[CANCER RESEARCH57, 4830-4837, November 1, 1997)

ABSTRACT

Intratumoral expression of cytochrome P450 2B1 sensitizes tumor cellsto the cytotoxic action of the alkylating agent prodrug cyclophosphamide

(CPA)and providesa novel strategyfor cancergene therapythat mayenhance the selectivity and the effectiveness of this class of antitumordrugs [L. Chen and D. J. Waxman, Cancer Res., 55: 581—589, 1995].P450-catalyzed drug metabolism is obligatorily dependent on electroninput from the flavoenzyme NADPH-P450 reductase (RED), which iswidely expressed in many cell types, including tumor cells. Here, weinvestigate the potential utility of combining RED gene transfer withCPA-based P450 gene therapy. Rat 9L gliosarcoma cells stably expressingeither basal or elevated (up to 10-fold increase) levels of RED, in thepresence or absence of P450 2B1, were selected and characterized. REDoverexpresslon substantially increased the sensitivity ofthese cells to CPA,but only when combined with P450 2B1 expression. An enhanced cytotoxicresponse was also obtained when recombinant adenovirus encoding P450

2B1 was used to deliver the P450 gene to RED-overexpressing tumor cells.CPA cytotoxicity was substantially decreased by the RED inhibitor diphenyleneiodomwn chloride or by the P450 inhIbitor metyrapone, providing evidence of its dependence on the catalytic contributions of bothprotein components of the P450 metabolic pathway. Conditioned mediafrom P450 2B1-expressing and RED-overexpressing twnor cells treatedwith CPA exhibited Increased formation of the primary 4-hydroxy metabolite and greater cell contact-independent bystander cytotoxic potential compared to tumor cells containing P450 2B1 and basal levels of RED.Evaluation of the impact of P450/RED combination gene therapy using as.c. solid tumor model/tumor excision assay revealed a dramatic 50—100-fold increase in tumor cell kill in vivo over that provided by liver drugactivation alone. These findings establish the importance of endogenousRED levels as a determinant of the sensitivity of tumor cells to CPA/P450genetherapyanddemonstratethestrikingtherapeuticeffectivenessof ananticancer prodrug activation strategy based on the combination of acytochrome P450 gene with the gene encoding RED.

INTRODUCTION

The therapeutic activity of many cytotoxic anticancer drugs islimited by a moderate therapeutic index that is associated with nonspecific toxicity toward normal host tissues. One novel approach toenhancing the selectivity of cancer chemothcrapeutics involves theapplication of gene therapy technologies to cancer treatment (1, 2). Inone such application, the phenotype of the target tumor cell is genetically altered to increase the tumor's drug sensitivity and responsiveness. One promising strategy involves the direct transfer of a “chemosensitization― or “suicide―gene encoding a prodrug activationenzyme to confer sensitivity to otherwise innocuous agents (3, 4).Ganciclovir/herpes simplex virus thymidinc kinase represents a pro

Received 6/1 1/97; accepted 9/5/97.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 in accordance with

18 U.S.C. Section 1734 solely to indicate this fact.L This work was supported in part by NIH Grant CA49248 (to D. J. W.).

2 Present address: Department of Human Genetics, Merck Research Laboratories,

WP26A-3000, West Point. PA 19486.3 Present address: Department of Drug Metabolism, Pfizer Central Research, Groton,

cr 06340.4 To whom requests for reprints should be addressed, at Department of Biology,

Boston University, 5 Cummington Street, Boston, MA 02215. Phone: (617) 353-7401;Fax: (617) 353-7404; E-mail: djw@bio.bu.edu.

totypic prodrug/cnzymc activation system that has been widely stud

ied, with respect to its potential applications in cancer gene therapy.Tumor cells transduced with the gene encoding herpes simplex virusthymidine kinase acquire sensitivity to the prodrug ganciclovir, aclinically proven agent originally designed for treatment of antiviralinfections (5, 6). In a second example, the bacterial gene cytosincdeaminase has been shown to sensitize tumor cells to the antifungalagent 5-fluorocytosine as a result of its transformation to 5-fluorouracil, a known cancer chemotherapeutic agent (7—9).Recent studiesusing these drug susceptibility genes have yielded promising results(e.g., Rcfs. 10—13),suggesting that this general approach may ultimately find clinical applications. It is likely, however, that the successful clinical application of drug susceptibility gene therapies tocancer treatment (14, 15) will ultimately require multiple strategies,perhaps including the use of distinct prodruglcnzyme activation systems or a combination of activation systems for treatment of differenttumor types.

The conventional cancer chemothcrapeutic agent CPA5 and itsisomer ifosfamide are alkylating agent prodrugs that undergo bioactivation catalyzed by liver P450 enzymes (16). The primary 4-hydroxymetabolite is formed in the liver and spontaneously decomposes, both

in circulation and within the target tumor cells, to yield acrolcin andan electrophilic mustard, which exhibits the DNA cross-linking andcytotoxic effects associated with the parent drug. However, the systemic distribution of CPA and ifosfamide and their alkylating metabolites inevitably results in several significant side effects, includingcardiotoxicity, renal toxicity, marrow suppression, and neurotoxicity(17—20).We have recently demonstrated that CPA treatment in combination with P450 gene transfer represents a unique prodrug/enzymeactivation system that shows significant promise for cancer genetherapy (21, 22). Using this prodrug/enzyme activation system, tumorcells can be rendered highly sensitive to CPA or ifosfamide in vitro bytransduction of CYP2BJ, which encodes a liver P450 enzyme that

exhibits a high rate of CPA and ifosfamide activation (23, 24). Thisenhanced chemosensitivity is also apparent in studies using a s.c.rodent solid tumor model and a human breast tumor grown in nudemice in vivo and is strikingly effective in spite of the presence of asubstantial liver-associated capacity for prodrug activation in theseanimals (21, 25). This P450-based approach also shows promise forgene therapy applications in the treatment of brain tumors (22, 26).

