hematopoietic progenitor cells stimulating factor ...

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1994 84: 2940-2945

All-trans retinoic acid directly inhibits granulocyte colonystimulating factor-induced proliferation of CD34+ human hematopoietic progenitor cells EB Smeland, L Rusten, SE Jacobsen, B Skrede, R Blomhoff, MY Wang, S Funderud, G Kvalheim and HK Blomhoff

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All-Trans Retinoic Acid Directly Inhibits Granulocyte Colony-Stimulating Factor-Induced Proliferation of CD34+ Human Hematopoietic Progenitor Cells By Erlend B. Smeland, Leiv Rusten, Sten E.W. Jacobsen, Bjsrn Skrede, Rune Blomhoff, Meng Yu Wang, Steinar Funderud, Gunnar Kvalheim, and Heidi Kiil Blomhoff In this study we examine the effects of retinoids on purified mediated. Retinoids also significantly inhibited G-CSF-inCD34+ human hematopoietic progenitor cells. All-trans reti- duced colonyformation in semisolid medium,with 88% inhinoicacid inhibitedgranulocytecolony-stimulatingfactor bition observed at a concentrationof retinoicacid of 1 pmoll (G-CSF)-induced proliferation of CD34+ cells in short-term L. However, we did not observe any effects of retinoic acid liquid cultures in a dose-dependent fashion with maximal on G-CSF-induced differentiation as assessed by morphol1 ogy and flowcytometry. Similar to previous findings using inhibition of 72% at aconcentrationofretinoicacidof pmol/L. Although no significant effects were observed on total bone marrow mononuclear cells, we observed a stimuor granulocyte-macrophage CSF (GM-CSFI- interleukin-3lation of GM-CSF-induced colony formation after 14 days. stem cell factor (SCF)-induced proliferation, the combinaWe also observed a stimulatory effect of low doses of retiwere all inhibited. tions of G-CSF and each of these cytokines noic acid(30 nmol/L) on blast-cell colonyformation on stroMoreover, retinol(3 pmol/L) and chylomicron remnant retimal cell layers. Taken together, the data indicate that vitanyl esters (0.1 pmol/L) in concentrations normally found in min A present in human plasma has inhibitory as well as human plasma alsohad inhibitory effects. Single-cell experi- stimulatory effects on myelopoiesis. 0 1994 by The American Society of Hematology. ments showedthat the effects of retinoic acidwere directly

V

ITAMIN A plays an important role in embryogenesis and in the regulation of growth and differentiation in various cell types."3 Retinol (ROH) is the major retinoid in plasma, and is transported from liver to extrahepatic target cells as retinol bound to retinol-binding protein.' Newly absorbed retinol is also present as retinyl esters in chylomicrons and their remnants (CMR). After uptake into target cells, retinol can be oxidized to retinoic acid (RA). Retinoids have been found to exert profound effects on the growth and maturation of hematopoietic cell^.^.^ Thus, retinoids induce monocytic and/or granulocytic differentiation in various myeloid cell line^.^.^ As RA promotes dramatic inhibition of proliferation along with induced differentiation in acute promyelocytic leukemia both in vitro and in V ~ V O , ~all-trans ~ " ~ RA is currently used for therapy in this Interestingly, it has recently been shown that this type of leukemia is characterized by a translocation [t(15;17)] that involves the gene encoding the RA receptorCY (RAR-a).I6.I7 Conflicting data exist regarding the effects of retinoids on normal hematopoiesis in vitro, although RA has generally been found to stimulate the growth of CFTJ-GMS.~*'"~~ Thus, RA has been suggested to both inhibitz3," and to stimulate" granulopoiesis. Because vitamin A can promote a mixture From the Department of Immunology and the Department of Tu-

