Mechanism of Hb F stimulation by S-stagecompounds. In vitro studies with bone marrowcells exposed to 5-azacytidine, Ara-C, orhydroxyurea.

R Galanello, … , G Stamatoyannopoulos, TPapayannopoulou

J Clin Invest. 1988;81(4):1209-1216. https://doi.org/10.1172/JCI113437.

The in vitro effect of S-stage-specific drugs on the fetal hemoglobin (Hb F) potential oferythroid precursors and progenitors was tested by exposing bone marrow cells to 5-aza-2'-deoxycytidine, Ara-C, or hydroxyurea in suspension cultures and reculturing the cells indrug-free clonal cultures. Analysis of Hb F in the erythroblasts present at the end ofsuspension cultures and in the erythroid colonies formed from treated progenitors showedthat 1 X 10(-9)-5 X 10(-8) M 5-aza-2'-deoxycytidine produced a concentration-relatedincrease in the proportion of Hb F-positive erythroblasts, of Hb F-positive erythroid CFU(CFUe) colonies, and at the higher doses used, an increased Hb F expression in erythroidburst-forming unit (BFUe)-derived colonies. Preincubation of bone marrow cells with Ara-Cproduced significant megaloblastic changes by the end of the 2-d incubation and increasedthe proportion of Hb F-positive erythroblasts, CFUe colonies, and e-clusters, but BFUe-derived progeny was unaffected. Hydroxyurea failed to produce significant changes in Hb Fat the range of concentrations used. The data raise the possibility of more than onemechanism underlying the stimulation of Hb F by S-stage drugs.

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Mechanism of Hb F Stimulation by S-Stage CompoundsIn Vitro Studies with Bone Marrow Cells Exposed to 5-Azacytidine, Ara-C, or Hydroxyurea

R. Galanello, G. Stamatoyannopoulos, and Th. PapayannopoulouDivisions of Hematology and Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195

Abstract

The in vitro effect of S-stage-specific drugs on the fetal hemo-globin (Hb F) potential of erythroid precursors and progeni-tors was tested by exposing bone marrow cells to 5-aza-2'-de-oxycytidine, Ara-C, or hydroxyurea in suspension cultures andreculturing the cells in drug-free clonal cultures. Analysis ofHb F in the erythroblasts present at the end of suspensioncultures and in the erythroid colonies formed from treated pro-genitors showed that 1 X 10-9-5 X lo-8 M5-aza-2'-deoxycyti-dine produced a concentration-related increase in the propor-tion of Hb F-positive erythroblasts, of Hb F-positive ery-throid CFU(CFUe) colonies, and at the higher doses used, anincreased Hb F expression in erythroid burst-forming unit(BFUe)-derived colonies. Preincubation of bone marrow cellswith Ara-C produced significant megaloblastic changes by theend of the 2-d incubation and increased the proportion of HbF-positive erythroblasts, CFUe colonies, and e-clusters, butBFUe-derived progeny was unaffected. Hydroxyurea failed toproduce significant changes in Hb F at the range of concen-trations used. The data raise the possibility of more thanone mechanism underlying the stimulation of Hb F byS-stage drugs.

Introduction

Recently, several pharmacologic agents have been used tostimulate fetal hemoglobin (Hb F)' synthesis in vivo. The ini-tial studies involved treatment of baboons (1) or patients witheither Cooley's anemia (2-4) or sickle cell anemia (3-7) with5-azacytidine. The animals or the patients responded withconsiderable stimulation of y-chain synthesis, which was ini-tially attributed to the hypomethylation of y-globin genes(1-6). Subsequent studies (8-14), however, called attention tothe fact that the drug treatments triggered acute-phase regen-eration kinetics, previously known to be associated withheightened Hb F formation in vivo (15, 16), and suggested thatstimulation of Hb F synthesis by 5-azacytidine, as well as byother compounds is mainly indirect and cell kinetic in origin(8-14). The contribution of this mechanism to the in vivo

Address correspondence to Dr. Papayannopoulou, Dept. of Medicine,Div. of Hematology, RM-1O, University of Washington, Seattle, WA98195.

Received for publication 16 March 1987 and in revised form 19October 1987.

1. Abbreviations used in this paper: BFUe, erythroid BFU; CFUe,erythroid CFU; e-cluster, erythroid cluster; Hb F, fetal hemoglobin.

stimulation of Hb F by 5-azacytidine or hydroxyurea has,however, been questioned by other investigators (6, 17).

To investigate further the mechanisms of induction of HbF by cell cycle-specific drugs, we applied an experimental sys-tem consisting of exposure of bone marrow cells to S-stagecompounds for 42-60 h in suspension culture, followed bycloning of these cells in a drug-free environment. This systemallows testing of in vitro induction of Hb F by a drug and, inaddition, permits an assessment of the level of erythroid celldevelopment on which the effect of the drug is exerted and ofassociated changes in proliferation/maturation parameters.We find, by a variety of approaches, that 5-aza-2'-deoxycyti-dine increased Hb F in erythroblasts, erythroid clusters (e-clusters), erythroid CFU(CFUe), and erythroid burst-formingunit (BFUe) colonies, while Ara-C stimulates Hb F expressionin CFUe colonies, e-clusters, and erythroblasts. We find nosuch effects in the concentrations of hydroxyurea we used. Themechanism by which these compounds exert their in vitroeffect could be direct (i.e., demethylation of y-globin genes) orindirect, through changes in the kinetics of in vitro maturation(i.e., megaloblastosis), or through progenitor cell selection andeffect on the rate of their cell cycling. The data raise the possi-bility that these drugs, in addition to their triggering of acuteregeneration kinetics in vivo, can have other effects, whichmay account for the different levels of Hb F achieved in vivo,or the rapidity of Hb F-reticulocyte response in some patients.