The P450 system consists of two protein components, the hemecontaining P450 and the flavoprotein RED, both embedded in thephospholipid bilayer of the endoplasmic reticulum. RED is a FADand FMN-containing flavoenzyme, encoded by a single gene (27, 28),which catalyzes the transfer of electrons required for all microsomalP450-dependent enzyme reactions, including drug metabolism (29,30).Detailedstudiesof theelectrontransfermechanismhaveestablished that a total of two electrons from NADPH arc transferred, firstto FAD and then to FMN, before being transferred, one at a time, tothe P450 hemeprotein (31). P450, in turn, uses these reducing equiv

5 The abbreviations used are: CPA, cyclophosphamide; P450, cytochrome P450; RED,

NADPH P450 reductase; FAD, flavm adenine dinucleotide; FMN, flavin mononucleotide;AADP, 3-aminopyridine adenine dinucleotide; DPI, diphenyleneiodonium; Xli.', 2,3-bis[2-methoxy-4-mtro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide; MO!, multiplicityof infection.

4830

Potentiation of Cytochrome P450/Cyclophosphamide-based Cancer Gene Therapyby Coexpression of the P450 Reductase Gene1

Ling Chen,2 Li J. Yu,3 and David J. Waxman4

Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215

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P450 REDUCTASE ENHANCEMENT OF CANCER GENE THERAPY

alents for the hydroxylation of its substrates. Previous studies usingthe CPA/P450 prodrug activation system have been based on thepremise that RED gene transfer is unnecessary because RED is widelyexpressed in the target tumor cells. Indeed, P450-based cancer gene

therapy in combination with CPA treatment is strikingly effectivewithout concomitant transfer of RED (21, 22, 25, 26). In liver cells,which express levels of P450 that arc —50—100-foldhigher thanachieved in our intratumoral P450 gene expression studies (21), theRED component can, under certain conditions, be rate-limiting withrespect to P450-catalyzed enzymatic reactions (32). Titration andreconstitution studies suggest that the rate-limiting nature of REDshould only be manifest at higher levels of P450 (33, 34). Here, weinvestigate the potential for enhancement of the therapeutic activity ofCPA/P450-based gene therapy by cotransfer of the RED gene. Wereport that, despite the expectation that RED would not be limiting inengineered tumor cells that express low P450 levels (32, 33), REDoverexpression leads to a substantial augmentation of P450-mediatedCPA chcmoscnsitivity, both in vitro and in vivo. These findingsenhance the attractiveness of P450-based prodrug activation genetherapy strategies and may facilitate the application of CPAIP4SOgene therapy to cancer therapeutics.

MATERIALS AND METHODS

Chemicals. CPA, ifosfamide, and AADP were obtained from SigmaChemical Co. (St. Louis, MO). 4-Hydroperoxy-CPA was obtained from NovaPharmaceutical Corporation (Baltimore, MD). Metyrapone and DPI chloridewere purchased from Aldrich Chemical Co. (Milwaukee, WI).

Cell Lines. Rat 9L gliosarcoma parental cells (35), 9L transfectants P3 andP17, which stably express P450 gene CYP2BI, and the 9L transfectant 9L-lacZ, which stably expresses the Escherichia coli @3-ga1actosidasegene (21),were grown in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine,50 units/mI penicillin, 50 @g/mlstreptomycin, and 3.79 glliter NaHCO3, withthe pH adjusted to 7.2. Cells were maintained in a humidified atmosphere of5% CO2/95%air.The parental9L and CYP2B1-expressing cells were cotransfected with a rat RED expression plasmid (p450-Red-CMV, kindly provided

by Dr. Gregorio Gil, University of Massachusetts, Worcester, MA) and plasmid pREP4 (Invitrogen, CA) in a molar ratio of 10:1 using Lipofectin (LifeTechnologies, Inc.) according to the manufacturer's instructions. The plasmidpREP4 contains a hygromycin resistance gene. Cell clones that were resistant

to hygromycin B (300 @.sg/ml)were cloned, propagated, and evaluated.In Vitro Cytotoxlclty Assays. For the colony formation assay, cells at

concentrations ranging from 200 to 20,000 per well were plated in duplicate in30-mm tissue culture plates and treated with CPA at the indicated drugconcentrations. Seven days later, plates were stained with crystal violet, andthe number of colonies with >50 cells was counted. The survival fraction wasexpressed as the number of colonies in each treated group compared to

untreated controls. For the growth inhibition assay, cells seeded in 96-wellplates at 1000cells/well were treated with the indicated concentrations of CPAfor 4—5days. Corresponding controls received no drug treatment. Cell survivalwas determined by an Xli' colorimetric assay, a cell proliferation assay thatmeasures mitochondrial dehydrogenase activity of viable cells (36).

