|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
CHEMOKINES
From the Laboratoire d'Immunologie Cellulaire, Centre
de Transfusion Sanguine des Armées Jean Julliard, Clamart Cedex,
France; Institut National de la Santé et de la Recherche
Médicale, Unité 268, Institut André Lwoff,
Hôpital Paul Brousse, Paul Villejuif, France; Centre de Recherche
du Service de Santé des Armées, maquis du
Grésivaudant, La Tronche Cedex, France.
The stromal cell-derived factor 1 (SDF-1) chemokine has various
effects on hematopoietic cell functions. Its role in migration and
homing of hematopoietic progenitors is currently well established. Previously it was shown that SDF-1 stimulates myeloid progenitor proliferation in synergy with cytokines. Results of this study indicate
that SDF-1 alone promotes survival of purified CD34+ cells
from human unmobilized peripheral blood (PB) by counteracting apoptosis
as demonstrated by its capacity to reduce DNA fragmentation, annexin-V+ cell number, and APO2.7 detection and to
modulate bcl-2 homolog protein expression. The study demonstrates that
SDF-1, produced by sorted CD34+CD38+ cells and
over-released in response to cell damage, exerts an antiapoptotic
effect on CD34+ cells through an autocrine/paracrine
regulatory loop. SDF-1 participates in the autonomous survival of
circulating CD34+ cells and its effect required activation
of the phosphotidyl inositol 3 kinase (PI3-K)/Akt axis. Cell sorting
based on Hoechst/pyroninY fluorescences shows that SDF-1 production is
restricted to cycling CD34+ cells. SDF-1 triggers
G0 quiescent cells in G1 phase and, in synergy
with thrombopoietin or Steel factor, makes CD34+ cells
progress through S+G2/M phases of cell cycle. By assessing sorted CD34+CD38 Hematopoiesis consists of a complex process
in which hematopoietic progenitors (HPs) migrate, proliferate, and
generate a large number of lineage-committed blood cells. This process
is closely dependent on stromal cells and their secreted cytokines and
chemokines. An adjusted balance between self-renewal and
differentiation is necessary to maintain an adequate number of HPs,
which have long-term multilineage repopulating potential, and of mature
blood cells.1,2 Apoptosis is a naturally occurring process
involved in the homeostasis regulation of hematopoiesis.3
Whereas its role in control of mature compartment homeostasis is well
documented, molecular mechanisms underlying HP survival are still
unclear.4,5 Endogenous cell death regulatory proteins and
exogenous growth factors have been reported to participate in apoptosis
regulation.3 Actually, cytokines such as interleukin-3
(IL-3), thrombopoietin (Tpo), granulocyte-macrophage colony-simulating
factor (GM-CSF), Flt-3, and Steel factor (SF) have been described as
survival factors,6-10 but less is known about a possible
role for chemokines in HP survival.2,11,12
Stromal cell-derived factor 1 (SDF-1) is a CXC chemokine known
to be an effective chemotactic factor for progenitors and mature blood
cells.13-15 It is constitutively produced by bone marrow (BM) stromal cells and by other cells including CD34+
cells.16 SDF-1 was initially characterized as a pre-B
cell-stimulating factor. SDF1 gene knockout experiments have
demonstrated several defects including impaired
myelopoiesis.17,18 Recent studies have also reported a
crucial role for this chemokine in stem cell engraftment and
myelopoiesis.19-21 We previously demonstrated that SDF-1
enhanced proliferation and differentiation of unmobilized peripheral
blood (PB) CD34+ cells in synergy with cytokines. From
these results, we have proposed that exogenous SDF-1 could act as a
survival factor for CD34+ cells.22 Recently,
studies reporting the protecting effect of SDF-1 on normal
hematopoietic cells or leukemic B lymphocytes strengthened this
hypothesis.23-25
In the present study, we attempted to determine the mechanisms
whereby SDF-1 participated in CD34+ cell survival. For this
purpose, we investigated whether SDF-1 protected HPs from spontaneous
apoptosis and whether it interfered with cell cycle progression. Our
results showed that SDF-1 reduced DNA fragmentation,
annexin-V+ cell number, and APO2.7 expression, and
modulated apoptotic regulatory proteins of bcl-2 family in
CD34+ cells undergoing spontaneous
apoptosis.3,26,27 We showed that SDF-1 produced by PB
CD34+CD38+ cells played a role in their
autonomous survival through phosphotidyl inositol 3 kinase (PI3-K)/Akt
signaling. We also demonstrated the survival effect of SDF-1 on sorted
CD34+CD38 Mononuclear cell preparation and purification of
CD34+ cells
CD34+ cell culture conditions for analysis of
apoptosis, survival, and clonogenicity
The effect of exogenous or endogenous SDF-1 on apoptosis, cell
survival, and clonogenicity was evaluated by adding either recombinant
human SDF-1 Involvement of the PI3-K/Akt and mitogen-activated protein (MAP) kinase/MEK pathways in the antiapoptotic effect of SDF-1 on CD34+ cells was examined by using LY294002 and PD98059 specific inhibitors (10-100 µM), respectively (France Biochem, Meudon, France).28,29 Equivalent dilutions of dimethyl sulfoxide (DMSO) were used as controls. Assessment of DNA fragmentation by "sub-G1 peak" detection CD34+ cells (1 × 105/mL) were cultured for 4 days under apoptosis-inducing conditions with or without SDF-1 (0.01-0.5 ng/mL) and prepared for cell cycle analysis. Cells (5 × 104) were fixed in 70% ice-cold ethanol at 20°C for 30 minutes and were incubated for 15 minutes in the dark
at 4°C in phosphate-buffered saline (PBS) containing propidium iodide
(PI, 0.4 mg/mL; Molecular Probes Europe, Leiden, The
Netherlands). The PI fluorescence from fractional DNA content
of apoptotic cells produced a "sub-G1 peak" on the DNA
content frequency distribution histogram that defined the proportion of
apoptotic cells.30
Detection of membrane and mitochondrial apoptosis markers The percentage of cells undergoing apoptosis was determined using annexin-V and mitochondrial APO2.7 antigen detection assays.31,32 Briefly, CD34+ cells were incubated for 6 to 24 hours under apoptosis-inducing conditions with or without SDF-1 (0.01-0.5 ng/mL). In some experiments, an anti-rhSDF-1
antibody (5 ng/mL) was added to the medium. Cells (1 × 105) were incubated at room temperature in the dark
for 15 minutes in 200 µL buffer containing either
fluorescein-conjugated human annexin-V (5 µL, Pharmingen, BD
Biosciences, Le Pont de Claix, France) plus PI (5 µg/mL,
Molecular Probes) or R-phycoerythrin (PE)-conjugated APO2.7 antibody
(Beckman Coulter, Le Pont de Claix, France).
Annexin-V+PI and
annexin-V+PI+ cells correspond to apoptotic and
necrotic cells, respectively.