Methods

Experimental approach. After density separation of aspirated bonemarrow cells, the mononuclear interphase cells were washed and in-cubated in the media described below (see suspension cultures). Exper-imental samples contained the compound under testing, while controlscontained media without that compound. After 42 (and in some ex-periments, after 60) h of incubation, the samples were replated insemi-solid media (plasma clots or methylcellulose) in the absence ofthe drug. Colonies were counted in these clonal cultures, and Hb F wasanalyzed, as described later. With this approach, we tested whetherpreexposure, in the suspension culture, of bone marrow erythroid pro-genitors for 42-60 h to 5-aza-2'-deoxycytidine, to Ara-C, or to hy-droxyurea influences Hb F expression in the erythroid colonies pro-duced by these progenitors in clonal cultures done in a drug-free envi-ronment. To examine whether Ara-C or 5-aza-2'-deoxycytidineinfluenced Hb F expression on the morphologically recognizable (atthe end of suspension culture) erythroid cells, an aliquot from thesuspension culture was used for erythroblast purification (see "pan-ning") and Hb F expression in these erythroblasts was assessed afterlabeling with anti-# and anti-y-chain antibodies. In the present stud-ies, 5-aza-2'-deoxycytidine was used instead of 5-azacytidine because ofits higher potency and lack of incorporation into RNA(18). In addi-tion, for the clonal cultures, instead of FCS, we have used a treatedserum preparation which largely abrogates Hb F activation in vitro(unpublished data).

Suspension cultures. Mononuclear cells were isolated after layeringthe samples on a cushion of Ficoll and diatrizoate salts (LSM, Bionetics

In Vitro Effects of S-Phase Compounds on HumanBone Marrow 1209

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/88/04/1209/08 $2.00Volume 81, April 1988, 1209-1216

Laboratory Products, Litton Bionetics, Inc., Charleston, SC) at a den-sity of 1.077-1.080 g/ml and centrifugation at 400 g for 40 minat 22°C.

Suspension culture media consisted of 20% FCS (Armour Phar-maceutical, Kankakee, IL), 1% BSA (Sigma Chemical Co., St. Louis,MO), erythropoietin (Terry Fox Laboratory, Vancouver, British Co-lumbia, Canada) (0.5 IU/ml), and the desired concentration of thecompound under testing. Bone marrow mononuclear cells suspendedin the above media at a final concentration of 5 X 106 cells/ml wereincubated at 37°C in a highly humidified CO2 incubator for 42-60 h.

The three compounds under study, 5-aza-2T-deoxycytidine (SigmaChemical Co.), Ara-C (Upjohn Co., Kalamazoo, MI), or hydroxyurea(Sigma Chemical Co.) were dissolved immediately before their use inthe suspension cultures. Thereafter, every 12 h the cells were spun, andmost of the supernate media were removed and replaced by an equalvolume of fresh medium, to which newly dissolved drug was added.

Immune adherence (panning). For immune adherence, previouslydescribed methodology (19) was adopted. Briefly, after suspensionculture, the cells were washed, resuspended in PBS containing 2.5%FCS, and the cell suspension was gently added to a polystyrene sterilepetri dish previously coated with a 1:200 dilution (in 0.05 MTris, pH9.5) of monoclonal antibody Ep-l, an antibody specific for erythroidprogenitors and erythroblasts (20). After 70 min incubation at 4°C ona level surface, the plates were washed three times with cold PBScontaining 1% FCS, and the nonadherent cells were aspirated. Theadherent cells were recovered by pipetting vigorously the entire surfaceof the plate with PBS plus 1% FCS. The so-recovered adherent cellswere > 97% erythroid. Wehave previously shown by cell sorting andcomplement-dependent cytotoxicity experiments (20) that Ep-l anti-gen is present in most of the BFUe, in virtually all CFUe, and in allerythroblasts. Therefore, apart from erythroblasts, erythroid progeni-tors (mainly CFUe) are also present in these Ep-l-adherent prepara-tions, but their numbers are low compared with the population oferythroid precursors (unpublished data). Detached cells from Ep-1-coated plates were used for smear preparation. Smears were eitherstained with Wright-Giemsa or they were labeled with fluorescent an-tiglobin chain monoclonal antibodies.

Clonal cultures. Bone marrow cells from the suspension cultureswere washed and inoculated (1-3 x l0 cells/ml) in methylcelluloseand plasma clot supporting media using previously described methods(21, 22). After 3-4 d in culture, plasma clot preparations were stainedwith benzidine for colony counting, or they were doubly labeled withfluorescent antiglobin (ey and ,B) monoclonal antibodies for analysis ofHb F expression. e-clusters were aggregates of cells composed of two toseven well-hemoglobinized cells. CFUe-derived colonies were thosecomprised of 8 to 64 well-hemoglobinized cells. Colonies derived fromBFUe were counted on day 12 to 14 methylcellulose cultures.

Analysis of cells and colonies by immunofluorescence. Cytocentri-fuge smears of erythroid cells enriched by immune adherence were airdried, fixed in methanol for 18 min, rinsed in PBS, and subsequently indistilled water. The areas containing the fixed cells were overlaid with afew microliters of anti-y monoclonal antibody and incubated for I hat 37°C in a humidifed chamber. After washing in PBS and distilledwater, the preparations were dried, covered with anti-mouse IgGF(ab')2 conjugated to FITC (Tago Inc., Burlingame, CA), and incu-bated, washed, and dried, as described above. The cells were thendoubly labeled with an anti-ftl-globin monoclonal (or, when indicated,with anti-#' monospecific) antibody directly conjugated to rhodamine.After incubation for 1 h at 37°C in a humidified chamber, the prepara-tions were washed and dried. The doubly labeled preparations wereviewed in a Zeiss Universal fluorescent microscope with the aid ofappropriate filters. To assess Hb F expression in erythroid colonies,flattened plasma clots were fixed and labeled with the anti-,8 and theanti-y-chain antibodies, as above.

