Proc. Natl. Acad. Sci. USAVol. 88, pp. 2351-2355, March 1991Medical Sciences

Normal and leukemic hematopoietic cells manifest differentialsensitivity to inhibitory effects of c-myb antisenseoligodeoxynucleotides: An in vitro study relevantto bone marrow purging

(leukemia/hematopoiesis/antisense DNA/protooncogene/transplantation)

BRUNO CALABRETTA*tt, ROBERT B. SIMSt, MAURO VALTIERI§, DANIELE CARACCIOLO¶, CEZARY SZCZYLIK*,DONATELLA VENTURELLI*t, MARIUSZ RATAJCZAK II, MILOSLAV BERAN**, AND ALAN M. GEWIRTZII,ttltDepartments of *Pathology and tInternal Medicine, and tFels Cancer Research Institute, Temple University School of Medicine, Philadelphia, PA 19140;§Istituto Superiore di Sanita, Laboratorio di Ematologia, Rome, Italy; 11stituto di Medicina ed Oncologia Sperimentale, Sezione di Ematologia, Torino,Italy; **Department of Medicine, M. D. Anderson Hospital, Houston, TX 77030; and Departments of IlPathology and ttInternal Medicine,University of Pennsylvania, School of Medicine, Philadelphia, PA 19104

Communicated by Peter C. Nowell, November 29, 1990

ABSTRACT The c-myb protooncogene is preferentiallyexpressed in hematopoietic cells, and its encoded protein, Myb,is required for hematopoietic cell proliferation. To analyze therelative Myb dependence of normal and leukemic humanhematopoietic progenitor cells, normal bone marrow cells,several types ofleukemic blast cells, and 1:1 mixtures ofnormaland leukemic cells were cultured in the presence ofc-myb senseor antisense oligodeoxynucleotides; cell viability and cloningefficiency were then assessed. c-myb sense oligomers had neg-ligible effects on normal and leukemic cells. In contrast, c-mybantisense oligomers strongly inhibited or completely abolishedclonogenic growth of a T-cell leukemia line, 78% (18 of 23) ofprimary acute myelogenous leukemia cases examined, and 4 of5 primary chronic myelogenous leukemia (CML) cases in blastcrisis. In three of the latter patients, polymerase chain reactionanalysis of a 1:1 mixture ofc-myb antisense-treated normal andCML cells revealed a complete absence of ber-abl expression,suggesting that the CML clonogenic units had been completelyeliminated from the cultures. At antisense doses that inhibitedleukemic cell growth, normal hematopoietic progenitor cellssurvived. Thus, normal and leukemic hematopoietic cells showdifferential sensitivity to the toxic effects of c-myb antisenseDNA. Perturbation of c-myb function with antisense oligode-oxynucleotides might eventually form the basis for a molecularapproach to leukemia therapy, perhaps most immediately as exvivo bone marrow purging agents.

The protooncogene c-myb is preferentially expressed inhematopoietic cells (1). Its encoded protein, Myb, binds to aspecific nucleotide consensus sequence (2) and has beenidentified in a variety of malignant (3-5) and normal (6, 7)hematopoietic cell types. Downregulation of c-myb expres-sion is associated with increasing hematopoietic cell matu-ration (1, 8-10), and overexpression ofc-myb has been shownto inhibit mouse erythroleukemia cell differentiation in re-sponse to erythropoietin (11) and dimethyl sulfoxide (12).Use of c-myb antisense oligodeoxynucleotides has directlydemonstrated that c-myb gene function is required for theproliferation of normal (13) and malignant (14) human hema-topoietic cells. We reasoned that synthetic antisense oligo-mers targeted to c-myb mRNA might prove useful antileu-kemic agents ifleukemia cells were more dependent on c-mybfunction for proliferation and/or cell maintenance than theirnormal counterparts.

Here we provide in vitro evidence that normal and leuke-mic hematopoietic cells manifest differential sensitivity toinhibition of c-myb gene function with antisense oligodeoxy-nucleotides. When normal human marrow mononuclear cellswere cocultured with different types ofleukemia blast cells inthe presence of c-myb antisense DNA, colony formation byleukemic progenitor cells was either greatly diminished orcompletely abolished, whereas normal colony formation wasonly moderately impaired. These findings suggest that per-turbation ofc-myb function with antisense oligodeoxynucleo-tides might eventually form the basis for a molecular ap-proach to leukemia therapy, perhaps most immediately as exvivo bone marrow purging agents.

