[CANCER RESEARCH 56, 1331-1340. March 15. 1996]

Sensitization of Human Breast Cancer Cells to Cyclophosphamide and Ifosfamideby Transfer of a Liver Cytochrome P450 Gene1

Ling Chen, David J. Waxman,2 Dongshu Chen, and Donald W. Kufe

Division of Cancer Pliarrnacology, Dana-Farber Cancer Institute, Han'artl Medical School. Boston. Massachusetts 02115 [L. C., D. C., D. W. K.]. and Division of Cell and

Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215 [L. C., D. J. W.j

ABSTRACT

The cancer chemotherapeutic agent Cyclophosphamide (CPA) and itsisomer ifosfamide (IFA) are alkylating agent prodrugs that require metabolism by liver cytochrome P450 (P450) enzymes for antitumor activity.The therapeutic effectiveness of these oxazaphosphorines is limited by thehematopoietic, renal, and cardiac toxicity that accompanies the systemicdistribution of liver-derived activated drug metabolites. Transfer of a livercytochrome P450 gene, CYP2BI, into human breast MCF-7 cancer cells is

presently shown to greatly sensitize these cells to oxazaphosphorine toxicity as a consequence of the acquired capacity for intratumoral CPA andIFA activation. Thus, CPA and IFA were highly cytotoxic to MCF-7 cells

following stable transfection of CYP2BJ but exhibited no toxicity to parental tumor cells or to a ß-galactosidase-expressing MCF-7 transfectant.

This cytotoxicity could be appreciably blocked by the CYP2B1 inhibitormetyrapone. Cell cycle analysis revealed that CPA arrested the CYP2B1-expressing cells, but not CYP2B1-negative cells, at G2-M phase. A strongbystander cytotoxicity effect that does not require direct cell-cell contactwas mediated by CYP2Bl-expressing MCF-7 cells on non-CYP2Bl cells.

Intratumoral CYP2B1 expression conferred a distinct therapeutic advantage when treating MCF-7 tumors grown in nude mice with CPA, asrevealed by a 15-20-fold greater in vivo cytotoxicity, determined by tumorexcision/colony formation assay, and by the substantially enhanced anti-tumor activity, monitored by tumor growth delay, for CYP2B1-expressingMCF-7 tumors as compared to CYP2B1-negative control tumors. These

enhanced therapeutic effects were obtained without any apparent increasein host toxicity. To evaluate the extent to which a CPA/P4SO gene therapystrategy may be generally applicable to other tumor cell types, a replication-defective recombinant adenovirus carrying the CYP2B1 gene drivenby the cytomegalovirus (CMV) promoter Ad.CMV-2Bl was constructedand used to infect a panel of human tumor cell lines. Ad.CMV-2Bl

infection rendered each of the cell lines highly sensitive to CPA and IFAcytotoxicity, with substantial chemosensitization seen at multiplicities ofinfection as low as 10. The CPA/P450 prodrug activation system may thusserve as a useful paradigm for further development of novel cancer genetherapy strategies that utilize drug susceptibility genes to significantlypotentiate the antitumor activity of conventional cancer chemotherapeuticagents.

INTRODUCTION

Gene therapy using drug susceptibility genes is emerging as apromising strategy for cancer chemotherapy. This approach to cancertreatment is based on the premise that a significant therapeutic advantage can be gained by direct transfer to tumor cells of a drugsusceptibility gene, or "suicide gene." which encodes an enzyme that

can catalyze the intratumoral activation of an anticancer prodrug ( 1,2). Widely studied model systems that have shown some promise are

Received 9/21/95; accepted 1/17/96.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1This investigation was supported in part by NIH Grant CA49248 (to D. J. W.) and by

Army Grant #DAMD 17-94-J-4394. The content of the information does not necessarily

reflect the position or the policy of the government, and no official endorsement shouldbe inferred.

•¿�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.

based on the introduction of genes such as HSV-rk3' or bacterial CD,

which render mammalian cells sensitive to the otherwise nontoxicantiviral agent and nucleoside analogue ganciclovir (3, 4) or to theantifungal drug 5-fluorocytosine (5-7), respectively. This general

strategy may be extendable to conventional anticancer drugs, in particular those nontoxic prodrugs that are subject to enzymatic activation by endogenous drug-metabolizing enzymes (8). Transfer of genesencoding such prodrug-activating enzymes directly into tumor cells

may conceivably augment the endogenous activity associated withextratumoral (e.g., liver) drug metabolism, thereby providing for amore effective anticancer effect (9).

The oxazaphosphorine CPA and its isomer IFA are cell cycle-

independent alkylating agents that have a broad spectrum of activityagainst a variety of neoplasms, including breast cancer (10). CPA andIFA are both devoid of alkylating activity and must first be activatedin the liver by cytochrome P450 (CYP) enzymes to manifest theirlatent cytotoxic potential. The therapeutic efficacy of these drugs islargely dependent on the patient's liver enzyme function with respect

to prodrug activation and on the tolerance of host tissues to thesystemic distribution of activated metabolites formed in the liver.However, administration of agents that induce liver P450 enzymes,such as phénobarbitalor prednisone (11). accelerates the rate but notthe net extent of CPA activation and appears to have little impact ondrug efficacy (12-14). Recently, the specific P450 enzymes that

catalyze the activation of CPA and IFA have been identified in bothrat and human liver. The P450 enzymes designated CYP2B1,CYP2C6, CYP2C11, and CYP3A1 contribute to the activation ofCPA and IFA in adult rat liver (15, 16), whereas the correspondinghuman gene products CYP2B6 and CYP3A4, in addition to severalCYP2C enzymes, have been identified as the catalysts of oxazaphosphorine activation in human liver (17, 18). CPA and IFA are hydroxy-lated by these P450s at C-4 to yield 4-hydroxy-CPA and 4-hydroxy-

IFA, respectively, which exist in equilibrium with the correspondingring-opened aldophosphamides. These primary metabolites then undergo spontaneous ß-eliminationto yield acrolein and an electrophilic

mustard (phosphoramide mustard or ifosphoramide mustard), which isthe therapeutically active. DNA-alkylating metabolite (10).

