Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Desplat, V.
Right arrow Articles by Ivanovic, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Desplat, V.
Right arrow Articles by Ivanovic, Z.

Stem Cells 2002;20:347-354 www.StemCells.com
© 2002 AlphaMed Press

Hypoxia Modifies Proliferation and Differentiation of CD34+ CML Cells

Vanessa Desplata, Jean-Luc Faucherb, François Xavier Mahona, Persio Dello Sbarbac, Vincent Pralorand, Zoran Ivanovicd

a Laboratoire de Greffe de Moelle, Université Bordeaux 2, Bordeaux, France;
b Laboratoire d'Hématologie, CHU de Limoges, Limoges, France;
c Dipartimento di Patologia e Oncologia Sperimentali, Universita di Firenze, Firenze, Italia;
d Laboratoire Universitaire d'Hématologie, Université Bordeaux 2, Bordeaux, France

Key Words. Hypoxia • CML cells • CD34+ cells • Differentiation • PAF-R

Zoran Ivanovic, Ph.D., Laboratoire Universitaire d'Hématologie, 146 rue Léo Saignat, 33076 Bordeaux cedex, France. Telephone: 33-05-5757-1611; Fax: 33-05-5651-4218; e-mail: Zoran.Ivanovic{at}hemato.u-bordeaux2.fr


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously showed that hypoxia (1% O2) favors the self-renewal of murine and human normal hematopoietic stem cells. This study represents the first attempt to characterize the effects of hypoxia on the maintenance of chronic myeloid leukemia (CML) progenitors. CD34+ cells isolated from apheresis products of CML patients were incubated in hypoxia (1% O2) and normoxia (20% O2). After 8 days of culture, their proliferation, capacity for colony-forming-cell (CFC) generation in secondary cultures (pre-CFC), and phenotype (CD34 and platelet-activating factor receptor [PAF-R]) were compared with those of normal cells, and tyrosine phosphorylation in CML cells was measured. Hypoxia inhibits the proliferation of CD34+ cells and preserves the pre-CFC capacity and cell-surface CD34 expression of CML cells better than normoxia. The PAF-R expression, which was absent on freshly isolated cells, was detected at the cell surface in both populations after 8 days of culture, but with a lower percentage of positive cells in CML cell cultures. Incubation in hypoxia suppressed the PAF-R expression of normal cells and increased it in CML cells, resulting in a similar expression in the two populations. These effects could be linked to inhibition by hypoxia of the tyrosine hyperphosphorylation of cellular proteins, a major hallmark of CML cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Severe hypoxia (0.9%-1% O2) has been shown to favor the self-renewal of murine and human hematopoietic stem cells [1, 2]. Hypoxia appears, indeed, to maintain stem cells in a state where a limited proliferation is allowed, but not its coupling to differentiation toward committed progenitors and to the consequent clonal expansion. This confirms hypoxia as a key feature of the "stem cell niches" in vivo [3], where the balance between differentiation and self-renewal is in favor of the latter. This phenomenon is of major potential interest for the in vitro maintenance and expansion of hematopoietic stem cells to be used for bone marrow transplantation.

Early hematopoiesis is regulated by cytokines, but lipid mediators such as platelet-activating factor (PAF), a potent inflammatory mediator, could also play a role in the maturation of normal CD34+ cells [4]. PAF acts through a specific transmembrane receptor (PAF-R) that belongs to the superfamily of G-protein-coupled receptors [5]. The PAF-R gene produces two different species of mRNA (i.e., transcripts 1 and 2) [6]. Their transcription is directed by two distinct and tissue-specific promoters that are each translated in a unique transmembrane PAF-R. Indeed, PAF-R transcripts 1 and 2 are found together in several organs, while circulating leukocytes express only the PAF-R transcript 1 [6]. We previously have shown that the PAF-R, which is absent from the cell surface of freshly isolated normal blood CD34+ progenitors, appears during the course of differentiation in culture [7].

Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy characterized by a reciprocal translocation between chromosomes 9 and 22 that creates a bcr-abl hybrid gene encoding for a fusion protein with deregulated tyrosine kinase activity [8]. This oncoprotein induces abnormal proliferation, inhibition of apoptosis, tyrosine hyperphosphorylation of a number of proteins, and altered adhesion of CD34+ cells cultured at 20% O2 [9]. The effects of the BCR-ABL oncoprotein on the expression of surface PAF-R in CML CD34+ cells in the course of their differentiation, as well as the influence of hypoxia on their proliferation and differentiation, remain unknown. These issues were investigated here by comparing apheresis-collected CD34+ cells from CML patients with those from lymphoma patients in complete remission (considered as control cells).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Cell Cultures
Cells were recovered from frozen vials of apheresis products collected for autologous transplantation from CML patients at the time of diagnosis or from lymphoma patients in complete remission, as previously reported [3, 7]. CD34+ cells were isolated by immunomagnetic cell sorting (MiniMacs; Miltenyi Biotec; Paris, France; http://www.miltenyibiotec.com), resuspended in Iscove's-modified Dulbecco's medium (IMDM) containing antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; GIBCO; Cergy Pontoise, France; http://www.lifetech.com), and supplemented with 10% fetal bovine serum (FBS; GIBCO) and 10% IMDM conditioned by cells of the 5637 human bladder carcinoma cell line as the source of hemopoietic growth factors [7]. Purified CD34+ cells (>85%) were incubated for 8 days in primary liquid culture (LC1) (3 x 104 cells/ml) at 37°C in a fully humidified atmosphere, either in normoxia (20% O2) in a CO2-regulated incubator (5% CO2, 95% air) (Jouan; Saint-Nazaire, France; http://www.jouan.com) or in hypoxia (1% O2) in hermetic incubator chambers (Billups-Rothenberg; Del Mar, CA; http://www.brincubator.com) initially flushed for 45 minutes with a preformed gas mixture (1% O2, 5% CO2, 94% N2). In some experiments, the CD34+ CML cells were incubated for 24 hours at 20% or 1% O2 with or without the inhibitor of Bcr-Abl tyrosine kinase, STI571 (2 or 5 µg/ml) (Novartis; Basel, Switzerland; http://www.pharma.novartis.com). Viable cells were then counted and used for flow cytometry. In some experiments, the colony forming cells (CFCs, namely: colony-forming units-granulocyte-macrophage + BFU-E + CFU-Mix) present in the cell suspension at time zero and after 8 days of LC1 were detected by culturing cells in semisolid methylcellulose-based culture medium (Stem-{alpha} ID; Stem-{alpha}, Saint-Clémént-les-Places, France) for 14 days at 20% O2. In order to detect, in LC1, the primitive progenitors (pre-CFCs) capable of generating clonogenic progenitors in secondary liquid cultures (LC2), cells were replated in cytokine-supplemented (interleukin-1 [IL-1], IL-3, IL-6, stem cell factor [SCF], GM-CSF, G-CSF, and FLT3 ligand) serum-free AG medium (Stem-{alpha}) and incubated for 3 weeks with a weekly demi-depopulation and addition of fresh medium. Cells from LC2 were plated in methylcellulose to detect their CFC capacity by means of the Stem-{alpha} ID kit.

The 5637 cells, used as a positive control for the expression of PAF-R transcripts 1 and 2, were grown in RPMI 10% FBS with antibiotics at 37°C in 5% CO2 in air.

RT-PCR Analysis of PAF-R Transcripts
Total RNA extracted from blood CD34+ cells was reverse-transcribed (RT) as previously described [10]. The cDNA was amplified by polymerase chain reaction (PCR) as follows: 1 minute at 94°C, 1 minute at 56°C, 1.5 minutes at 72°C for 35 cycles. The PCR reaction was performed in a total volume of 50 µl containing 75 mM Tris-HCL (pH 9.0), 20 mM (NH4)2SO4, 0.01% Tween 20, 1.5 mM MgCl2, 0.4 mM of each dNTP, 0.3 µM sense primer, 0.6 µM antisense primer (0.3 µM for ß-actin), and 0.1 U/ml DNA polymerase (Eurogentec; Seraing, Belgium; http://www.eurogentec.com). The human PAF-R transcript 1 sense primer was 5'-GACAGCATAGAGGCT GAGGC-3', the transcript 2 sense primer was 5'-CCTGAGCTCCCCGAGAAGTCA-3', and the common antisense primer was 5'-TAGCCATTAGCAATGACCCC-3'. These primers amplify 225- and 269-bp fragments for transcripts 1 and 2, respectively (the two sense primers and the antisense primers were used in the same PCR reaction). The constitutive expression of ß-actin [10] in all samples represented a positive control of reverse transcription. As a negative control, the PCR reaction was performed with all reagents except the cDNA. PCR products were electrophoresed on a 2% agarose gel (Eurobio; Les Ulis, France; http://www.eurobio.fr) visualized by ethidium bromide staining.