Erythroblasts were considered to be F positive if they were labeledwith anti-y-chain FITC. They were F negative if they were labeledonly by the anti-,BA (or the anti-#') antibody. The e-clusters and CFUecolonies were considered as A positive when all the cells were ,B-glo-

bin-positive and 'y-negative; they were considered as F positive whentwo or more cells of a colony were y-globin-positive, while all the cellsof the colony were # positive. (Colonies containing only one F-positivecell were very few, and they were not included since, occasionally, itwas not clear whether a single F-positive erythroblast was part of asingle colony or represented superimposition of a free backgrounderythroblast.) Colonies that appeared "young" (i.e., few, large cells)and were negative in Hb A were not considered as CFUe. Frequenciesof F-positive e-clusters or F-positive CFUe were calculated aftercounting at least 50 (range from 50 to over 500) colonies in eachcategory. All counting was done by one person. Measurements of Fcolonies from multiple clots inoculated with the same cells have shownthat the fluorescent method of F analysis has < 5%experimental error.

The sensitivity of the fluorescent antibody detection method hasbeen assessed with previous experiments. Studies of cells of knownhemoglobin content (23) or with known proportion of an abnormalhemoglobin fraction (24) indicate that fluorescent anti-globin chainantibodies can detect at least 0.1 pg of hemoglobin per red cell.

Globin biosynthesis. For measuring Hb F production in e-clustersand CFUecolonies, 6 to 14 clots (depending on the number of coloniesper clot) were incubated at 37°C for 6 h in the presence of leucine-freemedia, 10% FCS, and 100 gCi/ml of [3H]leucine. At the end of theincubation, cells were lysed, and hemoglobin was extracted by centrif-ugation at 8,000 rpm. For measuring globin biosynthesis in BFUecolonies, fully hemoglobinized erythroid bursts were lifted from theplates, pooled, and incubated with [3H]leucine (100-200 MCi/ml) inleucine-free medium. Cell lysates were processed for isoelectric focus-ing of globin chains in the presence of NP40, ,B-mercaptoethanol, and 8Murea, as described before (25). The relative synthesis of globin chainswas estimated from densitometric tracings of the resultant fluoro-grams.

Analysis of data. Numbers of erythroid progenitors recovered aftersuspension culture and frequencies of F-positive colonies at variousconcentrations of cell cycle-specific drugs are presented in Table I. Tocorrect for the variation between experimental samples (which is espe-cially profound in the case of clonogenic assays; see Table I), the resultsin each drug concentration were expressed as percent of the valuesobtained when the same cells were cultured in the absence of cellcycle-specific drugs. Linear regression analysis was used to test thestatistical significance of the results.

Results

Effects of 5-aza-2'-deoxycytidine. Bone marrow cells fromnormal volunteers were cultured in suspension, either in theabsence of 5-aza-2'-deoxycytidine (controls) or in the presenceof a given concentration of this compound. At the end of the42-h incubation period, the proportion of viable cells in thecontrol and in the 5-aza-2'-deoxycytidine-treated cultures was74.3 and 67.6% (respectively) of the inoculated cells. After theincubation in the suspension cultures, the bone marrow cellswere inoculatd in plasma clot or methylcellulose cultures(done in the absence of 5-aza-2'-deoxycytidine), and numbersof erythroid colonies, as well as Hb F expression, were mea-sured.

As shown in Table I, incubation with 5-aza-2T-deoxycyti-dine affected the clonogenic potentials of erythroid progeni-tors. While there was only a minor decrease in e-clusters andCFUewhen I X I0-8-I X I09 M5-aza-2'-deoxycytidine werepresent in culture, at 5 X 10-8 M, erythroid progenitors werereduced to 10-43% of control values (Fig. 1). No colonies weregrown in the clonal cultures when the bone marrow cells hadbeen exposed to 1 X 10-6 M5-aza-2'-deoxycytidine during thesuspension culture.

The effect of 5-aza-2'-deoxycytidine on Hb F expression ine-clusters and, separately, in CFUe colonies was assessed by

1210 R. Galanello, G. Stamatoyannopoulos, and Th. Papayannopoulou

Table I. Erythroid Progenitors in Bone Marrow Cells Cultured in Suspension for 42 h*in the Presence or Absence of Cell Cycle-specific Compound

Hb F expression in colonies

Numbers of erythroid progenitors per 10 e-clusters CFUeConcentration innoculated cells

Bone in suspension Number %F Number %FCompound tested marrowt culture e-clusters CFUe BFUe evaluated positive evaluated positive

M

5-Aza-2'-deoxycytidine A

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1,352±16 619±1011,112±220 569±1231,058±72 616±46

568±90 416±65578±74 368±24347±12 203±26

1,022±142 129±16593±11 64±14568±4 64±16

177±62 108±36139±35 89±1676±33 28±11

358±21 324±1900 00

20 552 5.125 269 10.426 379 25.5

85 270 21.178 176 25.645 183 57.9

54 352 23.062 350 46.627 203 75.3

109

200

550 34.2273 49.8436 73.4

231 50.0195 55.4186 81.2

102 39.285 50.677 87.0

96 33.3 141 53.9120 75.0 179 79.356 82.1 25 88.9

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312±62 333±81349±33 403±62

90±17 92±31

750±95 580±120782±194 641±69581±7 499±113

358±21 324±19302±40 315±50

568±90 416±65389±76 192±21347±24 176±16

1,022±142 124±16600±80 198±31227±75 47±8

129 18.6114 26.343 34.9

178 13.1248 15.7

62 22.6

124 44.3119 50.474 67.6

68 44.178 42.356 48.2

281 32.0 193 36.8173 52.0 112 51.8

85 270 21.175 157 22.370 179 34.7

231 49.882 52.4

189 62.4

54 352 23.0 102 39.259 371 27.2 177 40.231 173 58.8 55 58.2

0 903±14 404±80X 10-6 750±14 320±10X l0o- 617±42 340±103

0 947±161 170±71X 10-6 654±189 184±23X lo0- 584±5 140±3

0 1,617±127 127±19lo-, 1,287±66 114±9

0 1,427±113 107±9lo-, 1,160±38 114±19

68 214 14.0175 14.8

52 158 13.9

47 216 20.9287 17.8

42 226 17.7

9 267 20.111 271 20.3

13 299 24.19 211 22.7

64 53.171 54.983 56.6

139 65.593 70.182 74.2

60 48.375 53.3

42 64.327 62.9

In Vitro Effects of S-Phase Compounds on HumanBone Marrow 1211

Ara-C

Hydroxyurea I-15;)5;)

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1-211

K-1

* Except for bone marrow B-2. * All bone marrow donors were persons without a hemoglobinopathy, < 1% Hb F, and < 2-5% F cells. § Thebone marrow suspension culture lasted for 60 h (see text). 11 I-2 and K-2 were cultured in the presence of untreated FCS. All other bone mar-row cultures were done with charcoal-treated FCS.

