transport of the antitumor antibiotic ci-920 into l1210 leukemia

[CANCER RESEARCH 44, 3366-3370, August 1984]

Transport of the Antitumor Antibiotic CI-920 into L1210 Leukemia Cells

by the Reduced Folate Carrier System

David W. Fry,1 Judith A. Besserer, and Theodore J. Boritzki

Warner-Lamben/Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105

ABSTRACT

CI-920 is a structurally novel antitumor antibiotic which has

activity against a wide spectrum of tumor cells in vitro and iscurative in L1210 leukemia in vivo. Several lines of evidenceindicate that this drua penetrates l 1210 relis via the reducedfolate carrier system. Reduced folates (100 Â«M)including leuco-vorin and 5-methyltetrahydrofolate completely protected L1210cells from growth inhibition by CI-920. Protective effects were

not observed, however, with folie acid, a compound which istransported by a process distinct from that for reduced folates.CI-920 was a potent inhibitor of methotrexate influx exhibiting a

mixture of competitive and noncompetitive inhibition and havinga K, (slope) of 30.0 //M and a K, (intercept) of 58.8 UM. Theinhibition appeared to be irreversible since, after cells werepreincubated with drug, the inhibitory effects persisted after cellswere washed in drug-free media. The irreversibility could beeliminated, however, by dithiothreitol, suggesting that CI-920

may interact with a thiol which is essential to this transportsystem. Cells made 71-fold resistant to CI-920 by continuousexposure to increasing concentrations of this drug were 245-fold cross-resistant to methotrexate but were collaterally sensi

tive to the lipophilic antifolate trimetrexate and contained normallevels of dihydrofolate reductase. This mutant cell line had aseverely impaired reduced folate carrier system exhibiting methotrexate influx rates of less than 1% of control cells. Finally,inhibition of methotrexate influx by a number of CI-920 analogues

showed that the intact lactone ring and the presence of thephosphate ester were required for maximum interaction with thecarrier system and that the degree of inhibition correlated withrelative antitumor potency. These observations are compatiblewith the concept that CI-920 utilizes the folate carrier system

and could be of fundamental importance for understanding thecytotoxicity and selectivity of CI-920.

INTRODUCTION

CI-920 is a recently discovered antitumor antibiotic that was

isolated from the fermentation beer of a previously undescribedsubspecies of Streptomyces pulveraceus (subspecies fosfreus;ATCC 31906) (31, 32). The structure of this novel agent hasbeen characterized (20) and is shown in Chart 1 along with someof its analogues. This drug exhibits antitumor activity against awide spectrum of tumor cells in vitro and excellent activity againstL1210 and P388 in vivo (25, 26). Previous studies have shownthis drug to be a potent inhibitor of macromolecular synthesis,and it may act directly at the level of DNA and RNA polymerases(8).2

The present paper provides evidence to indicate that CI-920

1To whom requests for reprints should be addressed.2 D. W. Fry. T. J. Boritzki, and R. C. Jackson, submitted for publication.

Received January 9,1984; accepted May 9,1984.

penetrates L1210 cells via the reduced folate carrier, a systemthat has been well characterized with reference to methotrexatetransport (4, 11, 12, 16, 18, 30). A growing body of evidenceindicates that this carrier system is an important determinant ofcytotoxicity and selectivity for antifolates (13, 24, 28, 29) andthus may be relevant in the same context for CI-920. The

significance of these observations is discussed both in relationto chemotherapy and at a molecular level in light of the novelstructure of CI-920.

MATERIALS AND METHODS

Chemicals. CI-920, PD 113,270, PD 113,271, and PD 114,027 were

isolated as the sodium salts from fermentation cultures as described byStampwala et al. (31 ). PD 114,631 was prepared by alkaline phosphatasetreatment of CI-920 (20) and PD 116,250 by acetylation of CI-920 (20).[3',5',9-3H]Methotrexate was obtained from Moravek, City of Industry,

CA, and purified by anion-exchange high-pressure liquid chromatography

on a Whatman Partisil PSX 10/25 SAX column (0.46 x 25 cm) with anammonium phosphate gradient starting with 5 HIMand pH 2.8 and endingat 0.5 M and pH 4.8. Unlabeled methotrexate, leucovorin, dihydrofolate,NADPH, thymidine, and hypoxanthine were obtained from Sigma Chemical Company, St. Louis, MO. Trimetrexate (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline) was obtained from Warner-

Lambert Co., Ann Arbor, Ml.Growth Inhibition Assays and Selection of CI-920-resistant Cells.