Flow Cytometry

PKH26 Staining   The stable membrane fluorescent dye PKH26 (Sigma; St. Louis, MO; http://www.sigmaaldrich.com) was used to study the proliferative history of CD34+ cells by measuring, by flow cytometry, the fluorescence intensity of these cells ("0 division") and its decrease after 8 days of liquid culture, as previously described [11]. Briefly, after 5 minutes of incubation with PKH26 (2 µM), purified CD34+ cells were washed extensively and resuspended in culture medium. Simple PKH26 labeling and double PKH26/CD34 labeling were performed.

PAF-R Staining   Purified CD34+ cells at time zero and after 8 days of culture were first incubated for 30 minutes at 4°C with an anti-PAF-R mouse monoclonal antibody (Spi-Bio; Massy, France; http://www.spibio.com), washed in phosphate-buffered saline containing 5% AB serum, and then incubated with a fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (GAM) antibody (Dakopatts; Glosturp, Denmark; http://www.dako.com) for 45 minutes at 4°C in the dark (indirect labeling). After washing, the cells were incubated with a Cyanin-5-Phycoerythrin (Cy5PE)-labeled anti-CD34 monoclonal antibody (Immunotech; Marseille, France; http://www.immunotec.com) for 30 minutes at room temperature in the dark (direct labeling) before flow cytometry.

Phosphotyrosine Staining   For the detection of phosphotyrosine (P-tyr), purified CD34+ cells were stained with a PE-labeled anti-CD34 monoclonal antibody (Becton Dickinson; Le-Pont-de-Claix, France; http://www.bd.com) as described above, washed, fixed in 1% paraformaldehyde for 10 minutes at 4°C, permeabilized with 0.3% saponin, and finally incubated with an anti-P-tyr monoclonal antibody (Santa Cruz; Tebu, France) for 1 hour at 4°C. The cells were then incubated with a secondary fluorescent antibody (FITC-GAM; Becton Dickinson), washed, and analyzed on an XL flow cytometer (Beckman Coulter; Miami, FL; http://www.beckman.com).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypoxia Reduces the Numbers of Viable Cells and Cell Divisions and Maintains the Number of Pre-CFCs in CML Cultures
We previously showed that hypoxia (<1% O2) impedes the numerical increase of normal cells in vitro [2, 12, 13]. In the present study, the increase in cell number was markedly higher in CML than in normal cell day 8 primary cultures (LC1s), both incubated in normoxia (Fig. 1Go). Together, these results indicate that hypoxia, when compared with normoxia, reduced CML cell growth by 3.3-fold and normal cell growth by 1.7-fold, and, therefore, that hypoxia was twofold more effective in inhibiting the growth of CML cells than that of normal cells.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Effects of hypoxia on expansion of normal and CML cells. *p <0.05 with respect to adequate values in normoxia. 3 x 104 cells per ml of liquid culture (LC) at time zero in all conditions.

