|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1668-1676
Expression and Function of Leptin Receptor Isoforms in Myeloid
Leukemia and Myelodysplastic Syndromes: Proliferative and
Anti-Apoptotic Activities
By
Marina Konopleva,
Adel Mikhail,
Zeev Estrov,
Shourong Zhao,
David Harris,
Gisela Sanchez-Williams,
Steven M. Kornblau,
Joan Dong,
Kay-Oliver Kliche,
Shuwei Jiang,
H. Ralph Snodgrass,
Elihu H. Estey, and
Michael Andreeff
From The University of Texas M.D. Anderson Cancer Center, Houston,
TX; and Progenitor, Inc, Menlo Park, CA.
 |
ABSTRACT |
The receptor for the gene product of the obesity gene, leptin, was
recently reported to be expressed on murine and human hematopoietic progenitor cells. Therefore, we studied the expression of the leptin
receptor, OB-R, in normal myeloid precursors, human leukemia cell
lines, and primary leukemic cells using reverse-transcriptase polymerase chain reaction. In normal hematopoiesis, OB-R was expressed in CD34+ cells. Normal promyelocytes
(CD34 33+ and
CD3413+) expressed only very low levels
of the short, presumably nonsignaling isoform. Both the long and short
isoforms of OB-R were expressed in 10 of 22 samples from patients with
newly diagnosed primary or secondary acute myeloid leukemia (AML), with
a higher incidence of the long isoform in primary AML (87.6% v
28.6%; P = .01). The incidence of OB-R expression was
higher in recurrent than in newly diagnosed AML (P < .001),
and samples from four patients with refractory AML showed strong
expression of both isoforms. Both OB-R isoforms were also expressed in
newly diagnosed and recurrent acute promyelocytic leukemia cells but
were essentially absent in samples of chronic or acute lymphocytic
leukemia. In vitro growth of myeloid leukemic cell lines and of blasts
from 14 primary AMLs demonstrated that recombinant human leptin alone
induced low level proliferation, significantly (P < .05)
increased proliferation induced by recombinant human granulocyte
colony-stimulating factor, interleukin 3, and stem cell factor in a
subset of AML and increased colony formation (P < .005).
Also, leptin reduced apoptosis induced by cytokine withdrawal in MO7E
and TF-1 cells. Serum leptin levels correlated only with body mass
index (P < .001) and gender (P = .03). Results
confirm the reported expression of leptin receptor in normal
CD34+ cells and demonstrate the frequent expression of
leptin receptors in AML blasts. While normal promyelocytes lack
receptor expression, leukemic promyelocytes express both isoforms. We
also demonstrate proliferative effects of leptin alone and in
combination with other physiologic cytokines, and anti-apoptotic
properties of leptin. These findings could have implications for the
pathophysiology of AML.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
MANY CYTOKINES have been shown to
regulate the survival, proliferation, differentiation, and function of
normal hematopoietic1 and leukemic cells.2 Two
groups recently cloned the leptin receptor from CD34-expressing
(CD34+) human progenitor cells3 and from
murine fetal liver and yolk sac-derived cell lines.4 The
receptor is expressed in primitive hematopoietic cells and transduces
signals from the obese (Ob) gene product, leptin.5
Leptin is involved in the regulation of fat metabolism in
mammals.6,7 It is expressed primarily in white adipose
tissue and is secreted as a nonglycosylated protein of 16 kD into the
circulation. The amount of leptin mRNA and serum levels of leptin are
highly correlated with body fat volume.8,9 The human leptin
receptor gene (OB-R) is localized on chromosome 1.10 Interestingly, the primary structure of OB-R
shows homologies to the signaling subunits of the interleukin
(IL)-6-type cytokine receptors, including gp130, and to receptors for
the leukemia inhibitory factor and the granulocyte colony-stimulatory
factor (G-CSF).5 mRNAs for four human and six murine splice
variants of OB-R have been identified, although the exact
significance of the various isoforms has yet to be
determined.4,6,7 The predominant OB-R mRNA found in
most tissues encodes a transmembrane protein with a short cytoplasmic
domain of 34 amino acid residues,5 referred to hereafter as
the short isoform. In the hypothalamus, an OB-R mRNA exists
that encodes a protein with an extracellular domain identical to that
of the short isoform, but with a longer cytoplasmic domain of 302 amino
acids.4,6,7 The long isoform was expressed in
hematopoietic tissues (murine and human fetal livers and
hematopoietic cell lines).4 The db mutation leads to the
production of an aberrant splice variant of the long isoforms transcript, resulting in a protein with a truncated cytoplasmic domain.7,11 The loss of the carboxy-terminal region in the mutated protein has been proposed to render OB-R inactive and generates the obese phenotype in db/db mice.11
Recent studies of the physiologic expression of OB-R and the
type of normal cells responsive to leptin suggest that leptin and
OB-R may function as a growth factor/receptor-ligand system in
hematopoietic stem and/or progenitor
cells.3,4,12,13 The potential role of OB-R in
primary hematopoietic malignancies has not been investigated. However,
considering the pattern of receptor expression in normal hematopoietic
progenitor cells, we hypothesized that the leptin gene product could be
an important, and perhaps a distinct, regulator of leukemia cell
proliferation. This hypothesis is supported by the reported presence of
OB-R transcripts in myeloid and some lymphoid T-cell cell
lines.3 A rise in the level of fetal leptin has recently
been reported in cord blood,14 suggesting that the
OB-R has a possible role in fetal development. Because leptin
is thought to regulate pluripotent stem cells, and because some
leukemias might be derived from the transformation of these cells, we
thought it of interest to analyze the expression of the OB-R
gene and its isoforms in human leukemic cells. Because leptin is
produced by adipocytes and stromal cells,3,15 which make up
a significant part of the bone marrow microenvironment, it could also
stimulate leukemic progenitors in a paracrine fashion. No data has been
reported on the effects of leptin on proliferation and clonal growth of
leukemic progenitors from patients with acute myeloid leukemia (AML).
