic region) and dimerization (leucine
zipper [ZIP]) domains by either deletions or nonsense codons. Also,
inframe deletions or insertions in the fork region located between the
leucine zipper and basic region, or within the leucine zipper,
disrupted the -helical phase of the bZIP domain. The inframe
deletion and insertion mutations abrogated the transcriptional
activation function of C/EBP on the granulocyte colony-stimulating
factor receptor promoter. These mutants localized properly to
the nucleus, but were unable to bind to the C/EBP site in the promoter
and did not possess dominant-negative activity. The mutations in the
MDS patient and one AML-M2 patient were biallelic, indicating a loss of
C/EBP function. These results suggest that mutation of C/EBP is
involved in specific subtypes of AML and in MDS, but may occur rarely
in other types of leukemias or nonhematologic malignancies.
(Blood. 2002;99:1332-1340)
© 2002 by The American Society of Hematology.
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Introduction |
The CCAAT/enhancer binding protein (C/EBP)
belongs to a family of proteins that possess a bipartite DNA-binding
domain composed of a positively charged basic (b) region that contacts the DNA and a leucine zipper (ZIP) in the C terminus that mediates dimerization.1 The less-conserved N terminus
contains regulatory and transactivation domains.2-5
C/EBP is expressed in a number of tissues, most prominently in the
highly differentiated cells of the liver, white and brown adipose,
lung, and myeloid-lineage cells.6-9 It has also been
detected in the adrenal gland, skin, pancreas, prostate, differentiated
enterocytes in the intestine, and, during follicular development, the
ovary.9-12
C/EBP is proposed to be a regulator of energy metabolism and
transcriptionally activates the promoters of energy-related genes such
as GLUT4 and PEPCK in hepatocytes and adipocytes.13-15 In
myeloid cells, C/EBP transcriptionally activates the promoters of
the myeloid-specific receptors for the growth factors macrophage colony-stimulating factor, granulocyte colony-stimulating
factor (G-CSF) and granulocyte-macrophage colony-stimulating
factor.16-18 Studies demonstrate that C/EBP is critical
for the process of terminal differentiation of adipocytes. C/EBP is
upregulated in adipocyte differentiation, and blocking its expression
halts differentiation of preadipocytes into adipocytes, while
overexpression induces differentiation and inhibits
proliferation.19-23 Also, overexpression of C/EBP
induces differentiation of myeloid leukemia cell lines and inhibits the
proliferation of a number of cell lines and tumor
cells.24-27 The inhibition of proliferation is partly due
to the ability of C/EBP to activate transcription and induce
posttranscriptional stabilization of the cyclin-dependent kinase
inhibitor p21 (WAF-1).28,29 These studies suggest a central role for C/EBP in the regulation of cell proliferation and differentiation.
Targeted inactivation of C/EBP in mice demonstrates its importance
in the proper development and function of liver, adipose, lung, and
hematopoietic tissues.8,30,31 Within 8 hours after birth,
the mice die of impaired glucose metabolism, and adipose metabolism is
altered with a failure of adipocytes to accumulate lipids.30,31 The lung shows hyperproliferation of type II
pneumocytes and abnormal alveolar structure, and histopathology of the
liver displays a structure resembling regenerative changes or
hepatocellular carcinoma.30-32 The null mice also display
impaired neutrophil development intrinsic to the hematopoietic
system resulting from a blockade in differentiation and the absence of
expression and signaling of the G-CSF and interleukin-6
receptors.8,33
The importance of C/EBP in cell growth and differentiation suggests
it may play the role of a tumor suppressor. A number of studies support
this hypothesis. Expression of C/EBP was transcriptionally downregulated in preneoplastic hepatic nodules and further decreased in
hepatocellular carcinomas in rat liver.34 Primary
hepatocytes from newborn C/EBP-null mice showed significantly higher
proliferative rates than wildtype mice, while cell lines from null mice
exhibited rapid growth and accumulation of chromosomal
abnormalities.35 These cell lines were capable of forming
nodules when inoculated into nude mice. In squamous cell carcinomas,
the expression of C/EBP was greatly diminished.36
Because of the early lethality of the C/EBP-null mice, determination
of whether these mice are susceptible to the development of tumors is
not possible. The heterozygous (+/ ) mice were not observed to suffer
from an increased rate of tumor development.31 In this
study, we tested the hypothesis that C/EBP may function as a tumor
suppressor that is mutated during tumorigenesis. We report the findings
from a screen of 408 patient samples representing 11 different cancers.
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Materials and methods |
Samples
Primary samples (408) from 11 types of human malignancies were
studied. These are summarized in Table
1.The following cell lines were examined:
HL60, Kasumi-1 [AML-M2, t(8;21)], Kasumi-5, KG-1, and NB-4 (AML-M3).
The soft-tissue sarcoma samples were generously provided by Jonathan
Said (Department of Pathology, UCLA School of Medicine). Genomic DNA
was extracted as described.37
Polymerase chain reaction-single-strand conformation polymorphism
analysis
The primers used to amplify the C/EBP genomic locus are
described in Table 2 and represented
graphically in Figure 1. The nucleotide numbering throughout this study is based on the published sequence available from EMBL/GenBank/DDBJ under accession number NM_004364.1. The primers were used in the following combinations to
amplify 4 overlapping regions (regions 1 through 4) of the genomic DNA:
(1) 1F plus 1R, 306 base pairs (bp); (2) 2F plus 2R, 309 bp; 3F plus
3R, 366 bp; and (4) 4F plus 4R, 266 bp. Platinum Taq DNA polymerase
(Gibco/BRL, Bethesda, MD) with 4% dimethyl sulfoxide was used to
amplify the fragments from regions 1, 2, and 4. To amplify region 3, Failsafe Taq DNA polymerase (Epicentre Technologies, Madison, WI) and
buffer K (supplied by the manufacturer) were used. The polymerase chain
reaction (PCR) conditions were as follows: 94°C, 3 minutes; 35 cycles
95°C, 30 seconds; either 66°C (region 1) or 64°C (regions 2, 3, and 4), 30 seconds; and 72°C, 1 minute. Shifted bands were excised
from the dried gel and eluted in double-distilled H2O,
reamplified, and gel purified for sequencing. The products were ligated
into a pCR2.1-TA cloning vector (Invitrogen, Carlsbad, CA) as described
by the manufacturer. Products were sequenced in both directions with an
ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit
(Perkin-Elmer, Foster City, CA) with the use of the region 1 through 4 PCR-single-strand conformation polymorphism (PCR-SSCP) primers or
primer-binding sites available in the plasmid (T7 and M13R) for the
cloned fragments. The sequencing reactions were analyzed by an ABI 377 sequencing machine.
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| Figure 1.
A schematic diagram of the C/EBP gene locus.
The gene does not contain introns; therefore, the coding region is
contained within one exon represented as an open box. The domains
discussed in the text are indicated by the shaded boxes. The position
of the primers used for PCR-SSCP analysis for each region (1 through 4)
are indicated by the arrows above (forward or sense primers) and below
(reverse or antisense primers) the schematic. Abbreviations: ADM,
activation domain modules; BR, basic region; and L-ZIP, leucine
zipper.
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Expression vectors
The entire coding region (nucleotide [nt] 132-1230) of
wildtype or mutant C/EBP was amplified with primers ATG-FIX
(5'-ggagaactctaactccaccatggagtcgg-3') and 4R under the previously
described PCR conditions for region 4. The C-nucleotide at the 3
position from the ATG was changed to an A-nucleotide to create a
perfect Kozak consensus sequence.38 The fragments were
gel-purified and cloned into the TA-cloning vector pCR2.1 (Invitrogen).
Sequencing was performed to verify the integrity of the insert.
The inserts were subcloned into the MIG retrovirus expression
vector (kindly provided by Dan Tenen, Harvard Medical School,
Boston, MA) with the use of EcoRI39 and the pCMV-Sport1
vector (Gibco/BRL). The orientation was determined by means of the
BglII and NotI restriction enzymes.
Cell lines, transfections, and analysis of protein
expression
NIH3T3 cells were maintained in Dulbecco modified Eagle medium
supplemented with 10% bovine serum. For cellular localization, cells
were plated at 70% confluency on a glass coverslip in a 35-mm dish.
The cells were transfected with 3 µg MIG, MIG-C/EBP mutant, or
pCMV-C/EBP wildtype plus pEGFPc with the use of 15 µL GenePORTER,
as described by the manufacturer (Gene Therapy Systems, San Diego, CA).
At 24 hours after transfection, cells were fixed in 10% formalin for 1 hour, washed twice in phosphate-bufferend saline (PBS), and
permeablized in PBS with 0.1% Triton X-100 for 5 minutes. The cells
were incubated with rabbit anti-C/EBP antibody (SC-61) diluted to
0.01 µg/mL (Santa Cruz Biotechnology, CA) for 1 hour, washed twice
with PBS, and incubated with a 1:200 dilution of tetrarhodamine
isothiocyanate-conjugated goat antirabbit immunoglobulin G (Sigma, St
Louis). The coverslips were mounted with Gel/Mount (Biomeda, Foster
City, CA) and examined by fluorescent microscopy. Total cell protein
was prepared from NIH3T3 cells transfected with pCMV,
pCMV-C/EBP42 wildtype (WT) or mutant expression vectors and lysed in 0.5% NP-40 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40). Western blot analysis was performed as described previously.40 The blots were incubated overnight with 0.2 µg/mL of either rabbit anti-C/EBP antiserum (Santa Cruz
Biotechology) diluted in PBS containing 5% powdered
milk.41 The primary antibodies were detected with either
donkey antirabbit-horseradish peroxidase (HRP) or antigoat-HRP
(1:5000). The complexes were developed with SuperSignal West Pico
Chemiluminescent Substrate (Pierce, Rockford, IL), as described by the
manufacturer, and detected by autoradiography.
