Effects of recombinant human tumor necrosis factor-α on the surface phenotype and the growth of human malignant glioma cell lines

(CANCER RESEARCH 46, 4357-4361, September 1986]

Effects of Recombinant Human Tumor Necrosis Factor a, Recombinant Human 7-

Interferon, and Prostaglandin E on Colony Formation of Human HematopoieticProgenitor Cells Stimulated by Natural Human Pluripotent Colony-

stimulating Factor, Pluripoietin a, and Recombinant Erythropoietinin Serum-free Cultures1

Li Lu,2 Karl Weite, Janice L. Gabrilove,3 Giao Hangoc, Edward Bruno, Ronald Hoffman, and Hal E. Broxmeyer

Departments of Medicine (Hematology/Oncology) [L. L., G. H., E. B., R. H., H. E. B.J, Microbiology and Immunology [L. L., H. E. B.], the Walther Medical ResearchInstitute, the Indiana Elks Cancer Research Center, Indiana University School of Medicine, Indianapolis, Indiana 46223, and Laboratories of Molecular Hematology{K. W.] and Developmental Hematopoiesis [J. L. G.], The Sloan Kettering Institute for Cancer Research, New York, New York 10021

ABSTRACT

The influences of pure human pluripotent colony-stimulating factor,highly purified pluripoietin or, pure recombinant human tumor necrosisfactor a, pure recombinant human -v-interferon, and natural prostaglandin

Ki (PGEi) were evaluated on colony formation of multipotential anderythroid progenitor cells in the presence of recombinant erythropoietinand hemin and on colony formation of granulocyte-macrophage progenitors in normal human marrow cultured in the presence or absence ofserum. Serum was replaced by bovine serum albumin, iron-saturatedtransform!, cholesterol, and calcium chloride. Increasing concentrationsof pluripotent colony-stimulating factor and pluripoietin a stimulatedincreasing numbers of colonies from nonadherent low-density T-lympho-cyte-depleted cells in the absence and presence of serum. Growth wasusually greater in the presence of serum and on a unit basis pluripoietina was more active than pluripotent colony-stimulating factor. Recombi-nant human tumor necrosis factor a and recombinant human 7-interferonsuppressed colony formation colony forming unit-granulocyte-macro-phage, burst forming unit-erythroid, and colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte; I'(,K, suppressed colony formation by colony-forming unit-granulocyte-macrophage, stimulated colonyformation by burst forming unit-erythroid, and had no effects on colonyformation by colony forming unit-granulocyte-erythroid-macrophage-me-gakaryocyte in both serum-containing and serum-free medium. The l'( ; I-,enhancing effects on erythroid colony formation required T-lymphocytes.Thus, results are similar using serum-containing and serum-free culturesof human bone marrow cells and serum-free defined culture medium canbe used to study the mechanisms of action of purified natural andrecombinant growth and suppressor molecules in vitro.

INTRODUCTION

The growth of multipotential, erythroid, and granulocyte-macrophage progenitor cells is influenced in vitro by specificstimulating and suppressing molecules (1-3). Some of the molecules implicated in the growth regulation of human hemato-poietic progenitor cells in vitro include PPO4 (4), PPO-a (5),

Received 3/31/86; accepted 5/16/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1These studies were supported by USPHS Grants CA 36740 and CA 36464

to H. E. B. from the National Cancer Institute and by a grant from the DeutscheForschungsgemeinschaft to G. H.

2To whom requests for reprints should be addressed, at Department ofMedicine (Hematology/Oncology), the Walther Medical Research Institute, theIndiana Elks Cancer Research Center, Indiana University School of Medicine,541 Clinical Drive, Indianapolis, IN 46223.

' Recipient of National Cancer Institute Clinical Investigator Award and a

Junior Faculty Fellowship Award from the American Cancer Society.4The abbreviations used are: CFU-GEMM, colony-forming unit-granulocyte,

erythroid, macrophage, megakaryocyte; BFU-E, burst-forming unit-erythroid;CFU-GM, colony-forming unit-granulocyte-macrophage; PPO, pluripotent colony-stimulating factor; PPO-a, pluripoietin a; rEPO, recombinant erythropoietin;rHuTNF-a, recombinant human tumor necrosis factor a; r11u11N -:. recombinanthuman -Hnterferon; n, natural; PGE, prostaglandin E; FBS, fetal bovine serum;CM, conditioned medium; IMDM, Iscove's modified Dulbecco's medium.