mor Biology, Institute for Cancer Research, The Norwegian Radium Hospital; andtheInstitute for Nutrition Research, University of Oslo, Oslo, Norway. Submitted September 20, 1993; accepted June 29, 1994. Supported by the Norwegian Cancer Society and the Norwegian Royal Council for Industrial and Scient@ Research. Address reprint requests to Erlend B. Smeland, MD, PhD, Department of Immunology, Institute for Cancer Research, Montebello, N0310 Oslo, Norway. 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. 0 1994 by The American Society of Hematology. 0006-4971/94/8409-02$3.00/0 2940

of direct and indirect effects in heterogeneous cell populations like total bone marrow (BM) mononuclear cells, we wanted to examine the effects of retinoids in a more restricted population of purified hematopoietic progenitor cells. In humans, most committed progenitor cells capable of forming colonies in semisolid culture assays have been found to reside in the CD34+ subpopulation of BM The CD34 population, which represents 1% to 4% ofBM mononuclear cells, also includes more primitive hematopoietic progenitor cells.26~29-32 Recent data have shown that enriched CD34+ marrow cells can reconstitute hematopoiesis in vivo in humans and nonhuman p r i r n a t e ~ .In ~ ~this . ~ ~study we used immunomagnetically purified CD34+ cells to examine the effects of retinoids on enriched populations of hematopoietic progenitor cells. We found that retinoids potently inhibited G-CSF-induced proliferation and colony formation ofCD34' cells. The effect was directly mediated as similar inhibition was observed in single-cell cultures. In addition, physiologic concentrations of retinol and CMR retinyl esters were found to exert similar effects. MATERIALS AND METHODS

Growth factorsand reagents. Purified recombinant human (rHu) granulocyte colony-stimulating factor (G-CSF) and rHu stem cell factor (SCF) were generously supplied by Dr Ian K. McNiece (Amgen Inc. Thousand Oaks, CA). rHu granulocyte-macrophage CSF (GM-CSF) and rHu interleukin-3 (L-3) were generously provided by Dr Steven Gillis (Immunex Corp, Seattle, WA). Unless otherwise indicated, all growth factors were used at predetermined optimal concentrations: rHuG-CSF (20 ng/mL), rHuGM-CSF (50 ng/mL), rHuIL-3 (20 ng/mL), and rHuSCF (50 ng/mL). Retinol (Sigma, St Louis, MO) and RA (Sigma) were solubilized in ethanol and kept dark bottled at -20°C. Chylomicron remnants were prepared as described previously." Cells. BM was obtained by iliac crest aspiration from normal adult volunteers with informed consent and the approval of the Ethics Committee of The Norwegian Radium Hospital. BM cells were collected in syringes containing preservative-free heparin (5,000 I U / 10 mL BM). Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (Lymphoprep; Nyegaard, Oslo, Norway). Positive selection of CD34' cells was performed as de~cribed.2~ Briefly, BM mononuclear cells were rosetted with Dynabeads M450 Blood, Vol84, No 9 (November l),1994: pp 2940-2945

From bloodjournal.hematologylibrary.org by guest on July 13, 2011. For personal use only. 2941