Cell cycle fractionation with PI and Ki67 The effect of SDF-1 on cell cycle progression was evaluated by culturing PB CD34+ cells in serum- and cytokine-free Stem-A medium (Stem-Alpha, St Clément les
Places, France) in the presence or absence of SDF-1 (0.01-1 ng/mL), Tpo, SF, and Flt-3 ligand (R & D Systems). At different time
points up to 72 hours, cells were analyzed for simultaneous expression
of Ki67 proliferation-associated nuclear antigen and DNA content. The
Ki67 antigen is expressed in cells entering G1 with
increasing expression during cell cycle but was undetectable in
G0 resting cells.33 Cells
(1 × 105) were fixed in 70% ice-cold ethanol,
permeabilized (OrthoPermeafix, OrthoDiagnostic System, Roissy en
France, France), immunostained using fluorescein isothiocyanate
(FITC)-labeled anti-Ki67 (Beckman Coulter) or its isotype IgG control
and washed before staining with PI. Specificity of the Ki67 antibody
binding was evaluated by using a FluoroTrol-DNAplus Cell
Cycle control kit (Bioergonomics, Clinisciences, Montrouge, France).
Intracellular detection of SDF-1, cyclins, and apoptosis-related Bcl-2 family proteins Intracellular expression of SDF-1, cyclins (D1-3 and E), and Bcl-2 family proteins (Bad, Bax, Bcl-2, and Bcl-xL) was detected by flow cytometry. CD34+ cells were cultured in serum- and cytokine-free medium (Stem-A or IMDM) in the presence
or absence of SDF-1 (0.05 and 0.5 ng/mL). At various time points up to
72 hours, 1 × 105 cells were harvested and incubated
with FITC- or PE-conjugated anti-CD38 monoclonal antibody (mAb) (clone
T16, Beckman Coulter) and with allophycocyanin (APC)-conjugated
anti-CD34 mAb (clone 581, Beckman Coulter) for 30 minutes at 4°C in
the dark. Cells were washed with PBS/2% human serum albumin
(HSA)/0.5% Ig, incubated in OrthoPermeafix, and labeled with the
following conjugated mouse antihuman antibodies: bcl-2-FITC (clone 124, Dako SA, Trappes, France), Bcl-xL-PE (clone 7B2.5,
Chemicon, Euromedex, Souffelweyersheim, France), Bad-FITC
(clone 48, Transduction Laboratories, BD Biosciences, Le Pont de Claix,
France), cyclins D1-, D2-, D3-FITC (clone G124-326, G132-43,
G107-565, from Pharmingen, BD Biosciences, respectively) or
unconjugated mouse antihuman antibodies: Bax (clone 4F11, Beckman Coulter), cyclin E (clone HE12, Pharmingen, BD Biosciences), or goat
antihuman SDF-1 antibody (R & D Systems). To avoid SDF-1 release during
permeabilization, cells were preincubated for 4 hours with 10 µg/mL
brefeldin-A (Sigma, Saint-Quentin Fallavier, France). Cells
labeled with anti-Bax, anticyclin E, anti-SDF-1 were subsequently
labeled with either a PE-conjugated goat antimouse (Caltag
Laboratories, Tebu, Saint-Quentin en Yvelines, France) or an
FITC-conjugated rabbit antimouse (Dako SA) or a PE- or
FITC-conjugated swine antigoat (Caltag Laboratories), respectively.
Cells were washed and analyzed by flow cytometry. Membrane-bound SDF-1
was estimated by omitting permeabilization before SDF-1 staining. Background fluorescence was assessed by using isotype-matched irrelevant mouse or goat immunoglobulins.
Flow cytometry analysis and cell sorting Analysis was performed using a Coulter Elite flow cytometer (Beckman Coulter). Cell debris was excluded from the analysis by gating on apoptotic and live cells on forward light scatter (FSC) versus side light scatter (SSC) dot plots. Stored events (5000-10 000) were analyzed with WinMDI (J. Trotter J, The Scripps Research Institute [TSRI], La Jolla, CA) or WinCycle software (Phoenix Flow Systems, San Diego, CA). Mean fluorescence intensity (MFI) was expressed in arbitrary units (AU).In some experiments, CD34+ subpopulations were sorted
according to their CD38 expression.22 Resting
(G0) or cycling (G1+S+G2/M) CD34+ cells were sorted based on Hoechst/pyroninY
fluorescences as described by Gothot et al.34,35 Figure
1 shows the gating strategy of cell
sorting and the purity of sorted fractions.
Western blotting analysis Cell lysis, protein separation, and transfer were performed as previously described.36 Separated proteins were incubated with an anticyclin D1 mAb (Zymed, Clinisciences, Montrouge, France), probed with a peroxidase-labeled goat antimouse antibody and revealed by chemiluminescence.36Statistical analysis Data were expressed as means ± SD. P < .05 was considered statistically significant (Student t test for paired samples).
Exogenous SDF-1 acts as a survival factor in counteracting PB CD34+ cell apoptosis We have previously reported that SDF-1 displayed a stimulatory effect on PB CD34+ proliferation in synergy with cytokines and have suggested that SDF-1 might act as a survival factor.22 This promoting effect was mainly demonstrated on CD34+ cells purified after an overnight incubation on a plastic support (Inc+CD34+). In the present study, we investigated whether SDF-1 could counteract CD34+ cell apoptosis. We first determined the appropriate experimental conditions inducing optimum spontaneous apoptosis in PB CD34+ cells.8 We quantified the percentage of subdiploid cells37 and analyzed the expression of annexin-V and APO2.7 in response to SDF-1.38 We studied whether SDF-1 altered the expression of Bcl-2 family proteins involved in apoptosis regulation.1,3,27SDF-1 decreases the sub-G1 peak, annexin-V,
and APO2.7 expression.
Freshly isolated Inc
The antiapoptotic effect of SDF-1 was further confirmed by evaluating the expression of APO2.7 protein, a mitochondrial marker of early apoptotic events.32 As shown in Figure 4, APO2.7 expression level was strongly reduced from 450.5 ± 62.4 AU to 110.3 ± 24.6 AU (n = 3; P = .001) after 6 hours in the presence of SDF-1; this effect was specific because it was totally abolished by addition of a neutralizing anti-SDF-1 antibody (Figure 4).
Expression of Bcl-2 family proteins is modulated by SDF-1.
We evaluated whether the antiapoptotic effect of SDF-1 went
through modulation of Bad, Bax, Bcl-2, and Bcl-xL
expression. We showed that freshly isolated PB
Inc+CD34+ cells expressed Bcl-2 and
Bcl-xL proteins (42.7 ± 5.6 AU and 21.6 ± 4.8 AU,
respectively; Figure 5A). We
observed a bimodal expression profile in which a small proportion of
CD34+ cells expressed low level of Bcl-2 (15% ± 6.3%,
12.7 ± 2.6 AU) and Bcl-xL (29.3% ± 5.2%,
7.1 ± 3.4 AU), whereas the majority of CD34+ cells
displayed high levels of these proteins (84.8% ± 4.2%, 48.1 ± 3.8 AU and 72.6% ± 6.1%, 28.9 ± 4.6 AU). After 72 hours under apoptosis-inducing conditions, the global expression of Bcl-2 and Bcl-xL cell death suppressors was increased up to
90.6 ± 5.3 AU (P = .03) and 83.5 ± 3.8 AU
(P = .001), respectively, with disappearance of the
bimodal profile (Figure 5A). Addition of SDF-1 (0.05 ng/mL) maintained
the global expression levels of Bcl-2 and Bcl-xL at their
initial values (39.2 ± 5.1 AU and 28.5 ± 5.6 AU, respectively,
n = 3; Figure 5A). Expression of Bad and Bax cell death promoters,
undetectable in freshly isolated Inc+CD34+
cells, increased up to 41.2 ± 6.1 AU and 150.6 ± 5.8 AU,
respectively, after 72 hours under apoptosis-inducing condition
(P < .001; n = 3; Figure 5B). Addition of SDF-1 (0.05 ng/mL) reduced their expression levels to 25.5 ± 2.8 AU,
P = .03 and 36.5 ± 5.2 AU, P = .006,
respectively (n = 3; Figure 5B).