Therefore, the aims of this study were (1) to investigate the
expression patterns of the different receptor isoforms in purified
myeloid progenitors from normal bone marrow, myeloid leukemic cell
lines, and primary AML; (2) to investigate the mitogenic potential of
recombinant leptin on the proliferation and clonal growth of human
acute leukemia cells of myeloid origin, alone and in combination with
other hematopoietic growth factors; and (3) to assess whether the
biologic effects of leptin could be associated with the expression of
distinct OB-R isoforms. Results indicate the presence of a
functional leptin receptor capable of transducing proliferative and
survival signals in the majority of patients with AML.
| |
MATERIALS AND METHODS |
Cell lines.
The OCI/AML3 and OCI/AML2 cell lines, originally established from AML
patients,16 were kindly provided by Dr M.D. Minden (Ontario
Cancer Institute, Toronto, Canada). HL-60, KG-1, K562, BV173, Raji,
Jurkat, and TF-1 cell lines were obtained from the American Type
Culture Collection (Rockville, MD). NB4 cells were kindly provided by
Dr M. Lanotte.17 In addition, HL-60-doxorubicin resistant
cells (HL-60-DOX)18 were also used. Cell lines were maintained in RPMI-1640 medium containing 10% fetal calf serum, 1%
L-glutamine, and penicillin-streptomycin. Cultures for MO7E cells
(provided by Dr H. Broxmeyer, Indiana University,
Indianapolis, IN) were supplemented with 10 U/mL of recombinant
granulocyte-macrophage colony-stimulating factor (GM-CSF)
(Schering-Plough, Kenilworth, NJ) and were maintained under conditions
similar to those described for the other cell lines. Cell density was
adjusted to a starting concentration of 0.5 × 106
cells/mL, and cells were cultured in 96-well well plates at 37°C for
48 hours in 5% CO2 with and without leptin in increasing
concentrations (10 to 300 ng/mL) for 72 hours. For costimulation
experiments, cells were cultured for 72 hours with or without leptin
(100 ng/mL) in combination with G-CSF, IL-3, and stem cell factor (SCF).
Human subjects.
Bone marrow or peripheral blood cells for in vitro studies were
obtained from patients with newly diagnosed and recurrent AML, and from
patients with advanced myelodysplastic syndrome (MDS) after obtaining
informed consent according to institutional policy. The mononuclear
cells were separated by Ficoll-Hypaque (Sigma, St Louis, MO) density
gradient centrifugation. For proliferation experiments, only samples
that contained more than 80% blasts were used.
The morphological subtypes of AML were defined according to the
French-American-British (FAB) classification. Patients with a history
of MDS or chemotherapy-induced AML were classified as having secondary AML.
Reagents.
Highly purified human recombinant leptin was purchased from R&D Systems
(Minneapolis, MN). Recombinant human (rH) G-CSF (Schering-Plough) was
added to cultures in concentrations of 100 U/mL. Recombinant IL-3
(Sandoz, East Hanover, NJ) and SCF (Amgen, Thousand Oaks, CA) were
added at 50 and 100 ng/mL, respectively.19
[3H]Thymidine uptake.
Triplicate samples of 1 × 105 cells suspended in 200 µL
RPMI were cultured in the presence or absence of recombinant leptin (100 ng/mL) in 96-well flat-bottom microtiter well plates (Costar, Cambridge, MA) in a humidified atmosphere of 5% CO2 in air
at 37°C. After 2 days, 0.1 µCi of [3H]thymidine
(specific activity, 6.7 Ci/mmol), (DuPont, Wilmington, DE) was added to
each well, and the cultures were incubated overnight. For costimulation
experiments, rHG-CSF or GM-CSF was added to cultures at 100 U/mL; IL-3
and SCF were added at 50 and 100 ng/mL, respectively. The cells were
then deposited onto harvester filters using a semiautomatic cell
harvester. Radioactivity was measured in a scintillation counter and
expressed as counts per minute. The stimulation index (SI) was
calculated as the ratio of the leptin-treated and control samples in
counts per minute.
Clonogenic assay.
The clonogenic assay was performed as previously
described.20 Briefly, OCI/AML2 cells were cultured in 0.8%
methylcellulose (Fluka Chemical Corp, Ronkoncoma, NY), 10% fetal
bovine serum, and Iscove's modified Dulbecco medium (IMDM). Leptin was
added in increasing concentrations (10 to 300 ng/mL) at the initiation of culture. Triplicate culture mixtures were placed in 35-mm Petri dishes (Nunc Inc, Naperville, IL) and maintained at 37°C with 5%
CO2 in air in a humidified atmosphere. Colonies were
counted after 7 days using an inverted microscope. A colony was defined as a cluster of more than 40 cells.
AML blast colony assay.
A previously described method was used to assay AML blast colony
formation.21,22 Briefly, 1 × 105
T-cell-depleted nonadherent low-density bone marrow cells were plated
in 0.8% methylcellulose in IMDM supplemented with 10% fetal bovine
serum and 15 ng/mL rHGM-CSF. Leptin was added at the initiation of
cultures at concentrations ranging from 10 to 300 ng/mL. Duplicate cultures were incubated in 35-mm Petri dishes for 7 days at 37°C in a
humidified atmosphere of 5% CO2 in air. AML blast colonies were microscopically evaluated on day 7 of culture. A blast colony was
defined as a cluster of 20 or more cells. The leukemic origin of these
colonies was previously shown by cytogenetic analysis.23
Apoptosis assays.
Apoptosis was analyzed in cytokine-dependent MO7E and TF1 cell lines
after 48 and 72 hours of GM-CSF withdrawal in the presence or absence
of leptin using the acridine orange DNA/RNA technique.24 Samples were measured in a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Briefly, aliquots (80 µL) of cell suspension were mixed with 100 µL of a solution containing 0.1% (vol/vol) Triton X-100 (Sigma Chemical Co), 0.05 mol/L HCl, 0.15 mol/L NaCl, and 8 µg/mL acridine orange (Polysciences, Warrington, PA). DNA and RNA
fluorescence was measured within 5 minutes of staining. The percentage
of cells in the subG1 peak defined the proportion of apoptotic cells.
RNA isolation and reverse transcription.
RNA was isolated according to the single-step acid guanidinium
thiocyanate-phenol-chloroform method.25 Purified RNA
samples were quantitated using a DU-640 spectrophotometer (Beckman
Instruments, Fullerton, CA). A reverse-transcription kit was used to
synthesize cDNA according to the manufacturer's instructions
(Boehringer-Mannheim, Indianapolis, IN). One microgram of the total RNA
template was used per 10 µL of reverse transcriptase (RT) reaction.