100

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500

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0

o 200

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Figure 1. Effect of5-aza-2'-deoxycytidineon (A) the clonogenicpotential of erythroidprogenitors, and (B) theexpression of Hb F inerythroid clones grownin plasma clot cultures.Normal bone marrowcells were grown for 42h in suspension culturescontaining the concen-trations of 5-aza-2'-de-oxycytidine shown inthe figure. The cellswere subsequently re-plated to clonal culturesdone with drug-freemedia. Counts of ery-throid colonies (or of F-positive colonies) ineach drug concentrationare expressed as percentof control counts ob-tained when the samecells were cultured (in

the suspension cultures) in the absence of 5-aza-2'-deoxycytidine (seetext). Findings in (e) e-clusters, (o) CFUe colonies, and (A) BFUecolonies.

labeling the plasma clot preparations with anti-3 and anti-y-chain monoclonal antibodies and analyzing the colonies as

described in Methods. In the control samples, the range of HbF-positive e-clusters was from 5 to 35% and of Hb F-positiveCFUefrom 34 to 55%. As shown in Fig. 1 B, 5-aza-2'-deoxycy-tidine increased the F-positive e-clusters and CFUecolonies inall concentrations used; this result is statistically highly signifi-cant (P < 0.001). However, there was no correlation betweenthe degree of increment of F-positive colonies and the dose of5-aza-2'-deoxycytidine used (r = 0.23, not significant), sug-gesting that maximal induction of Hb F was achieved by thelower concentration of 5-aza-2'-deoxycytidine used in our cul-tures. Note that the frequency of F-positive colonies was in-creased even in concentrations of 5-aza-2'-deoxycytidine (1X l0-9-l X 10 -8 M), which only slightly decreased the numberof erythroid colonies (compare Fig. 1, A and B).

Globin biosynthesis was measured in pooled CFUe ande-cluster colonies and separately in BFUe-derived colonies.The y/,y + ,3 synthetic ratios in control cultures ranged from0.08 to 0.13 in the CFUeand 0.10 to 0.28 in the BFUe colo-nies. y/,y + #3 ratios were measured in the clonal cultures ofcells preexposed to various concentrations of 5-aza-2'-deoxy-cytidine, and the results in these experimental cultures were

expressed as percent of the y/y + # ratio of the control culturesdone in the absence of 5-aza-2'-deoxycytidine. The data in Fig.2 A show that 5-aza-2'-deoxycytidine increases y-chain biosyn-thesis in erythroid colonies produced by e-clusters and CFUein a concentration-related fashion (r = 0.7954; P < 0.01). Asshown in Fig. 2 B, low concentrations of 5-aza-2'-deoxycyti-dine (5 x l0-9-l X 10-9 M) do not appear to stimulate Hb Fsynthesis in BFUe-derived colonies. However, preincubationof bone marrow with 1 x 10-8-5 X 10-8 M5-aza-2'-deoxycyti-dine increased Hb F production in six of seven BFUe cultures,

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Figure 2. Effect of5-aza-2'-deoxycytidineon Hb F expression.Globin chain biosyn-thetic measurementsdone on (A) pooledpreparations of CFUecolonies and e-clustersfrom plasma clot cul-tures, and (B) BFUecolonies from methyl-cellulose cultures. -Globin biosynthesis inthe experimental cul-tures is expressed aspercent of y-globin bio-synthesis in control cul-tures of the same cellsdone in the absence of5-aza-2'-deoxycytidine.

ne Culture conditions areas in Fig. 1.

suggesting that the increment of Hb F expression is related tothe concentration of the drug in the media (r = 0.5649; P< 0.025).

Bone marrow from a normal person (case B, Table I) wasused for the following experiment. The cells were incubated for42 h in suspension culture in the presence of 5 X 10-9 or 3X 10-8 M5-aza-2'-deoxycytidine, part of the cultured cells was

plated in plasma clots, while incubation of another part con-tinued (after new suspension media containing 5-aza-2'-deoxy-cytidine were added). At 60 h, these latter samples were platedin plasma clots. Measurement of the number of colonies andthe frequency of F-positive colonies in the plasma clot culturesallowed an assessment of whether prolongation of the time ofexposure of progenitor cells to 5-aza-2'-deoxycytidine duringsuspension culture increases Hb F expression in coloniesformed by the so-exposed progenitors. As shown in Fig. 3,

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42h 60h 42h 60hDuration of Suspension Culture

Figure 3. Comparisonof the effects of 42-hsuspension culture tothe effects of a 60-h sus-

pension culture of bonemarrow cells in thepresence of 5-aza-2'-de-oxycytidine. Controlswere cultures of thesame cells in the ab-sence of 5-aza-2'-deoxy-cytidine. (Top) Effectson the clonogenic po-tential of erythroid pro-genitors. (Bottom) Ef-fect on Hb F expressionin erythroid colonies.(-) e-clusters, (o) CFUecolonies, and (A) BFUecolonies.

1212 R. Galanello, G. Stamatoyannopoulos, and Th. Papayannopoulou

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exposure of bone marrow cells to 5-aza-2'-deoxycytidine for 60h increased F-positive colonies more than the exposure to5-aza-2'-deoxycytidine for 42 h. The increment was higher inthe bone marrow cells incubated with 3 X 10-8 Mcomparedwith 5 X 10-9 M5-aza-2'-deoxycytidine.