RETINOIDS INHIBIT G-CSF-INDUCEDPROLIFERATION

(Dynal, Oslo, Norway) directly coated with the anti-CD34 monoclonal antibody (MoAb) BI-3C5 for 45 minutes at 4°C on an apparatus that provided both gentle tilting and rotation (1 to 2 X IO7 BM mononuclear cells/mL; bead-to-cell ratio l:]). Rosetted cells were attracted to a samarium cobalt magnet and nonrosetting cells (CD34cells) were removed by pipetting, and rosetted cells were washed ( ~ 7 ) Detachment . of beads from positively selected cells was performed by incubation with anti-Fab antiserum (DETACHaBEAD, Dynal) at a concentration of 35 mg/mL in 0.3 mL for 45 minutes at room temperature. Beads were removed by attachment to a magnet ( ~ 2 and ) isolated cells were washed in medium (X2) and counted. The punty of cells isolated by this method was reproducibly greater than 90% CD34+ cells, as determined by flow cytometric analyses.*' Flow cyrometry. Direct immunofluorescence was performed according to standard techniques with phycoerythrin (PE)-labeled HPCA-2 (CD34; Becton Dickinson, San Jose, CA). An isotypematched, PE-conjugated irrelevant MoAb served as control (Serotec, Oxford, UK). To block unspecific binding via Fc receptors, aggregated human IgG (Dakopatts, Copenhagen, Denmark) was included at a concentration of 100 pg/mL. Flow cytometric analyses were performed on a FACScan flow cytometer (Becton Dickinson) equipped with an argon-ion laser tuned at 488 nm. Data acquisition and analysis were performed using Lysis I1 software (Becton Dickinson). Cell cultures in liquid medium. Cells were grown in triplicates (5 x IO3 cells/0.2 mL) in round-bottomed mictrotiter wells (Costar, Cambridge, MA) in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10%fetal calf serum (FCS), penicillin, and streptomycin and different cytokines in the absence or presence of retinoids. Cells were pulsed with 1 pCi of 3H-thymidine (The Radiochemical Centre, Amersham, UK) for the last 24 hours of a 6-day incubation, harvested on a Skatron cell harvester (Lier, Norway), and counted on a liquid scintillation counter. Cell phenofyping. Morphologic analysis of stimulated cells was performed by differential counts on May-Griinwald-Giemsa-stained cytocentrifuge cell preparations. Colony formation in semisolid media. Cells (1 X IO3 CD34' cells per tissue-culture grade 35-mm Petri dish) were plated in a volume of 1 mL IMDM (GIBCO, Paisley, UK) supplemented with 20% FCS (Sera-lab, Sussex, UK), 1.2% methylcellulose, 5 X mol/L 2-mercaptoethanol, 300 m g L L-glutamine, 66 mg/L penicillin, 1 0 0 mg/L streptomycin, and recombinant human growth factors as indicated. Colonies (>40 cells) were counted using an inverted microscope after 1 or 2 weeks of incubation at 37°C and 5% CO2 in air. Single-cell proliferation assay. CD34' cells were seeded in Terasaki plates (Greiner, Frickenhausen, Germany) at a concentration of 1 cell per well (300 wells per group) in 20 pL IMDM containing 20% FCS, extra L-glutamine, penicillin, and streptomycin. Wells were scored for proliferation (> 10 cells) after 2 weeks of incubation at 37°C and 5% CO2 in air. Blast colony formation on stromal feeder layers. The blast colony cultures were set up essentially as described by Gordon et al.36 First, feeder layers of stromal cells were prepared by plating 2 X IO' BM mononuclear cells in 10 mL of IMDM medium supplemollL mented with 10% FCS, 10% horse serum, and 5 X hydrocortisone sodium succinate (Sigma; No. H-4881) in 50 mL culture tissue flasks. The stromal cultures were fed weekly by complete replacement of the medium, as described above, until a confluent layer of stromal cells covered the base of each dish. In the second step, CD34' cells (2 X IO4 cells) in 10 m1 of RA containing medium were added to the irradiated stromal layers. After 2 hours of incubation at 37°C in 5% humified CO2 in air, unattached cells were removed, and the medium was replaced by 10 mL of 0.3% agar in IMDM medium containing RA and 10% FCS. The number of colo-

T

T

I

Fig 1. Effects of all-trans RA onproliferation of CD34+cellsin short-term liquid cultures. Cells were cultured in triplicate 15 x lo3 cells/0.2 mL) in microtiter wells with the indicated stimuli in the absence (0)or presence of 30 nmollL (81and 1 pmol/L 1. of RA. Cells were pulsedwith ['HI-thymidine from day 5 to day 6. Data are presented as mean (SEMI of six experiments. 'Statistical dgnificance ( P < ,051 using the paired Wilcoxon test.

nies (>20 cells) with blast cell morphology was counted after 1 week of culture. RESULTS

