[CANCER RESEARCH 50. 3921-3927, July 1, 1990]

Elevated Pentose Cycle and Glucuronyltransferase in Daunorubicin-resistantP388 Cells1

Teresa Gessner,2 Lurine A. Vaughan, Blake C. Beehler, Christopher J. Bartels, and Raymond M. Baker

Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263

ABSTRACT

Anthracycline resistance of P388 daunorubicin-resistant cells cannotbe accounted for merely by differences in drug uptake and retention;protection against intracellular drug was also indicated.

Cytotoxicity of daunorubicin may be partially due to the formation offree radicals and reactive oxygen species (hydrogen peroxide, hydroxylradical, singlet oxygen, and Superoxide aniónradical). Protection againstfree radicals and peroxides is largely dependent upon the availability ofreduced glutathione, which in turn requires NADPH for its continualregeneration. Pentose phosphate cycle (also called hexose monophos-phate shunt) is known to provide NADPH for maintenance of glutathione.Activities of the two NADPH-producing dehydrogenases of the cycle,glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40%higher (P < 0.05) and activity of the cycle in intact cells was 2-fold higherin the resistant than the sensitive cells. The cycle was as active in thesecells as it is known to be in macrophages, indicating a very effectiveprotection against oxidative stress, free radicals, and alkylating electro-philes. Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiplestructurally unrelated drugs.

Efflux of daunorubicin may be aided by further metabolism to glucu-ronides. Daunorubicinol, a known active metabolite of daunorubicin, canbe metabolized to a glucuronide by the cells and eliminated into thesurrounding medium. Glucuronidation of daunorubicinol was evidencedby (a) release of daunorubicinol following glucuronidase hydrolysis ofmedia from cell incubations with 1.8 MMdaunorubicin and (b) productionof radioactive glucuronide when cell homogenates were incubated withUDP-[l4C]glucuronic acid plus daunorubicinol. Glucuronyltransferase ac

tivity with a broad substrate specificity was found in the cells. Usingmodel substrates, 1-naphthol and o-aminophenol, it was determined thatglucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells. Elevated glucuronyltransferase couldcontribute to daunorubicin and multidrug resistance.

INTRODUCTION

One cause of cancer therapy failure is the emergence ofresistance to a drug which was initially effective against thetumor. Moreover, the refractoriness which develops often encompasses cross-resistance to structurally unrelated agents (i.e.,multidrug resistance). Drugs which elicit emergence of themultidrug-resistant phenotype are generally natural productsor their derivatives, and they include the anthracycline antibiotics Adriamycin (doxorubicin) and daunorubicin (1-6).

One known mechanism of multidrug resistance involves adecrease in drug accumulation that is associated with an increased level of the M, 170,000 membrane glycoprotein termedP-glycoprotein (3-5). However, in highly resistant cells, decreased drug accumulation usually cannot account for the totalobserved resistance to anthracyclines (6-8). Additionally, mechanisms of resistance to the intracellular drug are observed in

Received 1/3/89; revised 3/8/90.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 USPHS Grants CA21071 and CA24538 fromthe National Cancer Institute, and BRSG SO7-RR05648-22 from BiomédicalResearch Grant Program. Division of Research Resources, NIH.

2To whom requests for reprints should be addressed, at Grace Cancer Drug

Center, Roswell Park Cancer Institute, 666 Elm Street, Buffalo, NY 14263.

anthracycline-resistant lines (8-12). Prominent among themare glutathione-centered pathways and mechanisms protectingagainst peroxidations (9-11).

Murine lymphocytic leukemia cell lines, such as P388, havebeen extensively studied in evaluations of chemotherapeuticregimens. Daunorubicin- and Adriamycin-resistant sublines ofP388, P388DAU and P388ADR, that are differentially cross-resistant to the two anthracyclines (13) represent interestingmodels of resistance to these two closely related and widelyused anticancer drugs. Both P388DAU and P388ADR areknown to accumulate less of the anthracyclines (7, 13-16);however, the differences in drug retention cannot account forall of the resistance (6, 7, 13, 14), even when drug retention isenhanced by calcium antagonists, e.g., in P388ADR (7, 16).Moreover, DNA damage in P388ADR does not relate directlyto intracellular ADR3 content (17), even when verapamil mod

ulation is applied (18). In the P388ADR cell line, multifactorialresistance has been observed. Thus, elevated P-glycoprotein(19-21), DNA repair, glutathione transferase (21, 22), glutathione content, and glutathione peroxidase (19) have beenobserved, but a decrease has been noted in a potentially activating mixed function oxidase pathway (23).