EPO (6), HuIFN-T (7-9), TNF (10, 11), and PGE (12-16).The complementary DNA and/or genes have been cloned andexpressed for human EPO (17, 18), HuTNF (19-21), andHuIFN-7 (22).

Studies evaluating the regulation of hematopoiesis in vitrohave been performed mainly in the presence of serum, whichcontains complex mixtures of proteins and other defined andundefined molecules which vary between lots, and have includeduse of crude preparations of stimulating molecules. This makesit difficult to rule out potential influences of factors other thanthose one is attempting to evaluate. Culture conditions for thegrowth of hematopoietic cells under serum-free conditions havebeen reported (23-29) but these studies did not always usepurified growth-stimulating molecules. With serum-free defined culture conditions and purified regulatory factors it isnow possible to more precisely evaluate the influence of specificregulatory molecules on the growth of hematopoietic progenitorcells in vitro.

In this report we compare the effects of nPPO, nPPO-a,rEPO, rHuTNF-a, rHuIFN-f, and nPGE, on colony formationin vitro by hematopoietic progenitor cells present in low-densityor nonadherent low-density T-lymphocyte-depleted normal human bone marrow grown either in the presence of FBS or inthe presence of defined medium containing bovine serum albumin, iron-saturated transferrin, cholesterol, and calciumchloride.

MATERIALS AND METHODS

Cells and Cell Separation Procedures

Bone marrow cells were obtained by aspiration from the posterioriliac crest of healthy volunteers who had given informed consent.Unseparated nucleated huffy coat cells were obtained by centrifugationof whole marrow aspirates at 1SOOrpm for 10 min and aspiration ofthe WBC which overlay the RBC layer. Low-density cells (< 1.077 g/ml) were obtained after separation on Ficoll-Hypaque (Pharmacia FineChemicals, Piscataway, NJ) at 1500 rpm for 30 min, washed threetimes, and resuspended in IMDM (Gibco, Grand Island, NY) containing 10% FBS (Hyclone; Sterile Systems, Inc., Logan, UT). Cells werefurther separated into nonadherent and adherent cells after incubationon plastic tissue culture dishes (Falcon 3003; Falcon Plastics, Div.,Becton Dickinson and Co., Rutherford, NJ) for 90 min at 37°Cunder

5% CO2. Nonadherent cells were collected by gently swirling the dishesand slowly removing the suspension cells. Nonadherent cells preparedin this manner routinely contained <2% a-naphthyl acetate esterase-positive cells. Nonadherent low-density cells were further separatedinto erythrocyte rosette-positive and negative populations as describedelsewhere (15). The erythrocyte rosette positive fraction containedapproximately 95% T-cells as determined using the OKTlla (anti-sheep RBC) pan T-cell antibody. The nonadherent low-density T-lymphocyte-depleted marrow cell fraction usually contained <5%

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HEMATOPOIETIC REGULATION IN SERUM-FREE CULTURES

OKT1 la-positive cells and were treated with OKT1 la and complementin order to remove the remaining T-lymphocytes. All cells were washedthree times in lMI >\I without serum before resuspending in the appropriate medium.

Colony Assays

CFU-GEMM and BFU-E. The colony assays for CFU-GEMM andBFU-E have been described (14, 15, 30). Cells were plated at theconcentrations listed in the table legends in 35-mm tissue culture dishescontaining a I ml mixture of 1MDM, 1% methylcellulose, 30% FBS ascontrol, or a serum-free mixture (described below) in place of FBS, 5%medium conditioned in the absence of serum by human bladder carcinoma cell line 5637 (5637 CM) (4, 31), or different concentrations ofpure PPO (4) or highly purified PPO-a (5) as stimulators, and 5 x 10~5

M 2-mercaptoethanol. Hemin (Eastman Kodak Co., Rochester, NY)was added at a final concentration of 0.1 HIM(15, 30). Two units ofrecombinant EPO (Amgen, Thousand Oaks, CA) were added to theculture medium. Indomethacin ( 10 " M) was used to prevent endogenous prostaglandin production (12). Dishes were incubated at 37'C in

a humidified atmosphere of 5% CO2 and low (5%) oxygen tension.Low oxygen tension was maintained using an Oxyreducer (RentingBioinstruments, Rochester, NY). Conditions of low oxygen tensionwere used to increase colony formation (32, 33) and the sensitivity ofcells to respond to inhibiting and stimulating molecules (8, 15, 34).Colonies of CFU-GEMM and BFU-E were scored in the same dishesfrom three plates per point after 14 days of incubation.