Retinoids inhibit G-CSF-induced [3H]-thymidineincorporation of CD34+ cells in short-term liquid cultures. BM mononuclear cells represent a very heterogeneous cell population allowing a mixture of direct and indirect effects when assessing the effects of biologically active substances. Previous studies on the role of retinoids in hematopoiesis have mostly investigated the effects on unfractionated BM mononuclear cells. Because most progenitors in human BM express the CD34 antigen, we took advantage of a recently developed method for isolation of humanCD34' cellsz9 using immunomagnetic beads andan anti-Fab antiserum (DETACHaBEAD) to study the effects of retinoids on a restricted population of immature hematopoietic cells. We first studied the effects of all-trans RA on CSF- and SCF-induced proliferation of CD34+ cells in liquid cultures (Fig l). All-trans RA alone had no effects on the [3H]thymidine incorporation of CD34+ cells. G-CSF-induced proliferation was inhibited in a dose-dependent waywith maximal inhibition of 72% at 1 pmol/L of all-trans RA and significant effects were also observed at physiologic levels (30 nmoliL) of all-trans RA (P< .05). In contrast, all-trans RA did not significantly influence the proliferation induced by IL-3, GM-CSF, or SCF. However, when GM-CSF, IL3, or SCF were combined with G-CSF, all-trans RA potently inhibited r3H]-thymidine incorporation (Fig 2). The RA-mediated inhibition was evident from the onset of the cultures, and cell counting at various time points up to day 14 after stimulation confirmed the inhibitory effects of all-trans RA on G-CSF-induced proliferation of CD34' cells (data not shown). Furthermore, single CD34+ cell proliferation assays showed that the inhibitory effects of all-trans RA on G-CSFinduced proliferation of CD34+ progenitors were directly mediated (Table 1). Effect of retinol and retinyl esters bound to chylomicron remnantparticles (CMR). Although effects of all-trans RA on hematopoiesis have been well described, generally retinol


2

SMELAND ET AL

100

,

I

Table 2. Effects of Retinoids and CMR Retinyl Ester on DNA Synthesis of CD34+ Cells in Short-Term Liquid Cultures [ W T h v m i d i n e Incorporation (cpm)

1

L

8

l1

.-

40

.E

30

5

20

5

10

T -

O

z

Exp 1

Exp 2

Exp 1

Exp 2

1,709 1,123 1,087 1,260 233

1,421 3,222 3,436 2,751 599

2,227

1,221

5,195 2,574 2,919 2,699 1,968 6,761

36,047 21,560 15,130 16,140 22,595 30,554

50

No addition RA ( 100 nmol/L)

C

E

G-CSF

Medium

,

T

RA (30 nmol/L)

ROH (3 pmol/L) CMR-RE (0.1 pmol/L) CMR (0.1 pmollL) G-CSF+IL-3 G-CSF+GM-CSF G-CSF G-CSF+SCF

Fig 2. Effects of all-trans RA on G-CSF-induced proliferation of CD34+ cells in liquid cultures. Cells were culturedas described in the legend to Fig 1 with the indicated cytokines in the absence (0) or presence of 30 nmol/L ( W , 100 nmol/L (U), or 1 pmol/L (m) of RA. Data are presented as mean (SEMI of six experiments.

has been found to have less or no effects on hematopoietic progenitor cell^.^^".^' However, as shown in Table 2, retinol also inhibited G-CSF-induced proliferation in short-term liquid cultures, albeit to a smaller degree than all-trans RA. Moreover,wealso wanted tostudytheeffects of retinol bound to its physiologic carriers. As shownin Table 2, physiologic levels of CMR-bound retinyl esters (0.1 pmol/L) also inhibited G-CSF-induced proliferation, again indicating that thisisaphysiologiceffect of retinoids. In contrast, CMR alone did not significantly modulate G-CSF-mediated proliferationat equal concentrations, althoughCMRalone tended to inhibit proliferation of CD34+ cells at higher concentrations. Effects of retinoidson in vitro colony formation. The majority of committed colony-forming cells (CFCs) in BM reside in the CD34' In agreement the with ["HI-thymidine incorporation data, we observed a dose-dependent inhibition of G-CSF induced day7 colony formation (CFU-G) by all-rrans RA (Fig 3). Maximal inhibition of on average 87% was obtained at 1 pmol/L of all-trans RA (n = 6, P < .OS). In addition, retinol (3 ,umol/L) also significantly inhibited G-CSF-induced colony formation (Fig 3). The effects of all-trans RA were completely reversible after exposure of CD34' cells to 100 nmol/L of RA for 24hours (data not shown). This suggests that the observed inhibition was not caused by toxic effects of RA. Previous work has established that RA stimulates the formation of CFU-GM colonies.'*-*' In thepresentstudy we