With respect to daunorubicin resistance specifically, it caninvolve a decreased accumulation of DAU and ADR inP388DAU (13, 15), Ehrlich ascites (24), and Chinese hamsterovary cells (25), but a decreased drug accumulation is not anabsolute prerequisite (26, 27). In Chinese hamster cells, somechanges in patterns of membrane glycoproteins have been noted(28), including elevation of P-glycoprotein (25). A decreasedactivity of daunorubicin-metabolizing aldoketoreductase andincreased metabolism to daunorubicin aglycones have beenobserved in human myelocytic drug-resistant cell lines (26, 27).

Preliminary experiments to the present study have indicatedthat lower amounts of daunorubicinol but higher amounts ofdaunorubicin aglycones may be produced by P388DAU thanP388 cells (29). However, there appeared to be no significantdifferences in the requisite reductases to account for such aneffect.4 An increased production of the aglycones could be due

to a higher availability of the reducing cofactors in the resistantcells, through an increased activity of the pentose phosphatecycle. The interrelationship of these pathways is shown schematically in Fig. 1. Since the pentose phosphate cycle couldcontribute to multidrug resistance (see "Discussion"), its activ

ity was compared in P388 sensitive and daunorubicin-resistantcells.

The present study demonstrates that the P388DAU cell lineexhibits about 2-fold higher activity of the pentose phosphatecycle and about 4-fold higher glucuronyl transferase activity,both of which may contribute to multidrug resistance (see"Discussion"). While this work was in progress (30), a report

appeared about an increased activity of the cycle in a human

3The abbreviations used are: ADR, Adriamycin; DAU, daunorubicin; PBS,phosphate-buffered saline (136 mM NaCI, 2.5 mM KCI, 6.5 mM Na2HPO4, 1.5min KH2PO4, pH 7.3); BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea; STR, strep-tozotocin; 5FU, 5-fluoruacil; HPLC, high performance liquid chromatography.

4 Unpubished observations.

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RENTÓSE CYCLE AND GLUCURONYLTRANSFERASE IN P388

Pintose PhosphatePathRibulose

.5-phojphJRibos«-5-PiXG-6-P

lCO,~^N^—

NADPHt,

P Gluconol«'l«*ïflrog«n««

V_NADP*;6-P-Gkjconot«

¡l,-—NADPHGlu<M«-6-P-lMhy4n)g«ncMJ

V_NADP*

•tucoae—«

2 H,0

DAUNORUBICINx s "!

FlavOp' rtduclOM !

—^ DAUNORUBICIN'^^ 0,

7- Deoïydaunorubicinone

+ Daunoiamin«

FADH,

Fig. 1. Interrelationships between daunorubicin redox cycling, pentose phosphate pathway, and tricarboxylic acid (TCA) or Kreb's cycle, showing generation

of NADPH and its utilization for regeneration of reduced glutathione duringdetoxication of Superoxide via hydrogen peroxide. S.O.D.. Superoxide dismutase;GS//, reduced glutathione; G-S-SG.oxidized glutathione; redactase and peroxidase,glutathione reducíaseand glutathione peroxidase respectively; flavop.reduclase.NADPH-dependent flavoprotein reductases. e.g., cytochrome P450 reducíase;G-6-P, glucose-6-phosphate; Ribose-5-P. ribose-5-phosphate.

breast cancer cell line (11) resistant to Adriamycin. Preliminarycommunications of this work have been presented (30, 31).

MATERIALS AND METHODS

Materials. Daunorubicin and Adriamycin (doxorubicin) hydrochlo-ride salts were obtained from Sigma Chemical Co. (St. Louis, MO), aswere D-glucose, glucose-6-phosphate, 6-phospho-D-gluconic acid, D-isocitric acid, NADP, NADPH, UDP-glucuronic acid, 1-naphthol, 4-methylumbelliferone, saccharic acid 1,4-lactone, and /J-glucuronidaseType BIO and LII. orrAo-Aminophenol was from Eastman Kodak Co.(Rochester, NY). Pharmaceutical preparations were bischlorethylnitro-sourea (Carmustine) from Bristol Laboratories and streptozotocin(Zanosar) from Upjohn. D-[l-14C]Glucose (59 mCi/mmol), D-[6-'4C]glucose (58.5 mCi/mmol), UDP-[U-'4C]glucuronic acid (307 mCi/mmol), and l-[l-'4C]naphthol (20.1 mCi/mmol) were from Amersham

Corporation (Arlington Heights, IL). Cell culture medium RPMI 1640and fetal calf serum were from GIBCO (Grand Island, NY). Otherreagents and solvents were from Baker Chemical Co. (Phillipsburgh,NJ). The authentic reference compounds daunorubicinol, 7-deoxydau-norubicinolone, 7-deoxydaunorubicinone, and daunorubicinone weregifts from Dr. S. Penco of Farmitalia Carlo Erba (Milan, Italy).