In all experiments, we used L1210 mouse murine leukemia cells grownas a suspension culture in RPMI 1640 supplemented with 5% fetalbovine serum and gentamicin (50 Â¿ig/ml).Dilutions of test compounds in20 n\ water were placed into wells of 24-well Linbro plates (1.7 x 1.6 cm,

flat bottom) followed by the addition of L1210 cells (3 x KP/ml) in 2 mlmedia. Plates were incubated for 3 days at 37Â°in a humidified atmos

phere containing 5% CO2 in air. Cell growth was determined by cellcount with a Coulter Model ZM electronic cell counter (Coulter Electronics, Inc., Hialeah, FL). Protection experiments were performed in anidentical manner except protecting agents were included along with thedrug. L1210 cells were selected for resistance to CI-920 by exposingthem initially to 1 ^M CI-920 at 3 x 104/rnl, allowing the cells to grow to5 x 10s, and then reseeding them at the lower density in fresh media

containing a 50% higher concentration of CI-920. This procedure wasrepeated until the cells grew at a maximum rate (24-hr doubling time) in30 MMCI-920 and were maintained at this concentration for 3 months.

Methotrexate Transport Studies. Cells were washed by centrifugaron in transport buffer containing 136 mM NaCI, 4.4 mw KCI, 1.1 mwKH2PO4, 1 mM MgCI2, and 20 mM A/-(2-hydroxyethylpiperazine)-A/'-2-ethanesulfonic acid at pH 7.4 and resuspended at approximately 107/mlin the same buffer. Cells were agitated and maintained at 37Â°with a

shaking water bath. Fluxes were initiated by exposing the cells to tritiatedmethotrexate (specific activity, 694 ,iCi///moi) and abolished at designated intervals by injecting the cells into 10 volumes of ice-cold 0.9%

NaCI solution (saline). The cells were washed twice by centrifugationwith 5 volumes of ice-cold saline, and the pellet was digested in 1 ml of

Protosol (New England Nuclear, Boston, MA). Radioactivity was determined in a Beckman Model 6800 liquid scintillation counter, and countingefficiencies were determined with quenched standards. Efflux rates weredetermined by exposing cells to 5 UM titriated methotrexate for 1 hr,

3366 CANCER RESEARCH VOL. 44

Research. on January 30, 2018. © 1984 American Association for Cancercancerres.aacrjournals.org Downloaded from

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Transport of CI-920

Â«l

H

H

OH

H

H

H

R2

P03-

P03-

PÃœ3-

P03-

H

PÃ›3-

R3

H

H

H

H

H

COCH3

OH

H

OH

OH

OH

OCOCH3

CI-920

PD 115,270

PD 113,271PD 111,027*

PD llt,631

PD 116,250

'OPENED LACTONE

Chart 1. Chemical structures of CI-920,2-H-pyran-2-one, 5,6-dihydrc-6-[3,6,13-trihydroxy-3-methyl-4-(phosphonoxy)-1,7,9,11 -tridecatetraenyl]-, monosodium salt

(NSC 339638) and its analogues.

washing twice with excess ice-cold saline, and resuspending into 37Â°

transport buffer with or without CI-920.Assay for DihydrofoÃate ReducÃase. Approximately 107 cells were

washed in saline and resuspended into 1 ml 0.05 M Tris-chloride, pH 7.2.

The suspension was frozen and thawed 3 times and centrifuged at100,000 x g for 30 min at 4Â°.The supernatant was used as the enzyme

preparation. In a 3-ml quartz cuvet, 2.5 ml of the above Tris buffer, 0.02ml of 5 Â¿Â»MNADPH, and 0.1 ml enzyme were mixed and warmed to 37Â°.

The reaction was started by addition of 0.05 ml of 2 mu dihydrofolate,and the decrease in absorbance was monitored at 340 nrn with a GaryModel 219 spectrophotometer.