Primers for the long and short isoforms of OB-R and primers for
 2-microglobulin were
synthesized on an oligonucleotide synthesizer (Model 392; Applied
Biosystems, Foster City, CA). Oligonucleotide primers (F, forward; R,
reverse) used for expression analysis by RT polymerase chain reaction
(PCR) were as follows: primers that recognize all forms amplify a
conserved sequence in the extracellular domain of the receptor. The
human OB-R: F 5'-GTCAGAAGATGTGGGAAA-3' (nucleotides 2266-2283)
and R 5'-GTGCCCAGGAACAATTCTT-3' (nucleotides 2828-2846). The long
isoform F 5'-GCTATTTTGGGAAGATGT-3' (nucleotides 2800-2817) and R
5'-TGCCTGGGCCTCTATCTC-3' (nucleotides 3281-3298); and the short
isoform (6.4 sequence) F 5'-GACTCATTGTGCAGTGTTCAG-3' (nucleotides 2204-2221) and R 5'-TGGCACATTGGGTTCATC-3' (nucleotides 2905-2923) are
shown in Fig 1. The expected PCR products
are 579 base pairs (bp) for OB-R, 501 bp for the OB-R
long isoform, and 720 bp for the short isoform. The primers for
2-microglobulin were F
5'-ACCCCCACTGAAAAAGATGA-3' and R 5'-TATCTTCAAACCTCCATGATG-3'. In all
experiments, PCR was performed for 35 cycles. A
32P-labeling PCR method was used to detect the radioactive
products.26 The radioactive products were detected on the
Betascope 603 (Betagen, Waltham, MA) after exposing the gel to Kodak
film. The quality of each cDNA was determined by amplification of
2-microglobulin.
View larger version (14K):
[in this window]
[in a new window]
| Fig 1.
OB-R map: (OB-RL), long
form of leptin receptor; (OB-RS), short (6.4 variant) form. The short form of the receptor represents a splice
variant of the receptor, which has a unique cytoplasmic domain
following the lysine residue at position 891 (stops 5 amino acids after
Lys891). The reverse primers for the long and short
isoforms of the receptor were designed to anneal to the unique
sequences in the cytoplasmic portion of the receptor.  forward
primer;  reverse primer; SF, short form; LF, long form; ECD,
extracellular domain; TM, transmembrane domain.
|
|
Flow cytometry and FACS-sorting.
Mononuclear cells from normal bone marrow donors were isolated by
density gradient centrifugation (Sigma), followed by two washes in
phosphate-buffered saline. Cells were incubated with HPCA-2/CD34,
Leu-M9/CD33, and Leu-M7/CD13 and IgG controls (Becton Dickinson, San
Jose, CA), respectively, for 30 minutes on ice, as recommended by the
manufacturer, and were subsequently washed twice with
phosphate-buffered saline. Cells displaying greater fluorescence
intensity than their controls were considered positive. Promyelocytic
cells were sorted based on forward and side scatter characteristics and
CD33 positivity. Other cells were sorted according to their specific
immunophenotype on a FACS Vantage (Becton Dickinson) equipped with an
argon-ion laser (Spectra Physics, San Jose, CA) operated at 488 nm and
300 mW. Fluorescent signals were detected using 530/30-nm and 585/40-nm
bandpass filters. Sorting was performed using R mode and Lysis II
software (Becton Dickinson) as previously described.27 All
cells were kept on ice during the sorting procedure. An aliquot of
sorted cells was reanalyzed for purity.
Serum leptin levels.
Serum leptin levels were measured in 56 patients with MDS or AML and in
five normal controls. Serum samples were drawn between 8 and 9 AM after an overnight (12-hour) fast. Leptin levels were determined by radioimmunoassay for human leptin (Linko Research Inc, St
Charles, MO) as previously described.28 Briefly, patient samples were assayed in duplicate by a completely homogeneous assay
using a rabbit antihuman leptin antibody; human leptin was used for
both the standard and the tracer. The assay is sensitive down to 0.5 ng/mL; its limit of linearity is 100 ng/mL. Random samples that showed
leptin immunoreactivity by radioimmunoassay were confirmed to contain
bioactive leptin by assaying them on engineered leptin sensitive cell
lines (data not shown). The degree of adiposity was determined as the
body mass index (BMI), calculated as weight in kilograms divided by
height in meters squared.29
Statistical analysis.
The statistical significance of differences in measured qualities was
determined with the two-tailed Student's t-test. A P factor of .05 or less was considered statistically significant. Unless
otherwise indicated, average values were expressed as means ± SEM.
The chi-square test was used to compare the expression of OB-R
with clinical features.
The leptin levels in serum were analyzed after logarithmic
transformation to eliminate the impact of skewing and variance heterogeneity. Multiple regression analysis was used to examine whether
diagnosis (MDS and primary or secondary AML), patient status (new
diagnosis, recurrence, resistance, complete remission, or partial
remission), treatment, age, sex, and blood counts influenced the
relationship between leptin and BMI.
| |
RESULTS |
Expression of OB-R in normal hematopoiesis.
Because it was shown that leptin can stimulate normal myeloid
progenitors, we first analyzed the expression of OB-R isoforms in purified myeloid subsets from adult human bone marrow. Both long and
short OB-R isoforms were detected in bone marrow mononuclear (n = 3) and FACS-sorted CD34+ progenitor cells (n = 5)
(Table 1). Both isoforms were expressed in
purified CD34+ cells with perhaps a higher expression of
the short isoform (Fig 2A and B). However, sorted
CD34CD33+ and
CD34CD13+ cells did not have the long
isoform and showed only weak expression for the short isoform by RT-PCR
(n = 3, Fig 2A), suggesting rapid downregulation of OB-R
expression during myeloid differentiation. Cord blood mononuclear cells
(n = 3) had lower expression of OB-R than normal adult bone
marrow, and normal peripheral blood samples (n = 3) showed low levels
of expression for both isoforms (Table 1).
View larger version (46K):
[in this window]
[in a new window]
| Fig 2.
(A) Expression of OB-R in normal bone marrow
(NBM), normal peripheral blood (NPB) and sorted myeloid progenitors
(CD34+, CD3433+,
CD3413+) from adult bone marrow. The
MO7E cell line served as a positive control. (B) OB-R
expression in FACS-sorted CD34+ cells from normal bone
marrow, AML, and chronic myeloid leukemia in blast crisis
(CML-BC). 2-M, 2-microglobulin;
OB-RL, long isoform (501 bp);
OB-RS, short isoform (720 bp); M,
molecular-weight marker.
|
|
Expression of leptin receptor in human leukemia cells.
We then studied the expression of OB-R mRNA in leukemia cell
lines and primary leukemia cells. OB-R transcripts were
detectable at significant levels in all myeloid and lymphoid cell lines
studied (Table 2). The highest levels were detected in
MO7E (Fig 2A) and K562 cell lines. Interestingly, both isoforms were
expressed in the majority of cell lines studied.
OB-R mRNA expression was analyzed in 49 AML and 6 MDS samples.
Examples of mRNA expression are shown in Fig
3. Overall, both isoforms were detected in 10 of 22 samples of newly diagnosed AML. The long isoform was detected in 15 of
22 (57.7%), and the short isoform in 13 of 22 (59.1%) samples. Newly
diagnosed cases of AML were classified as primary (n = 15) and
secondary (after MDS or chemo-/radiotherapy for the primary tumor,
n = 7). The OB-R long isoform was more frequently expressed
in primary rather than in secondary AML (13/15 v 2/7;
P = .01); no significant difference in the expression of the
short isoform was found (9/15 v 4/7). The observed difference
could not be attributed to the blast count, which did not differ
significantly between the two groups (78.3 ± 3.4 v
82.1 ± 3.9, respectively; P = .2). OB-R
transcripts were not limited to a specific AML FAB subtype (Fig
4). In MDS, expression of OB-R was similar to
that seen in secondary AML (long isoform 3/6; short isoform 4/6) (Table
3). Only the short isoform was detected in three AML and
two MDS cases, which showed no particular pattern with regard to
morphology, immunophenotype, or chromosomal abnormalities compared with
other samples. In five samples, the short isoform was not detected and
the level of expression of the long isoform was consistently low.
OB-R expression was observed in both CD34+ and
CD34 (n = 2) AML samples.
View larger version (82K):
[in this window]
[in a new window]
| Fig 3.
Expression of long and short isoforms of OB-R by
RT-PCR in AML (N 2,3,5-9,11,13, 15,16) and MDS (N 1,4,10,12,14)
samples. PV, polycytemia vera.
|
|
View larger version (22K):
[in this window]
[in a new window]
| Fig 4.
OB-R expression in different FAB subtypes of
newly diagnosed AML. Long ( ) and short ( ) OB-R
isoforms were identified using specific primers as described in
Materials and Methods. Amplification and detection was performed using
32P-dCTP incorporation. Results are shown as percentages of
positive cases in each category. The long isoform only was detected in
one case classified as FAB M0, and both isoforms were present in one
case classified as FAB M5 (not shown).
|
|
No correlation was found with cytogenetics, blast count, or response to
chemotherapy with regard to OB-R expression. In 39 AML samples
tested (excluding M3), samples from 26 patients had more than 80%
blasts, 11 had 50% to 80% blasts, and two had 46% and 31% blasts,
respectively, before density gradient separation. Of nine AML samples
tested in which the blast count exceeded 90% (before gradient
separation), the long isoform of OB-R was expressed in seven
samples, and the short isoform in eight samples. Four samples from
patients with refractory AML who were studied after initial
chemotherapy were found to express high levels of OB-R. OB-R
was also expressed in all 13 recurrent AML samples tested (Table 3).
Of interest, OB-R was expressed in all samples analyzed from 10 patients with acute promyelocytic leukemia (APL), five of whom were in
relapse (Fig
5A,
Table 3). In seven cases, both the long and short isoforms were
expressed, and in three additional cases, OB-R was detected by
primers that did not discriminate between the short and long isoforms.
In two APL cases, no detectable CD34+ cells were identified
by FACS analysis, but OB-R was detected. In three cases, a high
expression of OB-R in peripheral blood promyelocytes was found.
Both OB-R isoforms were detected in sorted CD3433+ promyelocytes of one newly diagnosed
APL sample (data not shown). In contrast, in sorted myeloid cells from
patients in complete remission, OB-R was expressed only in the
bone marrow CD34+ compartment, but not in
CD3433+ promyelocytes (Fig 5B).
View larger version (44K):
[in this window]
[in a new window]
| Fig 5.
(A) Expression of both isoforms of OB-R in two
patients newly diagnosed with APL and one patient with recurrent APL
compared with expression in normal bone marrow (NBM), FACS-sorted
normal myeloid progenitors, peripheral blood (PB), and MO7E cells (as
positive control). In one APL sample (lane 3) OB-R expression
was detected in peripheral blood containing 37% promyelocytes.
(B) OB-R expression of FACS-sorted
CD34+33 and
CD3433+ APL cells in complete remission
(CR). OB-R was identified only in the CD34+
compartment of NBM. In contrast, in a sample from a patient with newly
diagnosed APL containing 90% promyelocytes and no CD34+
cells, the level of OB-R expression corresponded to that seen
in NBM, which was used as a control.
|
|
We also studied the expression of OB-R in some other samples
from hematologic malignancies. Only the short isoform was found in
three samples of CD34+ cells from patients with chronic
myeloid leukemia in blast crisis (Fig 2B). Leptin receptor expression
was detected in none of five peripheral blood samples from patients
with advanced chronic lymphocytic leukemia. In samples from four
patients with acute lymphocytic leukemia and one with APL, only very
low-level expression of the short isoform was detected.
Effect of rH leptin on human leukemia cell proliferation.