Tumor Lines and Preparation of Cell Suspensions. Murine lympho-cytic leukemia cell lines P388-945 and P388DAU-1224, which aresensitive and resistant to daunorubicin, respectively, were obtained fromThe National Cancer Institute, Frederick Cancer Research Facility,DCT Tumor Repository. The cell lines were maintained in vivo inascitic form by passaging i.p. in DBA/2 female mice. The host micewere killed instantaneously by cervical dislocation and the cells (50 to200 x IO6cells/mouse) were harvested in about 6 ml of ice-cold PBS.The cells at such tumor burdens had 95-100% viability. The cells werecounted and appropriately diluted and a known number were injectedi.p. for further passage (approximate doubling time was estimated tobe in the range of 12-15 h). Tumor resistance was checked periodicallyby determining the prolongation of the life span of mice inoculatedwith IO6cells i.p. and treated the next day i.p. with 2 mg/kg DAU or

5 mg/kg ADR. Response to other drugs was similarly evaluated atdoses given in the figure legends. To compare the effectiveness of thedrugs, i test statistics were applied to the mean survival days, in theexperiment where the drugs were tested contemporaneously on animalsfrom the same batch.

Freshly harvested cells were rapidly isolated as follows. All manipulations were done at 0-4°C.The suspensions were centrifuged at 750x g for 5 min at 0-4°Cand the supernatants were decanted. To clear

the suspensions of any contaminating RBC a brief hypotonie shock was

applied by resuspending the cell pellet in 1 ml of ice-cold water,vortexing for 10 s, rescuing with 9 ml of ice-cold PBS, and immediatelyrecentrifuging. The cell pellets were again resuspended in cold PBS.Aliquots of the cells were counted in a hemocytometer and the viabilitywas determined by trypan blue exclusion. Meanwhile, the remainder ofthe suspension was recentrifuged as before. The resulting cell pelleteither was frozen at -80°C for later use in enzyme assays or was

resuspended in an appropriate medium and used immediately for druguptake or glucose utilization experiments. Routinely, the cell suspensions had >90% viable cells.

Determination of Drug Retention in Cells and Metabolites in Media.Freshly harvested tumor cells, processed as above, were suspended (10*

cells/ml) in RPMI 1640 medium containing 10% fetal calf serum andwere incubated at 37°Cfor 30 min. Then DAU or ADR was added and

the cells were incubated for 15-60 min with 1.8 pM drug. Aliquots ofthe incubations were centrifuged at 4°Cfor 5 min at 750 x g. Theresulting cell pellets were rinsed with ice-cold PBS, centrifuged at 4°Cfor 5 min, and then either stored at -20°Cfor analysis or resuspendedin drug-free medium (2.5 x IO6 cells/ml), incubated at 37°Cfor an

additional 60 min, and processed and stored as above. Just beforeanalysis, the frozen cell pellet was resuspended in 50 n\ water, followedimmediately by extraction with 150 p\ of 0.6 N HC1 in ethanol ormethanol, centrifugation, and rapid analysis of the extract in the HPLCsystem already described (32). Spiked biological samples give 108 ±5% recovery of anthracyclines by this method (32). All the work withthe anthracyclines was performed in the dark or under UV filters (32).All glassware was siliconized.

Incubation media were kept frozen at —80°Cuntil analyses, which

were performed on the media concentrated by lyophiiization. Aliquotsof the concentrates were adjusted to pH 4.5 and incubated with ß-glucuronidase (33) overnight at room temperature, and the reactionswere stopped with 3 volumes of methanol. The centrifuged methanolicextracts were analyzed by HPLC for peak identification, in the twosolvent systems already described (32).