RESULTS

Protection Experiments. A number of agents were tested fortheir protective effects on growth inhibition by CI-920. As shown

in Table 1, L1210 cells grew at 96% of the control rate in thepresence of 1 JUMCI-920 and 100 MM concentrations of thereduced folates leucovorin or 5-methyltetrahydrofolate as compared to 38% with CI-920 alone. Folie acid, however, had no

effect, nor did thymidine, hypoxanthine, or their combination,suggesting the protective effects were not due to folate repletionor restoration of those metabolites requiring folate cofactors fortheir synthesis.

Inhibition of the Reduced Folate Carrier System by CI-920.Since CI-920 did not appear to produce an antifolate effect, yetreduced folates protected tissue culture cells against the cyto-

toxic effects of this drug, an alternative explanation was proposed in which reduced folates were blocking its entry into cells.This possibility was also supported by the fact that protectionwas not afforded by folie acid, a compound thought to entercells by a transport route different from that utilized by reducedfolates (23, 27). The interaction of CI-920 with the reduced folate

carrier system was examined by its effect on methotrexatetransport. As suspected, CI-920 was a potent inhibitor of meth

otrexate influx. Chart 2 shows a double reciprocal plot of influxrate versus methotrexate concentration at 3 different concentrations of CI-920. The pattern of interaction was a mixture between

competitive and noncompetive inhibition. Analysis of the data by

the method of Cleland (3) shows a K, (slope) of 30.1 Â±0.1 (S.E.)UM and a KÃŒ(intercept) of 58.8 Â±0.1 /Ã•M.

Since a strong component of noncompetitive inhibition wasobserved, the reversibility of the inhibition was studied as shownin Table 2. Methotrexate influx was measured in either thepresence or the absence of 100 UMCI-920 added at 0 time or incells that had been preincubated for 30 min with CI-920 and thenwashed 2 times with saline and resuspended into drug-free

media prior to influx measurements. The pretreated cells retaineda severely inhibited influx rate even though CI-920 was removed

Table1Protection of L1210 from growth inhibition by CI-920

Cells were seeded at 3 x 10'/ml in the presence of 1 MMCI-920 with or withoutadditions. Cell growth was determined after 3 days as described in 'Materials andMethods."

Additions8 Growth rate" (% of control)

CI-920 (1 MM)

+ Leucovorin+ 5-Methyltetrahydrofolate+ Folte acid+ Thymidine+ Hypoxanthine+ Thymidine + hypoxanthine38.6

Â±2.4C

96.0 Â±1.696.9 Â±1.043.6 Â±1.443.0 Â±1.142.0 Â±2.339.7 Â±1.7

"The concentration of all additions was 100 MM except for thymidine which

was 10 MM.No effect on growth was caused by these agents alone.6 Percentage of the control growth rate determined in the absence of CI-920.c Mean Â±S.E. of 4 experiments.

0 02 0.4 06 0.8 1.0[MTX]e'

Chart 2. Double reciprocal plot of the extracellular methotrexate concentration([MTXJ.) versus influx rates in L1210 cells exposed to different concentrations ofCI-920. Influx was measured over 3 min; each value represents the mean of 5

separate experiments. Lines were fitted to the measured values by the method ofleast squares. A, control; B, 25 MMCI-920; C, 50 MMCI-920; D, 100 M" CI-920.

Table2Irreversible inhibition of methotrexate influx by CI-920 and its prevention by

dithiothreitol

Additions Methotrexate influx (% of control)*

Dithiothreitol(HIM)0.0

1.00.01.0CI-920

(MM)0.0

0.0100100Additions

present6

duringinflux100

101 .7 Â±0.6"

23.0 Â±6.326.7 Â±6.530

min incubation"

with additions butabsent duringflux100

98.0 Â±1.837.6 Â±7.091 .8 Â±0.2

* Methotrexate influx was measured in L1210 cells over an interval of 3 min andan extracellular concentration of 1 MMas described in 'Materials and Methods."

* Additions were introduced simultaneously with methotrexate.c Cells were preincubated with additions for 30 min at 37Â°,washed twice in ice-

cold saline, and resuspended into buffer containing no additions. Influx wasmeasured as above.