Endogenous SDF-1 participates in the autonomous survival of PB CD34+ cells Inc
Endogenous SDF-1 maintains PB CD34+ cell survival.
We further investigated whether intracellular SDF-1 played a role in
the control of CD34+ cell autonomous survival by adding an
anti-SDF-1 neutralizing antibody to the culture. Table
3 shows that the percentage of surviving
cells was higher in Inc
cells from 25.5% ± 5.1%
to 48.4% ± 6.8% (P = .002; n = 3), whereas no
difference was observed in Inc+
cells (Figure 7).
SDF-1 antiapoptotic effect is mediated by the PI3-K pathway The PI3-K/Akt and MAP kinase proteins are reported to be the 2 main pathways involved in hematopoietic cell survival.28,39-41 We examined whether SDF-1 could prevent CD34+ cells from undergoing apoptosis through these signaling pathways by using LY294002- and PD98059-specific inhibitors (Figure 8). When Inc+ or IncCD34+ cells were cultured for 24 hours
under apoptosis-inducing conditions, the percentage of
annexin-V+ cells was 31% ± 3.6%
and 10.5% ± 3.2%, respectively. In
presence of SDF-1 (0.5 ng/mL), this percentage decreased from
31% ± 3.6% to
8.1% ± 3.4% for Inc+
(P = .002; n = 5) and from
10.5% ± 3.2% to
5.4% ± 1.8% for Inc cells
(P = .05; n = 5), confirming the antiapoptotic effect of exogenous SDF-1 (/ ). Addition of LY294002 (25 µM) alone
significantly increased the percentage of annexin-V+ cells
to 58.7% ± 6.8% and
46.5% ± 4.8%, respectively, for
Inc+ and Inc cells, as compared to untreated
control cells (P < .02, n = 3;  /), demonstrating
the role of the PI3-K in the autonomous survival of CD34+
cells. Addition of LY294002 to SDF-1 inhibited the survival effect of
SDF-1 because it increased the percentage of annexin-V+
cells from 8.1% ± 3.4% and
6.4% ± 3.8% to
59.1% ± 6.4% and
47.5% ± 2.3% for Inc+ and
Inc cells, respectively (/ cells significantly increased
from 10.5% ± 3.2% to
37.8% ± 5.8% (P < .002;
n = 3; / /CD34+ cells.
Addition of LY294002 to anti-SDF-1 did not modify the percentage of
annexin-V+ cells as compared to LY294002 treated cells
( ), suggesting that the survival effect of endogenous SDF-1 did
not go through a pathway different from PI3-K. In contrast, PD98059
(20-100 µM) alone had no effect on the percentage of
annexin-V+ cells as compared to untreated control cells
( ). Addition of PD98059 to SDF-1 or anti-SDF-1 did not modify
the percentage of apoptotic cells (  /
SDF-1 promotes survival of sorted clonogenic CD34+ progenitors We further evaluated the effect of exogenous or endogenous SDF-1 on clonogenic hematopoietic progenitor survival. This was performed by preincubating Inc+ or Inc sorted
CD34+ cells with either SDF-1 or anti-SDF-1 antibody for
72 hours under apoptosis conditions before plating in methylcellulose.
SDF-1 pretreatment (0.1 ng/mL) significantly gave 165.2% ± 21.3%
more erythroid burst-forming units (BFU-Es), 253% ± 18.4% more
granulocyte colony-forming units (CFU-Gs), 410% ± 26.5% more
macrophage colony-forming units (CFU-Ms), 225% ± 48.2% more
granulocyte-macrophage colony-forming units (CFU-GMs), and
100% ± 39.4% more CFU-Mix colony formation by
Inc+-sorted CD34+CD38 and
375.2% ± 36.3% more BFU-Es, 125.8% ± 31.1% more CFU-Ms,
150% ± 26.3% more CFU-GMs, and 350% ± 86.3% more CFU-Mix
colony formation by Inc+-sorted
CD34+CD38+ cells than was recorded for
untreated control cells (P < .05, n = 3; Figure
9A,B).
Addition of anti-SDF-1 (10 ng/mL) significantly gave 64% ± 12.3%
fewer BFU-Es, 24.5% ± 9.6% fewer CFU-Gs, 52.6% ± 5.6% fewer CFU-Ms, 86.4% ± 4.6% fewer CFU-GMs, and 88.9% ± 6.5% fewer
CFU-Mix colony formations by Inc SDF-1 triggers G0 CD34+ cells into cycle Our demonstration of a role for SDF-1 as an antiapoptotic factor incited us to investigate its effect on cell cycle progression. We have previously reported that SDF-1 increased the percentage of PB Inc+CD34+ cells in S+G2/M phases22; this effect was not observed in IncCD34+ cells. To analyze mechanisms
underlying such a difference, we studied the role of SDF-1 in the early
phases of the cell cycle. Therefore, we attempted to discriminate
G0 from G1 phases by using the Ki67 expression
assay. When freshly isolated, 28.3% ± 5.8% of
IncCD34+ cells did not express Ki67 (Figure
10A) and therefore were considered to
be in the G0 phase. In contrast, the majority of
Inc+CD34+ cells were in the G1
phase because 93.9% ± 2.1% of them expressed Ki67 (Figure
11A). As expected, the percentage of
cells in S+G2/M phases was low in both populations, with a
higher proportion in Inc+ cells as compared to
Inc cells (5.2% ± 0.4% and 1.9% ± 0.9%,
respectively, n = 3; Figures 10 and 11). Treatment with SDF-1
decreased the percentage of Inc cells in G0
from 22.3% ± 2.8% to 5% ± 0.7% (P < .0001,
n = 4; SDF-1: 0.5 ng/mL) and triggered them into cycle because their percentage in G1+S+G2/M phases increased from
78% ± 3.8% to 93.8% ± 1.6% (P < .0005, n = 4;
Figure 10B). The SDF-1 effect was dose dependent and maximal for 0.5 ng/mL (Figure 10B). SDF-1 increased the percentage of
Inc+CD34+ cells in S+G2/M phases
from 14% ± 0.2% to 28.8% ± 0.7% (P < .0001, n = 4; SDF-1: 0.05 ng/mL; Figure 11B). Therefore, SDF-1 triggered the
progression of CD34+ cells from G0 into
cycle when cells were initially quiescent, whereas it
stimulated the transition of cells already engaged in G1 to
S+G2/M phases.
We further analyzed the synergistic effect of SDF-1 with early acting
cytokines, such as Tpo, SF, and Flt-3 ligand, on CD34+ cell
cycle progression. Tpo or SF alone increased the percentage of cells in
S+G2/M without affecting the G0 compartment.