Wesubsequently tested whether 5-aza-2'-deoxycytidine in-duces Hb F expression in erythroblasts. Bone marrow cellsfrom an Hb S homozygote (patient W.H.) were cultured in thepresence of 1 X 10-8 or 3 X 10-8 M5-aza-2'-deoxycytidine, aswell as in the absence of the drug. After 42 h in suspensionculture, erythroblasts were separated by immune adherence,smears were prepared, they were doubly stained with anti-yFITC and anti-,8 rhodamine, and the proportion of F erythro-blasts determined. 6%of the reticulocytes of patient W.H. wereF reticulocytes, and 29% of his red cells were F cells. In thesuspension culture done in the absence of 5-aza-2'-deoxycyti-dine, 22% of erythroblasts were positive for Hb F. In the sus-pension culture done in the presence of 1 X 1o-8 and 3 X 1o-8M5-aza-2'-deoxycytidine, 36.7 and 44.8% of the erythroblasts(respectively) were positive for Hb F.

Effects ofAra-C. Bone marrow samples from normal vol-unteers were cultured, in suspension, in the absence of Ara-C(controls), or in the presence of various concentrations of thiscompound (Table I). At the end of the 42-h incubation, thecells were plated in plasma clot and in methylcellulose culturesdone in the absence of Ara-C. In these clonal cultures, weassessed whether preincubation of bone marrow cells withAra-C influenced the clonogenic potentials of progenitors orthe Hb F expression in erythroid colonies.

Effects of Ara-C on the clonogenic potentials of erythroidprogenitors are summarized in Fig. 4 A. Suspension culture ofbone marrow cells for 42 h in the presence of 5 X 10-10 MAra-C did not decrease the clonogenic potential of erythroidprogenitors (indeed, it may have slightly increased growth ofe-clusters and CFUe). Erythroid progenitors were, however,reduced when the cells were cultured for 42 h in the presenceof 3 X 10-8-l X l0-9 MAra-C.

'I Is Is Is,N\0 Nd\

Concentration (M) of Ara C inSuspension Culture

Figure 4. Effect ofAra-C on (A) the clono-genic potential of ery-throid progenitors, and(B) Hb F expression inerythroid colonies. Datain each experimentalculture are expressed aspercent of control cul-tures done in the ab-sence of Ara-C. Find-ings in (e) e-clusters, (o)CFUecolonies, and (A)BFUe colonies.

Fig. 4 B presents the results of evaluation of Hb F expres-sion in CFUecolonies and, separately, in e-clusters by immu-nofluorescence. In control cultures, frequency of F-positivee-clusters ranged from 13.1 to 32%, while the frequency ofF-positive CFUeranged from 36.8 to 49.8%. The frequency ofF-positive colonies was higher in the Ara-C preincubated cul-tures, compared with their controls, in 12 of 14 experiments.However, the stimulation of Hb F expression was less pro-nounced than that observed in the 5-aza-2'-deoxycytidine-treated samples (compare Fig. 4 B to Fig. 1 B). Regressionanalyses showed correlation between increment in F coloniesand concentration of Ara-C in the media (r = 0.7028; P< 0.0025).

Results of globin biosynthesis in CFUe colonies and e-clusters are presented in Fig. 5 A. In the control cultures, y/y+ ,B biosynthetic ratios ranged from 0.05 to 0.13 for CFUe(and e-clusters) and 0.05 to 0.20 for BFUe colonies. In six outof nine experiments, preincubation of bone marrow cells withvarious concentrations of Ara-C increased Hb F biosynthesisin CFUecolonies and e-clusters, compared with the values ofcontrols. Regression analyses failed to reveal a statistically sig-nificant correlation between Hb F synthesis and concentrationof Ara-C in the media (r = 0.3908, not statistically significant).Ara-C did not influence Hb F biosynthesis in BFUe colonies(Fig. 5 B; r = 0.1712, not statistically significant).

Frequencies of F-positive colonies were determined at both42 and 60 h after suspension cultures of a normal bone mar-row (case B, Table I) in the presence of 1 X 10-9 and 3 X 1o-8MAra-C (Fig. 6). This experiment showed that prolongationof suspension culture from 42 to 60 h increased F colonieswhen cultures were done in the presence of 3 X 10-8 MAra-C,but not in cultures done with 1 X 10-9 MAra-C.

Wealso examined whether Ara-C influences Hb F expres-sion in erythroid cells maturing during suspension culture.Homozygous Hb S bone marrow cells (from patient W.H.)were cultured in the presence of 1 X 10-8 or 3 X 10-8 MAra-C.At the end of the 42-h incubation, erythroblasts were enrichedby immune adherence and stained with anti-y and anti-,8-chain FITC. Both concentrations of Ara-C increased F eryth-roblasts by - 1.4-fold, compared with control suspension cul-tures. Ara-C had a striking effect on morphology of erythro-blasts; 70-80% of erythroblasts were overtly megaloblastic,

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In Vitro Effects of S-Phase Compounds on HumanBone Marrow 1213

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while megaloblastic erythroblasts were rare in the control cul-tures. In 5-aza-2'-deoxycytidine-treated cultures, 15% ofthe erythroblasts were megaloblastic.

Effect of hydroxyurea. There was no reduction of erythroidprogenitor cell numbers when the bone marrow cells werecultured for 42 h in suspension in the presence of 1 X l0-6 1X 10-8 Mhydroxyurea (data not shown). Slight reduction(20-40% of control values) was observed when bone marrowcells were preincubated for 42 h in the presence of 5 X 10-5-5X 10-6 Mhydroxyurea (Fig. 7 A). In control cultures, frequen-cies of F-positive e-clusters and F-positive CFUe ranged from

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14.0 to 24.1% and 48.3 to 65.5%, respectively. As shown inFig. 7 B, exposure, during suspension culture, of erythroidprogenitors to 5 X l0-5-5 X 10-6 Mhydroxyurea essentiallyproduced no increment in F-positive e-clusters or CFUecolo-nies. By globin biosynthesis, 8 of 12 biosynthetic determina-tions of Hb F expression in e-clusters and CFUe coloniesshowed slightly higher values in the experimental samplescompared with controls (Fig. 8 A), but the results were notstatistically significant. Measurements in BFUecultures (Fig. 8B) showed that 10 of 18 experimental samples had higher, 4had the same, and 4 had lower y/'y + (3 ratios compared withcontrols; these differences were not statistically significant.