Pentose Phosphate Path (Hexose Monophosphate Shunt) Activity.Freshly harvested P388 cells, cleaned of RBC as described above, wereused. Utilization of [1-'4C]- and [6-'4C]glucose by the cells was evaluatedusing the method of Varnes et al. (34). The cells at 1 x IO6 cells/mlwere suspended in modified Kreb's buffer containing either 2.5 or 5.6HIM ['4C]glucose (1-2 ¿tCi/ml)and incubated at 37°Cfor 1 h. Thegenerated I4CO2was trapped in 5% KOH and counted in a scintillation

counter.Dehydrogenases. Glucose-6-phosphate, 6-phosphoglucomate, and

isocitrate dehydrogenase activities were determined spectrophotomet-rically in lysates of cells, according to published methods (35-37). Thecells, stored for up to 24 h at -80°C, were suspended in 0.1 M Tris-

HC1 buffer and lysed by freeze-thawing 4 times. The reactions werecarried out in a cuvette at room temperature, and changes in absorbanceat 340 nm were monitored in a dual-beam Perkin Elmer Lambda 3UV/visible spectrophotometer attached to a chart recorder. Extinctioncoefficient of 6.22 x IO3 liter- mol"' -cirT1 at 340 nm was used for

NADPH.UDP-Glucuronyltransferase Activity. The transferase activity was

determined in the presence of Lubrol WX as described previously (33,38, 39). The particulars of the concentrations used are given in thetables and figures. For quantitative experiments two model substrateswere used, o-aminophenol and 1-naphthol, which probe for the type ofglucuronyltransferase that becomes elevated in response to carcinogentreatment (40, 41). When o-aminophenol was the acceptor substrate,the incorporation of the label from UDP-['4C]glucuronic acid wasdetermined by the method of Bansal and Gessner (33); the sum of O-and AAglucuronides is listed. When l-['4C]naphthol was the labeledsubstrate, l-['4C]naphthol glucuronide was separated by high pressure

liquid chromatography using the system of Merrick and Selkirk (42)and quantified with the aid of a Radiomatic radioflow detector, asdescribed by Gessner and Byczkowski (43).

RESULTS

Properties of the P388 and P388DAU Cells. The sensitivityof P388 and P388DAU cells to the various drugs administered

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RENTÓSE CYCLE AND GLUCURONYLTRANSFERASE IN P388

in vivo is shown in Fig. 2. Anthracycline resistance was checkedperiodically with in vivo administration of DA U or ADR. Testscarried out contemporaneously on the control and drug-treatedanimals are depicted by the same symbol in Fig. 2. Tumorresponsiveness to 5FU, BCNU, and STR was also tested. Fig.2 shows that treatment of P388 hosts with DAU or ADRprolonged the survival of the hosts by 4 to 5 days and 6 to 7days, respectively (P < 0.002 and 0.001). Similar treatment ofP388DAU hosts produced no significant prolongation of survival when DAU was administered and only a trend towards 1-day prolongation after ADR treatment. Considering the doubling time of the cells to be about 15 h and applying Skipper's

cell kill kinetics (44), one can deduce that 4-5-day improvementin survival represents about 2-log kill. Thus, the sensitive cellsbut not the resistant ones underwent such a kill. Therefore, theresults show that the P388DAU cell line was highly resistantto both DAU and ADR. Additionally, Fig. 2 shows that BCNUtreatment produced a 7-8-day (P < 0.001) increase in the lifespan of the P388 hosts and about a 4-day increase (P < 0.001)in that of the P388DAU hosts, indicating that P388DAU cellswere partially cross-resistant also to BCNU. Nevertheless,BCNU was the most effective drug tested against the resistantcells. Treatment with 5FU or STR produced about a 2-dayincrease (P < 0.002 or 0.01) in the life span of the P388 hosts.In P388DAU hosts, 5FU was similarly effective (P< 0.02), butSTR virtually lost its effectiveness.

Fig. 3 shows intracellular accumulation of DAU and ADRin cells that were exposed to the drugs at l ßg/ml(1.8 ^M) forup to 1 h. Retention of the drugs by the cells was determinedfollowing incubation of drug-loaded cells for l h in drug-freemedium. It was ascertained that the unchanged drugs constituted >90% of the intracellularly located anthracycline compounds recognizable by the HPLC analysis. It can be seen thatthe sensitive cells accumulated 2-3 times greater amounts ofthe drugs than did the resistant ones. Both cell lines retainedbetween 50 and 80% of their accumulated drugs after l hexposure to drug-free medium (Fig. 3). The retained drug levelswere about 3-4 times higher in the sensitive than the resistantcells.