Transcriptional activation assays
For transcriptional activation assays, NIH3T3 cells were plated
in a 12-well dish at 50% to 70% confluency. For each triplicate, the
plasmids were prepared as a master mix of pG-CSF receptor-luciferase, pRLSV40, a renilla luciferase expression vector (Promega, Madison, WI),
C/EBP expression vector, and empty vector, for a final total of 3 µg DNA. The combinations and amounts of expression vectors are
indicated in the figure legends. The plasmids in 0.5 mL Opti-MEM (Gibco/BRL) were mixed with 15 µL GenePorter in 0.5 mL Opti-MEM and
incubated 45 minutes; then, 0.33 mL were aliquoted to each well of the
12-well plate (1 µg DNA per well). The reporter plasmid pG-CSF
receptor-luciferase was generously provided by Dan
Tenen.16 At 24 hours after transfection, cells were
harvested in passive lysis buffer, and luciferase activity was measured
by means of a dual luciferase assay (Promega).
Electrophoretic mobility shift assays
A double-stranded oligonucleotide containing the C/EBP site (bp
57 to 38; 5'-aaggtgttgcaatccccagc-3') of the human G-CSF receptor
promoter was end-labeled with  32P adenosine triphosphate
by means of T4 polynucleotide kinase as described by the manufacturer
(Gibco/BRL).16 The labeled oligonucleotide (1 ng per
reaction) was mixed with equal volumes of total cell extract from
NIH3T3 cells transfected with expression vectors for the different
C/EBP proteins and buffer B (20% glycerol, 20 mM Hepes [pH 7.9],
50 mM NaCl, 2 mM MgCl2, and 1 mM dithiothreitol). Poly (dI-dC)
(Amersham Pharmacia Biotech, Piscataway, NJ) and bovine serum albumin
(Sigma) were added to 50 µg/mL and 300 µg/mL final concentrations,
respectively. Reactions were incubated at room temperature for 30 minutes and analyzed by gel electrophoresis through a 4%
polyacrylamide gel with the use of a Tris-glycine buffer (50 mM Tris,
400 mM glycine, 1 mM EDTA, with pH adjusted to 8.5). For supershifts,
0.2 µg antibody was added to the reaction 30 minutes prior to
addition of the probe. Gels were exposed to Kodak XO-Mat film
(Rochester, NY).
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Results |
Identification of C/EBP polymorphisms
PCR-SSCP analysis using the region 3 primers revealed 3 variant
patterns for the entire collection of samples (data not shown). Sequence analysis indicated that 1 pattern was from the wildtype sequence while the other 2 were due to nucleotide changes that were
silent and would not affect the predicted amino acid sequence. The
frequency of these changes suggested the existence of polymorphisms (Table 1). Polymorphism 1 (P1) (55 of 408 or 13.5%) consisted of an
836T>G transversion, and polymorphism 2 (P2) (9 of 408 or 2.2%) consisted of transversions at 3 different sites in the same allele (836T>G, 839C>G, and 902G>T) (Table 1). To verify that these
changes were polymorphisms, we examined 30 matched tumor and normal
samples from nonsmall cell lung cancers by PCR-SSCP (data not shown).
For each tumor with a shift, the corresponding shift was present in the
matched normal sample, indicating the existence of a polymorphism. P1
was present in 10.6% (24 of 227) of hematologic and 17% (31 of 181)
of nonhematologic cancers (Table 1). In contrast, P2 was present in
0.4% (1 of 227) of hematologic and 4.4% (8 of 181) of nonhematologic
cancers (Table 1).
Absence of C/EBP mutations in nonhematologic malignancies and
their presence in acute myelogenous leukemia and acute myelogenous
leukemia
A summary table of the mutations described below indicates the
type of cancer, the genotype of each patient, the mutation, and its
effect on the amino acid sequence of the predicted protein (Table
3). We examined 181 nonhematologic human
tumors and found 2 mutations: a 551G>A for one prostate cancer and a
1087G>A in a nonsmall cell lung tumor (Table 3). These silent changes
occurred in the third position of the codon and were not predicted to
alter the amino acid sequence.
Of 227 hematologic cancers, a total of 10 mutations were identified
among 1 acute myelogenous leukemia (MDS) and 7 acute myelogenous leukemia (AML) patient samples (Table 3). The MDS sample, a refractory anemia with excess of blasts in transformation (RAEB-t), possessed biallelic mutations. Abnormally migrating bands for regions 2 and 4 (Figure 2A, lane 3) were observed.
Sequence analysis revealed that one allele acquired a 37-bp duplication
(nt 483 to 419) in region 2 that was predicted to result in a
frameshift and truncation of the protein (Figure
3). The other allele acquired a nonsense mutation (1015G>T) that would introduce a stop codon in region 4. Both
of these mutations are predicted to encode C/EBP proteins that lack
the bZIP domain (Table 3). Also, biallelic mutations were detected in
the AML-M2 sample F3820 (Table 3). Abnormally migrating bands for
regions 1 and 4 were observed (Figure 2B, lane 3). Sequence analysis
revealed that one allele acquired a deletion of the nucleotide C381 in
region 1 that would produce a frameshift at amino acid Phe77
(Table 3). This allele would potentially generate a messenger RNA
capable of synthesizing a shortened 30-kd form of C/EBP, but not the
42-kd C/EBP (Table 3). The second allele acquired a 24-bp
duplication in region 4 (Figure 3) that would introduce an 8-amino acid
inframe duplication within the first leucine zipper conserved among the
bZIP family (Figure 4; Table 3).
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| Figure 2.
PCR-SSCP analysis of the C/EBP gene locus.
(A) Abnormally migrating bands in lane 3 of both panels for the MDS
(RAEB-t) patient F3881. Region 2 is a 37-bp duplication and region 4 is
a 1015G>T. The other lanes are derived from genomic DNA samples of
other MDS patients and display bands with normal mobility. (B)
Abnormally migrating bands in lane 3 of both panels for the AML-M2
patient F3820. Region 1 is a 1-bp deletion of C381, and region 4 is a
24-bp duplication of nt 1062 through 1085. The other lanes are derived
from genomic DNA samples of other AML patients and display bands with
normal mobility. (C) Abnormally migrating bands for region 4 are
detected in lanes 1 (patient F3901), 10 (patient J2), 11 (patient J3),
and 14 (patient J6). Lane 1 is a 3-bp deletion of nt 1083 through 1085;
lane 10 is a transversion of 1001C>A; lane 11 is 15-bp duplication of
nt 1083 through 1097; and lane 14 is a 3-bp duplication of nt 1083 through 1085. The other lanes are derived from genomic DNA samples of
other AML patients and display bands of normal migration. The arrows at
the left and right of each panel indicate the positions of the
shifted bands.
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| Figure 3.
Sequences of the C/EBP duplication and deletion mutants found in the
MDS and AML patients.
Panel A represents an alignment of a portion of region 2 of the WT
sequence of C/EBP with the sequence from the MDS patient F3881.
Panel B represents an alignment of a portion of region 4 of the WT
sequence with the AML patient samples F3820 (24-bp duplication), F3901
(3-bp deletion), J3 (15-bp duplication), and J6 (3-bp duplication). The
duplications are in bold text, and the duplicated regions are indicated
by underlined italics. The codons for the alanine at position
134 (Ala134) in region 2 and the lysine at position 312 (Lys312) in region 4 are underlined and indicated above and
below the nucleotide sequence, respectively.
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| Figure 4.
Predicted amino acid sequence comparison of AML region-4
mutations with WT C/EBP and the family members C/EBP , C/EBP ,
and C/EBP .
The predicted amino acid sequences from residues 272 through 354 for
the AML region-4 mutants were aligned with that of WT human C/EBP,
C/EBP, C/EBP, and C/EBP. The consensus sequence was derived by
Vinson et al1 for 11 bZIP family members. The consensus
basic (B) residues and leucine residues in each amino acid sequence are
highlighted in bold text above the consensus sequence. The first
conserved leucine is a threonine for C/EBPs. The duplicated amino acid
residues for samples F3820, J3, and J6 are indicated by bold text. The
Arg305Pro change in sample K-6 is indicated by the shaded box. The
basic, fork, and leucine zipper regions are indicated above and below
the alignments. The fork region is an invariant 7-amino acid residue
spacer region between the first leucine in the zipper and the conserved
basic region.1 The alignments were performed by means of
AlignX (Vector NTI Suite v.6.0; Informax, Bethesda, MD).
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The AML patient samples F3901 (M4), J6 (M2), and J3 (M2)
acquired a 3-bp deletion, 3-bp duplication, and 15-bp duplication, respectively, in region 4 (Figure 2C, lanes 1, 11, 14; Figure 3; Table
3). These would generate either an inframe deletion (in sample F3901)
or a duplication of 1 (J6) or 5 (J3) amino acids in the first leucine
zipper (Figure 4). The AML sample J2 possessed a 1001C>A nonsense
mutation that would replace the Tyr284 with a termination codon (Figure
2C, lane 10; Table 3). The AML-M2 sample K-6 showed an abnormally
migrating band in region 4 by SSCP (data not shown), and sequence
analysis revealed a 1063G>C transversion. This missense mutation
created an amino acid residue change of Arg305Pro in the fork region
between the basic and the leucine zipper regions (Table 3; Figure 4).
The AML-M4 sample F4431 possessed the same silent mutation 551G>A in
region 2 as described for the prostate cancer (Table 3). Each of the
AML-M2 samples lacked the t(8;21) translocation that produces an
AML-ETO fusion (Table 3). From the PCR-SSCP analysis, it is difficult to conclude whether these samples possess a wildtype allele because potential contamination by normal cells with the subsequent
amplification of the wildtype allele by PCR may occur. However, sample
J6 lacks a wildtype pattern upon shorter exposure of the
autoradiograph, suggesting it possesses only the mutant allele (Figure
2C, lane 14, and data not shown). Also, in a cell line derived from
sample K-6, a wildtype and mutant allele were identified, indicating that the mutation is heterozygous (H. Asou et al, submitted September 2001).
In addition to patient samples, 5 AML cell lines were examined
for C/EBP mutations. These were HL60, Kasumi-1 [M2,
t(8;21)], Kasumi-5, KG-1, and NB-4 (M3). All cell lines were normal.
Wildtype and mutant C/EBP proteins localize in the
nucleus
Four of 7 mutations present in our samples involved inframe
deletion or duplication of odd numbers of amino acid residues within
the first 2 conserved leucines of the zipper domain (Figure 4). The
remaining mutations would terminate or shift the translation reading
frame and produce proteins lacking the bZIP domain. To determine the
impact of the inframe alterations on the function of the C/EBP
protein, we cloned expressible complementary DNAs for the 3-bp deletion
and the 15- and 24-bp duplications into the retrovirus vector MIG that
allows coexpression of green fluorescent protein (GFP) with
the gene of interest. Each vector was transiently transfected into
NIH3T3 cells, and the nuclear localization was determined by
immunofluorescent microscopy (Figure 5).
Both the wildtype and mutant proteins localized in the nucleus. These
results indicate that the mutations do not impair the protein
localization of C/EBP to its normal cellular compartment.
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| Figure 5.
Locus of WT and mutant C/EBP.
WT and mutant forms of C/EBP localize in the nucleus. NIH3T3 cells
were transfected with the MIG retrovirus constructs expressing the
mutant samples F3901, J3, and F3820. The negative control
cells were transfected with empty MIG, and the positive control cells
were cotransfected with pCMV-C/EBP42 and pEGFPc. The MIG
retrovirus constructs express GFP along with the gene of interest,
thereby allowing identification of transfected cells. All
GFP+ cells, except for the empty vector control (MIG),
showed nuclear staining for wildtype or mutant C/EBP whereas all
GFP cells were negative for C/EBP expression.
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Deficient transcriptional activation by mutant C/EBP
proteins
The ability of these mutant proteins to activate transcription
from the G-CSF receptor promoter hooked up to a luciferase reporter
gene was tested (Figure 6). The
pCMV-Sport1 expression vector was used to express the wildtype and
mutant proteins in these experiments (Figure 6A). The wildtype C/EBP
protein activated the transcription 15-fold over the empty expression
vector alone (Figure 6B). Each of the mutant proteins activated the
reporter only 2-fold over the empty expression vector, indicating a
greater than 80% reduction in transactivation function (Figure
6B).
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| Figure 6.
Transcriptional and DNA-binding activity in C/EBP
mutants.
C/EBP mutants demonstrate severely impaired transcriptional and
DNA-binding activity. (A) Western blot analysis of WT and mutant
(F3901, J3, and F3820) C/EBP proteins expressed in NIH3T3 cells from
the pCMV-Sport1 vector. Similar expression levels for each protein were
noted. (B) The pG-CSF receptor promoter-luciferase reporter construct
(1 µg) was cotransfected with either empty (), WT (50 ng), or
mutant expression vector (100 ng). Relative firefly luciferase activity
was measured and normalized to renilla luciferase activity. The fold
change is indicated on the y-axis. The graph presents data from a
duplicate experiment performed in triplicate. (C) Electrophoretic
mobility shift assays (EMSAs) were performed with the use of the total
cell lysates in panel A. An end-labled double-stranded oligonucleotide
representing the C/EBP site in the G-CSF receptor promoter was
incubated with the lysate in the absence () or presence (+) of 0.2 µg rabbit anti-C/EBP antibody (Ab).
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Deficiency in DNA binding of mutant C/EBP proteins
One possible explanation for the loss of transactivation potential
is the inability of the mutant proteins to bind their cognate site in
the G-CSF receptor promoter. The DNA-binding activity of the mutant
proteins was tested by electrophoretic mobility shift assay (EMSA) with
the use of a double-stranded oligonucleotide containing the C/EBP site
from the G-CSF receptor promoter and the total cell lysates from
transiently transfected cells (Figure 6A). Although the expression of
the wildtype and mutant proteins was similar, the wildtype C/EBP
showed significantly higher binding activity than the mutants (Figure
6C, lanes 1, 3, 5, 7, 9). The bound DNA/protein complexes were
supershifted with rabbit anti-C/EBP antibody, verifying that the
binding activity belonged to C/EBP (Figure 6C, lanes 2, 4, 6, 8, 10). The mutants revealed a greater than 90% reduction in binding
compared with the wildtype C/EBP. These results indicate the
mutations abrogated the DNA-binding function of C/EBP.
Mutant forms of C/EBP do not inhibit transcriptional activation
by wildtype C/EBP
To determine if any of the mutant proteins could interfere with
the transactivation function of wildtype C/EBP, the wildtype protein
was coexpressed with increasing amounts of each mutant (Figure
7). The mutants were unable to activate
transcription or interfere with the ability of the wildtype C/EBP to
activate transcription. These results suggest that the mutant C/EBP
proteins lost their transactivation potential and were unable to impair wildtype C/EBP transcriptional activation of the G-CSF receptor promoter. As a positive control, the same transfections were performed with the 30-kd form of C/EBP. This shortened isoform of C/EBP attenuates transcriptional activation by
C/EBP42.42 It is potentially expressed in
cells from patient F3820 from the other mutant allele (C381 deletion,
Table 3). As expected, a dose-dependent reduction in
C/EBP42 transactivation was observed (Figure
7).
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| Figure 7.
Effect of C/EBP mutants on transcriptional activation
by wildtype C/EBP.
C/EBP mutants lack a dominant-negative effect on transcriptional
activation by wildtype C/EBP. NIH3T3 cells were transfected with
empty expression vector (400 ng) or pCMV-C/EBP42 (50 ng)
with C/EBP30 (100 ng) or with mutant samples F3901, J3,
and F3820 (100 ng). To test for a dominant-negative effect,
the cells were cotransfected with pCMV-C/EBP42 (50 ng)
and increasing amounts of the mutants (100 and 300 ng). Luciferase
activities were measured and normalized and fold changes
determined (y-axis).
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Discussion |
The growth-inhibitory and differentiation-promoting functions of
C/EBP make it an attractive candidate for a tumor suppressor. We
tested this by screening the C/EBP locus in 408 patient samples from
11 different malignancies. The only functionally significant mutations
were found in 1 MDS and 6 AML (M2 and M4) patients (Table 3). These
findings extend those of Pabst et al,43 who recently reported dominant-negative mutations in C/EBP for AML-M1 and AML-M2
lacking the t(8;21) in a screen of 137 AML patient samples. We did not
detect alterations in tumors derived from nonhematopoietic tissues that
express C/EBP. If abrogation of C/EBP function is important for
the development of these tumors, then it may occur via another mechanism.
The mutations in this report fall into 4 classes in regard to their
effect on the predicted protein: (1) termination of translation by
introducing a nonsense codon (samples J2 and F3881); (2) alteration of
amino acid sequence by introducing a missense codon (K-6); (3)
frameshift by either deletion or duplication of nucleotides and an
eventual termination (F3820 and F3881); and (4) inframe deletion or
duplication that removes or inserts additional amino acid residues
(F3901, F3820, J3, and J6). The nonsense mutations in samples J2 (AML)
and F3881 (MDS) would introduce termination codons before the bZIP
domain. This would create polypeptides that are unable to localize to
the nucleus, dimerize, and bind to DNA (Table 3).5,44 The
mutation of Arg305Pro in patient K-6 occurs in the fork region of bZIP
domain (Figure 4; Table 3). The inframe deletion and insertion
mutations of patients F3901, F3820, J3, and J6 occur within the first
conserved leucine finger (Figure 4; Table 3). Previous
structure/function studies indicate that these mutations would abrogate
C/EBP function.1,45
Comparison of the predicted amino acid sequences of 11 bZIP
family members reveals conservation between the basic and leucine zipper regions and the exact spatial register or phasing between these
regions, referred to as the fork (Figure 4).1 The phasing is important for a continued -helical structure that progresses from
the zipper into the basic region.1,45,46 The basic region starts exactly 7 amino acid residues amino-terminal to the first leucine zipper.1 When the phasing between these regions is altered by insertion or deletion of 2-, 4-, 5-, or 6-amino acid residues, sequence-specific DNA-binding is eliminated although dimerization is reported to still occur via the leucine
zippers.45,46 In contrast, a wide variety of mutants with
insertions of an integral number of -helical turns (7-amino acid
residues) were functional.46 The inframe deletion and
insertions identified in this study involve 1-, 5-, and 8-amino acid
residues (Table 3; Figure 4). These are predicted to disrupt the
conserved phasing of the bZIP domain. Consistent with this prediction,
we demonstrated that these mutants were unable to activate
transcription from the G-CSF receptor promoter because the proteins do
not bind the C/EBP site in the promoter (Figure 6).
We hypothesized that these mutant forms would function in a
dominant-negative fashion since they may heterodimerize with the wildtype as suggested by prior studies with similar
mutations.45,46 These mutants did not attenuate
transcriptional activation by wildtype C/EBP (Figure 7). This may be
due to a lack of heterodimerization between the wildtype and mutant
forms of C/EBP. The ability of these mutants to homodimerize and
heterodimerize is currently under investigation. The data from this
study are consistent with a model in which the mutations disrupt the
dimerization interface formed by the leucine zipper and the proteins
are unable to homodimerize or heterodimerize and bind to DNA. Of note,
the previous studies were performed by cross-linking bacterially
expressed and purified proteins whereas we expressed our mutant
proteins in cell culture.45,46
The absence of helical-destabilizing residues, such as proline and
glycine, in the bZIP domain in all family members is consistent with
the hypothesis that phasing is critical for the bZIP
domain.1 The Arg305Pro mutation found in K-6 is predicted
to introduce a break in the -helical structure of the fork region
and disrupt the phasing of the bZIP domain (Table 3; Figure 4). Neither
a homodimer nor a heterodimer formed with the wildtype polypeptide would bind to DNA. Preliminary studies indicate this mutant is unable
to activate transcription, but the DNA-binding, dimerization, and
dominant-negative properties are presently being investigated (data not shown).
Further studies are needed to determine how the different mutations
contribute to the development of leukemia. For 2 patients, F3881 and
F3820, the mutations were biallelic, and patient J6 appears to possess
only the mutant allele, suggesting that loss of C/EBP function is
involved in leukemogenesis. Interestingly, patient F3820 carried a
potentially dominant-negative mutation that would produce the p30 form
of C/EBP. This was the primary mutation reported by Pabst et
al.43 Because the C/EBP family members heterodimerize with
other bZIP family members, this dominant-negative function could extend
beyond inhibition of wildtype C/EBP to other C/EBP or CREB/ATF
proteins. Our remaining patient samples with mutations appear to be
heterozygous for the mutation, but we cannot rule out contamination of
our samples by normal cells. This would result in the wildtype allele
pattern and sequence in our SSCP and sequencing analyses. If the
patients are heterozygous for the mutation, then the decreased
expression of wildtype C/EBP may be sufficient to contribute to
leukemogenesis (haploinsufficiency). Alternatively, the mutants may
have gained functions not yet identified in this study. Recent reports
indicate that C/EBP exerts effects inhibiting growth and gene
expression independent of DNA-binding.47,48 The C/EBP
protein interacts with the p21 and CDK2 proteins, resulting in
decreased CDK2 activity and inhibition of cell
proliferation.47 The C/EBP protein disrupts E2F
complexes in several cell lines, and this correlates with
C/EBP-mediated growth arrest.49,50 Interaction of
C/EBP with E2F appears to be the mechanism by which it downregulates
c-myc expression during granulocytic differentiation.48 These protein-protein interactions appear to involve several
regions of the C/EBP protein, including the bZIP
domain.47 Perhaps the duplication and deletion mutations
in the bZIP domain may affect these interactions, thereby contributing
to leukemogenesis.
The absence of mutations in the nonhematologic malignancies was
surprising. In tissues such as liver and adipose, the lack of mutations
could be due to the small size of our collections of
hepatomas6 and liposarcomas.2 Further
screening of these tumors may be of interest. In the case of our fairly
extensive collection, one reason for the lack of mutations may be that
other mechanisms are blocking C/EBP function. A number of studies
indicate that various oncogenic proteins appear to target the C/EBP
family of proteins and abrogate their function. For example, some human myxoid liposarcomas have chromosomal translocations involving either
translocation liposarcoma-C/EBP homologous protein (CHOP) or EWS
(Ewing sarcoma)-CHOP.51-53 The fusion protein
prevents adipocyte differentiation by directly interfering with
C/EBP function that may regulate the levels of C/EBP that in turn
regulates the differentiation state of the cells.54 Muller
et al55 reported that the E7 oncoprotein of human
papillomavirus 16 overides C/EBP-mediated proliferation arrest.
