Download the gln223arg polymorphism of the leptin receptor gene and

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2006, 57, Supp 4, 375–383
www.jpp.krakow.pl
M. W¥SIK , E. GÓRSKA , K. POPKO , K. PAWELEC , M. MATYSIAK , U. DEMKOW
1
1
1
2
2
1
THE GLN223ARG POLYMORPHISM OF THE LEPTIN RECEPTOR
GENE AND PERIPHERAL BLOOD/BONE MARROW LEPTIN LEVEL
IN LEUKEMIC CHILDREN
1
Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age
and
2
Clinic of Pediatric Hematology, Warsaw Medical University, Warsaw, Poland
Leptin is an adipocyte-derived hormone regulating energy homeostasis and body
weight.
Leptin
also
plays
a
role
in
hematopoesis,
cell
cycle
regulation,
and
in
oncogenesis. The leptin receptor is a single transmembrane protein belonging to the
superfamily of cytokine receptors, structurally related to the hemopoietin receptor
family. The aim of the study was to evaluate bone marrow and peripheral blood
leptin level and frequency of distribution of leptin receptor gene polymorphism
Gln223Arg
in
children
with
acute
leukemia.
The
examined
group
included
92
children with acute leukemia (83 ALL and 9 AML) and 39 non-leukemic control
children. Leptin level was measured by ELISA method at the day of leukemia
diagnosis. Genomic DNA was isolated with the use of a column method and the
genotyping of DNA sequence variation was carried out by the restriction enzyme
analysis of PCR – amplified DNA. The samples were then electrophoresed on 2.5%
agarose gel. Leptin level in leukemic children was lower than in healthy children.
Bone marrow leptin level was significantly lower than that in the blood in leukemic
children
with
ALL-T
and
AML.
An
analysis
of
frequency
distribution
of
the
Gln233Arg polymorphism in the leptin receptor gene in leukemic children showed
lack of differences between the patients and controls. There was no difference in the
genotype
results
frequencies
indicate
a
between
possible
the
leukemic
relation
AML
between
the
and
ALL
leptin
groups
level
either.
and
The
leukemia
development in children. The effectory effect of the hormone seems not related to
Gln223Arg polymorphism of its receptor.
Key
w o r d s : Gln223Arg polymorphism, leukemia, bone marrow, leptin
376
INTRODUCTION
Leptin is a 16-kDa adipocyte-derived hormone regulating energy homeostasis
and
body
weight
proportional
inflammatory
to
by
body
reaction
adipose
mass,
(1-3).
tissue
but
(1-3).
may
Leptin
be
acts
Leptin
lowered
via
a
serum
concentration
rapidly
family
of
by
fasting
membrane
is
or
bound
receptors (RB) (2, 3). RB is a single transmembrane protein belonging to the
superfamily of cytokine receptors related to the hemopoietin receptor family (47).
RB
is
predominantly
expressed
in
the
hypothalamic
regions,
but
also
is
distributed ubiquitously in peripheral tissues (4, 6).
Leptin also plays an important role in immunity, hematopoesis, cell cycle
regulation, and susceptibility to infections. It can modulate hematopoiesis by
stimulating proliferation and inhibiting apoptosis of myelocytic and primitive
hematopoietic progenitor cells (4, 8, 9). This effect may by exerted by cell surface
receptors detected also on human hematopoietic cells and cells of other tissues
(10). RB is expressed in enriched hematopoietic stem cells and in a variety of
hematopoietic cell lines (11). In animal models, leptin is highly expressed in
cultured adipocytes isolated from bone marrow (8, 9). Moreover, leptin not only
promotes normal hematopoiesis, but also stimulates the growth and viability of
leukemic
cells,
which
suggests
a
role
for
leptin
in
the
pathogenesis
of
hematological malignancies (8, 9). Indeed, leptin may induce proliferation and
enhance survival of primary leukemic cells from patients with acute myeloid
leukemia (AML) (10). The presence of RB was demonstrated on primary AML
cells (10). The role of leptin and RB in the pathogenesis of some malignant
tumors is debatable. Clement et al (12) reported a mutation in the human RB
gene, where at the +1 position of intron 16, transition of glutamine to arginine was
observed. Recently, several other polymorphisms have been identified in the
leptin receptor genes and the transition of glutamine to arginine is associated with
leptin overproduction (13, 14). A hypothesis has been put forward that leptin and
RB
polymorphism,
associated
with
a
higher
leptin
serum
level
and
a
high
expression of leptin in adipocytes, may serve as genetic markers for cancer
disease (15).