Interestingly, although SDF-1 triggered G0 quiescent cells
in G1, it made them progress through the S+G2/M
phases in combination with Tpo or SF
(Table 4). In contrast, Flt-3 ligand alone neither affected CD34+ cell cycle status nor synergized with SDF-1.
SDF-1 up-regulates cyclin E and cyclin D1 expression in
CD34+ cells.
Cyclins D1 to D3 are known to be
restricted to G0/G1 phases, whereas cyclin E is
mainly expressed during late G1 and during G1/S
transition.42 We investigated the effect of SDF-1 on
cyclin expression in Inc
Endogenous SDF-1 and CXCR-4 expression are related to
CD34+ cell cycle status.
We further explored whether endogenous SDF-1 expression was
associated with a precise phase of the cell cycle and whether it might
control CD34+ cell cycling. SDF-1 and its receptor were
expressed in cycling G1+S/G2M cells; in
contrast, quiescent G0 Inc
Addition of anti-SDF-1 to Inc CD34+
cells inhibited their progression into cycle and maintained the
percentage of G0 cells at its steady-state value as
compared to untreated cells (P = .001, n = 5; Table
5), suggesting that SDF-1 released by
cycling cells triggered G0 cells into cycle through an
autocrine/paracrine mechanism.
Hematopoiesis is supported by a network of growth factors mainly produced by stromal cells within the hematopoietic microenvironment and by hematopoietic cells.43 Whereas a number of studies have dissected the ability of these factors to support proliferation and differentiation, less is known about their capacity to promote cell survival. Among a myriad of growth factors, only a few, such as Tpo, SF, and Flt-3, display survival activity by suppressing apoptosis and stimulating cell cycling.6-9,44,45 Chemokines are also reported to be involved in hematopoiesis. They promote cell migration and participate in the negative regulation of progenitor proliferation.11,46 In our group, we were particularly concerned to study the role of SDF-1 in hematopoiesis because it induced CD34+ progenitor mobilization.14 Our previous studies demonstrating that SDF-1 stimulated the proliferation of primitive circulating CD34+ cells suggested a role for this chemokine as a survival factor.22 To determine the mechanisms whereby SDF-1 acts on cell survival, we investigated whether SDF-1 protected CD34+ cells from spontaneous apoptosis and whether it interfered with cell cycle progression. Apoptosis is a complex process characterized by a series of controlled sequential events. Therefore, spontaneous apoptotic CD34+ cells resulting from a short-term incubation in serum- and cytokine-free medium were analyzed by using different appropriate methods.38 We showed that SDF-1 counteracted spontaneous apoptosis of CD34+ cells, as demonstrated by the reduction of APO2.7 expression and of annexin-V+ and sub-G1 percentages, according to the kinetics of the apoptotic process.32 We also investigated the effect of SDF-1 on Bcl-2 family proteins reported to be involved in the regulation of hematopoietic cell survival.1,7,27 Our results showed that freshly isolated PB CD34+ cells expressed Bcl-2 and Bcl-xL proteins, whereas Bad and Bax were not detected in these cells. These results fit with those reported in CD34+ isolated from BM or cord blood.3,47 When CD34+ cells underwent spontaneous apoptosis, expression of Bcl-2 and Bcl-xL was up-regulated. Whereas these results could not fit with some data showing that antiapoptotic cytokines such as IL-10, Flt-3, and SF increased Bcl-2 expression,7,10,12 they are in agreement with data from other groups suggesting that such Bcl-2 up-regulation could be one of the mechanisms allowing cells to escape from apoptosis.3,26 Therefore, prevention of Bcl-2 and Bcl-xL modulation in response to SDF-1 could indicate that CD34+ cells did not undergo apoptosis under these conditions. Lastly, our results showing a reduction in Bad and Bax cell death promoter expression in response to SDF-1 argued for the antiapoptotic effect of this chemokine on CD34+ cells. Inc+ cells were more responsive to exogenous SDF-1
than Inc Under apoptosis-inducing conditions, the up-regulation of CXCR-4
expression on both Inc Recent findings have indicated the involvement of PI3-K/Akt and MAP kinase pathways in cell survival and SDF-1 signaling.39-41,49 By using specific inhibitors, we demonstrated that exogenous SDF-1 displayed its antiapoptotic effect in CD34+ cells through activation of the PI3-K/Akt axis. This result is in agreement with the ability of SDF-1 to phosphorylate and activate PI3-K/Akt in CD34+ cells and in hematopoietic cell lines as demonstrated by Majka and coworkers, though in these studies the authors did not report any effect of SDF-1 on cell survival.40,50,51 Several hypotheses, including CD34+ cell source (BM versus PB) and SDF-1 doses (500 ng/mL versus 0.5 ng/mL), may account for such differences. Interestingly, we showed that specific inhibition of PI3-K/Akt by LY294002 affected the autonomous survival of CD34+ cells and that the antiapoptotic effect of endogenous SDF-1 did not go through a pathway different from the PI3-K/Akt axis. These results strongly suggested that endogenous SDF-1 participates in CD34+ autonomous cell survival through PI3-K/Akt signaling.52 In our experimental system, the MAP kinase/MEK pathway was required neither for autonomous CD34+ cell survival nor for the antiapoptotic effect of SDF-1. This may appear to contradict the findings of Lee and colleagues that SDF-1 was able to phosphorylate MAP kinases39; however, in their study, direct implication of such a pathway in CD34+ cell survival was not demonstrated. Moreover, conflicting data have been reported concerning the involvement of the MAP kinase/MEK pathway in adhesion protein activation and chemotaxis in response to SDF-1.50,51,53 Taken together these studies illustrate the complexity of SDF-1-mediated signaling, depending on studied functions and experimental conditions. Apoptosis and cell cycling are thought to be intimately linked processes involved in hematopoiesis homeostasis. When analyzed with a standard DNA histogram, up to 98% of PB CD34+ cells are in G0/G1 phases, whereas less than 85% of BM and cord blood CD34+ cells are quiescent.22,33,34 This conventional cell cycle analysis allows definition of cells in G0/G1 and S+G2/M phases but does not discriminate G0 from G1 phase.35 By using dual DNA/Ki67 staining we have demonstrated that exogenous SDF-1 was able to trigger resting G0 PB CD34+ cells into G1 and to make them progress through the cycle without executing a complete cell cycle. Such a triggering effect was confirmed by the overexpression of G0/G1-restricted D1 and E cyclins in response to SDF-1. To our knowledge, our study is the first reporting a role for a chemokine in the G0 to G1 transition in CD34+ hematopoietic progenitors. Very few studies have analyzed the role of growth factors in the control of early phase transitions. Among these factors, SF was reported to increase the percentage of CD34+ cells in S+G2/M without recruiting cells from G0.33 We show that Tpo and SF preferentially stimulate the transition from G1 to S+G2/M and, when combined with SDF-1, allow G0 cells to progress to the late phases of the cell cycle. These findings suggest that SDF-1 acts as a priming factor by recruiting quiescent cells from G0 into cycle and by rendering them responsive to further growth factor stimulation. As an attempt to explore the possible involvement of endogenous SDF-1 in CD34+ cell cycle regulation, we investigated whether its expression was related to cell cycle status. We showed that SDF-1 was expressed and secreted by sorted cycling cells but not by G0 resting cells and that its secretion was increased under apoptosis conditions. Because most of cycling cells expressed antigenic markers of CD34+CD38+ committed cells (data not shown), it could be suggested that the cycling and CD34+CD38+ cell populations (that produce SDF-1) overlap. We and others showed that CXCR-4 was expressed in both cycling and resting CD34+ cells,54 with a higher expression on sorted G1+S+G2/M cells. Under apoptosis-inducing conditions, CXCR-4 was up-regulated on both sorted cycling and resting cells. Addition of anti-SDF-1 maintained the percentage of G0 CD34+ cells at its steady-state value and inhibited their progression into cycle, suggesting that endogenous SDF-1 was able to trigger CD34+ cells into cycle. Together these results suggested that CD34+ cells are able to control their own cycling and survival status via endogenous factors such as SDF-1. In conclusion, our present study demonstrates a role for the SDF-1 chemokine in survival and cycling control of CD34+ progenitors and provides evidence for an autocrine/paracrine mechanism. We propose that SDF-1 could play a part in hematopoiesis homeostasis by participating in the autonomous survival of circulating progenitors. SDF-1 may also occur in alarm situations by protecting them in response to cell damage. Preliminary results showing that treatment of lethally irradiated mice with SDF-1 significantly increased survival from 0% in control untreated mice to 30% in their SDF-1-treated counterparts, 30 days after irradiation, argued for an in vivo SDF-1 survival effect. By acting on stem cell trafficking, progenitor cell survival and cycling, SDF-1 chemokine could be of major interest in cellular therapy settings after myeloablative aggression.