5 X I O 3 CD34' cells were cultured in triplicate0.2 mL of medium in microtiter wells and pulsed with 3H-thymidine from day 5 to day 6.

found a weak stimulation of day 14 GM-CSF-induced colonygrowth(Table 3), whereas no distinct colonies were apparent on day 7 on stimulation with rHuGM-CSF alone. In addition, we observed an RA-induced stimulation of day 14CFU-GM colonyformationinresponse to different growth factor combinations in six of eight experiments, al( P = .06, though this did notreachstatisticalsignificance data not shown). In contrast, and inagreementwiththe results presented in Fig 3, G-CSF-induced colony formation was strongly inhibited at day 7 at all concentrations of alltransRA tested, whereasthe effects of RA on G-CSFinduced colony formation at day 14 were less pronounced (Table 3). Although all-trans RA preferentially inhibited GCSF-induced colony formation at day 7, we also observed significant inhibition of day 14 colony formation in 11 experiments using concentrations of all-trans RA of 1 pmoVL ( P < ,001) or 100 nmol/L ( P < .05, data not shown). The CD34' population also contains, in addition to committed progenitors like CFU-GM, moreprimitive hematopoietic progenitor cells that are not detected in standard colony assays.'x~'4Several lines of evidence indicate that cells capa-

Table 1. Effects of All-Trans RA in Single-Cell Cultures ~~

No. of Positive Wells/

300 Cells

Fig 3. Effects of retinoids on G-CSF-induced colony formation (CFU-G) of CD34+ cells. CD34+ cells were cultured for 7 days in methylcellulose cultures as described in Materials and Methods at 1 x lo3 cellslplate with 20 ng/mL of rHu G-CSF alone (0) or in combination 55 G-CSF 38 with 30 nmol/L (B), 100 nmol/L (U)or 1 pmol/L ( W ) of all-trans RA G-CSF + RA (100 nmol/L) 22 10 or 3 pmollL of retinol (!B). Results are presented as mean (SEMI Single CD34- cells were plated in microtiter wells. Cultures were number of colonies from six separate experiments with triplicate scored for proliferation ( > l 0 cells) after 14 days at 37°C and 5% CO2 determinations.*Statistical significance ( P < .05, paired Wilcoxon in air. Median values from two representative experiments are shown.test). Exp 1