The aim of the present study was to evaluate the level of leptin in bone marrow
and in peripheral blood as well as the frequency of distribution of the Gln223Arg
polymorphism in the leptin receptor gene in children with acute lymphoblastic
(ALL) or myeloblastic (AML) leukemia.
MATERIAL AND METHODS
Study protocol was approved by the Warsaw Medical University Ethics Committee. Written
informed consent was obtained during the enrollment visit from the parents of all patients and
control subjects. All patients and controls were referred to the Hematology Clinic of the Academic
Children’s Hospital of Warsaw Medical University in Warsaw, Poland.
377
Subjects
The study group consisted of 92 children with acute leukemia of age ranging from 5/12 to 18
years (mean 12.3 ±7.4 years) for girls and 4-9 years (mean 5.7 ±6.6 years) for boys. Eight children
(2 girls and 6 boys) with acute lymphoblastic T leukemia (ALL-T), 44 children (18 girls and 26
boys) with acute lymphoblastic B leukemia (ALL-B), 32 children with common ALL and 8
children (2 girls and 6 boys) with acute myeloblastic leukemia (AML) were included in the study.
The
diagnosis
of
leukemia
was
based
on
bone
marrow
smear
and
flow
cytometry
immunophenotyping.
The control group consisted of 39 healthy children (16 boys, 13 girls) who were free from
hematological malignancies. There were no significant differences in the age and sex between the
children
with
Department
or
and
without
were
leukemia.
diagnosed
The
as
control
having
children
benign
were
referred
hematological
to
the
Hematology
disorders
(anemia,
trombocytopenia).
Leukemic and control children were divided into 4 groups according to body mass index (BMI):
Group 1 = 11.7-13.9 kg/m ; Group 2 = 14.0-15.1 kg/m ; Group 3 = 15.2-18.4 kg/m ; and Group 4
2
2
2
= 18.5-20.6 kg/m .
2
Laboratory assays
Blood and bone marrow samples were collected at the day of the leukemia diagnosis was made,
centrifuged and serum or bone marrow supernatants were stored at -40°C until use. The portions of
the
samples
remaining
after
routine
laboratory
examinations
were
used
for
further
immunoemzymatic and molecular analyses.
ELISA test. A commercial immunoenzymatic kit to detect leptin (Quantikine Human Leptin, R
& D, Minneapolis, USA) was used. The test is based on a solid double antibody sandwich ELISA.
Serum and bone marrow supernatants were added to microwells precoated with leptin. All samples
were
assayed
in
duplicates.
In
the
positive
cases,
antigen-antibody
complex
reacted
with
peroxidase-labeled antihuman IgG conjugate. Using H2O2/TMB as substrate, the enzymatic activity
of peroxidase was measured at 450 nm with the use of automated reading system Stat Fax 2100
(ALAB,
Warsaw,
Poland).
Standards
were
provided
for
the
generation
of
a
semi-logarithmic
reference curve, which all the results were referred to. Immunoenzymatic assays were performed
blindly by a laboratory technician.
PCR assay. Genomic DNA was isolated using Genomic Midi AX isolation kit with ionexchange membranes (A & A Biotechnology, Gdynia, Poland). One milliter of blood was diluted
with saline 1:1 v/v. After incubation for 30 min at 50°C with lysing buffer and proteinase K
solution, the mixture was vortexed vigorously. The sample was placed in a column and centrifuged.