Additionally, a 3- to 5-fold higher DAU accumulation in thesensitive than the resistant cells was also seen at other DAUconcentrations. Thus the intracellular DAU (in nmol/106 cells)

yA«*niJ

««*XXX000«

Contra T„2mg/kgDAU

T.Smg/kg T„5mg/kg T,6mg/lig T.SOmg/kgADR 5FU BCNU STR

Fig. 2. Survival of DBA2 mice inoculated i.p. with IO6 P388 or P388DAUcells and treated 24 h later i.p. with DAU (2 mg/kg), ADR (5 mg/kg), 5FU (5mg/kg), BCNU (6 mg/kg), or STR (50 mg/kg). Contemporaneous assays aredepicted by the same symbol.

after incubation of P388 cells with 1 or 0. l ¿IMDAU was 0.322±0.067 or 0.043 ±0.009, respectively, after 15 min and 0.576±0.025 or 0.049 ±0.007 after 60 min; the correspondingvalues in P388DAU cells were 0.079 ±0.014 or 0.012 ±0.009after 15min and 0.115±0.011 or 0.014 ±0.002 after 60 min.

NADPH Generation in P388 and P388DAU Cells. The majorenzymes that generate NADPH reducing equivalents duringglucose metabolism by a cell are glucose-6-phosphate dehy-drogenase, 6-phosphogluconate dehydrogenase, and isocitratedehydrogenase. Constitute levels of these enzyme activities weremeasured in the lysates of P388 and P388DAU cells. As canbe seen from Table 1, the resistant cells possessed significantlyhigher activities of these enzymes. Moreover, in both cell linesthe dehydrogenases of the pentose cycle predominated overisocitrate dehydrogenase, pointing to the prominence of thecycle in these cells.

Pentose Phosphate Path in P388 and P388DAU Cells. Relative activity of the pentose phosphate cycle was determinedunder aerobic conditions in P388 cells, sensitive and resistantto daunorubicin, by quantitating I4CO2production from glucosediscretely labeled at position 1. There was 2-fold higher activityof the cycle in the P388DAU than the P388 cells (see Table 2and Fig. 4). In both cell lines, the activity of Kreb's cycle (asindicated by 14CO2production from glucose labeled in position

6) was lower than that of the pentose cycle. The finding underscores the relative preeminence of the pentose cycle in P388cells, in comparison to liver (45). Nevertheless, the resistantcells cycled glucose through Kreb's cycle 3 times more actively

than did the sensitive ones (Table 2). Taken together, the resultsshowed that glucose metabolism was significantly higher in theresistant than the sensitive cells.

To determine to what extent the elevated pentose cycle activity in P388DAU cells results in an increased protection againstoxidative stress, cycling of glucose through the pentose pathwas compared in the presence and absence of 0.5 mivi H2O2.The presence of H2O2 increased the pentose cycle activity about2-fold in the sensitive P388 cells (Fig. 4). The resistantP388DAU cells, whose basal activity was already 2-fold higherthan that of the sensitive cells, responded with about a 70%further increment (Fig. 4). The results show that P388DAUcells can better protect themselves against H2O2, which may begenerated during redox cycling of an anthracycline (see Fig. 1).

Glucuronides and UDP-Glucuronyltransferase Activity. Glu-curonidation is another pathway of carbohydrate metabolismwhich is closely linked with redox cycling and detoxication ofquiñones(46). The redox cycle, which gives rise to toxic reactiveoxygen species, may be interrupted when quiñones, such asDAU, are further metabolized to excretable glucuronides.DAU-derived glucuronides were sought in the incubation media

of P388 and P388DAU cells (Fig. 5) by HPLC analysis ofextracts of the media, before and after hydrolysis with ß-glucuronidase. It can be seen that, following hydrolysis, muchdaunorubicinol appeared in the media of both the sensitive (Fig.50) and the resistant cells (Fig. 5G); approximately comparableamounts of daunorubicinol were found, 0.056-0.071 nmol/106

cells.In the presence of the specific /3-glucuronidase inhibitor sac

charic acid 1,4-lactone, the production of daunorubicinol wasinhibited by 45%, confirming that at least 45% of the releaseddaunorubicinol came from glucuronide conjugate. In additionto the daunorubicinol, the reductively generated aglycones ofdaunorubicinol and daunorubicin (Fig. 5, peaks 4 and 6, respectively) were found in the media. Peak 6, due to 7-deoxyrub-icinone, can be discerned in the media of P388DAU cells (Fig.

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RENTÓSE CYCLE AND GLUCURONYLTRANSFERASE [N P388

400p

Fig. 3. Intracellular levels of daunorubicin(A) and Adriamycin (fi). •. P388; O,P388DAU. , drug build-up in cells duringincubations of 10' cells/ml of RPMI 1640medium containing 10% fetal calf serum and1.8 nM drug; , decrease in drug content ofcells as a result of 60-min incubation in drug-free medium.