 
The intensity of PKH26 staining, reflecting the number of cell divisions, of total viable cells is shown in Figure 2Go. Over 8 days of incubation in LC1, normal cells underwent a decrease in PKH26 fluorescence with respect to time zero that was not affected by the oxygen percentage in the atmosphere (Fig. 2AGo). CML cells underwent a much higher number of divisions than normal cells both in normoxia and hypoxia (Fig. 2BGo), but this number was markedly lower in hypoxia than in normoxia. The high CD34+ fluorescence intensity of normal cells in day 8 LC1s was unaffected by the oxygen tension in the incubation atmosphere (Fig. 3Go), in keeping with the results shown in Figure 2AGo. In CML cells, hypoxia maintained CD34+ cells significantly better than normoxia. The loss of CD34+ CML cells in normoxia was apparently associated with a higher number of divisions than in hypoxia, as shown in Figure 2BGo.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 2. Effects of hypoxia on the proliferative history of normal and CML cells. Viable CD34+ cells were labeled with PKH26 at time zero (T0) and analyzed by flow cytometry after 8 days of culture in normoxia or hypoxia (LC1). A) Normal cells. B) CML cells. On the basis of the mean PKH26 fluorescence intensity measured at T0, corresponding to undivided cells, fluorescence intensity of day-8 cultures allowed for the assessment of the number of cell divisions.

 


View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Effects of hypoxia on CD34 expression with respect to the number of divisions of normal and CML cells. CD34+ cells, labeled with PKH26 at time zero, were labeled with anti-CD34 antibodies (CD34-CYS) after 8 days of LC1 in normoxia or hypoxia, and finally analyzed by flow cytometry. One representative experiment out of three is shown.

 
The number of CFCs persisting in day 8 CML cultures, as detected in clonal assays established directly at the end of the LC1, was markedly lower (50%) in hypoxia than in normoxia (Fig. 4AGo), a result well in keeping with our previous observations obtained with normal human [1] as well as murine [12] hematopoietic cells. However, when clonal assays were established with LC2-incubated cells, the number of CFCs detectable in an LC2 derived from an LC1 incubated in hypoxia was similar to that of normoxia at day 7 after replating (day 15 from time zero) and higher at day 22 after replating (day 30 from time zero), indicating that the maintenance of CML pre-CFCs in LC1 was better ensured in hypoxia than in normoxia. These results are similar to those obtained with normal human pre-CFCs (Fig. 4BGo).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Effects of hypoxia on the number of clonogenic progenitors (CFCs) and on pre-CFC activity in CML and normal cell cultures. After 8 days of LC1 in normoxia or hypoxia (day 8), CML (A) or normal (B) cells were washed and incubated in normoxia (LC2) for 1 (day 15) or 3 (day 30) additional weeks. Aliquots of cell cultures were harvested at days 0, 7, and 22 of LC2 and replated in semisolid cultures for a further 14 days to determine the number of CFCs in LC2. One representative experiment out of three is shown.

 
Hypoxia Affects the Expression of PAF-R in CML Cells
We previously reported that PAF-R is a differentiation marker of normal CD34+ cells [7]. We, therefore, explored the effects of hypoxia on CML cell differentiation by determining PAF-R expression in purified CD34+ CML cells. In contrast to the 5637 human bladder carcinoma cell line, used as a positive control, which expresses both PAF-R transcripts 1 and 2 (as detected by RT-PCR), the CML cells, as well as the normal CD34+ cells, were found to express only the PAF-R transcript 1 (Fig. 5AGo). At time zero, PAF-R, as detected by flow cytometry, was not expressed on either normal (as previously reported by Desplat et al. [7]) or CML CD34+ cells (data not shown). Figure 5BGo shows the results of labeling with anti-CD34 and anti-PAF-R antibodies of normal and CML cells from day 8 LC1s incubated in hypoxia and normoxia. In normoxia, PAF-R was expressed by 67% of normal and only 5% of CML cells. Incubation in hypoxia, when compared with normoxia, resulted in a loss of PAF-R expression (23% PAF-R+ cells) in normal cells and an increase (15% PAF-R+ cells) in CML cells. Thus, hypoxia brought percentages of normal and CML cells expressing PAF-R to comparable values.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 5. Effects of hypoxia on the expression of PAF-R in normal and CML cells. A) RT-PCR products were electrophoresed in a 2% agarose gel with ettidium bromide (BET) to determine PAF-R expression in CML and other cell populations. Lane 1 = 100 bp DNA size ladder; lane 2 = bladder carcinoma 5637 cells (positive control); lane 3 = normal CD34+ cells; lanes 4 and 5 = CD34+ CML cells. Sizes of PCR products are indicated by arrows. B) Cells from day 8 LC1s incubated in normoxia or hypoxia were labeled with anti-PAF-R (PAF-R FITC) and anti-CD34 (34-CYS) antibodies and analyzed by flow cytometry. The percentages represent 67% and 5% of PAF-R+/CD34+ cells in normal and CML cell cultures, respectively, at 20% O2, and 23% and 15% at 1% O2. One representative experiment out of three is shown.