Exp 2


RETINOIDS INHIBIT G-CSF-INDUCEDPROLIFERATION

day 7 colony formation induced by G-CSF usingnormal BM cells. However, in their study it was not shown whether the effect was directly mediated. In apparent contrast, van Exp 1 Exp 2 Bockstaele et alZ5found that all-trans RA weakly stimulated Growth Factor Day 7 Day 14 Day? Day 14 the formation of day 14 CFU-G colonies, using isolated 32 (3.6) 39 (4.0) 36 (2.5) 39 (4.0) G-CSF (20ng/mL) CD34+ cells stimulated with a combination of several cyto+RA 30 nmol/L 6 (0.6) 5 (1.2) 29 (3.5) 25 (4.6) kines, whereas the total number of colonies found, including 1 (1.0) 16 (2.6) 1 (0.6) 16 (3.8) +RA 1 pmol/L CFU-M and BFU-E colonies, were significantly depressed. However, it is difficult to directly compare the results of 0 (1.2) 22 0 (3.2) 19 GM-CSF (50 ng/mL) these studies because of the use of different cytokines or +RA 30 nmol/L 0 25 (4.0) 0 (3.5) 34 cytokine combinations. In addition, whereas we observed 0 (4.6) 18 0 29 (5.0) +RA 1 Mmol/L the most potent inhibition of G-CSF-induced colony formaCD34+ cells (2 x lo3 cells/dish) were cultured with growth factors tion at day 7, van Bockstaele et a1 scored CFU-G colonies in methyl cellulose, as described in Materials and Methods. Data reponly at day 14. resent mean of triplicates (SD). Two representative experiments are shown. Most available data suggest that retinoids also can stimulate normal hematopoietic progenitor cells. A stimulatory effect of RA on CFU-GM was first shown by Douer and Koeffler,I8 using BM mononuclear cells. This finding has ble of adhering to a stromal feeder layer and giving rise to and is in general blast colonies represent more primitive progenitor cells.26,30*36 later been confirmed by several gro~ps,'~-'' agreement with our results on GM-CSF-induced colony forWhen examining the capacity of CD34+ cells to generate mation of isolated CD34+ cells. Taken together, the data B1-CFCs, we found that all-trans RA at low concentrations therefore suggest that retinoids can have both stimulatory (30 nmoVL) consistently stimulated blast cell colony formaand inhibitory effects on committed progenitor cells, and tion (on average twofold, P < .05), whereas high concentrathat the effects on such cells are strongly dependent on the tions (1 FmoVL) were inhibitory (Table 4), indicating a bidiCSFs stimulating their growth. rectional, dose-dependent effect on blast-cell colony Few reports have examined the effects of retinoids on formation. more immature myeloid cells. A few reports have studied RA does not injuence G-CSF-induced difSerentiation of the effects ofRA on CFU-Mix and no clear effects were CD34' cells.CD34' cells were cultured in bulk liquid f o ~ n d . ' ~We , ~ found ~ that all-trans RA in low concentrations cultures with G-CSF with or without all-trans RA, and mor(30 nmol/L) stimulated blast colony formation on preformed phologic analyses were performed after culture for 3, 6, and stromal layers, whereas higher concentrations (1 pmol/L) 12 days. As can be seen in Table 5, no effects of all-trans were inhibitory. It is possible that the stimulatory effects RA on the relative frequency of blasts or different stages of observed on blast-cell colony formation in the present study myeloid cells were observed. In agreement with this we also are caused by RA-induced cytokine production in the prefound no difference in G-CSF-induced expression of the formed stromal cell layer. Interestingly, we have recently granulocytic antigen CD15 in the absence or presence of allshown in the murine system that primitive progenitor cells trans RA after 6 days of culture (data not shown). (Lin-Sca-l+) are profoundly and directly inhibited by allDISCUSSION trans RA and 9-cis RA in various assays, including high proliferative potential colony-forming cells colony formaRetinoids have been shown to influence growth and differtion!' Similarily, we have recently also found that RA inhibentiation of various hematopoietic cells. However, most its the growth of CD34+CD38- cells in single cell assays studies of the effects of retinoids on normal hematopoiesis (L. R., unpublished results, November 1992). Whether retihave used unfractionated BM mononuclear cells. As retinoids also can directly stimulate subpopulations of immature noids may have indirect effects, for instance via induced hematopoietic cells will require further studies. secretion of ~ y t o k i n e s , it ~ ~is. ~ important ~ to use highly enriched and more homogenous populations of hematopoietic progenitors for such studies. In this study, we have used highly purified CD34+ BM cells, which contain most of the Table 4. Effects of All-Trans RA on Blast Colony Numbers progenitor cells in the We observed a specific and No. of Colonies/2 x IO' CD34+ cells marked inhibition of G-CSF-induced [3H]-thymidine incorNo addition (25) 88 poration and colony formation. The effects of all-trans RA +RA (30 nmol/L) (49)' 178 on G-CSF-induced proliferation ofCD34' cells were di+RA (1 bmol/L) 64 (25)* rectly mediated, as all-trans RA also inhibited growth of CD34' cells (2 x IO') cells were added to irradiated, preformed CD34' cells in single-cell experiments. G-CSF is known to stromal layers. After 2 hours of incubation a t 37°C in 5% CO, humidisynergize with several other hematopoietic growth factors, fied atmosphere, unattached cells were removed, and the medium including SCF?' and we found that all-trans RA also powas replaced by 10 mL of 0.3% agar in IMDM medium containing tently inhibited G-CSF-induced proliferation induced by 10% FCS. The number of colonies with blast cell morphology were two-factor combinations of G-CSF and IL-3, GM-CSF, or counted after 1 week of culture. Data represent mean (SEMI of six SCF. Our results are in agreement with the findings of Tohda experiments. et al.24who showed a specific inhibition by all-trans RA on P < .05, paired Wilcoxon test. Table 3. Effects of All-Trms RA on G-CSF- and GM-CSHnduced Day 7 and 14 Colony Formation