CFU-GM. Human bone marrow cells were plated at different cellconcentrations in 1 ml of 0.3% agar culture medium (Difco Laboratories, Detroit, MI) that included McCoy's Medium 5A supplemented

with additional essential and nonessential amino acids, glutamine,serine, asparagine, sodium pyruvate (Gibco), and 10% prescreened heat-inactivated (56'C for 0.5 h) FBS, or the serum-free mixture as described

below with 10% serum-free 5637 CM or different concentrations ofPPO or PPO-a added as stimulators. Dishes were incubated at 37°Cin

a humidified atmosphere of 5% CO2 and 5% O2. Colonies were scoredafter 7 and 14 days of incubation because the colonies scored at day 7(day 7 CFU-GM) appear to be more mature progenitor cells than day14 CFU-GM (35, 36). Three dishes were scored per point.

Preparation of Serum-free Mixture

The serum-free mixture was prepared as described by others (28).The final concentrations per ml of the mixture contained 10 mg bovineserum albumin, 300 «igiron-saturated human transferrin, 7.8 ¿igcholesterol, and 280 ¿tgcalcium chloride. These ingredients were purchasedfrom Sigma Chemical Co. (St. Louis, MO).

Preparation of Serum-free 5637 CM

A serum-free culture medium mixture containing 10 * M hydrocortisone (Sigma), iron-saturated human transferrin (50 Mg/ml; Sigma)and insulin (5 Mg/ml; Sigma) were added to the cells in place of theFBS-containing medium. After 5 days of incubation at 37'C under 5%

CO2, when a confluent monolayer was obtained, this serum-free 5637CM was collected.

Materials

Pure rTNF-a and pure r!FN--y were generous gifts from Dr. H.Michael Sheppard (Genentech, Inc., South San Francisco, CA). Thespecific activity for rTNF-a using L-cells and actinomycin D, was 5.0x IO7 units/mg. These materials were kindly retitered by Dr. Berish

Rubin (The Lindsley F. Kimball Research Institute of the New YorkBlood Center, New York, NY) as described (37, 38). Actinomycin Dincreases the TNF assay sensitivity. When TNF-a was titrated againstL-cells in the absence of actinomycin D the specific activity was 1.0 xIO6 units/mg (11). The specific activity of rIFN--y was 5x10* units/mg. PPO and PPO-a were purified and titered for activity in a humanCFU-GM assay as described (4, 5). The PPO purification involvedsequential ammonium sulfate precipitation, ion-exchange chromatog-raphy, gel filtration, and reversed-phase high-performance liquid diromatography. The purified protein has a molecular weight of 18,000 insodium dodecyl sulfate-polyacrylamide gel electrophoresis both by silver staining and by elution of biological activity from a correspondinggel slice. It has an isoelectric focus point of 5.5. The specific activity ofthe PPO in the CFU-GM assay is 1.5 x 10" units/mg of protein (4).

The biological characteristics of PPO have been reported (4, 39). ThePPO-a purification scheme was similar to that for PPO. PPO-a has amolecular weight of 16,500 on sodium dodecyl sulfate-polyacrylamidegel electrophoresis and it has an elution profile different from that ofPPO on ion-exchange chromatography and reversed-phase, high-performance liquid chromatography. This material, although not purifiedto homogeneity, has a specific activity of 1.0 x 10" units/mg of proteinin a CFU-GM assay (5). The biological characteristics of PPO-a havebeen described (5). PGEi and indomethacin were purchased fromSigma.

Statìstica!Analysis

Levels of significance for comparisons between samples were determined using the Student distribution. The results are expressed as themean ±SE of three plates per point for each experiment.

Table 1 Growth of hematopoietic progenitors stimulated by PPO and PPO-a in serum-containing and serum-free culturesNonadherent low-density T-lymphocyte-depleted normal human bone marrow cells were plated at 5 x HI4 ml. The HI I I < I I ( ;i MM plates contained 2 units

rEpo/ml and 0.1 HIM hemin. This represents the data from 1 of 2 comparable complete experiments. Similar data for selected points were obtained in 7 otherexperiments. Results are expressed as mean colonies ±SE.