400

_ 300

O"o

B

S"o

Minutes

Table 1 Comparison of the activities of the chief NADPH-generating dehydrogenases of the P388 sensitive and daunorubicin-resistant cells

EnzymeGlucose-6-phosphate

dehydrogenase6-Phosphogluconate

dehydrogenaseIsocitrate

dehydrogenaseCellsP388

P388DAUP388

P388DAUP388

P388DAUActivity

(nmol/min/mgprotein)"21.9

±3.829.7±3.720.1

±0.328.7±1.411.6±

1.417.6 ±2.6Activity

Ratio (PValue)41.4

(P=0.01)l.4(/>

=0.01)1.5(/>=0.006)

•Mean ±SD of 5-8 determinations.*F values by Student's / test.

Table 2 Relative rates of glucose utilization via pentose cycle ("COJfrom[¡-"CJglucose) and Kreb's cycle ("COifrom [6-"C]glucose) in P388 sensitive

and daunorubicin-resislant cells

"CO2 release from

[l-MC]Glucose [6-'"C]Glucose

P388 activitynmol/107cells/h

nmol/mg protein/h

P388DAU activitynmol/107cells/h

nmol/mg protein/h

Ratio •P388DAUP388

P value*

20.3 ±4.4«21.6 ±4.7

45.3 ±2.248.2 ±2.3

2.2

2.3 x 10~

1.34 + 0.321.42±0.34

4.02 ±0.624.27 ±0.66

3.0

2.7 x 10-

" Mean ±SD of 3-6 determinations.*P values by Student's t test.

5, E-G) but appears undetectable in the P388 media (Fig. 5,B-D). In these experiments, the 0-glucuronidase treatment didnot conspicuously increase any of these other DAU-derived

peaks.The ability of P388DAU cells to glucuronidate daunorubici-

nol as well as some model substrates, 0-aminophenol and 4-methylumbelliferone, was tested in incubations of the cell ho-mogenates with UDP-[14C]glucuronic acid. The radioactive me

tabolites were separated on silica gel, and the region whereglucuronides migrate (33) is marked by arrowheads in Fig. 6.It can be seen that all the tested substrates yielded glucuronides,demonstrating that the cells possess glucuronyltransferase ofwide substrate specificity. Daunorubicinol appeared to be theleast effective substrate, but it was present only at 0.1 IHMconcentration, whereas the other acceptors were at 1 HIM.Considering that only Chromatographie quantities of daunorub-icinol were available, comparative quantitative studies were

300

è250gü¿200^

150tO

1001

50n.-[*1T

P388 P388+HA

P388DAU P388DAU+HA

Fig. 4. Effect of hydrogen peroxide on pentose phosphate cycle activity inP388 and P388DAU cells. The activity was determined from 14CO2productionby the cells incubated with 2.5 ITIM[l-MC)glucose, (2 /iCi/ml) for l h at 37'C

with or without 0.5 m\i H¡O2.Values were significantly different from the 100%control (P f=0.5).

carried out using the model substrates o-aminophenol and 1-['4C]naphthol, with labeling of different moieties of the glucu-ronide molecules. In the first experiment, glucuronidation of o-aminophenol was compared, using radioactively labeled donorsubstrate, UDP-[14C]glucuronic acid, for incorporation into o-

aminophenylglucuronide. The resistant cells were found to havea 3.6 times more active glucuronyltransferase than the sensitiveones (Table 3), when counts in the glucuronide region wereconsidered. In the second experiment, the acceptor substrate,l-['4C]naphthol, was labeled and glucuronidation was determined using 5 mM LJDP-glucuronic acid. Again, the counts inthe region of 1-naphthylglucuronide show that 4 times higher

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PENTOSE CYCLE AND GLUCURONYLTRANSFERASE IN P388

TIME OF RETENTION

Fig. 5. HPLC chromatograms showing daunorubicin metabolites in extractsof incubation media of P388 (B-D) and P388DAU (E-G) cells. A, referencecompounds: /. daunorubicinol; 2. daunorubicinolone; 3. daunorubicin; 4, 7-deoxydaunorubicinolone: 5. daunorubicinone; 6, 7-deoxydaunorubicinone. A' in

E and F, an unknown. B and E. metabolites in extracts of neutral media beforehydrolysis; C and F, controls that were incubated 20 h at 23°C;at pH 4.5. withoutij-glucuronidasc; D and G, metabolites after fi-glucuronidase hydrolysis for 20 hat 23°C.at pH 4.5. The metabolites were generated by cells (10* ml) incubated

for 60 min with I >iMdaunorubicin in 1640 RPMI medium containing 10% fetalcalf serum; 75% aqueous methanolic extracts were chromatographed. HPLC wasperformed on a jj-Bondapak-phenyl column with 32% acetonitrile in ammoniumformate, 1.0 M pH 4.0. as the mobile phase.