 
Hypoxia Reduces Tyrosine Hyperphosphorylation in CD34+ CML Cells
The above-listed effects of hypoxia on CML cells, especially the reduction of proliferation and the enhancement of PAF-R expression (Figs. 2, 3, and 5BGoGoGo), seem to consistently indicate that hypoxia suppressed the differences between normal and CML cells, evidenced after 8 days of incubation in normoxia. This suggests that hypoxia may suppress the BCR-ABL-dependent leukemogenic signals. Tyrosine hyperphosphorylation of cellular proteins is a characteristic of CML CD34+ cells and the consequence of the constitutively upregulated tyrosine kinase activity of BCR-ABL. Tyrosine hyperphosphorylation in CML cells has already been shown by immunoblotting [14]. Here, we determined the effects of hypoxia on tyrosine phosphorylation in our system by flow cytometry. As shown in Figure 6AGo, incubation in hypoxia for 24 hours (as well as for 8 days, Fig. 6BGo) resulted in a markedly lower tyrosine hyperphosphorylation of CML CD34+ cells in comparison with that at 20% O2. This reduction by hypoxia was comparable with that obtained with 5 µg/ml of STI571 (Fig. 6AGo, 20% O2), while a lower dose of STI571 (2 µg/ml) was much less active (not shown). The addition of STI571 did not affect tyrosine phosphorylation in hypoxia (Fig. 6AGo, 1% O2).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 6. Effects of hypoxia on protein tyrosine phosphorylation in CML cells. CD34+ CML cells from day 1 (A) and day 8 (B) cultures incubated in normoxia or hypoxia were labeled with anti-CD34 antibody, fixed, and permeabilized before incubation with anti-P-tyr antibodies. The P-tyr fluorescence intensity of CD34+ cells was then measured by flow cytometry. Top panels, normoxia; bottom panels, hypoxia. Values represent the percentages of P-tyr positive cells with respect to the isotypic controls. One representative experiment out of three is shown.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study represents the first attempt to characterize the effects of hypoxia on the maintenance of leukemic progenitors, from CML in particular. It is, therefore, of great interest to compare the results presented here with those previously obtained, essentially by our group, for normal cells. Several parameters related to cell growth and differentiation were evaluated. With respect to the maintenance of pre-CFCs, CML cells responded to hypoxia similarly to normal human [1] and murine [2] cells, i.e., hypoxia enhanced the maintenance of pre-CFC potential in CML cells (Fig. 4AGo). With respect to cell cycling and differentiation, we previously showed that, in hypoxia, in the presence of stem-cell-active factors, the maintenance of normal murine pre-CFCs was compatible with their proliferation [2]. Furthermore, a better maintenance of pre-CFCs in hypoxia was accompanied by an accelerated proliferation of normal CD34+ cells when mobilized peripheral blood mononuclear cells were incubated in the presence of SCF and IL-3 [1]. Here, hypoxia did not accelerate proliferation of normal CD34+ cells (Figs. 2 and 3GoGo), which probably reflects a lower (suboptimal) stimulation by the 5637-cell-conditioned medium in comparison with recombinant cytokines. On the other hand, hypoxia decreased the number of divisions and enhanced the maintenance of CD34+ CML cells, which in normoxia are more sensitive than normal cells to cytokine stimulation [15]. Thus, CML cells, but not normal cells, respond to hypoxia with a reduction in division number of total and CD34+ cells (Figs. 2 and 3GoGo) and by a relatively lower overall cell growth (Fig. 1Go). These results point to profound differences between normal and CML cells with respect to their response to hypoxia.