Assay of Activated CPA Metabolites In Conditioned Culture Medium.

Cells were plated at 1.5 X 106cells/30.mm dish and then incubated for 24 hwith 2 mM CPA in the presence of 5 nmi semicarbazide (stock solution ofsemicarbazide prepared in culture media, pH 7.4), which traps and stabilizesthe initial 4-hydroxy metabolite. In control experiments, semicarbazide at thisconcentration had no detectable effect on cell viability when it was incubatedwith the cells for up to 48 h. The CPA-conditioned medium from each cell linewas harvested at 24 h, and 0.3 ml of medium was used to determine 4-hydroxy.CPA levels by a fluorimetricassay (37). The detectionlimit was 0.3 nmol of4.hydroxy-CPA (1 @t@i4-hydroxy-CPA produced in 0.3 ml of cell culturemedium).

Limited Dilution Assay to Evaluate Bystander Killing Effect. 9L cellswere seeded in six-well plates at 1.5 X 106cells/well for 12 h and treated with2 ms@CPA for 48 h. The conditioned medium from each well was collected,

diluted 5—50%into fresh medium, and added into wells of a second set ofsix-well plates preseeded with parental 9L cells (l0@ cells/well). Cultures were

maintained for 7 days, and colonies were scored as described for the clonogenic assay.

In Vivo Cytotoxicity Assay. Tumor cells were grown s.c. as solid tumorsin female Fischer 344 rats ( 120—150 g). Each rat was inoculated by s.c.injection of one tumor cell line at the right thigh and a second tumor cell line

at the left thigh at 2 X 106cells/site. Rats were randomized and divided intothree groups. One group was injected with saline as a control. The other twogroups were treated with CPA given as a single i.p. injection 2 weeks aftertumor implantation at 50 mg/kg or 100 mg/kg body weight. At 24 h after CPAinjection, the rats were sacrificed and soaked briefly in 75% ethanol. Thetumors were excised, suspended in DMEM, and minced under sterile conditions. The tumor tissue was then incubated for I5 mm at 37°Cwith shaking ina solution of 500 units/mI of collagenase (Sigma) containing 0.2 mg/mI ofDNase (Sigma). The samples were filtered through a Cell Strainer (FisherScientific), washed twice with DMEM, and then suspended in DMEM sup

plemented with 10%FCS. The single cell suspensions were counted and platedat densities of 200, 2 X l0@,and 2 X l0@cells/well on a six-well plate, eachin duplicate, to determine cell viability by a colony formation assay. Cellcultures were changed to fresh medium after overnight incubation. Cells weregrown for 7 days, and colonies (>50 cells) were then stained with crystal violetand counted. Results are expressed as the surviving fraction ±SE ofcells fromdrug-treated groups compared to untreated controls. The untreated tumor cellsuspension had a plating efficiency (colony forming activity) ranging from 3.0

to 12.2% in individual experiments.Adenovirus-mediated CYP2BI Gene Transduction. A recombinant ad

enovirus, Ad.CMV-2Bl, carrying the CYP2BI gene, was constructed as de

scribed (25). Large-scale production of recombinant adenovirus was performed

by growth in 293 cells followed by purification by double cesium gradientultracentrifugation (38). The titer of purified adenovirus was determined in aspectrophotometer at 260 nm and by plaque assays. Ad.CMV-2BI was addedto tumor cells plated on 30-mm-diameter tissue culture plates at multiplicities

of infection (MOIs) as indicated in the figure legends. Twenty four h after viralinfection, the infected cells were trypsinized and replated at 500 cells/well onto

96-well tissue culture plates and treated with CPA. Four to 7 days after CPAtreatment, the number of surviving cells was determined using the Xli' assay.

Western Blot and RED Enzymatic Analysis. Microsomal proteins or celllysates prepared from cultured cells were electrophoresed through 10% SDS/

polyacrylamide gels (20 p@gof protein/lane), transferred to nitrocellulose, andthen probed (39) with polyclonal rabbit anti-CYP2B1 antibodies or with rabbitantirat RED antibodies (40, 41). Phenobarbital-induced rat liver microsomes ( I

lLg)were used as a positive control for CYP2BI. RED activity was assayed incell homogenates by the NADPH-dependent reduction of cytochrome C at 550nm (e = 21 mM@cm'; Ref. 40).