Ill)

O

1234 56Fig. 6. Autoradiograms of chromatograms of incubation extracts on silica gel

thin layer chromatography plates, showing [14C]glucuronides generated by

P388DAU cells. Lanes 2 and 4, controls. Lane 2, metabolites of control incubationwithout an acceptor substrate; lane 4, UDP-[14C]glucuronic acid without cell

homogenates. Arrowheads, region where glucuronides are located. Glucuronidesof: o-aminophenol (lane /), 4-methylumbelliferone (lane 3), and daunorubicinol(lanes 5 and 6). Homogenates of the cells were incubated with 7 UM (lane 5) or50 MMUDP-C'Clglucuronic acid (other lanes) and 0.1 HIMdaunorubicinol (lanes5 and 6) or 1 mM model acceptor substrates, o-aminophenol (lane /) or 4-methylumbelliferone (lane 3). Thin layer chromatography was performed onsilica gel with n-butanol:acetone:glacial acetic acid:ammonia (30%):water(70:50:18:1.5:60) as the solvent.

glucuronidation occurred in the incubations with the resistantas compared to the sensitive cells (Table 3). Under these conditions, the glucuronyltransferase activity was calculated to "Introduction," multifactorial resistance to anthracyclines has

amount to 1.7 and 7.9 nmol/mg protein/30 min in the sensitiveand resistant cells, respectively. Thus glucuronyltransferase activity was 4 times higher in the daunorubicin-resistant than inthe sensitive cells, as determined by either method.

DISCUSSION

Early studies on the P388DAU cell line characterized it ashaving 2 orders of magnitude resistance to DAU and ADR (13)and possessing cross-resistance to other anthracyclines. Acti-nomycin D, and vincristine but not 5FU (47). As typicallyfound in a multidrug-resistant cell line, it displayed an enhanceddrug efflux, quite similar to that of P388ADR cells (13, 14).The present results are in agreement with those observations.Additionally, they demonstrate some cross-resistance to nitro-soureas, thus extending the multidrug resistance characteristicsofP388DAU.

Among the mechanisms for drug inactivation and an enhanced drug efflux may be conversion to glucuronide conjugatesof drugs that are hydroxylated molecules, e.g., anthracyclinesand Vinca alkaloids. Glucuronides are readily excretable andcan be eliminated from cells, as is the case for hydroxylatedmetabolites of, for example, benzo(a)pyrene (48). Evidence wasobtained here that the P388 cells, both the sensitive and resistant ones, can glucuronidate daunorubicinol, a known activemetabolite of DAU, and eliminate it into the surroundingmedium. Both P388 and P388DAU cells, after 1-h incubationwith l /J.MDAU, yielded comparable amounts of daunorubicinolglucuronide in the media, judging from daunorubicinol yieldsof 0.06-0.07 nmol/106 cells obtained after /3-glucuronidase

hydrolysis. That P388DAU produced as much daunorubicinolglucuronide as P388, in spite of taking up to 5 times less drug,strongly suggests that an elevated glucuronyltransferase may besignificantly augmenting the metabolism of the anthracyclinein the resistant cells. For P388DAU, the eliminated metaboliteamounted to 60% as much anthracycline as was retained by thecells in the form of DAU, indicating that it can substantiallycontribute to DAU elimination.

Glucuronyltransferase activity able to glucuronidate a varietyof substrates was found in the cells, and its activity was 4 timeshigher in the resistant ones. This compares with only a 2-foldincrease in glucuronyltransferase activity of MCF7/ADR cells,which are 200-fold resistant to ADR (49). More studies areneeded to elucidate the exact role and the mechanism of elevated glucuronyltransferase in resistance to anthracyclines andmultidrug resistance.