The effect of rH leptin on proliferation of leukemia cell lines and
fresh AML samples by [3H]thymidine incorporation and
colony-forming ability was tested. Among six myeloid growth
factor-independent cell lines tested, only OCI/AML2 displayed a
significant proliferative response to leptin alone (P = .01;
SI = 1.7). Low enhancement of proliferative activity (SI = 1.3) was
found in NB4 (P < .05). No synergistic response was found
in the cell lines tested when leptin was combined with G-CSF, IL-3, or
SCF. In OCI-AML2 cells, the response to leptin was dose-dependent over
concentrations ranging from 10 to 100 ng/mL (Fig
6). Leptin also enhanced the colony-forming
ability of OCI/AML2 cells in a dose-dependent fashion (data not shown), with greater than 50% increase observed in colony-forming units in
leptin-treated cultures at 50 ng/mL (control, 323.5 ± 6.4; leptin,
519.5 ± 23.3 CFUs). The proliferative response in cell lines did
not correlate with the level of OB-R expression; eg, K562 cells
exhibited no proliferative response in spite of the high levels of
OB-R mRNA. Cytokine-dependent MO7E cells consistently responded
to leptin with increased proliferation (SI = 1.7) (Fig 6) but showed
no additive response when leptin was combined with other cytokines. The
maximal proliferative response was observed at a higher leptin
concentration than was required for OCI/AML2 cells.
View larger version (17K):
[in this window]
[in a new window]
| Fig 6.
Effect of leptin on proliferation of the OCI/AML2 ( )
and MO7E () cell line. MO7E cells were growth-factor starved for 18 hours. OCI/AML2 or MO7E cells were adjusted to the starting
concentration of 100,000 cells/mL and cultured with increasing
concentrations of rH leptin. After 48 hours of incubation, cultures
were pulsed for 12 hours with [3H]thymidine (0.1 µCi/well), harvested onto filter paper, immersed in scintillation
fluid, and quantitated in counts per minute. Each data point was
sampled in triplicate. Data from one of two experiments that yielded
similar results are shown.
|
|
The proliferative response to leptin was also investigated in 14 fresh
samples from patients with AML with a high blast count. Leptin alone at
100 ng/mL induced proliferation in three samples (SI > 1.5) (Table
4). Costimulatory effects of leptin and
other cytokines (G-CSF, IL-3, and SCF) were also evaluated. Leptin
additively enhanced IL-3-induced proliferation of AML cells in four
samples and enhanced the effect of SCF in three. The combination of
leptin and G-CSF yielded variable responses; three samples showed a
synergistic increase and four showed inhibition of the proliferative
response induced by G-CSF. Cytokine-stimulated proliferation was also
augmented by leptin in some samples that showed no proliferative
response to leptin alone. As in the cell lines tested, the
proliferative response to leptin did not correlate with the intensity
of expression of OB-R isoforms. AML cells from patient no. 5, in which only the long OB-R isoform was expressed, exhibited a
proliferative response to leptin combined with G-CSF, IL-3, and SCF and
enhancement of colony-forming ability. Despite high expression of both
OB-R isoforms, only one of four samples from patients with
recurrent AML responded to leptin alone, and one showed a synergistic
response when leptin was combined with G-CSF.
Leptin suppresses growth factor withdrawal-induced apoptosis of MO7E
and TF-1 cell lines.
Because various cytokines were found to promote cell survival, we
tested the effect of leptin on apoptosis of cytokine-dependent cell
lines after cytokine withdrawal. We incubated MO7E and TF-1 cells in
the presence or absence of 10% fetal calf serum and leptin (200 ng/mL)
for 3 days and then detected DNA fragmentation by flow cytometry.
Leptin-treated cells had a markedly decreased population of apoptotic
cells in both cell lines tested in serum-free and serum-containing
conditions. Results shown in Table 5 are representative of the two independent experiments conducted. No significant difference in proliferating cell fraction was documented (data not shown), which suggests a primarily anti-apoptotic, rather than proliferation-inducing, role for leptin in these cell lines.
Effect of leptin on the clonogenic leukemic cells from AML patients.
The effect of leptin on the proliferation of AML progenitor cells was
examined by colony-forming unit blast assays using fresh bone marrow
cells from five patients. In all patients, leptin treatment caused a
significant (P < .005) dose-dependent increase in the
proliferation of AML colony-forming cells; the optimum effect was
observed at plateau concentrations of leptin (50 to 100 ng/mL) (Fig
7). At higher concentrations (200 and 300 ng/mL), the effect of leptin was inhibitory.
View larger version (22K):
[in this window]
[in a new window]
| Fig 7.
Influence of leptin on myeloid leukemia clonogenic
progenitor growth of primary AML cells. Data represent results from
five different samples. FAB subtypes are indicated in the legend.
Results are expressed as the mean number of colonies in the presence of
increasing concentrations of leptin (10 to 200 ng/mL) compared with
control. Rel, relapsed; sec, secondary.
|
|
Serum leptin levels in patients with AML and MDS.
The serum leptin levels were tested in 56 patients with MDS and AML.
The mean of the logarithmic leptin levels in patients with AML and MDS
(1.633 ± 0.147) was not significantly different from that in normal
controls (1.89 ± 0.169). Serum samples studied showed significant
correlations between patients' BMIs and serum leptin levels
(r2 = 0.303; P < .001). Plasma levels were
higher in women (2.203 ± 0.255, n = 15) than in men
(1.424 ± 0.168, n = 41; P = .03). In multivariate
analysis, only BMI and gender were found to correlate with leptin serum
levels; no correlation with current treatment, patient status, blast
count, or blood counts was noted. In four patients with resistant AML,
serum levels were lower than in other patients, but the differences
were not statistically significant (P = .184).
| |
DISCUSSION |
Many cytokines are capable of stimulating a variety of biologic
responses in a wide spectrum of cell types. It is known that the
primary physiologic role of leptin is to control adipose tissue mass.
This is presumably mediated, at least in part, by signal transduction
through the leptin receptor in the hypothalamus.5 However,
because the leptin receptor exhibits functional pleiotropy, as do many
other cytokines, the leptin signaling pathway is also functional in
hematopoiesis. Leptin and the leptin receptor were recently shown to
play a role in normal myeloid development by exerting a stimulatory
effect on the GM precursors in adult human and murine bone
marrow.13,30 Leptin production was demonstrated by
hematopoietic stromal cell lines and bone marrow adipocytes, as well as
by extramedullary fat cells.3,15,30 It is possible that fat
cell content of the bone marrow microenvironment reflects the
requirement for leptin in hematopoietic development.