RESULTS

Establishment of@Tumor Cell Lines Overexpressing RED. Parental rat 9L gliosarcoma cells and CYP2B 1-expressing 9L cells (21)were cotransfected with an expression plasmid encoding rat RED anda plasmid containing a hygromycin resistance gene. Cell lines thatwere resistant to hygromycin B were selected and cloned. Westernblot analysis of 9L cell microsomes using a rabbit polyclonal antibodythat was specific to RED showed the ovcrexprcssion of a singleprotein band of approximately 80 kDa, corresponding to the molecularmass of purified RED, in samples prepared from the clonal cell lines(Fig. lA, bottom, Lanes 5—JOversus Lanes 1—4).CYP2B1 expressionwas undetectable in parental 9L cells but was readily detectable incells stably transfected with a CYP2B1 expression plasmid (Fig. IA,top, Lanes 3—10)and in the phcnobarbital-induccd liver microsomcpositive control (Lane 11). Analysis of RED enzyme activities mdicated up to 4—10-fold higher RED activity in cell lysates preparedfrom the RED transfcctants than in the corresponding parental cellline controls (Fig. 1B). Clonal cell lines chosen for further studiesincluded parental 9L cells, cell line 9L-R (Fig. 1B), derived fromCYP2B1 - parental 9L cells, line PR) 1, derived from the CYP2B 1

4831

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P450 REDUCTASE ENHANCEMENT OF CANCER GENE THERAPY

RED was less pronounced at higher CPA concentrations owing to thehigh degree of growth inhibition already obtained in the absence ofRED gene transfer. Overexpression of RED in CYP2B1@ cells alsoenhanced the chemosensitivity of the cells to ifosfamide, an isomer ofCPA that also undergoes P450-catalyzed drug activation (data notshown). In control experiments, each of the cell lines showed a similarintrinsic sensitivity to activated CPA when chemically activated drug

was presented to the cells in the form of4-hydroperoxy-CPA (data not

A. WesternBlot9L 2B1 2B1/RED PB

. - lac7 Iac7 P3 ‘@r'R11 PR6 PR1 PR4 PR5 PR7 @tsm

@ —. . —@— — — 4_—,.@.w @—. 2B1

P450 shown).

Reductase Effects of P450 and RED Enzyme Inhibition on CPA Sensitivity

of RED-overexpressing Tumor Cells. In our previous studies, theCYP2B1-selective enzyme inhibitor metyrapone was used to verify

A.9L

9L-R

P3

PR1 1

P17

PR7

Cell LinesFig. 1. Immunoblotting (A) and enzymatic analysis (B) of parental 9L cells and 9L cells

that express CYP2B1 and/or overexpress RED. A, microsomal proteins prepared fromindividual clonal cell lines (20 @gprotein/lane) were analyzed on a Westem blot probedwith polyclonal rabbit anti-CYP2B1 antibodies (top) or anti-RED antibodies (bottom).Individualstablecell lineswerepreparedby stabletransfectionof CYP2BIand/orRED,as indicated. Phenobarbital (PB)-induced rat liver microsomes (1 @.sg)were used as apositive control for CYP2B1 (lower band of doublet in Lane 11, top). Sample in Lane 2is a 9L cell line stably transfected to express the lacZ gene, whereas a 9IJ2BI/lacZ line(21) is shown in Lane 3. Line PR6, derived from P3, expresses RED at a level similar toPR11 (Lane6). LinesPR1,PR4.and PR5,all derivedfromP17,expressREDprotein(Lanes 7—9)and enzyme activity (data not shown) at a 1.5—2-foldhigher level than 9Lwild-type cells and were not characterized further. RED protein is not detectable in Lane11 because of the low loading of liver microsomal protein in this sample. B, RED enzymeactivity (rate of cytochrome C reduction) measured in 9L-derived cell lines. Cell linesoverexpressing RED are designated with an R (9L-R, PR-ll, and PR-7), those expressingCYP2BI are designated by a P (P3 and P17), and those expressing both RED andCYP2B1 are designated by PR (PRI I and PR7). Lines 9L-R, P3, and P17 were derivedfrom parental 9L cells, line PR11 was derived from P3, and line PR7 was derived fromP17.

B.—0— 9L

@ ..@.P3

@ ----A---- P17

—--.---- PR!!

‘,.-,

0.‘@ -.-.R-.-. 9L-R

Fig. 2. Survival ofthe parental 9L, 9L-R(RED@@), P3, P17 (CYP2Bl@), PRI1, andPR7(CYP2B1@RED@ @)ce1lsexposedtoCPA.A,colonyformationcytotoxicityassay.Cells were plated in duplicate in 30-mm tissue culture plates at 200, 2000, and 20,000cells/well and treated with CPA at the indicated drug concentrations. Seven days later,plates were stained with crystal violet, and the number of colonies was counted. Thesurvival fraction was expressed as the number ofcolonies in treated group compared to theuntreated control. B, growth inhibition assay. Cells (1000/well) seeded in 96-well plateswere treated with the indicated concentrations of CPA for 4 days. Corresponding controlsreceived no drug treatment. Cell survival was determined by an Xli' colorimetric assay.Data points, mean growth ratio (%), i.e., cell number (XTI' activity) in drug-treated platesas a percentage of the corresponding drug-free controls, for triplicate samples.

4832

B. ReductaseActivity

0 .@.@

5-, b@

I)

oEQ @,

cell line P3, and line PR7, derived from the CYP2BI@ cell line P17.Overexpression of RED did not significantly alter the growth rates ofthese cell lines (data not shown).