" Mean Â±S.E. of 3 experiments.

AUGUST 1984 3367



D. W. Fry et al.

before the influx measurement, indicating that the interaction ofCI-920 with the carrier was irreversible under these conditions

or if reversible was at least a relatively slow process requiringadditional time in drug-free media for reversal to occur. Also

shown in Table 2 is the effect of dithiothreitol on the inhibition ofmethotrexate influx by CI-920. Dithiothreitol had no effect on the

inhibition of methotrexate influx when the drug was presentduring the measurement. However, if the thiol-protecting agent

was present during preincubation in the pretreated cells, influxapproached control rates after the drug was washed away,suggesting that the irreversible component of inhibition mayinvolve an essential thiol group. In contrast, CI-920 had no

significant effect on methotrexate efflux when 100 /Â¿Mdrug wasincluded in the resuspension buffer as described in "Materialsand Methods" (data not shown).

Correlation between Transport Impairment and Resistanceto CI-920. A line of L1210 was selected for resistance to CI-920as described in "Materials and Methods." Chart 3A shows a

dose response for normal L1210 and the resistant strain hereinknown as L121 OCI-920r and illustrates a 71-fold resistance toCI-920. L121 OCI-920r was also 245-fold cross-resistant to meth

otrexate (Chart 3B). To determine whether resistance in thesecells was due to impaired transport, influx rates and net uptakefor methotrexate were measured in L121OCI-920r and compared

to normal L1210 (Chart 4). Even after 1 hr in the presence of 1U.Mextracellular methotrexate, the net accumulation of methotrexate did not approach the dihydrofolate reductase-bindingcapacity (4.8 pmol/107 cells). Influx rates in the resistant cells

were so slow that accurate measurements were difficult to obtainand were less than 1% of control when the extracellular concentration of methotrexate was 1 ^M. A significantly higher interceptwas noted in the resistant line; and although the reason for thisis unknown, it has been observed in other cell lines with impairedmethotrexate transport (19). Otherwise, the resistant cells contained a similar activity for dihydrofolate reducÃaseas the controlcells (1 .7 Â±0.1 versus 2.1 Â±0.3 nmol of dihydrofolate reducedper min per 107 cells for control and resistant cells respectively

too

80

Â£ 20

10"$ 10'5

CI-320 (M)

XÂ»

z 40LU

S 20

10-9 IO"8 IO'7 IO'6 IO'5

Â»ETHOTREXATE (M)

Chart 3. Dose-response curves for growth inhibition by CI-920 (A) and methotrexate (B) in normal L1210 (â€¢)and L1210CI-9201 (A). Each point represents the

mean of at least 2 separate experiments.

10 20 Â» 40Â«INUTES

50 60

Chart 4. Representative time course for the net uptake of methotrexate inL1210 (â€¢)and L121OCI-920' (A). Cells were incubated at 37Â°in the presence of 1

fiu methotrexate.

TablesEffect of CI-920 analogues on methotrexate influx

Methotrexate influx was measured with or without 50 Â¡Madditions added atzero time over 3-mm intervals as described in 'Materials and Methods." During this

interval, the mtraceiiuiar level of methotrexate did not exceed the dihydrofolatereductase-binding capacity; therefore, the influx measurements represent initialunidirectional rates.

Additions" (50Â»IM)CI-920

PD-1 13,270PD-1 13,271PD-1 14,027PD-1 14,631PD-1 16,250LeucovorinFolk: acidInflux

rate (% ofcontrol)36.6

Â±31 .5 Â±57 .2 Â±71 .2 Â±89.2 Â±84.7 Â±32.1 Â±80.0 Â±4.4*

13.46.77.85.91.46.31.2

" Structures are given in Chart 1.6 Mean Â±S.E. of 3 separate experiments.

under the conditions described in "Materials and Methods") and

were collaterally sensitive to trimetrexate, a lipophilic antifolwhich penetrates cells by a process distinct from that of thefolate carrier system (5).