MATERIALS AND METHODSCells. Normal bone marrow mononuclear cells (MNCs)

were aspirated from consenting volunteers and enriched forhematopoietic progenitors (13, 15). Human leukemia cellsutilized were (i) CCRF-CEM cells (T-cell leukemia lineobtained from the American Type Culture Collection), (ii)primary acute myelogenous leukemia (AML) blast cells [iso-lated from peripheral blood of patients diagnosed usingFrench-American-British Cooperative Group (16) criteria],and (iii) chronic myelogenous leukemia (CML) blast cells(Philadelphia chromosome-positive).

Oligodeoxynucleotides. Unmodified 18-base oligodeoxynu-cleotides were synthesized on an Applied Biosystems 380BDNA synthesizer and purified (13, 14). Oligomer sequenceswere based on the human c-myb cDNA sequence (17) andwere as follows: 5'-GCC-CGA-AGA-CCC-CGG-CAC-3'(MYB 1: codons 2-7; sense); 5'-GTG-CCG-GGG-TCT-TCG-GGC-3' (MYB 2: codons 2-7; antisense); 5'-ACT-GCT-ATA-TAT-GCT-GTG-3' (MYB 12: codons 6-11; antisense); 5'-GCC-ATG-GCC-CGA-AGA-CCC-3' [MYB 21; nucleotides(nt) -3 to +15; sense]; 5'-GGG-TCT-TCG-GGC-CAT-GGC-3' (MYB 22: nt -3 to +15; antisense); 5'-CGC-GTA-CCG-CAG-GAA-CCC-3' (MYB 24 "scrambled" sequencecorresponding to mRNA nt -3 to + 15).

Cell Culture and Oligomer Exposure. Adherent-cell- andT-lymphocyte-depleted normal MNCs (0.5-2 x 105 per ml)and leukemic blast cells (0.5-2 x 105 per ml) were placed in

Abbreviations: AML, acute myelogenous leukemia; CML, chronicmyelogenous leukemia; CFU-GM, granulocyte/macrophage colony-forming unit; MNC, mononuclear cell; RT, reverse transcription; nt,nucleotide(s).#To whom reprint requests should be addressed at: John MorganBuilding, Room 230, University of Pennsylvania, School of Med-icine, 36th Street and Hamilton Walk, Philadelphia, PA 19104.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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liquid suspension cultures (RPMI 1640 with 20o fetal bovineserum) alone or in a 1:1 mix. Treated cultures received 2-80Ag/ml (1 ,.g/ml = 0.35 AM) of c-myb sense (MYB 1, unlessotherwise indicated) or antisense oligomers (MYB 2, unlessotherwise indicated) at the start of the culture period. Asecond dose (25-50% of the initial dose) was added to themedium after =18 hr. Cells were either left in suspensionculture for 4-9 days, during which time cell counts andviability were periodically determined, or immediatelyseeded into duplicate methylcellulose (18) or plasma clot (13,15) cultures containing recombinant human interleukin 3 (20units/ml) and granulocyte/macrophage-colony-stimulatingfactor (5 ng/ml). Cells placed in semisolid cultures wereallowed to grow for an additional 10-12 days. The plates werethen scanned with an inverted microscope, and granulocyte/macrophage-colony-forming unit (CFU-GM)-derived colo-nies and clusters (13) were counted. Control cultures weremanipulated in an identical manner but did not containoligomers.

Cell Phenotyping. Primary AML blast cells and CCRF-CEM T-leukemia cells were morphologically identified afterhistochemical staining (modified Wright's stain; Sigma).CCRF-CEM cells were also immunochemically identified(15) with Leu-3a monoclonal antibody (Becton Dickinson).