We are indebted to Dr Loïc Niel for continuously supplying blood samples. We also thank Peggy Sanatine for her technical skill in flow cytometry.
Submitted April 2, 2001; accepted October 11, 2001.
Supported by grants from the DRET (Direction des Recherches Etudes et Techniques, Ministère de la Défense, no. 957) and the ANRB (Association Nouvelles Recherches Biomédicales).
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: Marie-Caroline Le Bousse-Kerdilès, INSERM U268, Institut André Lwoff, Hôpital Paul Brousse, 14, Ave Paul Vaillant Couturier, 94800 Villejuif, France; e-mail: lebousse{at}ujf.inserm.fr.
1.
Domen J, Cheshier SH, Weissman IL.
The role of apoptosis in the regulation of hematopoietic stem cells: overexpression of Bcl-2 increases both their number and repopulation potential.
J Exp Med.
2000;191:253-263
2.
Hodohara K, Fujii N, Yamamoto N, Kaushansky K.
Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK).
Blood.
2000;95:769-775
3.
Peters R, Leyvraz S, Perey L.
Apoptotic regulation in primitive hematopoietic precursors.
Blood.
1998;92:2041-2052 4. Orkin SH, Weiss MJ. Cutting red-cell production. Nature. 1999;401:433-436[CrossRef][Medline] [Order article via Infotrieve]. 5. De Maria R, Zeuner A, Eramo A, et al. Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1. Nature. 1999;401:489-493[CrossRef][Medline] [Order article via Infotrieve]. 6. Nicholls SE, Winter S, Mottram R, Miyan JA, Whetton AD. Flt-3 ligand can promote survival and macrophage development without proliferation in myeloid progenitor cells. Exp Hematol. 1999;27:663-672[CrossRef][Medline] [Order article via Infotrieve].
7.
Lisovsky M, Estrov X, Consoli U, et al.
Flt-3 ligand stimulates proliferation and inhibits apoptosis of acute myeloid leukemia cells: regulation of Bcl-2 and Bax.
Blood.
1996;88:3987-3997
8.
Borge OJ, Ramsfjell V, Cui L, Jacobsen SEW.
Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+CD38 9. Tavassoli M. Hematopoietic survival factors. Exp Hematol 1993;21:1511-1513[Medline] [Order article via Infotrieve].
10.
Carson WE, Haldar S, Baiocchi RA, Croce CM, Caligiuri MA.
The c-kit ligand suppresses apoptosis of human natural killer cells through the upregulation of bcl-2.
Proc Natl Acad Sci U S A.
1994;91:7553-7557
11.
Han ZC, Lu M, Li J, et al.
Platelet factor 4 and other CXC chemokines support the survival of normal hematopoietic cells and reduce the chemosensitivity of cells to cytotoxic agents.
Blood.
1997;89:2328-2335
12.
Weber-Nordt RM, Henschler R, Schott E, et al.
Interleukin-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells.
Blood.
1996;88:2549-2558
13.
Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T.
Signal sequence trap: a cloning strategy for secreted proteins and type 1 membrane proteins.
Science.
1993;261:600-603
14.
Kim CH, Broxmeyer HE.
In vitro behavior of hematopoietic progenitor cells under the influence of chemoattractants: stroma cell-derived factor-1, Steel factor, and the bone marrow environment.
Blood.
1998;91:100-110
15.
Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC.
The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood.
J Exp Med.
1997;185:111-120
16.
Aiuti A, Turchetto L, Cota M, et al.
Human CD34+ cells express CXCR4 and its ligand stromal cell-derived factor-1. Implications for infection by T-cell tropic human immunodeficiency virus.
Blood.
1999;94:62-73
17.
Nagasawa T, Kikutani H, Kishimoto T.
Molecular cloning and structure of a pre-B-cell-growth stimulating factor.
Proc Natl Acad Sci U S A.
1994;91:2305-2309 18. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996;382:635-638[CrossRef][Medline] [Order article via Infotrieve]. 19. Ponomaryov T, Peled A, Petit I, et al. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest. 2000;106:1331-1339[Medline] [Order article via Infotrieve].
20.
Peled A, Petit I, Kollet O, et al.
Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.
Science.
1999;283:845-848
21.
Peled A, Kollet O, Ponomaryov T, et al.
The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice.
Blood.
2000;95:3289-3296
22.
Lataillade J-J, Clay D, Dupuy C, et al.
Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival.
Blood.
2000;95:756-768 23. Gotoh A, Miyazawa K, Yaguchi M, Broxmeyer H. Priming with SDF-1 induces enhancement of cytokine-dependent survival effect and pronounced activation of MAP kinase signaling in hematopoietic cells [abstract]. Blood. 1998;92:200a.
24.
Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell'Aquila M, Kipps T.
Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1.
Blood.
2000;96:2655-2663 25. Kollet O, Grabovsky V, Franitza S, Lider O, Alon R, Lapidot T. SDF-1 induces survival, adhesion, and migration in 3D ECM like gels of murine CXCR-4null fetal liver cells via another GPCR [abstract]. Blood. 2000;96:65a.
26.
Barcena A, Park SW, Banapour B, Muench MO, Mechetner E.
Expression of Fas/CD95 and Bcl-2 by primitive hematopoietic progenitors freshly isolated from human fetal liver.
Blood.
1996;88:2013-2025
27.
Park JR, Bernstein ID, Hockenbery DM.
Primitive human hematopoietic precursors express Bcl-x but not Bcl-2.
Blood.
1995;86:868-876
28.
Haseyama Y, Sawada K-I, Oda A, et al.
Phosphatidylinositol 3-kinase is involved in the protection of primary cultured human erythroid precursor cells from apoptosis.
Blood.
1999;94:1568-1577
29.
Fichelson S, Freyssinier JM, Picard F, et al.
Megakaryocyte growth and development factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors.
Blood.