Normal CD34+ cells are characterized by extensive PAF-R expression during differentiation [7]. This was confirmed in the experiments reported here (Fig. 5BGo), where we found an abundant PAF-R expression at the end of LC1 in normoxia. Markedly reduced PAF-R expression in normal cells, demonstrated here in hypoxia, combined with a selection of more immature cells (Fig. 4BGo; previous data obtained with murine and human hematopoietic cells [1, 2, 12, 13, 16]) point to an inhibition of their differentiation.

In CML cells, hypoxia enhanced PAF-R expression, which is, as summarized above, a differentiation marker of normal cells. However, only a small subpopulation of CD34+ CML cells was found to express PAF-R after LC1 in normoxia. Assuming that PAF-R gene expression is normal in CML cells, at least two hypotheses can be proposed for this low membrane protein expression: A) autocrine PAF release downregulates PAF-R, and B) mechanisms constitutively active in CML cells inhibit membrane insertion of PAF-R, determining its intracellular retention, or downmodulate membrane-inserted PAF-R. Irrespective of the above, it is a fact that the response of CML cells to hypoxia was the opposite of that of normal cells: hypoxia markedly increased the percentage of PAF-R+ cells in CML cell cultures, while it decreased that in normal cells. This resulted in a rather similar expression of PAF-R in CML and normal cells in hypoxia. Thus, hypoxia restores PAF-R expression in CML cells that is defective in normoxia, probably inhibiting leukemogenic signals leading to this defective expression.

The above-described effects of hypoxia on PAF-R expression in CML cells are paralleled by those on tyrosine phosphorylation (Fig. 6Go), which is lower in hypoxia, possibly reflecting its interference with leukemogenic signaling of the Bcr-Abl fusion protein. The effect of hypoxia on tyrosine phosphorylation was comparable with that of STI571, a specific inhibitor of Bcr-Abl tyrosine kinase activity (Fig. 6Go). In this light, hypoxia would modulate the Bcr-Abl-dependent leukemic phenotype. This scenario is very well in keeping with the fact that hypoxia preserves the primitiveness of CD34+ CML cells better than normoxia (maintaining CD34 expression and pre-CFC potential [Figs. 3 and 4GoGo]), which could also be linked to a reduction of the Bcr-Abl-induced protein hyperphosphorylation. Indeed, hypoxia has been found to interfere with protein phosphorylation in several studies. Both tyrosine and serine/threonine phosphorylation seem inhibited by hypoxia [17]. In particular, hypoxia has been shown to induce transient mitogen-activated protein kinase (MAPK) activation, as well as a more sustained MAPK-specific phosphatase activity [18]. MAPK activation, a phenomenon regulated in part by tyrosine phosphorylation, is involved in the transcriptional response to hypoxia, and phosphorylation is an important way of regulating the activity of hypoxia-inducible factors [19]. These interesting phenomena have not been described before for CML cells. On this basis, it will be very interesting to test the possibility of modulating the composition of CML cell populations via incubation at low, or extremely low, oxygen tensions.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
V.D. was supported by a grant from the Ligue Nationale Contre le Cancer - Comité de l’Indre. P.D.S. was supported by grants from Ministero dell’Istruzione, dell’Università e della Ricerca (CoFin 2001) and from Associazione Italiana per la Ricerca sul Cancro (AIRC). We are grateful to the Ligue Nationale Contre le Cancer—Comités de la Corrèze et des Landes for funding our project.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ivanovic Z, Dello Sbarba PD, Trimoreau F et al. Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent. Transfusion 2000;40:1482–1488.[CrossRef][Medline]

  2. Ivanovic Z, Belloc F, Faucher J-L et al. Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34+ murine bone marrow cells. Exp Hematol 2002;30:67–73.[CrossRef][Medline]

  3. Dello Sbarba P, Cipolleschi MG, Olivotto M. Hemopoietic progenitor cells are sensitive to the cytostatic effect of pyruvate. Exp Hematol 1987;15:137–142.[Medline]

  4. Dupuis F, Gachard N, Allegraud N et al. Platelet-activating factor decreases the growth of human erythroid and myeloid CD34+ progenitors. Mediators Inflamm 1998;7:31–33.[CrossRef][Medline]

  5. Izumi T, Takano T, Bito H et al. Platelet-activating factor receptor. J Lipid Mediat Cell Signal 1995;12:429–442.[CrossRef][Medline]