Effect of CI-920 Analogues on Methotrexate Influx. Finally,the effect of a number of structural modifications of CI-920 (listed

in Chart 1) on the relative inhibition of methotrexate influx isshown in Table 3. Removal of the terminal hydroxyl of CI-920

(PD 113,270) had no significant effect on the inhibition of methotrexate influx compared to the parent compound whereasaddition of a hydroxyl at the y position of the lactone ring (PD113,271) considerably reduced its inhibitory effectiveness. Apparently, the intact lactone ring and the phosphate ester werenecessary for maximum interaction with the carrier system, sincethe removal of either (PD 114,027 and PD 114,631) greatlyreduced inhibition of methotrexate influx. Finally, the diacetatehaving ethyl esters at the terminal and intermediate position (PD116,250) was also a very poor inhibitor of methotrexate influx.

DISCUSSION

The experimental results clearly imply that CI-920 interacts

with the reduced folate carrier system of L1210 and mostprobably enters the intracellular compartment via this process.This conclusion is supported by 3 separate lines of evidence, (a)Both leucovorin and 5-methyltetrahydrofolate completely protected cultured cells from the inhibitory effect of CI-920. Protec-




Transport of CI-920

tion, however, was not afforded by folie acid, a compound whichenters the cell by a route distinct from that of reduced folates(23,27). (b) CI-920 was a potent inhibitor of methotrexate influx,an agent which utilizes this carrier system, (c) Cells made resistant to CI-920 by continued exposure to this drug were cross-

resistant to methotrexate and exhibited little capacity to transportthis antifolate.

A relationship between folates and CI-920 was noted, how

ever, only at the membrane carrier, since this drug caused noantifolate effects as demonstrated by the lack of protection byhypoxanthine and thymidine and no decrease in purines orthymidylate was observed in previous studies (8).2 In fact, themechanism of CI-920 is far removed from this class of antime-

tabolites in that it appears to directly inhibit macromolecularsynthesis (8).2

A kinetic analysis of the inhibition of methotrexate influx by CI-

920 showed a mixture between competitive and noncompetitiveinhibition. This differs from the purely competitive interactionsseen for other compounds structurally unrelated to folates butproposed to utilize this carrier system (18) and reflects theirreversible interaction observed between the carrier and CI-920.The irreversible inhibition of methotrexate influx by CI-920 was

evidently dependent on the interaction with an essential thiolsince dithiothreitol prevented permanent inhibition. This observation is quite reasonable since the reduced folate carrier possesses a sensitive thiol (17, 27) and CI-920 contains an a,ÃŸ-

unsaturated lactone, a component necessary for inhibition (Table3) which is capable of attacking nucleophilic groups via a Michaeladdition (1, 6). It is surprising that CI-920 has no effect on effluxsince it has been reported that low concentrations of the sulfhy-dryl-binding agent, p-chloromercuriphenylsulfonate, inhibit thisprocess in L1210 cells (17). However, the association of CI-920

with the transport carrier is possibly time dependent, and therequired interval may not have been fulfilled in these experiments.Table 3 also indicates that the phosphate ester is necessary formaximum interaction with the carrier system and is consistentwith previous reports which demonstrate inhibition of methotrexate influx by a number of phosphate-containing compounds (12,

18).The potent interaction of CI-920 with this carrier system was

also realized in the ability of the drug to select a mutant cellwhich exhibits a severely impaired transport rate for methotrexate. The selection for this transport defect is quite novel in thatthese cells were never exposed to methotrexate or any otherantifol and appears to be the sole modification since the cellswere totally sensitive to trimetrexate, a lipophilic antifol whichcircumvents the reduced folate carrier (5), and since dihydrofolatereducÃaselevels were similar to control levels.