In the P388DAU cell line studied here, as in the case of manyother drug-resistant cell lines (6, 7, 12, 13), the degree ofresistance exceeds that attributable to enhanced drug efflux. Towitness, comparable drug exposures of the resistant and sensitive cells resulted in only 3- to 5-fold lower levels of the drugin the resistant cells, but equitoxic exposures might be expectedto produce higher drug levels in the resistant than the sensitivecells. For instance, P388 cells, after exposure to DAU thatresulted in 2-log cell kill (13), i.e., 0.1 ng/m\ (0.18 /¿M)for 1 h,contained 0.091 ±0.006 nmol DAU/106 cells; however, theresistant P388 cells already contained 0.172 ±0.016 nmol/106after 1-h incubation with 1.8 ^M DAU, which, nevertheless,was less toxic because a 2-h incubation caused only 50% cellkill. This indicated that there was at least a 2-fold resistanceagainst intracellular drug.

A factor in the protection against the intracellular drug maybe an elevated pentose phosphate cycle. As pointed out in

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Expt.

RENTÓSE CYCLE AND GLUCURONYLTRANSFERASE IN P388

Table 3 Comparison of glucoronyltransferase activity of P388 cells that are sensitive and resistant to daunorubicin

Glucuronide (cpm)

Substrates P388 P388DAURatio •P388

P388DAUUDP-('4C]glucuronic acid, 0.1 HIM0-Aminophenol, 1 mM

UDP-glucuronic acid, 5 mM[14C]Naphthol, 1 mvi

501 ±126°

1754 ±61

1797 ±92

7082± 1410

3.6

4.0

" Mean ±SD of 2 or 3 determinations.

been observed in other cells and included resistance againstoxidative stress (11, 22), that arises due to production of freeradicals and reactive oxygen species during anthracycline redoxcycling (50). As shown in Fig. 1, the free radical of the drugDAU can be reductively converted to aglycone (notably 7-deoxydaunorubicinone, peak 6, Fig. 5) or trigger the productionof Superoxide anión and hydrogen peroxide. The detoxicationof the latter can occur via glutathione peroxidase of the gluta-thione redox cycle, which can also be elevated in anthracyclineresistance (22). The reducing equivalents for regeneration ofGSH come from NADPH, which in turn is regenerated in greatmeasure by the pentose phosphate cycle (51).

The level of the pentose phosphate cycle, along with itsNADPH-generating enzymes glucose-6-phosphate dehydroge-nase and 6-phosphogluconic acid dehydrogenase, were examined in P388 and P388DAU cells and about a 2-fold highercapacity of the cycle was found in P388DAU cells. Both thesensitive and the resistant cells had so prominent a pentosecycle that the activities of the cycle's dehydrogenases were of

the same order of magnitude as isocitrate dehydrogenase. Thiswas unexpected, since in the liver isocitrate dehydrogenaseactivity is 1 order of magnitude higher than glucose-6-phos-phate dehydrogenase activity (45). Moreover, the constitutivelevel of glucose-6-phosphate dehydrogenase in P388 cells wasas high as that of alveolar macrophages, known to have highconstitutive levels of antioxidant enzymes (52), that is, 21.9 ±3.8 and 29.7 ± 3.7 nmol/mg protein/min for P388 andP388DAU, respectively, and 19.5 ±2.0 nmol/mg protein/minin the macrophages. It can, therefore, be surmised that P388cells, like the macrophages, are well endowed to cope withoxidative stress.

Considering the high basal activity of the pentose phosphatecycle in P388DAU cells (48.2 ±2.3 nmol/mg protein/h; Table2), it is not surprising that less than a 2-fold further increasewas seen in the P388DAU cells stressed with H2O2 (Fig. 4). A4-fold increase in the pentose phosphate cycle activity (up to122 nmol/mg protein/h; see Ref. 11) was noted in similarlychallenged breast cancer cells, MCF-7/ADR, that were 200-fold resistant to Adriamycin. Paradoxically, those cells had adepressed glucose-6-phosphate dehydrogenase activity, but apparently this was offset by a lower Km for NADPH (11). Thus,increases in the cycle's activity imply a significantly higher

potential in resistant cells to detoxify reactive oxygen species,as well as free radicals and alkylating electrophiles, all of whichmay tax the pentose cycle-glutathione-regenerating system illustrated in Fig. 1. An elevated pentose cycle activity cancontribute to multidrug resistance of P388DAU cells and maybe a common protective mechanism in other multidrug-resist-

ant cell lines.

ACKNOWLEDGMENTS

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1990;50:3921-3927. Cancer Res   Teresa Gessner, Lurine A. Vaughan, Blake C. Beehler, et al.   Daunorubicin-resistant P388 CellsElevated Pentose Cycle and Glucuronyltransferase in

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