Effect of RED Overexpression on CPA Sensitivity of CYP2B1-positive and CYP2B1-negative Tumor Cells. We first testedwhether the overexpression of RED sensitized tumor cells to CPA.Multiple RED-overexpressing cell lines derived from CYP2B1 andCYP2B1@ cell lines were assayed for their sensitivity to CPA cyto

toxicity. As shown in Fig. 2, although the overexpression of REDalone did not sensitize 9L tumor cells to CPA (line 9L-R), overexpression of RED in CYP2B1@ cells substantially enhanced the cyto

toxic response (lines PR1 1 and PR7 versus lines P3 and P17). Thisenhanced cytotoxicity was evident at both low and high CPA concentrations when evaluated in a colony formation assay (Fig. 2A).Enhanced cytotoxicity of the RED-ovcrcxpressing cells was also

observed in a growth inhibition assay (Fig. 2B), although the effect of

CPA (mM)

0.5 0.75

CPA (mM)

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..z.

A'@

P450 REDUCTASE ENHANCEMENT OF CANCER GENE ThERAPY

that the overcxprcssion of CYP2B 1 enzyme activity per se is responsible for the chemosensitivity of CYP2B1-positive tumor cells to CPAand ifosfamide (21). In agreement with those findings, metyraponc at10 @.LMand higher concentrations significantly blocked the cytotoxiceffects of CPA toward CYP2B1@ cells (Fig. 3; P3 and P17 cell lines).By contrast, in RED-overexpressing (indicated by RED@@CYP2Bl@ cells, metyraponc at a concentration of 10 p.Monly slightlyinhibited the cytotoxic effect of CPA. Inhibition of CPA cytotoxicityin the RED-overexpressing cells required a much higher concentration

I)

0

0

— 0— 0

0

0

—0—— 9L

. 9L-R....o.-.. p3

----.---- PR!!

- - -@- - - P17

-@-@A-@-@PR7

p...-@@@ -@--

A.

0

.@

C,,

c( ,

CPA (1 mM) +AADP(0.5mM) +DPI(5@xM)

Fig. 4. Effects of reductase inhibitors AADP and DPI on CPA cytotoxicity inCYP2B 1@ and CYP2B 1@ RED@@@ cells. Cells (500/well) seeded in 96-well plates weretreated with 1 mat CPA in the absence or presence of the RED inhibitors AADP (0.5 mat)or DPI (5 psi). Corresponding controls received no drug treatment. Cell survival wasdetermined by an XT1' colorimetric assay. Cell survival data (Xli' activity in drug-treatedplates as a percentage of the corresponding drug-free controls) were normalized to 1 forthe CPA-treated samples. Columns, mean fold increases in cell survival observed in thepresence of CPA together with AADP or DPI, compared to CPA alone for triplicatesamples; results shown are representative of two to three independent experiments. AADPand DPI were added to the cells 30 mm prior to CPA treatment.

0

— ‘@)

Conditioned Media (%)Fig. 5. CPA-treated CYP2B1@ RED@@@ cells mediate a strong bystander cytotoxic

effect toward CYP2B 1 cells. Parental 9L cells seeded in 30-mm plates (l0@'cells/well)in duplicate were cultured with 5—50%of condition media collected from each of theindicated cell lines (1.5 X l0@)incubated with 2 mat CPA for 48 h as described in“Materialsand Methods.―Data points, mean number of viable parental 9L cells, determined using a hemacytometer based on trypan blue exclusion S days after treatment, forduplicate values.

1@

0.1•

0.01@

B.

I I I I0 10 100 500

MTP (p@M)

Fig. 3. Effect of P450 enzyme inhibitor Ml? on CPA cytotoxicity in CYP2B1@ andCYP2B1@ RED@@@ cells. A, colony formation cytotoxicity assay. Cells (200 and 2000cells/well) were plated in duplicate in 30-mm tissue culture plates and treated with I matCPAin the absenceor presenceof the indicatedconcentrationsof metyrapone(MTP).Seven days after treatment, plates were stained with crystal violet, and the number of

colonies was counted. The survival fraction is expressed as the number of colonies in eachtreatment group compared to the untreated control. B, growth inhibition assay. Cells(1000/well) seeded in 96-well plates were treated with 1 mat CPA in the absence orpresence of metyrapone (MTP), as indicated. Corresponding controls received no drugtreatment. Cell survival was determined by an Xl'!' colorimetric assay. Data points, meangrowth ratio (%). i.e., cell number (XTT activity) in drug-treated plates as a percentage ofthe correspondingdrug-freecontrols,for triplicatesamples.

—a---

..-..-o...-...

----A----

- - -A- - -

-.-.u-.-.

9L

p3

P17

PR! I

PR7

9L-R

0 10 100 1000

of metyrapone, both in colony formation assays (Fig. 3A) and ingrowth inhibition assays (Fig. 3B). To verify that the overexpressionof RED is, in fact, the cause of the enhanced chcmosensitization of theCYP2B1@ RED@@@ cells, we examined the effects of two RED

4833

MTP (jiM)

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P450 REDUCTASE ENHANCEMENT OF CANCER GENE ThERAPY