Discussion

The mechanisms whereby S-stage compounds stimulate Hb Fsynthesis in vivo remain controversial. In the case of 5-azacy-tidine, direct induction of y-gene expression by the demethy-lating action of this drug has been proposed ( 1-7). Studies inbaboons (8) and monkeys (14) have shown, however, that atleast in part, the induction of Hb F by 5-azacytidine could beexplained by the acute-phase regeneration kinetics triggered bythe drug treatment. Induction of Hb F in vivo has also beenobserved after treatment of patients or animals with other S-stage or M-stage compounds: cytosine arabinoside (11, 13),hydroxyurea (9, 13, 14, 17, 26), myleran (27), and vinblastin(12, 14). In several of these studies, daily measuremnents of Freticulocyte production have demonstrated that the inductionof F cell formation occurs during the stage of erythroid mar-row regeneration (8, 11-14). Such results are compatible withthe hypothesis that the postchemotherapy induction of F cellformation is due to premature commitment of earlier progen-itors or to cell cycle changes induced by the rapid erythroidregeneration kinetics that follow the cytoreduction phase ofthe drug treatment (10, 14). This hypothesis, however, hasbeen challenged by other investigators. Humphries et al. (6)reported that there is no consistent suppression of the ery-throid marrow in patients treated with 5-azacytidine, suggest-ing that the cytoreduction and the resultant erythroid regener-ation play a minor role in the stimulation of Hb F by thiscompound. Platt et al. treated two patients with hydroxyureaand observed induction of F cells in the absence of reductionin absolute reticulocytes (26); however, the changes in other

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1214 R. Galanello, G. Stamatoyannopoulos, and Th. Papayannopoulou

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parameters (such as white blood cell and platelet counts) indi-cated that there was bone marrow suppression after the hy-droxyurea treatment (26). In the study of Dover et al. (17),eight patients were treated with hydroxyurea, and all re-sponded with cytoreduction, as revealed by the decrease in thevalues of absolute reticulocytes; these authors observed that Freticulocyte response was higher in two patients having thelowest erythroid suppression, and suggested that a mechanismother than the postcytoreduction fast erythroid regenerationkinetics could underlie the stimulation of Hb F by hydroxy-urea.

In this study, we tested the influence of three S-stage com-pounds on erythroid progenitors in vitro by exposing the pro-genitor cells to these compounds for a period of 42-60 h andthen allowing them to generate colonies in a drug-free environ-ment. Studies of Hb F expression in the colonies generated inthe clonogenic assays allowed us to obtain information, first,on whether the preexposure of bone marrow cells to an S-stagecompound resulted in stimulation of Hb F in the progenyformed in a drug-free environment; and second, on the type oftarget cells on which this effect had been exerted. Previousstudies have shown that differentiation and maturation of ery-throid cells continues in the presence of erythropoietin duringsuspension culture (28). Wehave found that the relative pro-portion of proerythroblasts and basophilic erythroblasts, aswell as the number of CFUe, are substantially higher at the endof 42 h suspension culture (compared with the initial inocula),suggesting de novo generation of both proerythroblasts andCFUe (data not shown). Thus, formation of increased num-bers of F-positive erythroblasts at the end of suspension culturesuggests an effect of the S-stage compound either on erythro-blasts present during inoculation of the culture and/or an ef-fect on late erythroid progenitors (e-cluster-forming cells orlate CFUe) which generate erythroblasts during the suspensionculture time. Increase of Hb F-positive e-clusters in the clono-genic assay is interpreted to indicate an effect on e-cluster-forming cells present originally in the suspension culture orgenerated from CFUe's during suspension culture. Likewise,induction of Hb F in CFUe-derived colonies is expected toresult from action of the drug on CFUe present originally inthe suspension cultures, as well as on in vitro-generated CFUethrough BFUe maturation (during the time of suspension cul-ture).

Analyses of Hb F expression showed that 5-aza-2'-deoxycy-tidine had stimulated Hb F production in all types of coloniesformed (BFUe, CFUe, and e-clusters), as well as in erythro-blasts present at the end of the 42-h incubation period. Therewas increased Hb F synthesis in CFUecolonies, e-clusters, anderythroblasts, in the Ara-C-treated cultures, but the effectswere less pronounced than those of 5-aza-2'-deoxycytidine.There was no stimulation of Hb F in hydroxyurea-treated cul-tures; this negative result, however, may relate to the fact thatrelatively nontoxic concentrations of hydroxyurea were used.

Since it is known that 5-azacytidine induces the expressionof several inactive genes, the in vitro induction of Hb F by5-aza-2'-deoxycytidine can be attributed to demethylation ofy-globin genes of erythroblasts and progenitors, as previouslyproposed (1, 2). Alternatively, to explain the in vitro inductionof Hb F by 5-aza-2'-deoxycytidine, as well as by Ara-C, othermechanisms can be considered. An indirect mechanism in-volving recruitment of less mature progenitors with an activeHb F program has been proposed to account for the induction

of Hb F by S-stage-specific compounds in vivo (8-14). Thesame mechanism could account for our in vitro results. It isreasonable to assume that, in the presence of cytotoxic levels ofa cell cycle-specific drug in the suspension culture, the mostmature, rapidly dividing progenitors (29) will be preferentiallykilled, while the less frequently dividing immature progenitorswill be spared. As a result of such in vitro cell selection, bonemarrow samples preincubated with Ara-C (or with 5-azacyti-dine) will be enriched in noncycling immature erythroid pro-genitors. If among these progenitor cells the proportion of cellshaving a program allowing the Hb F expression is higher, anincreased proportion of F colonies will be formed when theseprogenitor cells are plated in clonal erythroid cultures. ThatHb F induction mainly occurs at doses reducing the total num-ber of colonies (Figs. 1, 2, 4, and 5) is compatible with theselection hypothesis.