We compared the expression of the long and short isoforms of the leptin
receptor in normal and leukemic myeloid cell compartments of bone
marrow. As described previously, OB-R expression was detected in adult bone marrow and to a lesser degree in cord blood
cells.3 In purified myeloid progenitors from normal adult
bone marrow, both OB-R isoforms were strongly expressed in
CD34+ cells, perhaps with the short isoform being
predominant, consistent with published results.4,12 In more
mature CD3433+ (promyelocytes) and
CD3413+ cells, we detected only low level
expression of the short isoform and no expression of the long isoform,
which suggests that OB-R expression is rapidly downregulated in
normal myeloid progenitors during differentiation.
Analysis of OB-R isoform expression in samples from patients
with hematologic malignancies suggests that OB-R has a possible role in the pathogenesis of some myeloid leukemia. Both OB-R
isoforms were expressed in the majority of samples from AML patients,
whereas all samples from patients with chronic lymphocytic leukemia
were negative, and those from patients with acute lymphocytic leukemia showed only low-level expression of the short isoform. Both
OB-R isoforms were expressed in both peripheral blood and bone
marrow cells from AML cases with a high (>90%) blast count
indicating that the observed OB-R expression originates in the
leukemic cells. Expression of the long, but not the short isoform was
found to occur significantly more often in primary AML than in
secondary AML or MDS. OB-R was found to be expressed in all
recurrent AML samples studied. Surprisingly, only the short isoform was
detected in three samples of CD34+ cells from patients with
chronic myeloid leukemia in blast crisis, which might suggest a
different function of OB-R in this disease.
Previous results showed that leptin is capable of functioning as a
proliferation factor in primitive murine and normal human hematopoietic
cells.11,12,30 Our studies demonstrate that among seven
myeloid growth-factor independent cell lines tested, only OCI/AML2
cells showed a significant proliferative response to leptin, a result
confirmed by an increase in the colony-forming ability of these cells.
However, leptin stimulated growth of cytokine-dependent cell lines in a
dose-dependent manner. A cohort of fresh AML leukemic cells (3 of 14 samples tested) exhibited a proliferative response to leptin alone, and
combinations of leptin with other hematopoietic growth factors induced
an additive proliferative response in 7 of 14 AML cases. SCF was shown
previously to act synergistically with leptin to stimulate GM
precursors in adult human and murine bone marrow.12,30 This
effect could be attributable to the upregulation of specific receptors
so that leukemic precursors become more responsive to the additional
factor. Alternatively, leptin could prevent apoptosis of leukemic
progenitor cells as was shown by us for growth-factor-dependent cell
lines. Leptin also exerted stimulatory effects on leukemic clonogenic
cells when concentrations of 50 and 100 ng/mL were used in combination with SCF and GM-CSF. At higher, perhaps nonphysiologic concentrations, leptin inhibited colony formation, as was demonstrated previously for
murine bone marrow cells.13
In other experimental systems, it was shown that the long OB-R
isoform has signaling capabilities of the IL-6-type cytokine receptors
and uses STAT proteins in the signaling cascade.31 The
truncated short form exerts a reduced signaling
repertoire.32,33 The ability to proliferate after
stimulation with leptin or leptin in combination with cytokines was
observed in eight of nine samples expressing both receptor isoforms and
in one sample in which only the long isoform was expressed. This data
does not allow a correlation between receptor isoforms and signaling in AML.
The documentation of leptin production by placenta during pregnancy and
by gestational trophoblastic neoplasms suggests a pathophysiologic role
of leptin in leptin-producing tumors.34 We analyzed serum
leptin levels in 56 patients with AML and MDS. The positive correlation
with BMI and gender, which was established earlier for normal
individuals,8,9 was confirmed in the leukemia patients
tested here. Multivariate analysis showed no correlation between leptin
levels and clinical parameters (diagnosis, treatment, patient status,
blast count, or peripheral blood counts). However, these data do not
necessarily rule out the possibility of leptin production by AML blasts
or bone marrow stroma, which would create a local high concentration of
leptin within the bone marrow microenvironment. This hypothesis
requires further investigation.
The recently reported interesting observation of increased BMI in
patients with APL35 suggests a possible role for leptin in
this disease. Our data demonstrate high OB-R long isoform
expression in leukemic, but not in normal promyelocytes. The expression
was also found in CD3433+ FACS sorted cells
in newly diagnosed APL, but was absent in normal promyelocytes and in
promyelocytes from a patient with APL in remission. Hence, it is
conceivable that the proliferation of leukemic promyelocytes is driven,
in part, by increased leptin levels of obese APL patients.
Alternatively, it may reflect the abnormal expression pattern of leptin
receptor on leukemic cells, and the observation that OB-R is
expressed in CD34 blasts from two non-APL cases is
consistent with this notion.
In conclusion, we found expression of the leptin receptors in normal
progenitor cells and in the majority of AML. Aberrant expression was
seen in leukemic promyelocytes. Leptin induced a proliferative signal
and stimulated the colony-forming ability of leukemic progenitors,
presumably through the long isoform of OB-R. Leptin acts as an
inhibitor of apoptosis in cytokine-dependent leukemia cell lines. Our
understanding of the biologic and pathophysiologic role of leptin thus
far indicates that it may be part of a redundant network of cytokines
that regulates growth and viability of normal and leukemic progenitor cells.
| |
FOOTNOTES |
Submitted March 31, 1998; accepted October 22, 1998.
Supported by Grants No. CA 55164, CA 49639, and CA 16672 from the
National Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Michael Andreeff, MD, PhD, Department of
Molecular Hematology and Therapy, The University of Texas M.D. Anderson
Cancer Center, 1515 Holcombe Blvd, Box 81, Houston, TX 77030; e-mail:
mandreef{at}notes.mdacc.tmc.edu.
| |
REFERENCES |
1.
Metcalf D:
Hematopoietic regulators: Redundancy or subtlety?
Blood
82:3515, 1993[Free Full Text]
2.
Löwenberg B, Touw IP:
Hematopoietic growth factors and their receptors in acute leukemia.
Blood
81:281, 1993[Free Full Text]
3.
Benett BD, Solar GP, Yuan JQ, Mathias J, Thomas GR, Matthews W:
A role for leptin and its cognate receptor in hematopoiesis.
Curr Biol
6:1170, 1996[Medline]
[Order article via Infotrieve]
4.