After washing, the elution buffer was added and the sample was centrifugation twice at 4000 rpm
for 2 min. The sample was then centrifuged with isopropanol at 13 500 rpm for 7 min. The
supernatant was removed and the pellet was washed with ethanol. After consecutive centrifugation
(13 500 rpm for 7 min), the supernatant was removed and the pellet was air-dried for 10 min.
Finally, pure DNA was dissolved in 200 µl of sterile water.
Genotyping
was
done
using
polymerase
chain
reaction
–
restriction
fragment
length
polymorphism analyses. Amplification was carried out in a 50 µl volume containing 300 ng
genomic DNA, 0.1 µM of each primer (forward 5` AAA CTC AAC GAC ACT CTC CTT 3` and
reverse 5` TGA ACT GAC ATT AGA GGT GA 3`) (DNA - Gdañsk, Gdañsk, Poland), 200 µM of
each dNTP (DNA -Gdañsk, Gdañsk, Poland), 3 mM of magnesium chloride (Applied Biosystems,
Warrington, UK), and 0.2U of Taq Gold polymerase (Applied Biosystems, Warrington, UK).
378
Thirty five cycles were conducted in a thermocycler, Mastercycler personal (Eppendorf,
Hamburg, Germany), under the following conditions: initial denaturation at 94°C for 4 min,
denaturation at 94°C for 30 s, annealing at 55°C for 35 s, extension at 72°C for 60 s, and a final
extension at 72°C for 10 min. The amplified PCR product (80 bp) was digested with the addition
of 2U Msp I (New England BioLabs, Beverly, MA, USA) overnight at 37°C. The digested
samples were separated by electrophoresis through a 4% agarose gel. Digestion of the 80 bp
product with Msp I produced fragments of the following sizes: 80 bp in the homozygote Gln/Gln
(A/A); 80, 57, and 23 in the heterozygote Gln/Arg (A/G); 57 and 23 in the homozygote Arg/Arg
(G/G).
Statistical analysis
Results of bone marrow and serum leptin level are expressed as means
±SD.
The results of
leptin level in different groups were compared by the U Mann-Whitney test. Statistical significance
was accepted at P<0.05. The Pearson test was used for the analysis of the correlations between the
BMI and leptin concentration. Frequency distribution analysis was performed with chi square test.
A commercial Statistica package was used for all statistical data elaboration.
RESULTS
The blood serum and bone marrow leptin levels in leukemic children were
lower than those in the control children (Table 1). The level of leptin in bone
marrow was significantly lower than that in the serum of both control BMI Group
4 and leukemic ALL-T and AML children (Table 1). Significant differences in the
leptin serum level were observed between the leukemic ALL-T and AML groups
and the control BMI Group 3 (Table 1).
Stars
indicate
significant
differences
between
the
corresponding
leptin
concentration in bone marrow and peripheral blood at P<0.05. The P values in the
right hand column indicate significant differences between the leukemic and
control children in a given group.
Table 1. Leptin level in peripheral blood and in bone marrow of leukemic children compared with
control children.
PERIPHERAL BLOOD
Controls - BMI Group 3
Leukemic - ALL-B
Controls - BMI Group 4
Leukemic - ALL-T
Leukemic - AML
n
33
15
9
5
5
Leptin concentration (µg/ml)
±11.81
±20.86
189.9 ±83.0
66.9 ±105.7
53.3 ±8.07
28.5
23.8
P
NS
0.05
0.004
BONE MARROW
Controls - BMI Group 3
13
Leukemic - ALL-B
15
Leukemic - ALL-T
5
Controls - BMI Group 4
Leukemic - AML
9
7
±6.45*
±10.7*
93.5 ±77.37
71.8 ±112.9
20.5 ±27.07*
19.6
9.8
0.05
NS
NS
379
In the control group comprised of healthy children, there was a significant
positive correlation between BMI and blood serum (Fig. 1A) or bone marrow
leptin level (Fig. 1B). Such a correlation was not seen in leukemic patients (data
not shown). In this group, however, the leptin concentration in blood serum was
higher than that in bone marrow in all the BMI groups studied (Fig. 2A). In the
group
of
compared
leukemic
with
children,
that
in
bone
leptin
concentration
marrow
only
in
in
relation
peripheral
to
the
blood
type
of
was
acute
leukemia, as these children belonged to the BMI Group3 and Group 4 (Table 1).