A. 9L/2B1+9L/lacZ(1:1) B. 9L/2B1+9L/lacZ+CPA

@,,

C. 9L/2B1/RED + 9L/lacZ (1:1) D. 9L/2B1/RED + 9L/lacZ + CPA@..‘.- .f@.@

•@: “@@ r :@ : 4

ISt?@ I@@ ‘@L@.4s:@@@ I- ¼II@ ,

t@: t. @;i; @â€:̃@‘ , ‘ ,,@@@ ‘ti., ,

5―@@@ t j@

. &@ , .. ,4@,js'@ @, ..:@@@

, ?@:*%I;@:@@&.@ ‘. ‘ ‘r@1 •.@

Fig. 6. Bystander toxicity of CPA-treated CYP2B 1@ RED@@@ cells determined in a coculture assay. CYP2B1@ cells (A) and CYP2B1@ RED@@@ cells (C) were mixed with equalnumbers of lacZ-marked 9L cells and cultured in the absence (A and C) or in the presence (B and D) of I mat CPA. Shown are 0.5% glutaraldehyde-fixed cells, 5 days after initiationof drug treatment, stained with 5-bromo-4-chloro-3-indolyl-j3-t-galactopyranoside for 4 h to visualize the 9L-IacZ cells (dark staining).

inhibitors: AADP, a competitive inhibitor of RED (42), and DPI, amechanism-based inhibitor of RED activity (43, 44). In the absence ofCPA, AADP at 0.5 m@ishowed no toxicity to the cells, whereas DPIat 5 @.LMshowed about 20% growth inhibition (data not shown). AADPand DPI partially rescued the CYP2B I@ and CYP2B 1@ RED@@cells from CPA toxicity, with the extent of this protection effect beingsomewhat higher in the case of the CYP2B1@ RED@@@ cells (Fig. 4).Overall, the CYP2B 1@ RED@@@ cell survival ratio was increased bythe RED inhibitors 2—3-fold(AADP) and 3—5-fold(DPI). Thus, theenhanced chemosensitivity of CYP2B1@ RED@@@ cells to CPA isdependent on the overexpression of RED enzyme activity.

Bystander Killing Potential of RED-expressing Tumor Cells.CPA-treated CYP2B1@ cells mediate a bystander killing of cocul

tured CYP2BI cells that does not require direct cell-cell contact(21). On the basis of this observation, we carried out a limited dilution

assay to evaluate the cytotoxic potency of conditioned media obtainedfrom each of the 9L tumor cell lines incubated with CPA. Conditionedmedium from the CYP2B1@ RED@@@ cell lines PR1 1 and PR7 wassubstantially more cytotoxic than was conditioned medium prepared

from the CYP2B 1@ cells P3 and P17 (Fig. 5). This increased formation of cytotoxic metabolites by the RED-overexpressing cells re

sulted in a greater bystander cytotoxicity of these cells toward cocultured 9L cells that do not express CYP2B1. As shown in Fig. 6, CPA

treatment resulted in a substantial, albeit incomplete, bystander cytotoxicity of CYP2B1@ cells toward CYP2B1 cells, which weremarked in this experiment with the lacZ gene and visualized by5-bromo-4-chloro-3-indolyl-f3-D-galactopyranoside staining (Fig. 6, Bversus A). However, coculture with CYP2B 1@ RED@@@ cells resulted in a nearly complete killing of the 9L/lacZ marker cells (Fig. 6,D versus C). CPA treatment essentially eliminated both the CYP2B1@

cells and the CYP2B1@ RED@@@ cells in the experiment shown (Fig.6, B and D, unstained cells).

Metabolism of CPA to 4-Hydroxy-CPA by RED-overexpressing

Tumor Cells. To ascertain whether the increased CPA sensitivity ofCYP2B 1@ RED@@@ cells was associated with an increase in the

conversion of CPA to its activated metabolites, culture medium wascollected from the tumor cell lines 24 h after incubation with CPA inthe presence of semicarbazide, which stabilizes the primary metabolitc 4-hydroxy-CPA. Analysis of the fluorescent product obtainedafter derivatization with 3-aminophenol revealed —3-foldhigher 1evels of 4-hydroxy-CPA formed by the CYP2B 1@ RED@@@ cell linePR! 1 compared to the corresponding parental CYP2B1@ control line

4834

£..4..@4( ..@..,t@ I

. ,. - z@. .‘

@ . .‘.‘... ..

.@@ : • :.@@ . . ‘. . ...

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P450 REDUCTASE ENHANCEMENT OF CANCER GENE THERAPY

,@\ this in vivo study but not detectable in cell culture (compare Figs. 2,

@ 3, and 8), is a reflection of drug activation by P450 enzymes expressed‘; in theliver. Moststrikingly,however,anadditional1-logof tumorc@ cell killing wasachievedin theCYP2Bl@RED@@@ tumors.Thus,;.E thecombinationofintratumoralCYP2BJgeneexpressionwithRED

@ gene transfer yielded a dramatic 50—100-fold overall increase in@ tumor cell kill in vivo over that provided by hepatic P450-catalyzed

.E drugactivationalone.@ DISCUSSION

@ Previous studies have shown that transduction of a mammalianC P450gene,CYP2BJ,intoeitherrodentorhumantumorcellsrenders4 thesecellshighlysensitivetoCPAandifosfamidetoxicity(21,22,

25). Here, we demonstrate that the therapeutic efficacy of the CPA/P450 system can be significantly enhanced by incorporation of asecond gene, that encoding RED. RED mediates the transfer of

Cell lines

P3 (Fig. 7). A smaller increase in 4-hydroxy-CPA levels was observedin the case of the PR7 cell line because of the higher basal 4-hydroxy

CPA level formed by the CYP2B1@ parental cell line P17 comparedto P3. This latter fmding is in agreement with our observation that lineP17 is somewhat more sensitive to CPA cytotoxicity than line P3(compare Figs. 2 and 3). The low level of apparent 4-hydroxylatedmetabolites seen in the case of 9L parental cells corresponds to afluorescence value at the limit of detection in this assay.