Cioffi J, Shafer A, Zupanic T, Smith-Gbur J, Mikhail A, Platika D, Snodgrass H:
Novel B219/OB receptor isoforms: Possible role of leptin in hematopoiesis and reproduction.
Nat Med
2:585, 1996[Medline]
[Order article via Infotrieve]
5.
Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, Monroe CA, Tepper RI:
Identification and expression cloning of a leptin receptor, OB-R.
Cell
83:1263, 1995[Medline]
[Order article via Infotrieve]
6.
Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM:
Positional cloning of the mouse obese gene and its human homologue.
Nature
372:425, 1994[Medline]
[Order article via Infotrieve]
7.
Lee G, Proenca R, Montez J, Carroll K, Darvishzadeh J, Lee J:
Abnormal splicing of the leptin receptor in diabetic mice.
Nature
379:632, 1996[Medline]
[Order article via Infotrieve]
8.
Maffei M, Hallas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S, Kern PA, Friedman JM:
Leptin levels in human and rodent: Measurement of plasma leptin and ob mRNA in obese and weight reduced subjects.
Nat Med
1:1155, 1995[Medline]
[Order article via Infotrieve]
9.
Considine RV, Sinha MK, Heiman MR, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF:
Serum immunoreactive leptin concentrations in normal weight and obese humans.
N Engl J Med
334:292, 1996[Abstract/Free Full Text]
10.
Winik JD, Stoffel M, Friedman JM:
Identification of microsatellite markers linked to the human leptin receptor gene on chromosome 1.
Genomics
36:221, 1996[Medline]
[Order article via Infotrieve]
11.
Chen H, Charlat O, Tartaglia LA, Woolf EA, Wen X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP:
Evidence that the diabetes gene encodes the leptin receptor: Identification of a mutation in the leptin receptor gene in db/db mice.
Cell
84:491, 1996[Medline]
[Order article via Infotrieve]
12.
Gainsford T, Willson TA, Metcalf D, Handman E, McFarlane C, Ng A, Nicola NA, Alexander WS, Hilton DJ:
Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells.
Proc Natl Acad Sci USA
93:14564, 1996[Abstract/Free Full Text]
13.
Mikhail AA, Beck EX, Shafer A, Barut B, Gbur JS, Zupancic TJ, Schweitzer AC, Cioffi JA, Lacaud G, Ouyang B, Keller G, Snodgrass HR:
Leptin stimulates fetal and adult erythroid and myeloid development.
Blood
89:1507, 1997[Abstract/Free Full Text]
14.
Matsuda J, Yokota I, Iida M, Murakami T, Naito E, Ito M, Shima K, Kuroda Y:
Serum leptin concentration in cord blood: Relationship to birth weight and gender.
J Clin Endocr Met
82:1642, 1997[Abstract/Free Full Text]
15.
Laharrague P, Larrouy D, Fontanilles AM, Truel N, Campfield A, Tenenbaum R, Galitzky J, Corberand JX, Penicaud L, Casteilla L:
High expression of leptin by human bone marrow adipocytes in primary culture.
FASEB J
12:747, 1998[Abstract/Free Full Text]
16.
Wang C, Curtis JE, Minden MD, McCulloch EA:
Expression of retinoic acid receptor gene in myeloid leukemia cells.
Leukemia
3:264, 1989[Medline]
[Order article via Infotrieve]
17.
Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R:
NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3).
Blood
77:1080, 1991[Abstract/Free Full Text]
18.
McGrath T, Center MS:
Mechanisms of MDR in HL60 cells: Evidence that a surface membrane protein distinct from a p-glycoprotein contributes to reduced cellular accumulation of drugs.
Cancer Res
48:3959, 1988[Abstract/Free Full Text]
19.
Lisovsky M, Estrov Z, Zhang X, Consoli U, Sanchez-Williams G, Snell V, Munker R, Goodacre A, Savchenko V, Andreeff M:
Flt3 ligand stimulates proliferation and inhibits apoptosis of acute myeloid leukemia cells: Regulation of Bcl-2 and Bax.
Blood
88:3987, 1996[Abstract/Free Full Text]
20.
Estrov Z, Cohen A, Gelfand EW, Freedman MH:
Synergistic antiproliferative effects on HL-60 cells: Deferoxamine enhances cytosine arabinoside, methotrexate, and daunorubicin cytotoxicity.
Am J Pediatr Hematol Oncol
10:288, 1988[Medline]
[Order article via Infotrieve]
21.
Buick RN, Till JE, McCulloch EA:
A colony assay for proliferative blast cells circulating in myeloblastic leukemia.
Lancet
1:862, 1977[Medline]
[Order article via Infotrieve]
22.
Estrov Z, Black RA, Sleath PR, Harris D, Van Q, LaPushin R, Estey EH, Talpaz M:
Effect of interleukin-1 beta converting enzyme inhibitor on acute myelogenous leukemia progenitor proliferation.
Blood
86:4594, 1995[Abstract/Free Full Text]
23.
Freedman MH, Chan HSL, Chang L:
Childhood erythroleukemia: Studies on pathogenesis using colony assays.
Am J Pediatr Hematol Oncol
8:2, 1986[Medline]
[Order article via Infotrieve]
24.
Andreeff M, Darzynkiewicz Z, Sharpless TK, Clarkson BD, Melamed MR:
Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA.
Blood
55:282, 1980[Abstract/Free Full Text]
25.
Chomocyznski P, Sacchi N:
Single-step method for RNA isolation by acid guanidinium thiocyonate-phenol-chloroform extraction.
Anal Biochem
162:156, 1987[Medline]
[Order article via Infotrieve]
26.
Zhao S, Consoli U, Arceci R, Pfeifer J, Dalton WS, Andreeff M:
Semi-automated PCR method for quantitating MDR1 expression.
BioTechniques
21:726, 1996[Medline]
[Order article via Infotrieve]
27.
Drach D, Zhao S, Drach J, Mahadevia R, Gattringer C, Huber H, Andreeff M:
Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype.
Blood
80:2729, 1992[Abstract/Free Full Text]
28.
Ma Z, Gingerich RL, Santiago JV, Klein S, Smith CH, Landt M:
RIA of leptin in human plasma.
Clin Chem
42:942, 1996[Abstract/Free Full Text]
29.