ColoniesDay

7CFU-GMAddition(units)No

addedfactorsPPO

(2000)PPO(1000)PPO(500)PPO(250)PPO(125)PPO(63)PPO-o(lOOO)PPO-a

(500)PPO-a(250)PPO-a(125)PPO-a(63)PPO-a(32)PPO-a

(16)+

serum5±2235

±9247±8156

±1679±233

±27±2145

±8138±681

±145±221

±16±20.7

±0.7-

serum0260

±9207±1199

±635±616

±20102

±1105±264±333

±213±20.7

±0.70Day

14CFU-GM+

serum7±

1287

±7268±7146±598±543

±19±2214±

11223±11215

±3102±249±519±43±2-

serum0147

±7136±571±433

±25±20114±5116±5107

±647±213±21

±10BFU-E+

serum4±

195

±583±255±427±313

±27±2101

±798±587

±140±224

±113±25±1-

serum028

±116±210±25±20016

±218±110±15±20.7

±0.700CFU-GEMM+

serum07.3

±0.95.3±0.92.3±0.91.0

±0.6004.3

±0.94.3±0.33

±0.62.3±0.90.7±0.300-

serum2.7

±0.71.7±0.300002.7

±0.92.3±0.31.3±0.70.3±0.3000

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HEMATOPOIETIC REGULATION IN SERUM-FREE CULTURES

day 7 CFU-CMday 14 CFU-CM

BFU-ECFU-GEMM

0.1 0.5 1.0 2.0 4.0

Number of Bone Marrow Cells Plated (xlO* )

day 7 CFU-CM •—•day 14 CFU-CM O—O

BFU-E A—ACFU-CEMM •—•

0 0.1 0.5 1.0 2.0 4.0Number of Bone Marrow Cells Plated (xlO4 )

Fig. I. Influence of increasing numbers of nonadherent low-density Ihmphocyte-depleted normal human bone marrow cells on colony formation byhematopoietic progenitor cells plated in the presence of a constant source of PPO(1000 units/ml) (top) or PPO-a (500 units/ml) (bottom). Data are from one oftwo representative experiments.

RESULTS

Influence of PPO and PPO-a on Colony Formation. Both PPOand PPO-a supported the growth of colonies from CFU-GEMM and BFU-E in methylcellulose in the presence of rEPO

and hemin and the growth of colonies from day 7 and day 14CFU-GM in agar using nonadherent low-density T-lympho-cyte-depleted normal human bone marrow cells (Table 1). Increasing concentrations of PPO and PPO-a stimulated increasing numbers of colonies in serum-containing and serum-freecultures, although growth in most cases was greater in serum-containing than in serum-free medium. PPO (1000 units) andPPO-a (500 units) support the maximum growth of all thehematopoietic progenitor cells. On a unit basis, PPO-a wasmore active than PPO in the stimulation of colony formation.As shown in Fig. 1, with constant levels of PPO and PPO-a,colony numbers increased with increasing numbers of cellsplated under serum-free conditions. No differences were detected in the morphology of colonies stimulated by PPO orPPO-a when cultures grown in the presence or absence ofserum were compared (data not shown).

Influence of rHuTNF-a, rHuIFN-7, and PGE, on ColonyFormation Stimulated by 5637 CM or PPO. The results weresimilar when serum-containing and serum-free cultures wereevaluated using low-density human bone marrow cells and werenot significantly different when 5637 CM or 100, 500, or 1000units of PPO were used (Table 2). rHuTNF-a (100 units) andrHuIFN-7 (100 units) suppressed colony formation by day 7CFU-GM, BFU-E, and CFU-GEMM. PGE, (IO'7 M) sup

pressed colony formation by day 7 CFU-GM, stimulated colonyformation by BFU-E, and had no significant effect on colonyformation by CFU-GEMM. The dosages of HuTNF-a,rHuIFN--y, and PGE| used in this study were those yielding

maximal effects (11, 14). A comparative analysis of the influence of rHuTNF-a, rHuIFN-7, and PGEi on colony formationby low-density versus nonadherent low-density T-lymphocyte-

depleted bone marrow cells grown in the presence of 1000 unitsPPO under serum-containing or serum-free conditions is shownin Table 3. Results were similar for rHuTNF-a and rHu!FN--ywhether low-density or nonadherent low-density T-Iymphocyte-depleted bone marrow cells were used and whether cells weregrown in the presence or absence of serum. Both moleculessuppressed colony formation by day 7 and 14 CFU-GM and by