Observations on the kinetics of F cell formation after achronic or an acute stimulation of erythropoiesis in patients orin anemic baboons suggest that rapid erythroid regenerationkinetics underlie the stimulated F cell formation in these con-ditions (10). Rapid erythroid regeneration may stimulate F cellformation by increasing the chance of premature terminalcommitment of earlier cells having an active Hb F program(10, 30). An alternative hypothesis is that the accelerated cy-cling, per se, activates the fetal globin program in progenitorsand precursors. This hypothesis, detailed before (10), assumesthat shortening of the erythroid cell cycle below a critical timeincreases the chance of y-gene activation and, hence, F cellformation. This hypothesis could also explain the induction ofHb F by cell cycle-specific drugs in vitro. Weassume thaterythroid progenitors and precursors that incorporate sub-lethal concentrations of S-stage compounds become temporar-ily arrested at the G,/S boundary or early S-phase, as otherstudies of the effect of S-stage compounds on proliferating cellssuggest (31). When the drug-treated bone marrow cells arereplated in a drug-free environment, some of the surviving andpartially synchronized erythroid progenitors are released fromtheir temporary arrest and begin to proliferate with a short-ened cycling rate, which may activate the expression the y-genes. Increased plating efficiency of leukemic cells exposedpreviously to sublethal doses of 5-azacytidine (32) can be citedin favor of changes in cell kinetics postdrug treatment.

In summary, the in vitro data that we have generated com-plement in vivo observations and provide arguments for con-sidering more than one mechanism of induction of Hb F pro-duction by cell cycle-specific drugs.

Acknowledgments

Wethank Betty Nakamoto and Sumiko Kurachi for excellent techni-cal assistance, and Doreen Loomans for typing the manuscript.

This work was supported by grant HL-20899 from National Insti-tutes of Health.

References

1. DeSimone, J., P. Heller, L. Hall, and D. Swiers. 1982. 5-azacyti-dine stimulates fetal hemoglobin synthesis in anemic baboons. Proc.Natl. Acad. Sci. USA. 79:4428-4431.

2. Ley, T. J., J. DeSimone, N. P. Anagnou, G. H. Keller, R. K.Humphries, P. H. Turner, N. S. Young, P. Heller, and A. W. Nienhuis.

In Vitro Effects of S-Phase Compounds on HumanBone Marrow 1215

1982. 5-azacytidine selectively increases y-globin synthesis in a patientwith lB+-thalassemia. N. Engl. J. Med. 307:1469-1475.

3. Ley, T. J., J. DeSimone, T. Noguchi, P. H. Turner, A. N.Schechter, P. Heller, and A. W. Nienhuis. 1983. 5-azacytidine in-creases -y-globin synthesis and reduces the proportion of dense cells inpatients with sickle cell anemia. Blood. 62:370-380.

4. Nienhuis, A. W., T. J. Ley, R. K. Humphries, N. S. Young, andG. Dover. 1985. Pharmacological manipulation of fetal hemoglobinsynthesis in patients with severe ,8-thalassemia. Ann. NYAcad. Sci.445:198-211.

5. Charache, S., G. Dover, K. Smith, C. C. Talbot, Jr., M. Moyer,and S. Boyer. 1983. Treatment of sickle cell anemia with 5-azacytidineresults in increased fetal hemoglobin production and is associated withnonrandom hypomethylation of DNAaround the -y-6-B3-globin genecomplex. Proc. Natl. Acad. Sci. USA. 4842-4846.

6. Humphries, R. K., G. Dover, N. W. Young, J. G. Moore, S.Charache, T. Ley, and A. W. Nienhuis. 1985. 5-Azacytidine acts di-rectly on both erythroid precursors and progenitors to increase produc-tion of fetal hemoglobin. J. Clin. Invest. 75:547-557.

7. Dover, G. J., S. Charache, S. H. Boyer, G. Vogelsang, and M.Moyer. 1985. 5-azacytidine increases Hb F production and reducesanemia in sickle cell disease: dose-response analysis of subcutaneousand oral dosage regimens. Blood. 66:527-532.

8. Torrealba de Ron, A., Th. Papayannopoulou, M. S. Knapp, M.Fu, G. Knitter, and G. Stamatoyannopoulos. 1984. Perturbations inthe erythroid marrow progenitor cell pools may play a role in theaugmentation of Hb F by 5-azacytidine. Blood. 63:201-2 10.

9. Letvin, N. L., D. C. Linch, G. P. Beardsley, K. W. McIntyre, andD. G. Nathan. 1984. Augmentation of fetal hemoglobin production inanemic monkeys by hydroxyurea. N. Engl. J. Med. 310:869-873.

10. Stamatoyannopoulos, G., R. Veith, R. Galanello, and Th. Pa-payannopoulou. 1985. Hb F production in stressed erythropoiesis:observations and kinetic models. Ann. NYAcad. Sci. 445:188-197.

11. Papayannopoulou, Th., A. Torrealba de Ron, R. Veith, G.Knitter, and G. Stamatoyannopoulos. 1984. Arabinosylcytosine in-duces fetal hemoglobin in baboons by perturbing erythroid cell differ-entiation kinetics. Science (Wash. DC). 224:617-619.

12. Veith, R., Th. Papayannopoulou, S. Kurachi, and G. Stama-toyannopoulos. 1985. Treatment of baboon with vinblastin: insightsinto the mechanisms of pharmacologic stimulation of Hb F in theadult. Blood. 66:456-459.