Philadelphia chromosome-positive CML blasts were de-tected in liquified methylcellulose cultures by reverse tran-scription-polymerase chain reaction (RT-PCR) amplificationof bcr-abl mRNA (19). In brief, total cellular RNA wasextracted from cells and reverse-transcribed with Moloneymurine leukemia virus reverse transcriptase (Bethesda Re-search Laboratories) and 0.1 Ag of a 3'-end primer corre-sponding to a c-abl exon 2 sequence (5'-GCT-TCA-CAC-CAT-TCC-CCA-TTG-T-3') (20). Resulting cDNA was am-plified with 2.5 units of Thermus aquaticus polymerase anda 5' primer corresponding to a bcr gene exon 2 sequence(5'-CAC-AGC-ATT-CCG-CTG-ACC-ATC-A-3') (20). TheRT primer and the 3' PCR primers were identical. AmplifiedDNA was electrophoresed, transferred to nitrocellulose, andthen detected by Southern hybridization with a 40-nt,[y-32P]ATP end-labeled probe that recognized a 325-nt se-quence within the amplified bcr-abl junction cDNA.

Statistical Analysis. Statistical significance of differences innumbers ofcolonies arising in the duplicate control, sense, orantisense plates was determined using the Student t test forunpaired samples. P values <0.05 were judged to be ofstatistical significance.

RESULTSEffect of c-myb Oligomers on T-Leukemia (CEM) Cell and

Normal Hematopoietic Progenitor Cell Growth. c-myb senseoligomers had negligible effects on CEM cell growth inshort-term suspension cultures (Fig. LA), while exposure toc-myb antisense DNA resulted in a daily decline in cellnumber (Fig. 1B). In comparison to untreated controls,antisense DNA inhibited growth by a factor of -100. Growthreduction was not primarily a cytostatic effect because (i) cellviability was reduced 70%o after exposure to the antisenseoligomers and (ii) CEM cell growth did not recover whencells were left in culture for an additional 9 days. In contrast,normal cell viability and numbers were unaffected by anti-sense oligomer exposure (Fig. 1). Growth inhibition was alsosequence-specific (Fig. 2).These experiments suggested that T-leukemia cells could

be killed at an antisense oligomer dose that permitted survivalof normal progenitor cells. However, they did not directlyaddress the oligomers' effects on CFU survival. This issue iscritical since recurrent leukemia derives from malignantCFUs, while normal CFUs are responsible for maintenanceand restoration of normal hematopoiesis. To address this

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FIG. 1. Growth of the CEM human T-cell leukemia line andnormal MNCs maintained in suspension culture for 4 days (11) in thepresence or absence of the c-myb sense (A) or antisense (B) oligo-mers. Cell number and viability (trypan blue exclusion) were as-sessed daily. Results are means + SD offour experiments. (A) In theabsence of the oligomers (control) or in the presence of the sensepreparation (MYB 1; 40 ,ug/ml initially and 10 Mg/ml after 18 hr), theT-leukemia cells continued to divide, whereas the numbers andviability ofthe normal cells remained essentially unchanged. c, CEMcontrol; *, CEM withMYB 1; a, MNC control; O, MNCs with MYB1. (B) Incubation of T-leukemia cells in suspension with c-mybantisense oligomers (MYB 2; 20 ug/ml initially and 5 pg/ml after 18hr) resulted in a daily decline in cell numbers (to 25-30%O of initialcells cultured) and viability (to -30%o). MNCs exhibited only a slightdecline in numbers (to -90%o of initial cells cultured) and viability(>90%o) over the same time period. c, CEM control; *, CEM withMYB 2; a, MNC control; O, MNCs with MYB 2.

question, marrow cells, T-leukemia cells, or a 1:1 mix ofthese cells was exposed to oligomers for 4 days in suspensionculture (5 x 104 cells per ml) and then transferred intomethylcellulose. Ten to 12 days later, total CFU-GM-derivedcolonies [an index of primitive CFU survival (21)] andclusters arising in the dishes were counted (13). Results froma typical experiment, repeated three times, are shown inTable 1. In control cultures, normal MNCs gave rise to 24 +4 (mean ± SD) CFU-GM-derived colonies, whereas anequivalent number of T-leukemia cells gave rise to coloniestoo numerous to count (>>1000 per plate). Sense oligomersdid not affect growth ofeither cell type. In contrast, exposureto an equivalent amount of antisense oligomers reducedcolony/cluster formation by leukemic T cells from >>1000per plate to a maximum of =2 colonies/clusters per 5 X 103leukemia cells plated. Normal MNC colony formation wasnot significantly perturbed at equivalent c-myb antisenseoligomer doses. This result was not unexpected, however,since the antisense concentration was =30% of the previ-ously reported normal MNC inhibitory dose (13).