Pabst et al43 reported in t(8;21) myeloid leukemias
(AML-M2) that it appears the AML-ETO fusion protein suppresses C/EBP
expression indirectly by inhibiting positive autoregulation of the
C/EBP promoter. Lodie et al56 reported that the
PML/RAR protein product resulting from the acute promyelocytic leukemia translocation t(15;17) physically interacts with C/EBP. This interaction interferes with DNA binding and transcriptional activation by C/EBP and, in turn, blocks granulocytic
differentiation.56
In summary, we identified mutations of C/EBP in AMLs of the M2 and
M4 subtypes and an individual suffering from MDS (RAEB-t) that
represents an early stage of leukemia. This report expands upon the
identification of mutations in AML-M1 and AML-M2
samples.43 In addition, a wide range of other leukemias
and solid tumor samples lacked C/EBP mutations. Of interest, the
previous report identified the majority of mutations resulting in the
overexpression of the dominant-negative p30 isoform of C/EBP; in
contrast, a majority of our mutations affected the bZIP domain and did
not appear to possess dominant-negative activity. The use of
conditional, tissue-specific knockout murine models and overexpression
of the identified mutant proteins will further clarify the role of
C/EBP in leukemogenesis.
| |
Acknowledgments |
We are grateful to Jonathan Said for generously providing
soft-tissue sarcoma samples for analysis. We thank Alexey Chumakov for
helpful discussions and advice.
| |
Footnotes |
Submitted December 1, 2000; accepted October 4, 2001.
Supported by National Institutes of Health grant CA26038-20, Joesph
Troy Leukemia Fund, Horn Foundation, Lymphoma Research Foundation of
America, and C. & H. Koeffler Fund. A.F.G. is a recipient of a Lymphoma
Research Foundation of America Fellowship. W.K.H. is a recipient of a
fellowship (HO2207/1-1) from the Deutsche Forschungsgemeinschaft.
H.P.K. holds the Mark Goodson endowed chair for Cancer Research and is
a member of the Jonsson Cancer Center.
A.F.G., W.-K. H., and S. K. contributed equally to this work.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Adrian F. Gombart, Cedars-Sinai Medical Center,
Division of Hematology/Oncology, UCLA School of Medicine, Davis Bldg
5065, 8700 Beverly Blvd, Los Angeles, CA 90048; e-mail:
gombarta{at}csmc.edu.
| |
References |
1.
Vinson CR, Sigler PB, McKnight SL.
Scissors-grip model for DNA recognition by a family of leucine zipper proteins.
Science.
1989;246:911-916[Abstract/Free Full Text].
2.
Friedman AD, McKnight SL.
Identification of two polypeptide segments of CCAAT/enhancer-binding protein required for transcriptional activation of the serum albumin gene.
Genes Dev.
1990;4:1416-1426[Abstract/Free Full Text].
3.
Nerlov C, Ziff EB.
Three levels of functional interaction determine the activity of CCAAT/enhancer binding protein-alpha on the serum albumin promoter.
Genes Dev.
1994;8:350-362[Abstract/Free Full Text].
4.
Williams SC, Baer M, Dillner AJ, Johnson PF.
CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity.
EMBO J.
1995;14:3170-3183[Medline]
[Order article via Infotrieve].
5.
Williamson EA, Xu HN, Gombart AF, et al.
Identification of transcriptional activation and repression domains in human CCAAT/enhancer-binding protein epsilon.
J Biol Chem.
1998;273:14796-14804[Abstract/Free Full Text].
6.
Scott LM, Civin CI, Rorth P, Friedman AD.
A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells.
Blood.
1992;80:1725-1735[Abstract/Free Full Text].
7.
Williams SC, Cantwell CA, Johnson PF.
A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro.
Genes Dev.
1991;5:1553-1567[Abstract/Free Full Text].
8.
Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG.
Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice.
Proc Natl Acad Sci U S A.
1997;94:569-574[Abstract/Free Full Text].
9.
Birkenmeier EH, Gwynn B, Howard S, et al.
Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.
Genes Dev.
1989;3:1146-1156[Abstract/Free Full Text].
10.
Antonson P, Xanthopoulos KG.
Molecular cloning, sequence, and expression patterns of the human gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha).
Biochem Biophys Res Commun.
1995;215:106-113[CrossRef] |
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Abstract
Introduction