Berdanier C:
Obesity, in
Munson PL,
Mueller RA,
Breese GR
(eds):
Principles of Pharmacology: Basic Concepts and Clinical Applications. New York, Chapman and Hall, 1995, p 1045.
30.
Umenoto Y, Tsuji K, Yang F, Ebihara Y, Kaneko A, Furukawa S, Nakahata T:
Leptin stimulates the proliferation of murine myelocytic and primitive hematopoietic progenitor cells.
Blood
90:3438, 1997[Abstract/Free Full Text]
31.
Baumann H, Morella KK, White DW, Dembski M, Bailon PS, Kim H, Lai C, Tartaglia L:
The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors.
Proc Natl Acad Sci USA
93:8374, 1996[Abstract/Free Full Text]
32.
Masuzaki H, Ogawa Y, Isse N, Satoh N, Okazaki T, Shigemoto M, Mori K, Tamura N, Hosoda K, Yoshimasa Y, Jingami H, Kawada T, Nakao K:
Human obese gene expression. Adipocyte-specific expression and regional differences in the adipose tissue.
Diabetes
44:855, 1995[Abstract]
33.
Chilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC:
Defective STAT signaling by the leptin receptor in diabetic mice.
Proc Natl Acad Sci USA
93:6231, 1996[Abstract/Free Full Text]
34.
Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T, Nakao K:
Nonadipose tissue production of leptin: Leptin as a novel placenta-derived hormone in humans.
Nature Med
3:1029, 1997[Medline]
[Order article via Infotrieve]
35.
Estey E, Thall P, Kantarjian H, Pierce S, Kornblau S, Keating M:
Association between increased body mass index and a diagnosis of acute promyelocytic leukemia in patients with acute myeloid leukemia.
Leukemia
11:1661, 1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
G. Trudel, M. Payne, B. Madler, N. Ramachandran, M. Lecompte, C. Wade, G. Biolo, S. Blanc, R. Hughson, L. Bear, et al.
Bone marrow fat accumulation after 60 days of bed rest persisted 1 year after activities were resumed along with hemopoietic stimulation: the Women International Space Simulation for Exploration study
J Appl Physiol,
August 1, 2009;
107(2):
540 - 548.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Samudio, M. Fiegl, T. McQueen, K. Clise-Dwyer, and M. Andreeff
The Warburg Effect in Leukemia-Stroma Cocultures Is Mediated by Mitochondrial Uncoupling Associated with Uncoupling Protein 2 Activation
Cancer Res.,
July 1, 2008;
68(13):
5198 - 5205.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Ablamunits, Y. Cohen, I. B. Brazee, H. P. Gaetz, C. Vinson, and S. Klebanov
Susceptibility to Induced and Spontaneous Carcinogenesis Is Increased in Fatless A-ZIP/F-1 but not in Obese ob/ob Mice.
Cancer Res.,
September 1, 2006;
66(17):
8897 - 8902.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Papathanassoglou, K. El-Haschimi, X. C. Li, G. Matarese, T. Strom, and C. Mantzoros
Leptin receptor expression and signaling in lymphocytes: kinetics during lymphocyte activation, role in lymphocyte survival, and response to high fat diet in mice.
J. Immunol.,
June 15, 2006;
176(12):
7745 - 7752.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Tabe, M. Konopleva, M. F. Munsell, F. C. Marini, C. Zompetta, T. McQueen, T. Tsao, S. Zhao, S. Pierce, J. Igari, et al.
PML-RAR{alpha} is associated with leptin-receptor induction: the role of mesenchymal stem cell-derived adipocytes in APL cell survival
Blood,
March 1, 2004;
103(5):
1815 - 1822.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. P. Cleary, S. C. Juneja, F. C. Phillips, X. Hu, J. P. Grande, and N. J. Maihle
Leptin Receptor-Deficient MMTV-TGF-{alpha}/LeprdbLeprdb Female Mice Do Not Develop Oncogene-Induced Mammary Tumors
Experimental Biology and Medicine,
February 1, 2004;
229(2):
182 - 193.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. S. Kim, J. S. Hong, S. W. Kim, J.-M. Koh, C. S. An, J.-Y. Choi, and S.-L. Cheng
Leptin Induces Apoptosis via ERK/cPLA2/Cytochrome c Pathway in Human Bone Marrow Stromal Cells
J. Biol. Chem.,
June 6, 2003;
278(24):
21920 - 21929.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. O. Iversen, C. A. Drevon, and J. E. Reseland
Prevention of leptin binding to its receptor suppresses rat leukemic cell growth by inhibiting angiogenesis
Blood,
December 1, 2002;
100(12):
4123 - 4128.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Matarese, V. Sanna, R. I. Lechler, N. Sarvetnick, S. Fontana, S. Zappacosta, and A. La Cava
Leptin Accelerates Autoimmune Diabetes in Female NOD Mice
Diabetes,
May 1, 2002;
51(5):
1356 - 1361.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. FAGGIONI, K. R. FEINGOLD, and C. GRUNFELD
Leptin regulation of the immune response and the immunodeficiency of malnutrition
FASEB J,
December 1, 2001;
15(14):
2565 - 2571.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Okuya, K. Tanabe, Y. Tanizawa, and Y. Oka
Leptin Increases the Viability of Isolated Rat Pancreatic Islets by Suppressing Apoptosis
Endocrinology,
November 1, 2001;
142(11):
4827 - 4830.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Miyazato, S. Ueno, K. Ohmine, M. Ueda, K. Yoshida, Y. Yamashita, T. Kaneko, M. Mori, K. Kirito, M. Toshima, et al.
Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction
Blood,
July 15, 2001;
98(2):
422 - 427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Fantuzzi and R. Faggioni
Leptin in the regulation of immunity, inflammation, and hematopoiesis
J. Leukoc. Biol.,
October 1, 2000;
68(4):
437 - 446.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. R. Golub, D. K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J. P. Mesirov, H. Coller, M. L. Loh, J. R. Downing, M. A. Caligiuri, et al.
Molecular Classification of Cancer: Class Discovery and Class Prediction by Gene Expression Monitoring
Science,
October 15, 1999;
286(5439):
531 - 537.
[Abstract]
[Full Text]
|
|
|
|
|
Top
Abstract
Introduction