Proc. Natl. Acad. Sci. USA 88 (1991)

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CONT 10-2.5pig/ml 20-5z.g/ml 40-10kig/ml 80-20j.ig-ml

OUGOMER DOSE

A dose-response effect was also apparent in 1:1 mixingexperiments (Table 1). At c-myb antisense oligomer concen-trations -<6 g/ml (time 0, 5 ,ug/ml; hour 18, 1 ug/ml),leukemia cell growth was florid and precluded positive iden-tification of normal cells. When total antisense dose wasincreased to 12.5 ,ug/ml, leukemia cell colony formation wasgreatly reduced and at least 50%o of the colonies present wereshown to be progeny of normal myeloid CFU. At antisenseoligomer doses 2 25 pg/ml, leukemia cells could no longer beidentified with certainty by morphologic, histochemical, orimmunochemical analyses.

Effect of c-myb Oligomers on Cloning Efficiency of PrimaryAML and CML Blast Cells. The possibility that resultsobtained with the T-leukemia cell line might not be applicableto primary blast cells obtained directly from patients wasconsidered. Primary blast cells, for example, are likely to bemore heterogeneous in maturation state and cell cycle activ-ity. The effect ofc-myb antisense oligomers on primary AMLblast cell cloning efficiency was therefore evaluated (Table2). Patient blast cells were isolated on Ficoll/Hypaque den-sity gradients and then placed in suspension culture. Cultureswere either untreated (control) or exposed to c-myb sense orantisense oligodeoxyribonucleotides (40 ,g/ml initially and10 ,ug/ml after 18 hr). After a total of -24 hr in suspension,

Table 1. Effect of c-myb oligomer exposure on colony/clusterformation by CEM human T-leukemia cells and normal MNCs

No. added Colony/Cells plated per ml Oligomer added* clustert

MNCs 5 x 104 None 24 ± 4MYB 1 (20; 5.0) 31 ± 4MYB 2 (20; 5.0) 30 ± 6

CEM 5 x 104 None TNTCMYB 1 (20; 5.0) TNTCMYB 2 (20; 5.0) 1 ± 1

MNCs + CEM 5 x 104 None TNTCof each MYB 1 (20; 5.0) TNTC

MYB 2 (2; 0.5) TNTCMYB 2 (5; 1.0) TNTCMYB 2 (10; 2.5) 41 ± 5MYB2 (20;5.0) 34±1

Cells were exposed to oligomers (see Fig. 1 legend for description)for 4 days in suspension cultures and then transferred to semisolidmedium. After 12 days in culture, colonies and clusters were countedin paired dishes with an inverted microscope.*Amounts (Jg/ml) added to the culture medium at 0 hr and 18 hr,respectively, are shown in parentheses.tTNTC, too numerous to count (>1000 colonies).

FIG. 2. Growth in 4-day sus-pension cultures in the presence offour different c-myb oligomerpreparations. CEM T-leukemiacells (105) were seeded into 500 Alof tissue culture medium contain-ing the indicated oligomer at 10,20, 40, or 80 ,ug/ml. Eighteenhours later, 25% of the time 0oligomer dose was added. Cellswere counted 4 days later. Ab-scissa indicates 0-hr and 18-hrdoses. Bars indicate cell counts(per ul) obtained with each sense(S), scrambled sequence (ScSeq),and antisense (AS) oligomertested (MYB 21, MYB 24, MYB12, and MYB 22). Each oligomerhad a paired control well (CONT)containing no oligomer.

cells (2-5 x 104 per dish) were transferred into plasma clot ormethylcellulose cultures supplemented with recombinant hu-man hematopoietic growth factors. After 10-12 days coloniesand clusters were counted. In each culture, the number ofcolonies or clusters arising in the untreated control disheswas assumed to represent maximal (i.e., 100%) growth forthat patient. Colonies or clusters arising in the antisense-treated dishes were then expressed as a percentage of this

Table 2. Effect of c-myb oligomers on primary AML cell colony/cluster formation

Colonies, % control Clusters, % control

Case Sense Antisense Sense Antisense1 86 18 (0.058) 60 37 (0.080)4 NG NG 90 28 (0.036)5 NG NG 70 22 (0.101)6 NG NG 79 22 (0.026)7 170 100 (0.423) 76 128 (0.502)8 92 11 (0.008) 96 46 (0.020)

10 NG NG 190 216 (0.034)11 45 14 (0.021) 58 21 (0.084)14 68 01 (0.152) 90 53 (0.071)15 66 81 (0.736) 100 100 (0.8%)16 NG NG 66 24 (0.001)17 NG NG 16 8 (0.023)18 NG NG 110 77 (0.164)19 113 116 (0.717) 91 91 (0.763)20 92 09 (0.051) 100 50 (0.009)21 94 00 (0.006) 90 06 (0.004)22 80 13 (0.001) 103 11 (0.015)23 63 06 (0.001) 74 27 (0.004)24 87 17 (0.002) 91 26 (0.018)25 100 00 (0.019) 107 38 (0.364)26 76 00 (0.009) 89 00 (0.001)27 79 21 (0.014) 59 18 (0.043)28 88 20 (0.009) 94 152 (0.096)Blast cells were isolated from the peripheral blood of AML

patients and exposed to sense or antisense oligomers. Colonies andclusters were enumerated and values were compared with growth incontrol cultures, which contained no oligomers. For each case, thenumber of colonies or clusters arising in the untreated control disheswas assumed to represent maximal (100%o) growth for that patient.The numbers of colonies or clusters arising in the oligomer-treateddishes are expressed as a percentage of this number. NG, no growth.The statistical significance (determined by Student's t test forunpaired samples) of the change observed in the antisense-treateddishes relative to the untreated control is given as a P value inparentheses.

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Table 3. Effect of c-myb oligomers on primary CML cell colony/cluster formation

Myeloid colony formation by CML blast cells

Case Control Sense Antisense

1 217, 120 (X = 169) 102, 173 (X = 138) 150, 92 (X = 121)2 94 67 183 453, 494, 507, 479 (X = 483) 488, 539 (X = 513) 49, 34 (X = 42)4 190, 322(X = 256) 233, 202(X = 218) 49, 86(X = 68)5 634, 549 (X = 592) 1340, 347 (X = 843) 223, 122 (X = 173)

Blast cells were isolated from peripheral blood of CML patients in blast crisis and exposed tooligomers as detailed in the text. Colonies arising in dishes containing cells exposed to sense andantisense oligomers were enumerated. Growth in control (untreated) dishes is also indicated. Valuesare given for duplicate or quadruplicate dishes (for case 2, only a single dish was available for scoring;duplicate was used for PCR analysis). Mean values (X) are indicated in parentheses.

number. The statistical significance (P value) of the changeobserved in the antisense-treated cultures relative to un-treated controls is shown in Table 2.Of the 28 cases examined, colony- and cluster-formation

data were obtainable in 16 and 23 cases, respectively (Table2). Five cases were unevaluable because neither colonies norclusters grew in control dishes. After antisense oligomerexposure no change, or an apparent increase, in leukemia cellcolony and/or cluster formation was observed in 5 of the 23total evaluable cases (nos. 7, 10, 15, 19, and 28). A decline ineither colony or cluster formation in comparison to growth inuntreated cultures was observed in 18 of the 23 (78%)evaluable cases. Of the 16 evaluable colony-formation cases,c-myb antisense DNA inhibited development in 14 (88%), andthis was statistically significant (P < 0.05) in 12 (75%). In theother 2 cases the P values were 0.058 (case 1) and 0.152 (case14). A decrement in cluster formation was observed in 19 outof 23 (83%) total evaluable cases. This decrement wasstatistically significant in 12 of 19 (63%) and of borderlinesignificance (P < 0.10) in 16 of 19 (84%). The residual numberof leukemic colonies and clusters found in the antisense-treated cases was 10 ± 8% (mean ± SD) and 26 ± 15%,respectively, of untreated control cultures. Accordingly,primary leukemic progenitor cell growth was markedly in-hibited by exposure to the c-myb antisense oligomers.

Similar experiments were carried out with blast cells fromfive CML blast-crisis patients (Table 3). Colony formationwas inhibited a mean of 79 ± 9% in the four cases (nos. 2-5)in which a significant decrement in colony formation wasobserved.c-myb Oligomers as Putative Bone Marrow Purging Agents.

The above experiments strongly suggested that leukemia cellgrowth could be preferentially inhibited after exposure toc-myb antisense oligodeoxynucleotides. We speculated thatantisense oligomers might be used as ex vivo bone marrowpurging agents. To test this hypothesis, normal MNCs weremixed (1:1) with primary AML (case 26, Table 2) or CMLblast cells and then exposed to the oligomers in a slightlymodified protocol designed to test the feasibility of a moreintensive antisense exposure. To this end, an additionaloligomer dose (20 ,ug/ml) was given just prior to plating thecells in methylcellulose. In control growth factor-stimulatedcultures, leukemia cells formed 25.5 ± 3.5 (mean ± SD)colonies and 157 ± 8.5 clusters per 2 x 105 cells plated.Exposure to c-myb sense oligomers did not significantly alterthese numbers (19.5 ± 0.7 colonies and 140.5 ± 7.8 clusters;P > 0.1). In contrast, equivalent concentrations of antisenseoligomers totally inhibited colony and cluster formation bythe leukemic blasts. Colony formation was also inhibited inthe plates containing normal MNCs, but only by -50o incomparison to untreated control plates (control colony for-mation, 296 ± 40 per 2 x 105 cells plated; treated colonyformation, 149 ± 15.5 per 2 x 105 cells).

Detection of Minimal Residual Leukemic Cells After Expo-sure to c-myb Antisense Oligomers. In antisense-treated co-

cultures of normal MNCs and primary AML blast cells (1:1ratio), only normal colonies could be identified. Neverthe-less, the possibility that the cultures contained residualleukemic CFUs could not be ruled out. We therefore con-ducted coculture studies with CML blast cells, since theyexpress the tumor-specific bcr-abl fusion gene, which iseasily detectable by RT-PCR. Accordingly, CML blast cells(5 X 104 per ml), normal MNCs (5 x 104 per ml), or a 1:1 mixof these cells was maintained in suspension cultures for 9days in the absence or presence of c-myb oligomers. Asbefore, oligomers were added to the cultures at their incep-tion (40 pug/ml) and after 18 hr in suspension (20 lug/ml). After9 days, the cultures were split into two aliquots. One wasused for RT-PCR detection of bcr-abl mRNA, the other forcell cloning in methylcellulose to directly assess normal andleukemic CFU survival. As expected, bcr-abl transcriptswere not found in the normal cell culture (Fig. 3, lane 1) butwere easily detected in the CML cells exposed to c-myb senseoligomers (lane 2). In comparison, extremely low expressionwas observed in CML cells exposed to the c-myb antisenseoligomers (lane 3). The parallel CFU-GM assays revealedthat normal cells survived the oligomer exposure with littledecrement in colony number. Normal control MNCs formed140 CFU-GM-derived colonies, while MNCs exposed toc-myb antisense oligomers formed 131. CML colony forma-tion accurately reflected the bcr-abl expression data: control

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FIG. 3. Detection ofbcr-ablmRNA transcripts in normal MNCs,CML blast cells, or 1:1 mixtures of each after exposure to c-myboligodeoxynucleotides. Total RNA was extracted from 10- cells andreverse-transcribed with a 3' primer specific for the c-abl exon 2. Theresulting cDNA was amplified by PCR with a primer pair specific forthe bcr-abl junction. Amplified DNA was detected by Southernblotting with a 40-nt c-abl probe. Lane 1, normal MNCs; lane 2, CMLblast cells maintained in suspension culture for 9 days after exposureto c-myb sense oligomers; lane 3, CML blast cells maintained insuspension culture for 9 days after exposure to c-myb antisenseoligomers; lane 4, a 1:1 mix of CML blast cells and normal MNCsexposed to c-myb sense oligomers in liquid suspension culture for 24hr and then transferred to methylcellulose culture for 12 days; lane5, a 1:1 mix of CML blast cells and normal MNC exposed to c-mybantisense oligomers in liquid suspension culture for 24 hr and thentransferred to methylcellulose for 12 days.

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CML cells formed 44 colonies, while antisense-treated cellsformed only 3 small clusters.

Finally, cultures of normal MNCs and CML cells (1:1 mix)that had been exposed to oligomers for 24 hr and then directlyplated into methylcellulose were also analyzed for bcr-ablexpression by RT-PCR. Transcripts were found in the dishescontaining cells exposed to c-myb sense oligomers (Fig. 3,lane 4), but only a faint band was demonstrable from cellsmixed 1:1 and exposed to the antisense oligomers (lane 5). Inaggregate, these'data suggest that highly efficient purging ofleukemic elements from normal marrow cells is possible.