The leptin concentration was higher in the blood in children with ALL-B and
ALL-T, whereas it was comparable in both blood and bone marrow in children
with AML (Fig. 2B).
An analysis of frequency distribution of the Gln223Arg polymorphism in the
leptin
receptor
gene
in
leukemic
children
is
presented
in
Fig.
3.
In
the
lymphoblastic leukemia patients, the G/A allele was more frequent, although the
difference did not achieve statistical significance. The wild type G/G was more
Fig. 1. Correlations between body mass index (BMI) and leptin concentration in blood serum (Panel
A) and in bone marrow (Panel B) in control, healthy children.
Fig. 2. Leptin concentration in peripheral blood and in bone marrow in relation to BMI in control,
healthy children (Panel A) and to the type of acute leukemia in leukemic children (Panel B).
380
Fig. 3. Frequency
distribution of the
Gln223Arg
polymorphism in the
leptin receptor gene in
control and leukemic
children.
frequent in the AML patients, but likewise the difference was not significant.
There were no differences in the genotype frequencies between the AML and
ALL-B groups. Nor were there any appreciable differences between the leukemia
and control groups. There was no correlation between the leptin level and the type
of polymorphism in the control group.
DISCUSSION
Although there is a lot of evidence about the potential role of leptin in
myeloproliferative
disorders,
the
relation
between
leptin
and
leukemia
is
unclear. Total leukocyte counts in healthy humans correlate positively with both
body fat and leptin (16) and increasing BMI correlates with the diagnosis of
acute promyelocytic leukemia (APL) (17). Furthermore, leukemic blast cells can
express a functional leptin receptor (10, 18, 19). In our study, we detected a high
leptin level in bone marrow of leukemic children, although it was lower than that
in control children. These results are in accord with the data of Gaja et al (20).
Those authors examined leptin concentrations in the plasma from peripheral
blood
and
in
bone
marrow
in
relation
to
BMI
in
patients
with
lymphoproliferative diseases. They showed that bone marrow and peripheral
blood leptin levels significantly correlated with each other as well as with BMI.
The authors also demonstrated a relation between blood and bone marrow leptin,
on the one side, and the percentage of bone marrow fat, on the other side. In
addition, they found a negative correlation between blood and bone marrow
leptin and bone marrow malignant infiltration (20). In the control group of our
study, a significant correlation between BMI and serum or bone marrow leptin
level also was found. This effect was not seen in leukemic patients, which
suggests that the mechanisms controlling leptin level in leukemic and nonleukemic persons are different. Adipocytes are the potential source of leptin in
381
bone marrow. These cells are the prevalent stromal cell type and they play a role
in hematopoiesis.
A positive correlation between adipocyte differentiation of stromal cells and
the ability to support the growth of lymphoid cells has been documented (21).
The molecular basis for the ability of adipocytes to support hematopoiesis is
unknown. Tabe et al (22) demonstrated that leptin produced by mesenchymal
stem cell (MSC)-derived adipocytes controls survival of APL cells (22). The
authors investigated the effects of leptin produced by BM adipocytes on APL
cells using a co-culture system with MSC–derived adipocytes. They observed
antiapoptotic
with
effect
dependent
phosphorylation
(STAT3)
and
of
on
signal
direct
cell-to-cell
transducer
mitogen-activated
protein
and
kinase
interactions,
activator
(MAPK)
of
associated
transcription-3
(22).
Alternatively,
leptin secretion by BM adipocytes in the vicinity of leukemic cells could play
a major role in the proliferation and survival of APL cells through paracrine
interactions
in
the
marrow
microenvironment
(22).
More
recent
data
have
indicated that the development and progression of nonsolid tumors, such as
leukemias, also may depend on the generation of new blood vessels from the
pre-existing vasculature (23-25). Inhibition of angiogenesis reportedly retarded
leukemic growth in mice (26, 27). Among its pleiotropic actions, leptin may
function as an angiogenic factor in various in vitro systems as well as in rodent
models
of
angiogenesis
(28,
29).