1999;94:1601-1613 30. Darzynkiewicz Z, Bruno S, Del Bino G, et al. Features of apoptotic cells measured by flow cytometry. Cytometry. 1992;13:795-808[CrossRef][Medline] [Order article via Infotrieve].
31.
Koopman G, Reutelingsperger CPM, Kuijten GAM, Keehnen RMJ, Pals ST, Van Oers MHJ.
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.
Blood.
1994;84:1415-1420 32. Koester SK, Roth P, Mikulka WR, Schlossman SF, Zhang C, Bolton WE. Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein-specific monoclonal antibody APO2.7. Cytometry. 1997;29:306-312[CrossRef][Medline] [Order article via Infotrieve]. 33. Gore SD, Amin S, Weng LJ, Civin CI. Steel factor supports the cycling of isolated human CD34+ cells in the absence of other growth factors. Exp Hematol. 1995;23:413-421[Medline] [Order article via Infotrieve].
34.
Gothot A, Van der Loo JCM, Clapp DW, Srour EF.
Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice.
Blood.
1998;92:2641-2649
35.
Gothot A, Pyatt R, MacMahel J, Rice S, Srour EF.
Functional heterogeneity of human CD34+ cells isolated in subcompartments of the G0/G1 phase of the cell cycle.
Blood.
1997;90:4384-4393
36.
Horvàth G, Serru V, Clay D, Billard M, Boucheix C, Rubinstein E.
CD19 is linked to the integrin-associated tetraspans CD9, CD81, and CD82.
J Biol Chem.
1998;273:30537-30543 37. Kroemer G, Bosca M, Zamzami N, Marchetti P, Hortelano S, Martinez-A C. Detection of apoptosis and apoptosis-associated alterations. In: J. Lafkints, ed. Immunology Methods Manual. Academic Press; 1997:1112-1125. 38. Pepper C, Thomas A, Tucker H, Hoy T, Bentley P. Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis. Leuk Res. 1998;22:439-444[CrossRef][Medline] [Order article via Infotrieve]. 39. Lee Y, You M, Cooper S, Hangoc G, Broxmeyer HE. Enhancement of hematopoietic progenitor cell survival in response to SDF-1 alone and in synergy with other cytokines may be mediated by the MAPK/P90RSK cascade through modulation of anti-apoptotic protein expression and pro-apoptotic Bad function [abstract]. Blood. 2000;96:676a. 40. Majka M, Ratajczak J, Gewirtz AM, Ratajczak MZ. PI-3K-AKT axis inhibits apoptosis in normal human CD34+ cells and megakaryoblasts and is efficiently activated by thrombopoietin (TPO) but not by IL-6, IL-11 or SDF-1 [abstract]. Blood. 2000;96:569a. 41. Minamiguchi H, Kimura T, Fujiki H, et al. Simultaneous activation of signals through c-MPL, c-Kit, and CXCR4 dramatically enhances proliferation and differentiation of CFU-Meg: possible role of PI3-K, PKC, and MAPK pathways [abstract]. Blood. 2000;96:123b. 42. Darzynkiewicz Z, Gong J, Juan G, Ardelt B, Traganos F. Cytometry of cyclin proteins. Cytometry. 1996;25:1-13[CrossRef][Medline] [Order article via Infotrieve].
43.
Gupta P, Blazar BR, Gupta K, Verfaillie CM.
Human CD34+ bone marrow cells regulate stromal production of IL-6 and G-CSF and increase the colony stimulating activity of stroma.
Blood.
1998;91:3724-3733 44. Murray LJ, Young JC, Luens KM, Scollay R, Hill BL. Thrombopoietin, Flt-3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+Thy-1+ cells into rapid division. Exp Hematol. 1999;27:1019-1028[CrossRef][Medline] [Order article via Infotrieve].
45.
Majka M, Janowska-Wieczorek A, Ratajczak J, et al.
Numerous growth factors, cytokines and chemokines are secreted by CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate hematopoiesis in an autocrine/paracrine manner.
Blood.
2001;97:3075-3085 46. Broxmeyer HE, Kim CH. Regulation of hematopoiesis in a sea of chemokine family members with a plethora of redundant activities. Exp Hematol. 1999;27:1113-1123[CrossRef][Medline] [Order article via Infotrieve]. 47. Josefsen D, Myklebust JH, Lomo J, Sioud M, Blomhoff HK, Smeland EB. Differential expression of Bcl-2 homologs in human CD34+ hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells. Stem Cells. 2000;18:261-272[CrossRef][Medline] [Order article via Infotrieve]. 48. Bockaert J. G-proteins and G-protein coupled receptors: structure, functions and interactions. Curr Opin Neurobiol. 1991;1:32-42[CrossRef][Medline] [Order article via Infotrieve].
49.
Ganju RK, Brubaker SA, Meyer J, et al.
The alpha-chemokine, stromal cell-derived factor-1
50.
Majka M, Janowska-wieczorek A, Ratajczak J, et al.
Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis.
Blood.
2000;96:4142-4151
51.
Wang J-F, Park I-W, Groopman JE.
Stromal cell-derived factor-1 52. Ratajczak MZ, Majka M, Wieczorek AJ. Human CD34+ cells, myeloblasts secrete numerous growth factors, cytokines and chemokines: evidence for intercellular crosstalk in hematopoiesis [abstract]. Blood. 2000;96:126b. 53. Majka M, Ratajczak J, Lee B, et al. The role of HIV-related chemokine receptors and chemokines in human erythropoiesis in vitro. Stem Cells. 2000;18:128-138[CrossRef][Medline] [Order article via Infotrieve].
54.
Glimm H, Oh I-H, Eaves CJ.
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0.
Blood.