DISCUSSIONThe results are consistent with the hypothesis that lymphoidand myeloid leukemic blast cell growth is highly dependenton c-myb gene function. Proliferation and cloning efficiencyof continuous human T-leukemia cells (Figs. 1 and 2), andprimary AML (Table 2) and CML (Table 3; Fig. 3) blast cellswere significantly inhibited by c-myb antisense oligodeoxy-nucleotides. In addition, we provided data suggesting thatleukemic hematopoietic cells are much more dependent thantheir normal counterparts on c-myb gene function: manynormal CFUs survived and remained functional after expo-sure to c-myb antisense oligomer doses that preferentiallyinhibited leukemic CFU survival and cloning efficiency (Ta-ble 1; Figs. 1 and 3).The mechanism whereby deprivation of c-myb function

leads to preferential inhibition of leukemia cell growth re-mains unclear. We have previously demonstrated that Mybprotein is required for G1/S transition by phytohemaggluti-nin-stimulated normal lymphocytes (7) and by actively cy-cling normal hematopoietic progenitor cells (18). Accord-ingly, c-myb function might be of greatest importance for cellcycle progression in proliferating cells. This hypothesis ex-plains the antisense-mediated inhibition of rapidly prolifer-ating T-leukemia cells, and the greater inhibition of leukemiccolony formation as compared with cluster formation (Table2). Differential sensitivity, on the other hand, might beexplained by our observations that c-myb function is strictlyrequired for development of normal hematopoietic progeni-tor cells at a specific stage of maturation and that require-ments at other developmental levels are less stringent (18).Thus, if leukemic blasts were blocked as a cohort at asusceptible maturation stage this might at least partly explainour findings. Alternatively, Kastan et al. (22) detected Mybprotein in cells that were not actively proliferating andshowed that Myb levels did not necessarily correlate with cellcycle activity. Consistent with these data is the demonstra-tion that a v-myb-encoded protein functions as a transcrip-tional activator for mim-1, which encodes a maturation-related myeloid granule protein (23). Therefore, since Myb islikely to exert a number of important but incompletelydefined cellular functions, the greater sensitivity of leukemiacells to Myb deprivation may reflect a generally lowertolerance for perturbation of c-myb function in leukemiacells. Elucidation of those genes whose expression is regu-lated by Myb may ultimately provide better insight into thisphenomenon.The ability to block gene function with antisense oligode-

oxynucleotides is a powerful research tool (24, 25). Sinceoverexpression of protooncogenes may be important in thedevelopment of many human malignancies (26), includingthose of the hematopoietic system (27), the antisense strategycould have significant therapeutic potential as well. Sinceonly -25%o of conventionally treated leukemia patients be-come long-term survivors (28, 29), the need for novel treat-ment approaches is compelling. Using antisense DNA for exvivo marrow purging might fulfill one such need and wouldcircumvent numerous technical problems related to the invivo administration ofthese compounds. Further, since many

more CFUs-GM survive oligomer exposure than appear tosurvive conventional chemical purging (30), an improvedengraftment rate might result. High-efficiency, and selective,leukemic cell killing will have to be demonstrated beforeantisense oligomers can be used clinically for purging (30).Nevertheless, we believe that the results reported here areprovocative, and we hope that they will serve to stimulate thedevelopment of in vivo models for testing the cancer thera-peutic potential of antisense DNA.

We thank Dr. J. K. DeRiel for synthesis of the oligodeoxynucle-otides, and Genetics Institute (Cambridge, MA) for kindly donatingthe recombinant human granulocyte/macrophage colony-stimulatingfactor and interleukin 3. The editorial assistance of Ms. Elizabeth R.Bien is gratefully acknowledged. This research was supported in partby grants from the National Cancer Institute (CA36896, CA01324, andCA46782), a grant from the American Cancer Society (CH-455), agrant from the W. W. Smith Charitable Trust, and a grant from theAssociazone Italiana Ricerca Sul Cancro. D.C. was supported by a G.Ghirotti Foundation Fellowship. D.V. was supported by NationalInstitutes of Health Training Grant CA094859. B.C. is a Scholar oftheLeukemia Society of America. A.M.G. is the recipient of a ResearchCareer Development Award for the National Cancer Institute.

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