Table 2 Effects ofrHuTNF-a, rHuIFN-y, and natural PGEt on colony formation from hematopoietic progenitor cells stimulated by serum-free 5637 CM or PPOunder serum-containing and serum-free conditions

Low-density human bone marrow cells are plated at lOVml. 5637 CM was plated at 10% (v/v) for CFU-GM and at 5% (v/v) for BFU-E/CFU-GEMM assays.% of change from control'

5637 CM PPO

100 units 500 units 1000 units

FactorsaddedDay

7 CFU-GMrHuTNF-a (100 units)rHuIFN-y (100 units)PGE,(10-7M)BFU-ErHuTNF-a

(100 units)rHuIFN-f( 100 units)PGE, (IO'7M)CFU-GEMM

rHuTNF-a( 100 units)rl lull N %(100 units)PGE, (IO-7 M)+

serum-91

±1*-54±6*

-49 ±1*-72

±ll*-59 ±0?+48 ±(f4-82

±\V4-62 ±Y4

-8 ±7—

serum-92

±1*-44 ±\tf4-41 ±T4-72

±9*-54±y4+55±5**-100

±0"-50 ±0+19 ±6+

serum-93

±6*-51±6"*-40

±i'4-76

±15*'-53 ±S0-*+49 ±6Ctrf—

serum-95

±2*-si±y4-49±2'4-67

±16^-54±if4

+44 ±6"+

serum-87

±10*'-41 ±(f4-35 ±1CJ/-74

±16M-53 ±3'+50 ±l"-80

±2<f-48 ±12-35 ±15-serum-92

±3*-49 ±r4-41 ±\c4-71

±8W-57 ±2"'+56 ±1"-100±0C

-63 ±3'

0±0+

serum-93

±3*-5^4»-^-45 ±\"4-82

±12M-62 ±4M+45

±(f4-78

±22e-*-56 ±1"

+3 ±3-

serum-96

±1*-51 ±6'-45

±1J-73

±14W-56 ±I"4+49

±y-82

±18"-50 ±0*

-2± 11" Mean percentage of change from medium control in absence of added factors ±SE for 2 complete experiments. The control mean numbers of colonies in the 2

experiments were (in the following order: 5637, +, -; 100 units; +, -; 500 units; +, -; 1000 units, +, -): for Day 7 CFU-GM, 114-475, 89-384; 139-158, 54-181;151-252,49-229; 145-418,65-377; for BFU-E, 41-57, 25-32; 17-48, 5-8; 31-55, 13-22; 66-84; 31-39; and for CFU-GEMM, 4-7,1-3; 0-0,0-0; 2-4, 1-3; 4-5,3-3.

* Significant from control, at P < 0.0001.CP<0.0\."P< 0.001.'P<0.05.

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Table 3 Influence ofmonocyle and T-lymphocyte depletion from human bone marrow cells on the effects ofrHuTNF-a, rHuIFN-y, and natural PGE, on colonyformation from hematopoietic progenitor cells stimulated by PPO

Low-density cells (105/ml) and nonadherent low-density T lymphocyte depleted cells (5 x l()4/ml) were plated in the presence of 10 '•M indomethacin and

stimulated with 500 units PPO/ml.

Serum Factors added

Colony nos."

Day 7 CFU-GM Day 14 CFU-GM BFU-E CFU-GEMM

Low-density cells

+ Control medium+ rHuTNF-a( 100 units)+ rHuIFN-7 (100 units)+ nPGE, (10~7 M)

— Control mediumrHuTNF-o (100 units)rHuIFN-7 (100 units)nPGE, (IO-7 M)

418 ±2443 ±4 (-90)*

189± 13 (-55)'236 ±16 (-44)c

377 ±819 ±5 (-95)*

212 ±10 (-44)'205 ±2 (-46)'

151 ±311 ±2(-93)*86 ±4 (-43)'78 ±2 (-48)'

179 ±89 ±2 (-95)*

92 ±4 (-49)c89 ±4(-51)*

Nonadherent low-density T-lymphocyte-depleted cells

+ Control medium 409 ±1+ rHuTNF-a (100 units) 25 ±4 (-94)*+ rHuIFN-y (100 units) 214 ±4 (-48)*+ nPGE, (10~7 M) + erythrocyte 214 ±6 (-48)*

rosette-positive populations(10%)