  6. Mutoh H, Bito H, Minami M et al. Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor. FEBS Lett 1993;322:129–134.[CrossRef][Medline]

  7. Desplat V, Besse A, Faucher JL et al. Expression of platelet-activating factor receptor transcript-1 but not transcript-2 by human bone marrow cells. STEM CELLS 1999;17:121–124.[Abstract/Free Full Text]

  8. Lugo TG, Pendergast AM, Muller AJ et al. Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science 1990;247:1079–1082.[Abstract/Free Full Text]

  9. Deininger WNM, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood 2000;96:3343–3356.[Free Full Text]

  10. Fixe P, Rougier F, Ostyn E et al. Spontaneous and inducible production of macrophage colony-stimulating factor by human bone marrow stromal cells. Eur Cytokine Netw 1997;8:91–95.[Medline]

  11. Traycoff CM, Kosak ST, Grigsby S et al. Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis. Blood 1995;85:2059–2068.[Abstract/Free Full Text]

  12. Ivanovic Z, Bartolozzi B, Bernabei PA et al. Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating ability together with the expansion of committed progenitors. Br J Haematol 2000;108:424–429.[CrossRef][Medline]

  13. Cipolleschi MG, Rovida E, Ivanovic Z et al. The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability. Leukemia 2000;14:735–739.[CrossRef][Medline]

  14. Mahon FX, Deininger MW, Schultheis B et al. Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 2000;96:1070–1079.[Abstract/Free Full Text]

  15. Bhatia R, Munthe HA, Williams AD et al. Chronic myelogenous leukemia primitive hematopoietic progenitors demonstrate increased sensitivity to growth factor-induced proliferation and maturation. Exp Hematol 2000;28:1401–1412.[CrossRef][Medline]

  16. Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hematopoietic stem cells. Blood 1993;82:2031–2037.[Abstract/Free Full Text]

  17. Braunton JL, Wong V, Wang W et al. Reduction of tyrosine kinase activity and protein tyrosine dephosphorylation by anoxic stimulation in vitro. Neuroscience 1998;82:161–170.[Medline]

  18. Laderoute K, Mendonca HL, Calaoagan JM et al. Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments. A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity. J Biol Chem 1999;274:12890–12897.[Abstract/Free Full Text]

  19. Minet E, Arnould T, Michel G et al. ERK activation upon hypoxia: involvement in HIF-1 activation. FEBS Lett 2000;468:53–58.[CrossRef][Medline]

Received March 18, 2002; accepted for publication May 10, 2002.



This article has been cited by other articles:


Home page
 Stem CellsHome page
S. Giuntoli, E. Rovida, A. Gozzini, V. Barbetti, M. G. Cipolleschi, M. Olivotto, and P. Dello Sbarba
Severe Hypoxia Defines Heterogeneity and Selects Highly Immature Progenitors Within Clonal Erythroleukemia Cells
Stem Cells, May 1, 2007; 25(5): 1119 - 1125.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
W. Liu, M. Guo, Y.-B. Xu, D. Li, Z.-N. Zhou, Y.-L. Wu, Z. Chen, S. C. Kogan, and G.-Q. Chen
Induction of tumor arrest and differentiation with prolonged survival by intermittent hypoxia in a mouse model of acute myeloid leukemia
Blood, January 15, 2006; 107(2): 698 - 707.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
Z. Ivanovic, F. Hermitte, P. B. de la Grange, B. Dazey, F. Belloc, F. Lacombe, G. Vezon, and V. Praloran
Simultaneous Maintenance of Human Cord Blood SCID-Repopulating Cells and Expansion of Committed Progenitors at Low O2 Concentration (3%)
Stem Cells, September 1, 2004; 22(5): 716 - 724.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. J. Davie, J. T. Crossno Jr., M. G. Frid, S. E. Hofmeister, J. T. Reeves, D. M. Hyde, T. C. Carpenter, J. A. Brunetti, I. K. McNiece, and K. R. Stenmark
Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells
Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L668 - L678.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Desplat, V.
Right arrow Articles by Ivanovic, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Desplat, V.
Right arrow Articles by Ivanovic, Z.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS