(CANCER RESEARCH 52. 1770-1774, April 1. 1992]

Role of Aldehyde Dehydrogenase in the Protection of Hematopoietic ProgenitorCells from 4-Hydroperoxycyclophosphamide by Inter leukin Ißand TumorNecrosis Factor1

J. Moreb,2 J. R. Zucali, Y. Zhang, M. O. Colvin, and M. A. Gross

Department of Medicine, Division of Medical Oncology, University of Florida, Gainesville, Florida 32610 [J. M., J. R. Z., Y. Z., M. A. G.J, and Johns Hopkins OncologyCenter, Baltimore, Mainland 21205 {M. O. CJ

ABSTRACT

Preincubation of human bone marrow cells with interleukin iß(IL-1)and tumor necrosis factor a (TNF-a) for 20 h can protect early progenitorcells from 4-hydroperoxycyclophosphamide (4-HC) toxicity. In this report, we have studied the mechanism for such protection. We examinedthe effect of the length of incubation time and found that preincubationfor at least 20 h with IL-1 and TNF-a is needed for significant protection.The addition of 2 ¿ig/mlcycloheximide, a protein synthesis inhibitor,during the 20-h preincubation completely abolished the protection observed for all colony-forming cells. In order to study the role of aldehydedehydrogenase (ALDH), an enzyme which inactivates 4-HC, we useddiethylaminobenzaldehyde, an inhibitor of ALDH. Diethylaminobenzal-dehyde was added during the last 10 min of the 20-h preincubation withIL-1 and TNF-a. Diethylaminobenzaldehyde prevented the protection ofcolony-forming cells from 4-HC. Finally, using the same protection assaysystem, we showed that a 20-h preincubation with IL-1 and TNF-a canalso protect early progenitor cells from phenylketophosphamide, ananalogue of 4-HC which is resistant to inactivation by ALDH. Fromthese studies, we conclude that preincubation with IL-1 and TNF-a forat least 20 h is required for the protection of early progenitor cells from4-HC. During that time period, protein synthesis, specifically aldehydedehydrogenase synthesis, is critical for the protection from 4-HC. Preincubation with IL-1 and TNF-a also protects early progenitors fromphenylketophosphamide. Because phenylketophosphamide cannot bemetabolized by ALDH, the reason for this protection must be due toother, as yet unidentified, mechanisms.

INTRODUCTION

4-HC3 is an active derivative of cyclophosphamide which has

been used clinically for ex vivo purging of residual tumor cellsand treatment with autologous bone marrow transplantation(1,2). The successful reconstitution of hematopoiesis with suchtreatment suggested that hematopoietic stem cells are relativelyresistant to 4-HC. Several in vitro studies have shown that thisrelative resistance may be due to the presence of a more abundant cytoplasmic ALDH, an enzyme responsible for the inactivation of 4-HC in the stem cells (3, 4). Recently, Kastan et al.(5) reported the highest levels of ALDH in CD34+ cells, in

comparison with more mature hematopoietic progenitors.We have previously reported that 20-h preincubation of nor

mal human bone marrow cells with IL-1 and/or TNF-« canprotect early progenitors from lethal doses of 4-HC (6, 7).

Received 10/17/91; accepted 1/23/92.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.

1This work was supported by National Institutes of Health Grant AI 24709.3To whom reprint requests should be addressed, at Department of Medicine.

Box J-277, J. H. Miller Health Center. University of Florida, Gainesville. FL32610.

3The abbreviations used are: 4-HC, 4-hydroperoxycyclophosphamide; ALDH,aldehyde dehydrogenase; DMSO. dimethyl sulfoxide; DEAB, diethylaminobenzaldehyde; CFC, colony-forming cell; Bl-CFC, blast colony-forming cells; PKP.phenylketophosphamide; FBS, fetal bovine serum; IL-1, interleukin 1; TNF-a,tumor necrosis factor a; MEM-«,minimum essential medium a.

These early progenitors give rise to Bl-CFC, which appear afterthe second week of methylcellulose culture. These Bl-CFCproliferate rapidly and give rise to the different single- andmultiple-lineage secondary colonies upon replating. On theother hand, we observe no similar protection from 4-HC fordifferent leukemic cell lines or fresh leukemic blast cells (7).

The mechanism for such differential protection is not known.In the present study, we report experimental data suggestingthat increased synthesis of ALDH within the 20-h preincubation with IL-1 and TNF-a may be responsible for the protectionof early hematopoietic progenitors from 4-HC. On the otherhand, preincubation with IL-1 and TNF-a can also protect earlyhematopoietic progenitors from PKP, a 4-HC analogue whichis not metabolized by ALDH. Thus, the possibility that differentmechanisms exist at the same time is discussed.

MATERIALS AND METHODS

Materials. Recombinant human interleukin 1/3(specific activity, >2x IO7units/mg) was generously supplied from E. I. DuPont de Nemours

& Company by Dr. Robert C. Newton. Recombinant tumor necrosisfactor a (specific activity, 2.4 x IO7units/mg) was a gift from Dr. Leo

Linn, Cetus Corporation (Emeryville, ÇA).Recombinant human interleukin 3 (specific activity, 5.3 x IO6units/mg) was received from Sandoz

Research Institute (East Hanover, NJ). Tissue culture erythropoietin(lot N-5638, containing 10,000 units/ml) was generously supplied byOrtho Biological Company (Raritan, NJ). Conditioned medium fromthe human bladder carcinoma cell line 5637 was prepared in ourlaboratory as previously described (8). The ALDH inhibitor DEAB wasobtained from Aldrich Chemical Co. (Milwaukee WI). 4-HC was prepared as described previously (9). PKP was prepared and kindly provided by S. Ludeman, Johns Hopkins Oncology Center, Baltimore,MD) (10). Cycloheximide was purchased from Sigma Chemical Co.(St. Louis, MO), and FBS was obtained from Hyclone (Provo, UT).

Human Bone Marrow Cells. Bone marrow samples were aspiratedfrom the posterior iliac crest of healthy adult volunteers who had givenwritten informed consent. Bone marrow mononuclear cells were obtained after centrifugation over Ficoll-Hypaque (Pharmacia, Upsala,Sweden) or enriched for hematopoietic progenitor cells, using elutria-tion centrifugation followed by negative selection using a panel ofmonoclonal antibodies and immunomagnetic microspheres, as previously described (7), for use in our experiments. All bone marrow cellswere kept frozen at —¿�70°Cuntil used.

Incubations. Preincubation of bone marrow cells before treatmentwith 4-HC was performed as previously described (6). Briefly, post-Ficoll-Hypaque mononuclear cells (5 x IO5 cells/ml) or enriched hematopoietic progenitors (3 x 10" cells/ml) were incubated in an uprightposition at 37°Cin a 5% COz humidified atmosphere. The standard

time of incubation was 20 h; however, in order to study the effect ofthe length of incubation time, mononuclear cells were preincubated for2, 8, 14, 20, 34, and 48 h before treatment with 4-HC. Each tubecontained 4-5 ml of mononuclear cell suspension in MEM-a supplemented with 6% FBS alone, as control, or with the presence of 10 ng/ml IL-1 and 5 ng/ml TNF-«. Studies to evaluate whether protein

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PROTECTION OF HEMATOPOIETIC PROGENITORS

synthesis is crucial for the protection from 4-HC were performed by-adding 2 Mg/ml cycloheximide to the cell suspension during the 20-hpreincubation with or without IL-1 and TNF-a.

To assay for any effect on CFCs within the 20-h preincubationperiod, bone marrow cells from the different experimental groups werecultured (IO4 cells/dish) at the end of each preincubation period andbefore addition of the 4-HC. These cells were cultured using the blastcell colony assay, as described below.

Studies with 4-HC, PKP, and DEAB. 4-HC and PKP were dissolvedin MEM-«every time just before use. A dose-response curve for 4-HCand PKP was determined for each bone marrow in advance of eachexperiment, as described previously (7). A second sample of the samebone marrow was thawed and incubated as described above, followedby a 30-min incubation at 37°Cwith the predetermined lethal dose of

4-HC or PKP. The cells were then washed twice and cultured using theblast cell colony assay, as described below.

DEAB was dissolved in 100% DMSO at 50 mM and then diluted inwater to 5 m\i (10% DMSO) before use. DEAB at 25 //M (0.02% finalconcentration of DMSO) was added to the cell suspensions for IO minbefore and also during the 30-min exposure to 4-HC or PKP, asdescribed above. To demonstrate the effect of DEAB alone. 25 ¿tMDEAB was used with a sublethal dose of 4-HC in each of theseexperiments.

Blast Cell Colony Assay. Following the 20-h preincubation and 30-min 4-HC or PKP treatment, the cells were washed twice with chilledculture medium to remove the chemotherapeutic factors and culturedin semisolid medium for the growth of hematopoietic CFCs, as previously described (6). Cultures consisted of MEM-«supplemented with1.2% methylcellulose (4000 CP; Fisher), 5 x IO"4M2-mercaptoethanol,1% bovine serum albumin (Sigma), 30% FBS, 10~6M methylpredniso-

lone sodium succinate, 1 unit/ml recombinant human erythropoietin,50 units/ml recombinant human interleukin 3, and 5% 5637 conditioned medium. Four or five replicate 1-ml cultures, in 35-mm culturedishes (Costar, Cambridge, MA), for each experimental group weremaintained in a 5% CO2 humidified atmosphere at 37°Cfor up to 5

weeks.Colonies were scored on an inverted microscope at weekly intervals

for 4-5 weeks and during that time period were observed specificallyfor the appearance of newly formed Bl-CFC. The identification of blastcell colonies was based on morphological characteristics, in accordancewith our previous experience, I.e., undifferentiated colonies appearingafter 14 days of culture and capable of giving rise to secondary coloniesupon replating (6, 7). These colonies appear randomly from day 14 today 35 of culture, with great variability from one bone marrow to thenext. Also, these colonies are very large and diffuse, which may renderother colony scoring difficult. Thus, the results reflect the maximumpossible number of colonies scored during the culture span. In contrast,the results of untreated bone marrow cultures were scored at day 14,since these cultures start to degenerate after 14 days of culture.

Statistical Analysis. Three to six replicates of each experiment wereperformed. Levels of significance for comparison between samples ineach experiment were determined using the two-tailed Student's / test.

The results are expressed as means ±1 SD of four or five plates/point.

RESULTS

Effects of IL-1 and TNF-a Preincubation on CFCs. There isno significant effect of 20-h preincubation with IL-1 and TNF-

80

.| 40

OoO 30

•¿�

Iz

20

10

48

Time in HoursFig. 1. Effects of different times of preincubation with culture medium alone

or 10 ng/ml IL-1 and 5 ng/ml TNF-« on protection from 4-HC. Bone marrowmononuclear cells (5x10* cells/ml) were preincubated for different lengths oftime with MEM-« alone (•)or IL-1 and TNF-a (D) before addition of 60 »jg/ml4-HC. The results are expressed as the mean ± 1 SD of the total number ofcolonies scored in four replicate culture dishes on day 14 of culture. Significantprotection (P < 0.01) was seen after 20-h incubation with IL-1 and TNF-a.Longer incubations (34 and 48 h) with both cytokines did not further increasethe protection observed.

a on the number of CFCs seen at day 14 of culture of normalbone marrow, in comparison with preincubation with mediumalone (control). The results in Table 1 reflect the summary offour experiments performed with four different bone marrows.

Effects of Preincubation on 4-HC Toxicity. Fig. 1 demonstrates the effects of variable preincubation times on the totalnumbers of colony-forming cells surviving the toxicity of 4-HC.Protection from 4-HC toxicity by normal early bone marrowhematopoietic progenitors requires preincubation with IL-1 andTNF-tt. Significant colony growth is not seen until at least 14h of preincubation with IL-1 and TNF-«. In fact, the late-appearing colonies (i.e., burst-forming units erythroid, mixedcolony-forming units, and Bl-CFC) are not seen unless the bonemarrow cells are incubated for at least 20 h with IL-1 and TNF-tt (data not shown). Increasing numbers of CFCs are protectedfrom 4-HC by 20-h preincubation with IL-1 and TNF-a, whichincrease only slightly with extended preincubation times of 34and 48 h.

Effect of Cycloheximide. The addition of 2 ng/m\ cycloheximide during the 20-h incubation with IL-1 and TNF-cv completely abolishes the protective effects of IL-1 and TNF-«fromthe toxicity of 4-HC (Table 2). Cycloheximide itself is not toxicto colony-forming cells during the 20-h incubation (data notshown). These data suggest that protein synthesis during thepreincubation time with IL-1 and TNF-a is necessary for theobserved protection.

Effect of DEAB on the Protection from 4-HC. In order tostudy whether the protective effects of IL-1 and TNF-a on earlyhematopoietic progenitors are due to an increase in the synthesis of ALDH in these progenitors, we have used DEAB as aninactivator of ALDH. Bone marrow mononuclear cells were

Table I Effect of 20-h preincubation with IL-1 plus TNF-a on the number of colony-forming cells of normal human bone marrow

Data represent mean ±1 SD of the number of colonies obtained in 16 replicate culture plates from four different experiments with four different bone marrows.Each culture plate contained IO4 bone marrow mononuclear cells, and colonies were scored at day 14 of culture. M. macrophage: CM. granulocyte/macrophage: Eo,

eosinophil: E. erythroid burst; Mix. mixed colony containing granulocytc. macrophage, megakaryocyte. and/or eosinophil; Bl. blast cell colony.

Number of colonies

M CM Eo Mix Bl Total

ControlIL-1 +TNF-ÎÕ

17±99 ±7

19±810±3

6±27±3

66 ±2087 ±24

9±511 ±6

1 ±11 ±1

118±24125+ 30

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Table 2 Effect ofcycloheximide addition on the protective effects from 50 i^g/ml4-HCofIL-l and TNF-a

Data represent the mean ±1 SD of the number of colonies obtained in fourreplicate culture plates on day 28 of culture, with each culture plate containing 3x 10*cells from an enriched bone marrow cell population. Values in parenthesesrepresent the percentage of recovery of CFCs at day 28 after 4-HC treatment, incomparison with total CFCs cultured from the same bone marrow without 4-HCtreatment. The results of cultures of non-4-HC-treated bone marrow were countedat day 14 of culture, with each culture containing IO3cells/plate. Abbreviations

are the same as used in Table I.

Number of colonies

ControlColony

typesMCMEoEMixBlTotal-4HC15±522131211731344181201

42+4-HC1

±21±1001

±103

±1 (0.05)1L-1

+TNF-a-Cyclo-

heximide.+4HC19±716

±35±337

±175±12±

183±5(1.4)+Cyclo-

heximide.+4-HC0000000

MEM 65 ug

IL-1 * TNF

Z IL-1*TNF»DEABMa

0 10 20 30 40

Number of Colonies

Fig. 2. Effects of DEAB, an inhibitor of aldehyde dehydrogenase, on protectionfrom 4-HC by IL-1 and TNF-a. Bone marrow mononuclear cells (5 x 10* cells/ml) were incubated with 10 ng/ml IL-1 and 5 ng/ml TNF-a, or MEM-a alone,for 20 h. For the last 10 min of the 20 h, 25 /IM DEAB was added. DEAB wasalso present during the 30-min exposure to 65 jig/ml (lethal dose) 4-HC, as wellas during exposure to 50 , r ml (sublethal dose) 4-HC. The results are from oneof four similar experiments and are expressed as mean ±1 SD of the total numberof colonies scored in four replicate culture dishes on day 35 of culture. DEABsignificantly reduced the number of colonies protected by 20-h incubation withIL-1 and TNF-n (P < 0.005) and also reduced the number of colonies scoredusing a sublethal dose of 4-HC (P < 0.005).

suggest that IL-1 and TNF-a may induce different mechanismsof protection, in early hematopoietic progenitors, against different cyclophosphamide derivatives.