Control medium 283 ±8rHuTNF-a ( 100 units) 17 ±2 (-94)*rHuIFN-7 (100 units) 147 ±5 (-48)*nPGE, (IO-7 M) + erythrocyte 155 ±4 (-45)*

rosette-positive populations(10%)

121 ±59 ±2 (-92)*

56 ±1 (-51)*58 ±1 (-52)*

86 ±37 ±2 (-92)*

46 ±1 (-47)c45 ±1 (-47)'

84 ±35 ±1 (-94)*

36 ±2 (-57)*126 ±2(+50)'

31 ±24 ±1 (-87)*

16 ±3 (-49)'48 ±3 (+52)'

100 ±68 ±1 (-92)*

39 ±4(-61)*136 ±6(+36)'

33 ±26 ±1 (-83)*

14 ±1 (-47)'55 ±4 (+68)'

5.3 ±0.90(-100)'2.3 ±0.3 (-56)1*

5.7 ±0.7 (+6)

3.3 ±0.70(-100)'1.7 ±0.3 (-50)'

3.7 ±0.3 (+10)

6.3 ±0.70(-100)*2.3 ±0.3 (-63)'5.0 ±1.2 (-21)

2.0 ±10 (-100)'0.7 ±0.3 (-65)1.7 ±0.7 (-18)

* Mean ±SE for 1 of 2 representative experiments; the percentages of changes from control medium are given in parentheses.* Significant from control, at P< 0.0001.'/>< 0.001.

BFU-E and CFU-GEMM. Since the enhancing effect of PGEon erythroid colony formation requires the presence of T-cells(15, 16, 40), the influence of PGEi on colony formation fromnonadherent low-density T-lymphocyte-depleted marrow wasassessed in the presence of 10% erythrocyte rosette-positivecells. PGE] suppressed colony formation by day 7 and day 14CFU-GM, enhanced colony formation by BFU-E, and had nosignificant effect on colony formation by CFU-GEMM in thepresence and absence of serum.

DISCUSSION

Advancements in cell separation procedures, purification ofmolecules, recombinant technology, and cell culture conditionshave allowed a better understanding of the cell-cell and molecule-cell interactions involved in the regulation of hematopoietic progenitor cells in vitro. However, most studies assessingthe effects of purified molecules on enriched populations ofcells have been done in the presence of a source of serum,leaving open the possibility that the actions of these purifiedmolecules are mediated by other unknown factors present inthe serum. The results presented here demonstrate that although in most cases colony growth was superior in the presence of serum, the purified molecules assessed for activitybehaved in a fashion in the presence of defined medium similarto that in the presence of serum (4-15). Thus, PPO and PPO-»manifested the capability of stimulating colony formationfrom CFU-GM, BFU-E, and CFU-GEMM in bone marrowdepleted of two known accessory cells, monocytes and T-lym-phocytes, in the absence of serum. While these studies do notrule out the possibility that PPO and PPO-o might not bestimulating these progenitor cells directly, it does eliminate the

possibility that their actions were mediated by unknown factorspresent in the serum. In the same way, it is not possible fromour data to eliminate the possibility that the suppressive effectsof rHuTNF-a and rHuIFN--y are being mediated through non-progenitor cells, non-T-lymphocytes, and non-monocytes, butthese effects do not require the presence of unknown serumfactors. The suppressing effects of PGE on CFU-GM and theT-lymphocyte-mediated enhancing effect of PGEi on erythroidcolony formation similarly did not require the presence ofunknown serum factors. The data reported here demonstratethat it is possible to study mechanisms of action of purifiedgrowth-stimulating and growth-suppressing molecules undermore precisely defined conditions.

ACKNOWLEDGMENTS

We wish to thank Stephanie Moore and Shirley Duke for excellentsecretarial assistance.

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Research. on January 26, 2016. © 1986 American Association for Cancercancerres.aacrjournals.org Downloaded from

1986;46:4357-4361. Cancer Res   Li Lu, Karl Welte, Janice L. Gabrilove, et al.   Serum-free Cultures

, and Recombinant Erythropoietin inαFactor, Pluripoietin Stimulated by Natural Human Pluripotent ColonystimulatingColony Formation of Human Hematopoietic Progenitor Cells

-Interferon, and Prostaglandin E onγRecombinant Human ,αEffects of Recombinant Human Tumor Necrosis Factor

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