~~

~

~

~~

~

From bloodjournal.hematologylibrary.org by guest on July 13, 2011. For personal use only. SMELAND ET AL

2944

Table 5. Effects of All-Trans RA on G-CSF-Induced Differentiation of CD34' BM Cells Blasts

Promyelocytes

53

0

23 5 1

18 19 8

64 7

16

0 0

2

6 12

2

9

Band Granulocytes

Segmented Neutrophils

2 34 15

4 37 10

10 58

ND 72 ND

4 39 16

3

ND

35 6

62

ND

Metamyelocytes Myelocytes

Peroxidase

G-CSF d 3 (1 exp) d 6 (3 exp) d 12 (3 exp)

G-CSF + RA d 3 (1 exp) d 6 (3 exp) d 12 ( 3 exd

0

5

0

70

CD34' cells were grown in liquid cultures in the presence of G-CSF (100 ng/mL) with or without RA (100 nmol/L). Themorphologic evaluation was performed on cytospin specimens stained with May-GrOnwald-Giemsa. Figures represent percentages. Freshly isolated CD34' cells contained >90% cells with blast-cell morphology.

Retinoids are known to induce terminal differentiation of several human and murine myeloid cell lines, especially cell lines expressing features of relatively mature cells (ie, HL60 and U-937).5-9Moreover, RA has been shown to induce granulocytic differentiation of acute promyelocytic leukemia cells in vivo and in vitro.''-16 RA-induced differentiation of cell lines is generally accompanied by reduced proliferation, but inhibited cell growth is not always accompanied by differentiati~n.~ However, the effects on differentiation of normal hematopoietic progenitors are controversial. Although the early inhibition of G-CSF-induced growth observed in this study (Table 1) suggests an inhibitory effect by all-trans RA onproliferation, we did not observe any significant effect of all-trans RA on G-CSF-induced differentiation of CD34+ cells in liquid cultures. Although this is in agreement with most previous st~dies,2'*~~" others have found inhibited granulocyticz3 or increased monocytic differentiati~n."~ However, it is difficult to directly compare the results of these studies, as different hematopoietic growth factors were used. Moreover, in most of these studies heterogeneous cell populations were used, and therefore possible effects on differentiation of small subpopulations of the cells could easily be overlooked. One of the main limitations in most in vitro studies using vitamin A is that the retinoids are administered to the cells solubilized in ethanol or Me,SO, and notbound to its physiological carriers. In vivo, however, retinol is transported in plasma bound to retinol-binding protein (RBP),' and is taken up by target cells via RBP-receptors.' Newly absorbed retinol is also present as retinyl esters in CMR, which are taken up by cells presumably via binding to low density lipoprotein (LDL) receptors or LDL receptor related protein (LW).' In this study we have observed effects of all-trans RA and retinol in physiological Similar effects were also observed using CMR-bound retinyl esters at concentrations of 0.1 ymoVL. Taken together, our experiments therefore suggest that the effects of retinoids on hematopoiesis are physiologically relevant. REFERENCES 1. Blomhoff R, Green MH, Berg T, Norum K: Transport and

storage of vitamin A. Science 250:399, 1990

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