Inhibition
of
the
angiogenic
process
in
hematopoietic tissues by targeting leptin activity might, therefore, represent a
novel therapeutic option in leukemia.
The presence of leptin RB receptors on primary AML cells, with the highest
expression of RB long isoform on acute promyelocytic leukemia (APL) cells,
has
been
observed
hematopoietic
reported
to
(10).
progenitor
induce
RB
cells
has
recently
expressing
proliferation
and
been
CD34
detected
antigen,
differentiation
Multiple isoforms of RB have been identified (5, 6).
in
on
and
these
human
leptin
cells
(4,
was
8).
In the present study, we
analyzed the frequency of leptin receptor gene polymorphism in leukemic and
control
children.
polymorphism
of
We
RB
failed
between
to
the
detect
significant
non-leukemic
and
differences
leukemic
in
groups.
the
No
differences in the genotype frequency were observed between the AML and
ALL groups. This polymorphism is strongly associated with obesity (12-14),
but it does not seem to be as much associated with leukemia development.
Other authors did find the association between leukemia development and
increased
RB
expression
(10).
Thus,
RB/leptin
interactions
may
play
a
pathophysiological role in leukemia.
In conclusion, the results of this study indicate a possible relation between the
leptin level and acute leukemia development in children. However, the effectory
function of the hormone seems not to be related to the Gln223Arg polymorphism
of its receptor.
382
REFERENCES
1.
Considine R, Shinha MK, Heiman ML et al. Serum immunoreactive-leptin concentrations in
normal-weight and obese humans. N Engl J Med 1996; 334: 292-295.
2.
Russell CD, Ricci MR, Brolin RE, Magill E, Fried SK. Regulation of the leptin content of obese
human adipose tissue. Am J Physiol Endocrinol Metab 2001; 280: E399-E404.
3.
Maffei M, Halaas J, Ravussin E et al. Leptin levels in human and rodent: measurements of
4.
Cioffi JA, Shafer AW, Zupancic TJ et al. Novel B219/OB receptor isoforms: possible role of
5.
Tartaglia LA, Dembski M, Weng X et al. Identification and expression cloning of a leptin
plasma leptin and OB RNA in obese and weight reduced subjects. Nat Med 1995; 1: 1155-1161.
leptin in hematopoiesis and reproduction. Nat Med 1996; 2: 585-589.
receptor, OB-R. Cell 1995; 83: 1263-1271.
6.
Tartaglia L. The leptin receptor. J Biol Chem 1997; 272: 6093-6096.
7.
Baumann H, Morella KK, White DW et al. The full-length leptin receptor has signaling
capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci USA 1996; 93: 83748378.
8.
Umemoto Y, Tsuji K, Yang FC et al. Leptin stimulates the proliferation of murine myelocytic
9.
Ghilardi N, Skoda RC. The leptin receptor activates Janus kinase 2 and signals for proliferation
and primitive hematopoietic progenitor cells. Blood 1997; 90: 3438-3443.
in a factor-dependent cell line. Mol Endocrinol 1997; 11: 393-399.
10. Konopleva M, Mikhail A, Estrov Z et al. Expression and function of leptin receptor isoforms in
myeloid leukemia and myelodysplastic syndromes: proliferative and anti-apoptotic activities.
Blood 1999; 93: 1668-1676.
11. Considine R, Shinha MK, Heiman ML et al. Serum immunoreactive-leptin concentrations in
normal-weight and obese humans. N Engl J Med 1996; 334: 292-295.
12. Clement K, Vaisse C, Lahlou N, Cabrol S, Pelloux V et al. Mutation in the human leptin
receptor gene causes obesity and pituitary dysfunction. Nature 1998; 392: 398-401.
13. Le Stunff C, Le Bihan C, Schork NJ, Bougneres P. A common promoter variant of the leptin
gene is associated with changes in the relationship between serum leptin and fat mass in obese
girls. Diabetes 2000; 49: 2196-200.