Adenovirus-medlated Transfer of CYP2B1 Gene to TumorCells with and without RED Overexpression. The studies dcscribed above were performed in isolated clonal cell lines. To furtherestablish the relationship of RED overexpression to the enhancedchemosensitivity of CYP2B1@ tumor cells, replication-defective recombinant adenovirus carrying the CYP2B1 gene (Ad.CMV-2B1) wasused to infect parental 9L and 9L-R cells at MOIs ranging from 50 to800. As shown in Fig. 8, cells infected with Ad.CMV-2Bl acquiredCPA sensitivity, with the effect most striking in the case of theRED-overexpressing tumor cells (Fig. 8B). In control experiments,infection of 9L cells with an adenovirus carrying the LacZ gene(bacterial @3-galactosithse) did not sensitize the cells to CPA (data notshown).

Chemosensifivity of RED-overexpressing 9L Tumors Treatedwith CPA in Vivo. To determine whether RED ovcrcxpression in

CYP2B1@ tumor cells translates into a therapeutic advantage in vivo,a tumor excision assay was used to quantitate drug toxicity induced invivo over a 24-h period following CPA treatment. CYP2B1@ andCYP2B1@ RED@@@9L tumors and parental CYP2BF 9L tumorswere grown s.c. in female Fischer 344 rats and then were treated withCPA. Twenty four h after CPA treatment, the tumors were excisedfrom the animals, dispersed to give a single cell suspension, and thenplated on culture dishes. The number of surviving tumor cells thatform colonies was then determined. As shown in Fig. 9, CPA at a doseof 100 mg/kg body weight induced up to a 5—10-foldgreater killing ofthe CYP2B1@ tumors compared to wild-type 9L tumors. The cytotoxicity of CPA toward parental 9L tumor cells, readily apparent in

9L P3 PR11 P17 PR7

Fig. 7. 4-Hydroxy-CPA levels in conditioned medium from cells cultured with CPA.Parental CYP2B1 cell lines P3 and P17, and their corresponding RED-overexpressingderivatives PR11 and PR7 were plated confluent (1.5 X l0@cells per 30-mm culture dish)so that the cell number remained relatively constant during the course of the experiment.Cellswereincubatedfor 24 h with2 matCPAin the presenceof 5 matsemicarbazide.Mediumwascollectedandthenassayedfor4-hydroxy-CPAusinga fluorometricassay,as described in “Materialsand Methods.―Limit of detection in this assay was —1j@M4-hydroxy-CPA.ThehigherCPA4-hydroxylaseactivityof lineP17comparedto lineP3is consistent both with the higher cytotoxic potential of CPA-conditioned media from P17cells(Fig.5)andwiththegreatercytotoxicityofCPAtowardtheP17cellsin vitro(Figs.2 and 3) and in vivo (Fig. 9).

—a--

--0---

....o....

- - -n- - -

-. -..-...

UI

Ad.CMV-2B(50)

Ad.CMV-2B(100)

Ad.CMV-2B(200)

Ad.CMV-2B(400)

Ad.CMV-2B(800)

—a--

-—0—j

....o-...

—6---

- - -@- -

-....-...

UI

Ad.CMV-2B(50)

Ad.CMV-2B(lOO)

Ad.CMV-2B(200)

Ad.CMV-2B(400)

Ad.CMV-2B(800)

CPA (mM)

Fig. 8. CPA sensitivity ofparental 9L cells (A)and RED-overexpressing 9L-R cells (B)following infection with a recombinant adenovirus carrying CYP2BI gene. Cells wereinfected with Ad.CMV-2Bl at MOIs ranging from 50 to 800 (parentheses, right).Uninfected cells (UI) were used as controls. At 24 h postinfection, cells were replated at500 cells per well in duplicate on 96-well plates. CPA was then added at concentrationsup to 2 mat. After 5 days of incubation, cell survival was determined by an Xli'colorimetric assay. Data points, mean growth ratio (%), i.e., cell number (Xli' activity)in drug-treated plates as a percentage of the corresponding drug-free controls, for duplicate samples.

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A. 9L+Ad.CMV-2B1

B. 9L-R+

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P450 REDUCTASE ENHANCEMENT OF CANCER GENE ThERAPY

expression. Thus, the production of cytotoxic CPA metabolites wasincreased in CYP2B 1@ cells that overexpress RED, as evidencedby the increased formation of 4-hydroxy-CPA and by the enhancedbystander killing of cocultured parental 9L cells. This bystandereffect, associated with a localized increase in activated CPA metabolites, may lead to more efficient tumor cell killing even whenonly a subset of tumor cells is transduced with the CYP2BJ gene.Overexpression of RED may greatly facilitate electron transferfrom cellular NADPH to P450. This is supported by our observation that the P450 inhibitor metyrapone was less effective inblocking CPA toxicity in CYP2B1@ RED@@@ cells than inCYP2B 1@ cells (Fig. 3). On the other hand, the RED inhibitor DPIeffected greater reversal of CPA cytotoxicity in CYP2B1@RED@@@ cells compared to CYP2B1@ cells, despite its inherenttoxicity to these cells (Fig. 4). A similar effect was seen with theRED inhibitor AADP. To our knowledge, this is the first demonstration in an intact cell system that RED inhibitors can protectcells from P450-mediated drug toxicity.