DISCUSSION

Based on our previous report in which we showed synergybetween IL-1 and TNF-a in the protection of early progenitorsfrom 4-HC (7), we used a combination of low doses of bothcytokines in all our experiments in the present study. Westudied the mechanism of the protective effects from 4-HC of20-h preincubation with IL-1 and TNF-a, in early hematopoietic progenitor cells. The results in Table 1 demonstratevery clearly that there is no increase in CFCs induced by IL-1and TNF-«prior to 4-HC treatment and, therefore, this cannotbe a mechanism for the protection from 4-HC. On the otherhand, we demonstrate (Fig. 1) that at least 20 h of preincubationwith IL-1 and TNF-a are required to allow for significantprotection of early progenitors. This is especially true for burst-forming units erythroid, mixed colony-forming units, and Bl-

CFC. Incubation for more than 20 h does not significantlyincrease the protection observed (P > 0.1). The addition of

Table 3 Effect of DEAB addition on the protective effects from 75 fig/ml 4-HC ofIL-1 plus TNF-a

The results of the 4-HC-treated cultures are expressed as mean ±I SD of totalnumber of colonies in five replicate culture dishes on day 21 of culture, with eachculture plate containing 5x10* bone marrow mononuclear cells. The results ofthe pre-4-HC cultures are expressed similarly, except that each culture platecontained IO4cells. Values in parentheses represent the percentage of recovery of

CFCs. Abbreviations are as in Table 1.

Number of colonies

ColonytypesM

GMEoEMixBlTotal-4

HC12±4

14 ±210±372 ±22

9±31 ±1

118±28Control+4-HC0.2

±0.500000

0.2 ±0.5 (0.008)IL-1+

DEAB,+4-HC0

000000+

TNF-a-DEAB,

+4-HC32

±4(5.1)18 ±3(2.6)6±2(1.3)

22 ±12(0.6)5 ±2(1.3)9 ±2(18.5)

89 ±6(1.6)

incubated with 10 ng/ml IL-1 and 5 ng/ml TNF-a for 20 h andthen incubated with 25 UMDEAB for 10 min before and duringthe 30-min presence of 4-HC. The cells were then washed twiceand cultured as before. The results are expressed as mean oftotal colonies/dish ±1 SD (Fig. 2; Table 3). DEAB preventsthe protection from the toxicity of 4-HC seen with IL-1 andTNF-a. All colony types are affected (Table 3). DEAB alone isalso able to turn a sublethal dose of 4-HC into a lethal dose(Fig. 2); however, no direct toxic effect of DEAB is seen on thecolony-forming cells (data not shown).

Effect of Preincubation on PKP Toxicity. Since PKP is notmetabolized by ALDH (10), we studied the protective effectsof preincubation with IL-1 and TNF-a on early hematopoieticprogenitor cells subjected to 30 min of PKP. Fig. 3 demonstrates that IL-1 and TNF-a preincubation produces significantprotection for colony-forming cells against a lethal dose of 10/ng/ml PKP. As expected, the protection of colony-forming cellsby IL-1 and TNF-a is not affected by incubation with DEAB,the ALDH inhibitor. All colony-forming cells are protected,with the exception of erythroid colonies (Table 4). These results

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IL-1«TNF*DEAB

100

Number of Day

20021 Colonies

Fig. 3. Effects of PKP on the protection of colony-forming cells by IL-1 andTNF-n. Two experiments are depicted (•.experiment 1; Ü,experiment 2). Inboth experiments, bone marrow mononuclear cells (5x10* cells/ml) were treatedfor 20 h with MEM-«alone or with 10 ng/ml IL-1 and 5 ng/ml TNF-a, in thepresence or absence of 25 MMDEAB added during the last 10 min of the 20-hpreincubation. All cultures were then exposed for 30 min to 10 ¿ig/mlPKP. Theresults of two of four similar experiments are shown and expressed as mean ±1SD of the total number of colonies scored from four or five replicate culturedishes on day 21 of culture.

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Table 4 Protective effect from 10 vg/ml PKP of preincubation with IL-1 andTÑF-a, in early progenitor cells

Data represent the mean ±1 SD of the number of colonies obtained in fivereplicate culture plates on day 21 of culture, with each plate containing 5 X 10*

bone marrow mononuclear cells. Values in parentheses represent the percentageof recovery of CFCs, in comparison to those scored from bone marrow cultureswithout treatment, with each plate containing 2 x IO4cells. Abbreviations are as

in Table 1.

