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Brief report
Expression of cyclic adenosine monophosphate response-element
binding protein in acute leukemia
Heather N. Crans-Vargas, Elliot M. Landaw, Smita Bhatia, George Sandusky, Theodore B. Moore, and Kathleen M. Sakamoto
Cyclic adenosine monophosphate response-element binding protein (CREB)
is a nuclear protein that regulates expression of genes that control cell proliferation, differentiation, and survival. To analyze CREB expression in leukemia cells,
we conducted Western blot analysis of
bone marrow cells obtained from patients
with acute lymphoblastic leukemia, patients with acute myeloid leukemia, and
patients without active leukemia. CREB
was expressed at a higher frequency in
bone marrow cells from patients with
acute lymphoid or myeloid leukemia than
in patients with leukemia remission or
without leukemia. Our results indicate
that CREB expression could be a useful
marker for leukemia in patients with acute
disease and suggest a role for CREB in
leukemogenesis. (Blood. 2002;99:2617-2619)
© 2002 by The American Society of Hematology
Introduction
Cyclic adenosine monophosphate (cAMP) response-element binding
protein (CREB) is a nuclear protein that regulates gene expression on
activation of cAMP-dependent or cAMP-independent signal-transduction pathways in cells.1-3 CREB binds the cAMP response element in the
promoter regions of target genes that regulate cell proliferation and
survival, such as bcl-2 and egr-1.4-6 Phosphorylation of CREB at serine
133 in response to growth factors modulates the function of CREB.1,7,8
Thus, CREB was proposed to be a critical regulator of growth
factor–induced gene expression leading to cell proliferation, differentiation, and survival.1,7-10
In studying signaling pathways activated by the hematopoietic
growth factor granulocyte-macrophage colony-stimulating factor (GMCSF), we previously found that CREB was phosphorylated in response
to GM-CSF stimulation of myeloid leukemia cells.6,11,12 In the current
study, to determine whether CREB is overexpressed in leukemia cell
lines and bone marrow specimens from patients with acute leukemia,
we conducted Western blot analysis to detect the presence of CREB
protein. We found that CREB was expressed in most leukemia cell lines
and in bone marrow from patients with acute leukemia but that CREB
levels were below the detection limit in normal bone marrow samples
and in bone marrow samples from patients without active leukemia. Our
results suggest that CREB could be a marker for leukemia.
Study design
Patient selection
We analyzed bone marrow samples from 26 patients with newly diagnosed
acute myeloid leukemia (AML), 20 patients with acute lymphoblastic
leukemia (ALL), and 25 patients without active leukemia. Included in the
From the Department of Pediatrics, Division of Hematology-Oncology,
Gwynne-Hazen Cherry Memorial Laboratories, and Mattel Children’s Hospital
at University of California Los Angeles (UCLA); the Department of Pathology,
UCLA School of Medicine and Jonsson Comprehensive Cancer Center; the
Department of Biomathematics, UCLA School of Medicine; the Department of
Pediatrics, Division of Hematology-Oncology, City of Hope National Medical
Center, Duarte, CA; and Eli Lilly Research Laboratories, Indianapolis, IN.
Submitted October 10, 2001; accepted November 27, 2001.
Supported by National Institutes of Health (NIH), National Cancer Institute
(NCI), grant CA68221; the Leukemia and Lymphoma Society of America; and
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
cohort of patients without active leukemia were 9 patients with ALL and 4
patients with AML in documented remission. The nonleukemia bone
marrow samples were from 4 patients with neutropenia, 4 patients with
idiopathic thrombocytopenic purpura (ITP), 2 healthy volunteers (donors of
bone marrow for transplantation), and 2 patients with lymphoma who
underwent bone marrow aspiration as part of an evaluation for metastasis.
The patients’ age ranged from 4 months to 80 years. The diagnoses of
leukemia were made according to standard morphologic criteria on the
basis of bone marrow aspirations and biopsies and immunohistochemical,
immunophenotyping, and cytogenetic studies. Among the patients analyzed, one patient with ALL had t(12;2). Among the patients with AML, 5
had abnormal cytogenetic findings, including 5q⫺, inv(16), t(15;17), extra
chromosome 11, and t(8;21). Informed consent to participation in the study
was obtained from all patients before bone marrow samples were obtained,
in compliance with the Helsinki protocol.
Western blot analysis
Mononuclear cells from bone marrow specimens were extracted by
Ficoll-Hypaque density gradient separation. Cells were lysed and Western
blotting was done with rabbit polyclonal anti-CREB antiserum (0.5 ␮g/mL;
UBI, Lake Placid, NY) and antiactin antiserum (60 ␮g/mL; Santa Cruz
Biotechnology, Santa Cruz, CA) as described previously.11
Immunohistochemical studies
Tissue cores were obtained from tumor areas of each donor block and
transferred to the recipient paraffin block by using a custom-made precision
instrument.13 Sections (5 ␮m) were cut with a microtome and placed on
positively charged slides. The slides were baked, deparaffinized in xylene,
and rehydrated through graded alcohols to water. Antigen retrieval was
done by immersing the slides in target retrieval solution (DAKO, Carpinteria, CA). Slides were blocked by using protein blocking solution (DAKO).
Washes were done in Tris-buffered saline and 0.05% Tween 20 (pH 7.4).
American Cancer Society grant RPG-99-081-01-LBC (K.M.S.). H.N.C.-V. is
supported by NIH Clinical and Fundamental Immunology Training Grant
AI07126-25. E.M.L. is supported in part by NCI grant CA-16042.
Reprints: Kathleen M. Sakamoto, Department of Pediatrics, Mattel Children’s
Hospital at UCLA, 10833 LeConte Ave, Los Angeles, CA 90095-1752; e-mail:
[email protected].
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.
© 2002 by The American Society of Hematology
2617
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2618
CRANS-VARGAS et al
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
Figure 1. Expression of CREB in leukemia cell lines and bone marrow samples from patients with acute leukemia. Shown are results of Western blot analyses of cell
lines and of bone marrow specimens from patients with and without leukemia. (A) Leukemia and nonleukemia cell lines were assessed for CREB expression by Western blot
analysis. Cells were grown to a density of 1 ⫻ 106 cells/mL, and lysates were prepared from 1 ⫻ 106 cells. Twenty micrograms of protein from cell lysates was loaded on a 10%
sodium dodecyl sulfate (SDS)–polyacrylamide gel. Blots were probed with anti-CREB antiserum (0.5 ␮g/mL), stripped, and reprobed with actin (60 ␮g/mL) as the loading
control. The arrows represent CREB (43 kd) or the actin control (43 kd). (B) Mononuclear cells from bone marrow obtained from patients with leukemia or without active disease
were processed with the Ficoll-Hypaque method. Lysates were prepared from 1 ⫻ 106 mononuclear cells. Twenty micrograms of protein was loaded on a 10%
SDS-polyacrylamide gel. Western blot analysis was done with anti-CREB antiserum. Shown are results with positive controls (32Dcl3 cell lysates; lane 1), results with the AML
(lanes 2-5) and ALL (lanes 6-7) samples, and results with samples from patients without active leukemia or with normal bone marrow (lanes 11-13). (C) Western blot analysis of
lysates from bone marrow from patients with ALL and AML at diagnosis (lanes 2, 5, and 8), remission (lanes 3 and 7), and relapse (lane 4); lanes 2-4 are results from the same
patient with ALL at diagnosis, remission, and relapse, respectively. Lanes 6 and 7 are results from the same patient at diagnosis and remission, respectively. (D) Bone marrow
from 9 patients without leukemia was obtained and isolated as described above. Lane 1 shows results with the positive (plus sign) control (32Dcl3 cell lysates). All lanes had
equal protein loading on Ponceau S staining (data not shown). Densitometry measurements showed that the CREB-to-actin ratio ranged from 0.1 to 3.0 and the fold difference
between leukemia and nonleukemia samples ranged from 3.5 fold to 10 fold. (E) Immunohistochemical analysis with CREB antiserum (10 ␮g/mL) was done on bone marrow
biopsy specimens from (E) a representative patient with ALL and (F) a normal lymph node (⫻ 40 magnification). Nuclear staining of CREB was observed in leukemia blast cells
but not in normal lymphocytes. Staining was not detected with rabbit IgG (data not shown).
Slides were incubated with 10 ␮g/mL rabbit polyclonal anti-CREB
antibody (UBI). A biotinylated link antibody and a streptavidin-horseradish
peroxidase kit (LSAB2; DAKO) were used, along with a diaminobenzidine
chromogen and peroxide substrate, to detect the bound antibody complexes.
Counterstaining was done with hematoxylin. Light microscopy was used to
evaluate the intensity and localization of the staining.
Results and discussion
To determine whether CREB expression was increased in
leukemia cell lines, we first examined CREB protein levels in
the myeloid leukemia cell lines 32D, TF-1, KG-1, NFS-60, and
K562 and the lymphoid leukemia cell lines Jurkat and Raji.
Nonleukemia cell lines were also analyzed, including HeLa,
PC3, and FL5.12, which are cervical cancer, prostate cancer, and
myeloid cell lines, respectively. CREB was expressed in 6 of the
7 leukemia cell lines, with NFS-60 cells expressing decreased
levels of CREB (Figure 1A). PC3 cells expressed a highermolecular-weight band of 48 kd, which is most likely a protein
that cross-reacts with the CREB antibody. Nonhematopoietic
cell lines, including HeLa, also had high levels of CREB
expression. Thus, we found that most myeloid and lymphoid
leukemia cell lines overexpress CREB.
To assess CREB protein levels in patients with acute leukemia,
we conducted Western blot analysis with cell lysates prepared from
bone marrow samples. We observed CREB expression in bone
marrow samples from 16 of 20 patients with ALL (80%) and 17 of
26 with AML (65%) (Table 1). Figure 1B shows a representative
Western blot of lysates from ALL or AML patients with expression
of CREB. In contrast, CREB expression was detected in only 1 of
25 patients without active leukemia (4%; Figure 1B and 1D). The
increased frequency of CREB expression in leukemias compared to
controls was statistically significant (P ⬍ .001 for both AML and
ALL by Fisher exact test). Thus, CREB was expressed at detectable
levels more frequently in bone marrow from patients with acute
leukemia. The one patient in the nonleukemia group with CREB
expression had ITP. We also immunoblotted membranes with
activating transcription factor (ATF)–1 antibody. Both CREB and
ATF-1 are members of the CREB-ATF family of transcription
factors.1 ATF-1 was not expressed in bone marrow from patients
with leukemia, suggesting that CREB expression was specific and
not due to cross-reactivity with a related protein family member
(data not shown).
To assess CREB levels during therapy, we conducted Western
blot analysis with lysates from the same patients at diagnosis,
remission, and relapse. In 2 patients with ALL, CREB expression
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
CREB EXPRESSION IN LEUKEMIA
Table 1. CREB expression in bone marrow samples studied
Positive for CREB
(no. of patients)
Negative for CREB
(no. of patients)
AML
17
9
ALL
16
4
AML remission*
0
4
ALL remission†
0
9
Neutropenia
0
4
ITP
1
3
Lymphoma
0
2
Healthy
0
2
Diagnosis
Leukemia
No leukemia
CREB indicates cyclic adenosine monophosphate response-element binding
protein; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; and ITP,
idiopathic thrombocytopenic purpura.
*Includes remission samples from 2 patients with AML who were positive for
CREB at diagnosis.
†Includes remission samples from 3 patients with ALL who were positive for
CREB at diagnosis.
was elevated at diagnosis but below detectable levels at remission
(Figure 1C, lanes 2-7). In one of these patients, CREB expression
was observed again at relapse, although levels were lower than
those at diagnosis (Figure 1C, lane 4). In one patient with AML,
CREB expression was also increased at relapse but not during
remission (data not shown).
To determine whether CREB was expressed specifically in the
blast cells from patients with acute leukemia, bone marrow biopsy
2619
specimens were analyzed by immunohistochemistry. A representative biopsy sample (Figure 1E,F) showed nuclear staining with
CREB antiserum in more than 90% of lymphoblasts in the bone
marrow but less frequently in normal lymphocytes in the lymph
node. Control antibody (rabbit IgG) did not show CREB expression
(data not shown).
CREB has been reported to play a role in the proliferation,
differentiation, and survival of cells. Antisense oligonucleotides to
CREB were found to induce death of human leukemia cells and
bone marrow cells from patients with AML and chronic myeloid
leukemia.14 In the current study, we did not observe translocations
involving the CREB locus at 2q32.3-q34, except in one ALL
patient with t(12;2) who also had positive CREB results.15 This
finding suggests that other mechanisms are involved in increased
CREB expression in acute leukemia. Understanding the role of
CREB in leukemogenesis will provide new insights into how
transcription factors regulate cell proliferation during normal and
neoplastic hematopoiesis.
Acknowledgments
We thank Richard Pan and Phuong Kim Vu for technical assistance
and Jorge Vargas, Patricia Mora-Garcia, Deepa Shankar, and Mike
Lin for critical reading of the manuscript.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2002 99: 2617-2619
doi:10.1182/blood.V99.7.2617
Expression of cyclic adenosine monophosphate response-element binding
protein in acute leukemia
Heather N. Crans-Vargas, Elliot M. Landaw, Smita Bhatia, George Sandusky, Theodore B. Moore and
Kathleen M. Sakamoto
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