2000;96:4185-4193
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. Dominici, V. Rasini, R. Bussolari, X. Chen, T. J. Hofmann, C. Spano, D. Bernabei, E. Veronesi, F. Bertoni, P. Paolucci, et al. Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation Blood, September 10, 2009; 114(11): 2333 - 2343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Grundler, L. Brault, C. Gasser, A. N. Bullock, T. Dechow, S. Woetzel, V. Pogacic, A. Villa, S. Ehret, G. Berridge, et al. Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration J. Exp. Med., August 31, 2009; 206(9): 1957 - 1970. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, E. T. Gonzalez, S. S. Iyer, V. Mac, A. L. Mora, R. L. Sutliff, A. Reed, K. L. Brigham, P. Kelly, and M. Rojas Use of Senescence-Accelerated Mouse Model in Bleomycin-Induced Lung Injury Suggests That Bone Marrow-Derived Cells Can Alter the Outcome of Lung Injury in Aged Mice J Gerontol A Biol Sci Med Sci, July 1, 2009; 64A(7): 731 - 739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Foguenne, I. Di Stefano, O. Giet, Y. Beguin, and A. Gothot Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of {alpha}4 integrin- and CXCR4-mediated engraftment in NOD/SCID {beta}2-microglobulin-null mice Haematologica, February 1, 2009; 94(2): 185 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Arduise, T. Abache, L. Li, M. Billard, A. Chabanon, A. Ludwig, P. Mauduit, C. Boucheix, E. Rubinstein, and F. Le Naour Tetraspanins Regulate ADAM10-Mediated Cleavage of TNF-{alpha} and Epidermal Growth Factor J. Immunol., November 15, 2008; 181(10): 7002 - 7013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Barbieri, A. Bajetto, R. Stumm, A. Pattarozzi, C. Porcile, G. Zona, A. Dorcaratto, J.-L. Ravetti, F. Minuto, R. Spaziante, et al. Overexpression of Stromal Cell-Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas Clin. Cancer Res., August 15, 2008; 14(16): 5022 - 5032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Penn and A. A. Mangi Genetic Enhancement of Stem Cell Engraftment, Survival, and Efficacy Circ. Res., June 20, 2008; 102(12): 1471 - 1482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nie, Y.-C. Han, and Y.-R. Zou CXCR4 is required for the quiescence of primitive hematopoietic cells J. Exp. Med., April 14, 2008; 205(4): 777 - 783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Rezzoug, Y. Huang, M. K. Tanner, M. Wysoczynski, C. L. Schanie, P. M. Chilton, M. Z. Ratajczak, I. J. Fugier-Vivier, and S. T. Ildstad TNF-{alpha} Is Critical to Facilitate Hemopoietic Stem Cell Engraftment and Function J. Immunol., January 1, 2008; 180(1): 49 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Jin, Y. Tabe, S. Konoplev, Y. Xu, C. E. Leysath, H. Lu, S. Kimura, A. Ohsaka, M.-B. Rios, L. Calvert, et al. CXCR4 up-regulation by imatinib induces chronic myelogenous leukemia (CML) cell migration to bone marrow stroma and promotes survival of quiescent CML cells Mol. Cancer Ther., January 1, 2008; 7(1): 48 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Juarez, R. Baraz, S. Gaundar, K. Bradstock, and L. Bendall Interaction of interleukin-7 and interleukin-3 with the CXCL12-induced proliferation of B-cell progenitor acute lymphoblastic leukemia Haematologica, April 1, 2007; 92(4): 450 - 459. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Zheng, S.-h. Oh, Y. Jung, and B. E. Petersen Oval Cell Response in 2-Acetylaminofluorene/Partial Hepatectomy Rat Is Attenuated by Short Interfering RNA Targeted to Stromal Cell-Derived Factor-1 Am. J. Pathol., December 1, 2006; 169(6): 2066 - 2074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zeng, I. J. Samudio, M. Munsell, J. An, Z. Huang, E. Estey, M. Andreeff, and M. Konopleva Inhibition of CXCR4 with the novel RCP168 peptide overcomes stroma-mediated chemoresistance in chronic and acute leukemias Mol. Cancer Ther., December 1, 2006; 5(12): 3113 - 3121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wimmer, S. K. Khaldoyanidi, M. Judex, N. Serobyan, R. G. DiScipio, and I. U. Schraufstatter CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes Blood, December 1, 2006; 108(12): 3722 - 3729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Foudi, P. Jarrier, Y. Zhang, M. Wittner, J.-F. Geay, Y. Lecluse, T. Nagasawa, W. Vainchenker, and F. Louache Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvironment in CXCR4-/- chimeric mice Blood, March 15, 2006; 107(6): 2243 - 2251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Burger and T. J. Kipps CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment Blood, March 1, 2006; 107(5): 1761 - 1767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Muguruma, T. Yahata, H. Miyatake, T. Sato, T. Uno, J. Itoh, S. Kato, M. Ito, T. Hotta, and K. Ando Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment Blood, March 1, 2006; 107(5): 1878 - 1887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Infantino, B. Moepps, and M. Thelen Expression and Regulation of the Orphan Receptor RDC1 and Its Putative Ligand in Human Dendritic and B Cells J. Immunol., February 15, 2006; 176(4): 2197 - 2207. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Haneline, H. White, F.-C. Yang, S. Chen, C. Orschell, R. Kapur, and D. A. Ingram Genetic reduction of class IA PI-3 kinase activity alters fetal hematopoiesis and competitive repopulating ability of hematopoietic stem cells in vivo Blood, February 15, 2006; 107(4): 1375 - 1382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Woerner, N. M. Warrington, A. L. Kung, A. Perry, and J. B. Rubin Widespread CXCR4 Activation in Astrocytomas Revealed by Phospho-CXCR4-Specific Antibodies Cancer Res., December 15, 2005; 65(24): 11392 - 11399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Gerli, C. Vanelli, O. Turri, M. Erario, A. Gardellini, M. Pugliano, and M. L. Biondi SDF1-3'A Gene Polymorphism Is Associated with Chronic Myeloproliferative Disease and Thrombotic Events Clin. Chem., December 1, 2005; 51(12): 2411 - 2414. [Full Text] [PDF]
|
|
|
|
|
|
C. L. Semerad, M. J. Christopher, F. Liu, B. Short, P. J. Simmons, I. Winkler, J.-P. Levesque, J. Chappel, F. P. Ross, and D. C. Link G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow Blood, November 1, 2005; 106(9): 3020 - 3027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Dalakas, P. N. Newsome, D. J. Harrison, and J. N. Plevris Hematopoietic stem cell trafficking in liver injury FASEB J, August 1, 2005; 19(10): 1225 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-J. Lataillade, D. Clay, C. David, L. Boutin, B. Guerton, M. Drouet, F. Herodin, and M.-C. Le Bousse-Kerdiles Phenotypic and functional characteristics of CD34+ cells are related to their anatomical environment: is their versatility a prerequisite for their bio-availability? J. Leukoc. Biol., May 1, 2005; 77(5): 634 - 643. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Bendall, R. Baraz, J. Juarez, W. Shen, and K. F. Bradstock Defective p38 Mitogen-Activated Protein Kinase Signaling Impairs Chemotaxic but not Proliferative Responses to Stromal-Derived Factor-1{alpha} in Acute Lymphoblastic Leukemia Cancer Res., April 15, 2005; 65(8): 3290 - 3298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Smith, P. A. Johanesen, M. K. Wendt, D. G. Binion, and M. B. Dwinell CXCL12 activation of CXCR4 regulates mucosal host defense through stimulation of epithelial cell migration and promotion of intestinal barrier integrity Am J Physiol Gastrointest Liver Physiol, February 1, 2005; 288(2): G316 - G326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Piovan, V. Tosello, S. Indraccolo, A. Cabrelle, I. Baesso, L. Trentin, R. Zamarchi, H. Tamamura, N. Fujii, G. Semenzato, et al. Chemokine receptor expression in EBV-associated lymphoproliferation in hu/SCID mice: implications for CXCL12/CXCR4 axis in lymphoma generation Blood, February 1, 2005; 105(3): 931 - 939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Jaleel, A. C. Tsai, S. Sarkar, P. V. Freedman, and L. P. Rubin Stromal cell-derived factor-1 (SDF-1) signalling regulates human placental trophoblast cell survival Mol. Hum. Reprod., December 1, 2004; 10(12): 901 - 909. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. De Falco, D. Porcelli, A. R. Torella, S. Straino, M. G. Iachininoto, A. Orlandi, S. Truffa, P. Biglioli, M. Napolitano, M. C. Capogrossi, et al. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells Blood, December 1, 2004; 104(12): 3472 - 3482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Marchesi, P. Monti, B. E. Leone, A. Zerbi, A. Vecchi, L. Piemonti, A. Mantovani, and P. Allavena Increased Survival, Proliferation, and Migration in Metastatic Human Pancreatic Tumor Cells Expressing Functional CXCR4 Cancer Res., November 15, 2004; 64(22): 8420 - 8427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. C. Rombouts, B. Pavic, B. Lowenberg, and R. E. Ploemacher Relation between CXCR-4 expression, Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia Blood, July 15, 2004; 104(2): 550 - 557. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-C. Lee, T.-H. Lee, S. Avraham, and H. K. Avraham Involvement of the Chemokine Receptor CXCR4 and Its Ligand Stromal Cell-Derived Factor 1{alpha} in Breast Cancer Cell Migration Through Human Brain Microvascular Endothelial Cells Mol. Cancer Res., June 1, 2004; 2(6): 327 - 338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Dwinell, H. Ogawa, K. E. Barrett, and M. F. Kagnoff SDF-1/CXCL12 regulates cAMP production and ion transport in intestinal epithelial cells via CXCR4 Am J Physiol Gastrointest Liver Physiol, May 1, 2004; 286(5): G844 - G850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tavor, I. Petit, S. Porozov, A. Avigdor, A. Dar, L. Leider-Trejo, N. Shemtov, V. Deutsch, E. Naparstek, A. Nagler, et al. CXCR4 Regulates Migration and Development of Human Acute Myelogenous Leukemia Stem Cells in Transplanted NOD/SCID Mice Cancer Res., April 15, 2004; 64(8): 2817 - 2824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kahn, T. Byk, L. Jansson-Sjostrand, I. Petit, S. Shivtiel, A. Nagler, I. Hardan, V. Deutsch, Z. Gazit, D. Gazit, et al. Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation Blood, April 15, 2004; 103(8): 2942 - 2949. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. G. Kayali, K. Van Gunst, I. L. Campbell, A. Stotland, M. Kritzik, G. Liu, M. Flodstrom-Tullberg, Y.-Q. Zhang, and N. Sarvetnick The stromal cell-derived factor-1{alpha}/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas J. Cell Biol., November 24, 2003; 163(4): 859 - 869. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Laakko and R. L. Juliano Adhesion Regulation of Stromal Cell-derived Factor-1 Activation of ERK in Lymphocytes by Phosphatases J. Biol. Chem., August 22, 2003; 278(34): 31621 - 31628. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tarte, F. Zhan, J. De Vos, B. Klein, and J. Shaughnessy Jr Gene expression profiling of plasma cells and plasmablasts: toward a better understanding of the late stages of B-cell differentiation Blood, July 15, 2003; 102(2): 592 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. S. Zeelenberg, L. Ruuls-Van Stalle, and E. Roos The Chemokine Receptor CXCR4 Is Required for Outgrowth of Colon Carcinoma Micrometastases Cancer Res., July 1, 2003; 63(13): 3833 - 3839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Giron-Michel, A. Caignard, M. Fogli, D. Brouty-Boye, D. Briard, M. van Dijk, R. Meazza, S. Ferrini, C. Lebousse-Kerdiles, D. Clay, et al. Differential STAT3, STAT5, and NF-{kappa}B activation in human hematopoietic progenitors by endogenous interleukin-15: implications in the expression of functional molecules Blood, July 1, 2003; 102(1): 109 - 117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Papayannopoulou, G. V. Priestley, H. Bonig, and B. Nakamoto The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization Blood, June 15, 2003; 101(12): 4739 - 4747. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Broxmeyer, L. Kohli, C. H. Kim, Y. Lee, C. Mantel, S. Cooper, G. Hangoc, M. Shaheen, X. Li, and D. W. Clapp Stromal cell-derived factor-1/CXCL12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and G{alpha}i proteins and enhances engraftment of competitive, repopulating stem cells J. Leukoc. Biol., May 1, 2003; 73(5): 630 - 638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yahata, K. Ando, T. Sato, H. Miyatake, Y. Nakamura, Y. Muguruma, S. Kato, and T. Hotta A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow Blood, April 15, 2003; 101(8): 2905 - 2913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Herodin, P. Bourin, J.-F. Mayol, J.-J. Lataillade, and M. Drouet Short-term injection of antiapoptotic cytokine combinations soon after lethal gamma -irradiation promotes survival Blood, April 1, 2003; 101(7): 2609 - 2616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Hwang, J. H. Hwang, H. K. Chung, D. W. Kim, E. S. Hwang, J. M. Suh, H. Kim, K.-H. You, O-Y. Kwon, H. K. Ro, et al. CXC Chemokine Receptor 4 Expression and Function in Human Anaplastic Thyroid Cancer Cells J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 408 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Broxmeyer, S. Cooper, L. Kohli, G. Hangoc, Y. Lee, C. Mantel, D. W. Clapp, and C. H. Kim Transgenic Expression of Stromal Cell-Derived Factor-1/CXC Chemokine Ligand 12 Enhances Myeloid Progenitor Cell Survival/Antiapoptosis In Vitro in Response to Growth Factor Withdrawal and Enhances Myelopoiesis In Vivo J. Immunol., January 1, 2003; 170(1): 421 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Cottler-Fox, T. Lapidot, I. Petit, O. Kollet, J. F. DiPersio, D. Link, and S. Devine Stem Cell Mobilization Hematology, January 1, 2003; 2003(1): 419 - 437. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Murakami, W. Maki, A. R. Cardones, H. Fang, A. Tun Kyi, F. O. Nestle, and S. T. Hwang Expression of CXC Chemokine Receptor-4 Enhances the Pulmonary Metastatic Potential of Murine B16 Melanoma Cells Cancer Res., December 1, 2002; 62(24): 7328 - 7334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Kollet, I. Petit, J. Kahn, S. Samira, A. Dar, A. Peled, V. Deutsch, M. Gunetti, W. Piacibello, A. Nagler, et al. Human CD34+CXCR4- sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation Blood, September 26, 2002; 100(8): 2778 - 2786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fukuda, R. G. Foster, S. B. Porter, and L. M. Pelus The antiapoptosis protein survivin is associated with cell cycle entry of normal cord blood CD34+ cells and modulates cell cycle and proliferation of mouse hematopoietic progenitor cells Blood, September 18, 2002; 100(7): 2463 - 2471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ptasznik, E. Urbanowska, S. Chinta, M. A. Costa, B. A. Katz, M. A. Stanislaus, G. Demir, D. Linnekin, Z. K. Pan, and A. M. Gewirtz Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells J. Exp. Med., September 2, 2002; 196(5): 667 - 678. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
) or after incubation on a plastic
support (Inc+, 
) were incubated (1 × 105
cells/mL) for 24 hours under apoptosis-inducing culture conditions
(serum- and cytokine-free medium [IMDM]) in presence or absence of
SDF-1 (0.05 and 0.5 ng/mL) and processed for apoptosis assessment by
using the annexin-V apoptosis detection kit as indicated in
"Materials and methods." The role of endogenous SDF-1 was evaluated
by adding an anti-SDF-1 neutralizing antibody (10 ng/mL) to the
culture. Involvement of PI3-K/AKT and MAP kinase pathways was
evaluated by using LY294002- and PD98059-specific inhibitors at
concentrations varying from 10 to 100 µM. Data from 3 to 5 independent experiments are presented as mean percentage of
annexin-V+ cells ± SD. The antiapoptotic effect of
exogenous SDF-1 is shown in conditions 