13. Veith, R., R. Galanello, Th. Papayannopoulou, and G. Stama-toyannopoulos. 1986. Stimulation of F-cell production in patients withsickle cell anemia treated with cytarabine or hydroxyurea. N. Engl. J.Med. 313:1571-1575.

14. Letvin, N. L., D. C. Linch, G. P. Beardsley, K. W. McIntyre, G.Miller, and D. G. Nathan. 1986. Influence of cell cycle phase-specificagents on simian fetal hemoglobin synthesis. J. Clin. Invest. 75:1999-2005.

15. Dover, G. J., S. H. Boyer, and W. H. Zinkham. 1979. Produc-tion of erythrocytes that contain fetal hemoglobin in anemia. J. Clin.Invest. 63:173-176.

16. Papayannopoulou, Th., E. Vichinsky, and G. Stamatoyanno-poulos. 1980. Fetal Hb production during acute erythroid expansion.I. Observations in patients with transient erythroblastopenia andpostphlebotomy. Br. J. Haematol. 44:535-546.

17. Dover, G. J., R. K. Humphries, J. G. Moore, T. J. Ley, N. S.Young, S. Charache, and A. W. Nienhuis. 1986. Hydroxyurea induc-tion of hemoglobin F production in sickle cell disease: relationshipbetween cytotoxicity and F-cell production. Blood. 67:735-738.

18. Momparler, R. L., F. L. Momparler, and J. Samson. 1984.Comparison of the antileukemic activity of 5-aza-2'-deoxycytidine,

1-f,-D-arabinofuranosylcytonine and 5-azacytidine against L-12120leukemia. Leuk. Res. 8:1043-1049.

19. Cooper, K. D., K. Kang, T. L. Bush, and J. M. Hanifin. 1984.Direct depletion and purification of monoclonal antibody defined cellsfrom unfractionated human mononuclear leukocytes using antibodycoated polystyrene petri dishes. J. Clin. Lab. Immunol. 13:97-100.

20. Yokochi, T., M. Brice, P. S. Rabinovitch, Th. Papayannopou-lou, and G. Stamatoyannopoulos. 1984. Monoclonal antibodies de-tecting antigenic determinants with restricted expression on erythroidcells: from the erythroid committed progenitor level to the matureerythroblast. Blood. 63:1376-1384.

21. Papayannopoulou, Th., T. Kalmantis, and G. Stamatoyanno-poulos. 1979. Cellular regulation of hemoglobin switching: evidencefor inverse relationship between fetal hemoglobin synthesis and degreeof maturity of human erythroid cells. Proc. Natl. Acad. Sci. USA.76:6420-6424.

22. Papayannopoulou, Th., M. Brice, and G. Stamatoyanno-poulos. 1977. Hemoglobin F synthesis in vitro: evidence for control atthe level of primitive erythroid stem cells. Proc. Natl. Acad. Sci. USA.74:2923-2927.

23. Bigbee, W. L., E. W. Branscomb, H. B. Weintraub, Th. Pa-payannopoulou, and G. Stamatoyannopoulos. 1981. Cell sorter im-munofluorescence detection of human erythrocytes labeled in suspen-sion with antibodies specific for hemoglobin S and C. J. Immunol.Methods. 45:117-127.

24. Stamatoyannopoulos, G., P. E. Nute, Th. Papayannopoulou,T. McGuire, G. Lim, H. F. Bunn, and D. Rucknagel. 1980. Develop-ment of a somatic mutation screening system using Hb Mutants. IV.Successful detection of red cells containing the human frameshift mu-tants Hb Wayne and Hb Cranston using monospecific fluorescentantibodies. Am. J. Hum. Genet. 32:484-496.

25. Righetti, P. G., E. Gianazza, A. M. Gianni, P. Comi, B. Gig-lioni, S. Ottolenghi, C. Sechi, and C. Rossi-Bernardi. 1979. Humanglobin chain separation by isoelectric focusing. J. Biochem. Biophys.Methods. 1:45-46.

26. Platt, 0. S., S. H. Orkin, G. Dover, G. P. Beardsley, B. Miller,and D. G. Nathan. 1984. Hydroxyurea enhances fetal hemoglobinproduction in sickle cell anemia. J. Clin. Invest. 74:652-656.

27. Liang, C. C., D. P. Liu, P.C. Jia, Z. H. Ao, S. S. Chen, and K. G.Yang. 1987. Augmentation of fetal hemoglobin in anemic monkeys bymeleran. In Developmental Control of Globin Gene Expression. G.Stamatoyannopoulos and A. W. Nienhuis, editors. Alan R. Liss, Inc.,NewYork. pp. 467-477.

28. Dessypris, E. N., and S. B. Krantz. 1984. Effect of pure erythro-poietin on DNAsynthesis by day 15-human marrow BFIJe in short-term liquid culture. Br. J. Haematol. 56:295-306.

29. Gregory, C. J., and A. C. Eaves. 1978. Three stages of erythro-poietic progenitor cell differentiation distinguished by a number ofphysical and biologic properties. Blood. 51:527-537.

30. Stamatoyannopoulos, G., and Th. Papayannopoulou. 1979.Fetal hemoglobin and the erythroid stem cell differentiation process.In Cellular and Molecular Regulation of Hemoglobin Switching. G.Stamatoyannopoulos and A. W. Nienhuis, editors. Grune & StrattonInc., NewYork. 323-350.

31. Ishikura, H., T. Okazaki, T. Mochizuki, Y. Izumi, M. Tashima,H. Sawada, and H. Uchino. 1985. Effect of antimetabolites and thy-midine blockage on the induction of differentiation of HL-60 cells byretinoic acid or la,25-dihydroxyvitamin D3. Exp. Hematol. 13:981-988.

32. Motoji, T., T. Hoang, D. Tritchler, and E. A. McCulloch. 1985.The effect of 5-azacytidine and its analogues on blast cell renewal inacute myeloblastic leukemia. Blood. 65:894-901.

1216 R. Galanello, G. Stamatoyannopoulos, and Th. Papayannopoulou