14. Hoffstedt J, Eriksson P, Mottagui-Tabar S, Arner P. A polymorphism in the leptin promoter
region (-2548 G/A) influences gene expression and adipose tissue secretion of leptin. Horm
Metab Res 2002; 34: 355-359.
15. Snoussi K, Strosberg AD, Bouaquina N et al. Leptin and leptin receptor polymorphisms are
associated with increased risk and poor prognosis of breast carcinoma. BMC Cancer 2006; 6:
38-37.
16. Wilson CA, Bekele G, Nicolson M, Ravussin E, Pratley RE. Relationship of the white blood
cell count to body fat: Role of leptin. Br J Haematol 1997; 99: 447-451.
17. Estey E, Thall P, Kantarjian H, Pierce S, Kornblau S, Keating M. Association between increased
body mass index and a prognosis of acute promyelocytic leukemia in patients with acute
myeloid leukemia. Leukemia 1997; 11: 1661-1664.
18. Nakao T, Hino M, Yamane T, Nishizawa Y, Morii H, Tatsumi N. Expression of the leptin
receptor in human leukaemic blast cells. Br J Haematol 1998; 102: 740-745.
19. Hino M, Nakao T, Yamane T, Ohta K, Takubo T, Tatsumi T. Leptin receptor and leukemia. Leuk
Lymphoma 2000; 36: 457-461.
20. Gaja A, Chury Z, Pecen L, Frakova H, Jandakova E, Hejlova N. Bone marrow and peripheral
blood
leptin
levels
in
lymphoproliferative
infiltration. Neoplasma 2000; 47: 307-312.
diseases—relation
to
the
bone
marrow
fat
and
383
21. Friedrich C, Zausch E, Sugrue SP et al. Hematopoietic supportive functions of mouse bone
marrow
and
fetal
liver
microenvironment:
dissection
of
granulocyte,
B-lymphocyte,
and
hematopoietic progenitor support at the stromal cell clone level. Blood 1996; 87: 4596-4606.
22. Yoko Tabe, Marina Konopleva, Mark F et al. PML-RAR is associated with leptin-receptor
induction: the role of mesenchymal stem cell–derived adipocytes in APL cell survival. Blood
2004; 103: 1815-1822.
23. Padro T, Ruiz S, Bieker R et al. Increased angiogenesis in the bone marrow of patients with
acute myeloid leukemia. Blood 2000; 95: 2637-2644.
24. Scappaticci FA, Smith R, Pathak A et al. Combination angiostatin and endostatin gene transfer
induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid
tumors in mice. Mol Ther 2001; 3: 186-196.
25. Dias S, Hattori K, Heissig B et al. Inhibition of both paracrine and autocrine VEGF/VEGFR-2
signaling
pathways
is
essential
to
induce
long-term
remission
of
xenotransplanted
human
leukemias. Proc Natl Acad Sci USA 2001; 98: 10857-10862.
26. Sierra-Honigmann
MR,
Nath
AK,
Murakami
C,
et
al.
Biological
action
of
leptin
as
an
angiogenic factor. Science 1998; 281: 1683-1686.
27. Cao R, Brakenhielm E, Wahlestedt C, Thyberg J, Cao Y. Leptin induces vascular permeability
and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc Natl Acad Sci USA
2001; 98: 6390-6395.
28. Bouloumie A, Dexler HC, Lafontan M, Busse R. Leptin, the product of Ob gene, promotes
angiogenesis. Circ Res 1998; 83: 1059-1066.
29. Park HY, Kwon HM, Lim HJ et al. Potential role of leptin in angiogenesis: leptin induces
endothelial cell proliferation and expression of matrix metalloproteinases in vivo and in vitro.
Exp Mol Med 2001; 3 3: 95-102.
Author’s address: M. W¹sik, Department of Laboratory Diagnostics and Clinical Immunology
of Developmental Age, Warsaw Medical University, Banacha 1a St., 02-097 Warsaw, Poland;
phone: +48 22 5991000. E-mail: [email protected]