Number of colonies

ControlColony

typeCM

(M, CM. Eo)EMixBlTotal-PKP129±

1532 ±556 ±2

0216± 11+PKP2±3

000

2 ±3 (0.02)IL-

1 +TNF-a+DEAB,

+PKP205

±150

19 ±53±2

227 ±18(3.0)-DEAB,

+PKP202

+ 210.25 ±0.4

26 ±42±2

230 ±19(3.2)

cycloheximide, a protein synthesis inhibitor, during the 20-hpreincubation with IL-1 and TNF-a completely prevents anyprotection of early hematopoietic progenitors (Table 2). Theseresults suggest that protein synthesis during this 20 h of incubation is essential for the protective effect.

NAD-dependent aldehyde dehydrogenase has been shown toconfer antitumor drug resistance to cyclophosphamide in vivoand to 4-HC in vitro (I I, 12). DEAB was identified as a potent,partial, competitive inhibitor of the cytosolic (but not the mi-tochondrial) isozyme of ALDH (13). The kinetics of the inhibitory action of DEAB and its specificity for ALDH were testeddirectly by its ability to render cyclophosphamide-resistantLI210 leukemic cells sensitive to cyclophosphamide, using invitro survival assays (14). In the present study, we have shownthat the addition of 25 ßMDEAB can change a sublethal doseof 4-HC into a lethal dose (Fig. 2). We have also demonstratedthat DEAB is not cytotoxic by itself (data not shown). Furthermore, and most importantly, pretreatment with DEAB eliminates the protection seen against 4-HC after 20-h incubationwith IL-1 and TNF-a (Table 3; Fig. 2). These results suggestthat an increase in the ALDH level during the 20-h incubationwith IL-1 and TNF-a increases the resistance of early progenitors to lethal doses of 4-HC. Since the production of ALDHin these early progenitors, represented by CD34+ cells, has been

shown to be constitutive (5), it is possible that the presumedincrease in production induced by IL-1 and TNF-a is due to anincrease in transcription rate of the involved mRNA or prolongation of its half-life or both. The sequence of the humancytosolic ALDH gene has recently been published (15), and adirect approach to verify the sequence of events is now possible.In view of other reports concerning the ability of IL-1 and TNF-a to protect mice from several other chemotherapeutic drugs,such as cyclophosphamide or 5-fluorouracil, as well as lethalirradiation (16-19), it is likely that more than one mechanismof action is involved in the protective effects of these cytokines.Such a likelihood should not be surprising, given the multipo-tential effects both cytokines express (20, 21). The induction ofresistance to such a variety of lethal treatments could be partof a general response to a stress situation induced by IL-1 andTNF-a. Recently, it has been shown that TNF-a induces man-ganous Superoxide dismutase in cultured normal human cellsand thus increases their resistance to heat and radiation (22).Therefore, it is possible that more than one factor may beinduced to protect the early hematopoietic progenitors againstany one treatment. Indeed, we have shown that preincubation

with IL-1 and TNF-a can protect early progenitors from thetoxicity of PKP, a 4-HC analogue that is not metabolized byALDH (Fig. 3; Table 4). These results strongly support thepossibility that another mechanism of protection of hematopoietic progenitor cells by IL-1 and TNF-«is involved in thissituation, such as increased metabolism, increased efflux, increased DNA repair, etc., which needs further investigation. Aslong as none of these mechanisms are equally inducible intumor cells, such pretreatment with IL-1 and/or TNF-a mayhave clinical significance. We have previously reported that IL-1 and TNF-a did not protect leukemic blast cells from 4-HC(6, 7, 23). Thus, ALDH production may be induced to a lesserextent or not at all in these tumor cells, allowing for thedifferential protective effect of IL-1 and TNF-«towards earlyprogenitors in mixing experiments (7).

It is clear that more studies, at the molecular as well aspreclinical levels, are needed in order to understand fully therole of such cytokines in the protection of hematopoietic stemcells. These studies may open new directions in the research ofnormal stem cell drug resistance.

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