The level of cellular RED activity is presently shown to be criticalto the effectiveness of P450-mediated CPA activation. Accordingly,the basal RED activity level may not be sufficient in the case of manytumors to catalyze maximal electron transfer to CYP2B 1. Indeed,although RED activity is widely expressed in human tumor cells, largevariations in the level of enzyme expression are apparent in individual

tumor cell lines.6 The importance of endogenous RED activity wasunderscored in our studies by infecting 9L tumor cells with increasingamount of Ad.CMV-2Bl in parental and RED-overexpressing 9Lcells. RED-overexpressing 9L cells were more sensitive to CPA thanparental 9L cells when infected at the same Ad.CMV-2B1 viralmultiplicity. Moreover, the CPA cytotoxicity in Ad.CMV-2B 1-infected parental 9L cells was not further enhanced at MOIs above 200.In contrast, CPA cytotoxicity in RED-overexpressing 9L cells wasfurther enhanced at higher viral multiplicities.

Given the importance of RED activity level as a factor in determining the sensitivity of tumor cells to CYP2BI-dependent CPAactivation, factors that regulate endogenous levels of RED enzymeactivity and RED gene expression should be considered as potentiallyimportant modulators of intratumoral P450-catalyzed drug activation.These include thyroid hormone, which is essential for full expressionof RED in several tissues (32, 41), as well as various drugs and otherxenobiotics that can increase RED enzyme levels (45, 46). In the caseof P450-based cancer gene therapy applications, in which the goal isto achieve maximal intratumoral CPA activation with minimal systemic toxicity, the present findings suggest an additional approach toregulating hepatic bioactivation of CPA through the modulation ofRED enzyme levels/activities.

In conclusion, we have demonstrated that tumor cells with elevatedRED activity are highly responsive to CPA-based CYP2BJ genetherapy. P450-based cancer gene therapy may therefore be expected tobe greatly facilitated by using viral or other delivery vectors designedto transduce P450 and RED in combination. This could be achievedby use of internal ribosome entry site sequences to achieve coordinateexpression of P450 and RED on a bicistronic message (47, 48) or byconstruction of catalytically active P450-RED fusion proteins (49,50). Furthermore, P450-based cancer gene therapy may be particularly effective against tumor cells with a high endogenous level ofRED. Finally, up-regulation of RED activity in cancer cells by hormones or drug inducers may provide an additional approach to rendering tumor cells hypersensitive to the anticancer drug-activatingP450 genes.

C.—

I

Fig. 9. Evaluation of cytotoxic effects of CYP toward CYP2B1 @andCYP2B1RED@@@ 9L tumors grown in vivo.Female Fischer 344 rats were inoculated with parental9L. CYP2B1@,and CYP2BI@ RED@@@tumorcells by s.c. injection of2 X l0'@tumorcells/0.2 ml in the hindleg as described in “Materialsand Methods.―At 2 week after tumorimplantation, each animal received a single injection of CPA (50 or 100 mg/kg bodyweight) or saline as control. Twenty four h later, tumors were excised, and single cellsuspensions were prepared for assay of colony formation activity. Data points, meansurvival fractions as compared to control groups. for 4 or 5 determinations; bars, SE.

electrons from NADPH to P450 and is required for all microsomalP450-catalyzed enzyme reactions. RED is widely expressed in manycell types, including tumor cells, and this widespread expression ofRED provides an important underlying basis for P450-based cancergene therapy. Here, we demonstrate, however, that, despite its significant basal expression in tumor cells, RED gene transfer substantiallyaugments the CPA sensitivity of target tumor cells transduced withCYP2BJ and thus provides for a more efficacious anticancer prodrugactivation system.

The further chemosensitization of CYP2B 1-expressing tumor cellsby RED gene transfer has several important implications for P450-based cancer gene therapy. First, it appears likely from our presentfindings that the level of RED activity in a given tumor target will bean important determinant of the effectiveness of P450-based genetherapy in the absence of exogenous, cotransfected RED. Second,tumors that have moderate or low levels of RED activity are primetargets for incorporating RED into any P450-based gene therapystrategy. Some therapeutic enhancement may also be anticipated,however, even in tumor cells with comparatively high levels ofendogenous RED expression. RED is known to be potentially ratelimiting for P450-catalyzed oxidative metabolism in liver cells as aconsequence of the large molar excess of P450 over RED in this tissue(32, 33). However, here we demonstrate that, even in P450-transfected tumor cells, where RED is constitutively expressed at a comparatively high level and can supply electrons needed for P450-catalyzed CPA bioactivation, a substantial enhancement of P450metabolic activity can nevertheless be achieved through elevated RED 6D. J. Waxman,unpublishedresults.

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50

CPA (mg/kg)

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1997;57:4830-4837. Cancer Res   Ling Chen, Li J. Yu and David J. Waxm  Gene

P450 ReductaseCancer Gene Therapy by Coexpression of the Potentiation of Cytochrome P450/Cyclophosphamide-based

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