The ramifications of these observations are severalfold: (a)this observation is unique in that it is the first antitumor agentother than an antifolate that has been shown to utilize thereduced folate carrier; (b) whereas previous reports have shownthis carrier system to be quite specific both structurally andstereochemically for folates and folate analogues (2, 30), thesedata provide further evidence that this carrier is capable oftransporting compounds very dissimilar in structure to folates assuggested in more recent reports (18); (c) perhaps most important are the possible effects on chemotherapeutic efficacy. Anextensive literature has been generated on both the characterization of this carrier system and its influence on the cytotoxicity

and selectivity of the antifolates which utilize this process, whichhas been the subject of several reviews (13, 15, 28). Thecorrelation between cytotoxicity and membrane transport forantifolates has been suggested to involve several concepts.Depending on whether or not influx of the drug is rate limiting, itmay determine the rate at which the drug interacts with its targetsite. However, more importantly, transport parameters controlthe intracellular level of drug that can be achieved or govern theperiod for which free drug is sustained. This may determine notonly the magnitude and duration in which the target is suppressed (14, 21, 22, 33, 34) but also the extent to which thedrug is metabolized (7,10) should this be an important determinant of its efficacy. Whether or not these concepts can beextrapolated to CI-920 is not clear yet, since many questions

remain unanswered concerning the transport of this agent. Forexample, the intracellular disposition of CI-920 has yet to be

determined and, although attempts have been made to detectintracellular drug by high pressure-liquid chromatography and

UV detection, these methods were evidently too insensitive. Also,it is not known whether CI-920 utilizes the energy-dependent

efflux mechanism used by methotrexate (9, 11). Finally, sinceCI-920 appears to irreversibly inhibit the carrier, this may even

tually affect its own cellular uptake which in turn may dramaticallyinfluence its chemotherapeutic efficacy.

Nevertheless, there are implications that the reduced folatecarrier system influences the chemotherapeutic behavior of CI-

920. For example, the relative inhibition of methotrexate influxby CI-920 and its analogues (Table 3) correlates very well with

both the in vitro and in vivo activity of these compounds againstL1210 (25, 26). In addition, the spectrum of antitumor activity inmice for CI-920 is very similar to that of methotrexate, suggesting

that those tumors containing the methotrexate carrier are moresusceptible to CI-920 (25, 26).

REFERENCES

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1968.2. Chelto, P. L, Sirotnak, F. M., Dorick, D. M., and Donsbach, R. C. Therapeutic

relevance of differences in the structural specificity of the transport systemsfor folate analogs in L1210 tumor cells and in isolated murine intestinal epithelialcells. Cancer Res., 37:4297-4303,1977.

3. Cleland, W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol.,63A-103-138, 1979.

4. Dembo M., and Sirotnak, F. M. Antifolate transport in L1210 cells. Kineticevidence for the non-identity of carriers for influx and efflux. Btochim. Biophys.Acta, 448: 505-516,1976.

5. Diddens, H., Niethammer, D., and Jackson, R. C. Patterns of cross-resistanceto the antifolate drugs trimetrexate, metoprine, homofolate. and CB3717 inhuman lymphoma and osteosarcoma cells resistant to methotrexate. CancerRes., 43: 5286-5292,1983.

6. Friedman, M., Gavins, J. F., and Wall, J. S. Relative nucleophilic reactivities ofamino groups and mercaptide ions in addition reactions with a,/3-unsaturatedcompounds. J. Am. Chem. Soc., 87: 3672-3682,1965.

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8. Fry, D. W., Bontzki, T. J., and Jackson, R. C. Biochemical studies on the novelanticancer agent, PD 110,161. Proc. Am. Assoc. Cancer Res., 24:320,1983.

9. Fry, D. W., White, J. C., and Goldman, I. D. Effects of 2.4-dimtrophenol andother metabolic inhibitors on the bidirectional carrier fluxes, net transport, andintracellular binding of methotrexate in Ehrlich ascites tumor cells. CancerRes., 40: 3669-3673,1980.

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12. Goldman, I. D. The characteristics of the membrane transport of amethopterinand the naturally occurring fotetes. Ann. NY Acad. Sci., 786:400-422,1971.

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32. Tunac, J. B., Graham, B. J., and Dobson, W. E. Novel antitumor agents CI-920, PD 112,270 and PD 113,271.1. Taxonomy, fermentation, and biologicalproperties. J. Antibiot., 36:1595-1600,1983.

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1984;44:3366-3370. Cancer Res David W. Fry, Judith A. Besserer and Theodore J. Boritzki Leukemia Cells by the Reduced Folate Carrier SystemTransport of the Antitumor Antibiotic CI-920 into L1210

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transport of the antitumor antibiotic ci-920 into l1210 leukemia

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