In addition to their widespread use in conventional cancer chemotherapy, oxazaphosphorines can be used in high dose regimens to treatmetastatic breast cancer when combined with autologous bone marrow transplantation (19-21). In this setting as well, the therapeutic

efficacy of these drugs is limited by host toxicity as a result of thesystemic distribution of activated drug metabolites that have significant cytotoxic side effects, including cardiac and renal toxicity. Inaddition, IFA can have significant neurotoxic side effects, which aremediated by its /V-dealkylation metabolites (22, 23). Since tumor cellstypically do not express detectable levels of CPA- or IFA-activating

CYP enzymes, we have considered the possibility that transduction oftumor cells with an oxazaphosphorine-activating P450 gene may

1The abbreviations used are: HSV-tk, herpes simplex virus thymidine kinase; CD,

cytosine deaminase; CPA, Cyclophosphamide; IFA, ifosfamide; P450 or CYP. cytochromeP450: CYP2B1. CYP form 2B1; XTT, 2,3-bis[2-methoxy-4-nitro-5-sultbphenyl|-2//-tetrazolium-5-carboxanilide inner salt: CMV, cytomegalovirus; 4HC, 4-hydroperoxy-CPA; ßGal,ß-galactosidase.

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['450 BASCO CASfKR (iliNh TllliKAI'Y

sensitize the tumor cells to CPA and IFA by a mechanism thatinvolves direct, intracellular prodrug activation. Consistent with thispossibility, stable transduction of the gene encoding the oxazaphos-phorine-activating P450 form CYP2B1 (15) into rat 9L gliosarcoma

(24) and C6 glioma cells (25) renders these rodent brain tumor celllines highly sensitive to CPA and IFA. However, the extent to whichCYP2B1 gene transfer confers a therapeutic advantage when treatingtumors of non-central nervous system origin and the potential appli

cability of this strategy to human cancer cells in an in vivo situation isuncertain. In the present report, we have evaluated the therapeuticimpact of transferring the CYP2B1 gene into human MCF-7 breast

cancer cells when combined with oxazaphosphorine treatment in vitroand m vivo. In addition, we have constructed a replication-defective

recombinant adenovirus carrying the CYP2BI gene that facilitatesefficient transduction of tumor cells with CYP2B1. Breast cancer andother tumor cell types transfected with CYP2BI are shown to acquirehigh sensitivity to CPA. without a detectable increase in host toxicity.

MATERIALS AND METHODS

Chemicals. CPA was purchased from Sigma Chemical Co. (St. Louis.MO). IFA was obtained from Bristol-Myers Squibb (Princeton, NJ). 4-Hy-

droperoxy-CPA was obtained from Nova Pharmaceutical Corporation

(Baltimore, MD). Metyrapone was purchased from Aldrich Chemical Co.(Milwaukee, WI).

Cell Culture. Human tumor cell lines were obtained from American TypeCulture Collection (Rockville. MD). Cells were grown as monolayers in theculture media recommended by American Type Culture Collection, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100

units/ml penicillin, and 100 ng/ml streptomycin. Cells were maintained in a5% CO,/95% air humidified atmosphere.

Stable Transfection of MCF-7 Cells. MCF-7 cells were cotransfectedwith a plasmid containing a rat CYP2B1 cDNA (pMT2-CYP2Bl; kindly

provided by Dr. Milton Adesnik. New York University, New York. NY) andpSV2Neo at a molar ratio of 20:1 using Lipofectin (GIBCO-BRL) accordingto the manufacturer's instructions. pSV2Neo contains a neomycin phospho-

transferase gene, which confers resistance to G418. pCMV-ßGal.Neo. which

contains the neomycin phosphotransferase gene and the Escherichia coli IticZgene (24), was used to transfect MCF-7 cells to establish a cell line that stably

expresses ßGal.Stable transfectants were cloned under selection in 1 mg/miG418 (GIBCO-BRL). Cell lines resistant to G418 were cloned, propagated,

and evaluated.Cytotoxkity Assay. To test for drug sensitivity. 4 X K)4tumor cells were

plated in each well of a 6-well tissue culture plate (Falcon 3046) in duplicate.

Drugs were added approximately 20 h after seeding. Cells were allowed togrow for 6 to 7 days after drug treatment, and the final viable cell number wasthen determined by trypan blue exclusion and cell counting. Cells were rinsedwith PBS. dispersed using trypsin-EDTA (GIBCO-BRL), and then counted

with a hemocytometer. In some cases, cell number was assessed using acolorimetrie cell proliferation assay (XTT assay) that measures the mitochron-

drial dehydrogenase activity of viable cells (26).Flow Cytometric Analysis. Following drug treatment under conditions

indicated in the text, cells were trypsinized, centrifuged. resuspended in PBS.and then gently vortexed while 80% ethanol was added to a final concentrationof 40%. The fixed cells were treated with RNase A (10 jug/ml) at 37°Cfor 30

min and then resuspended in a solution of 50 ng/ml propidium iodide. Cellcycle analysis was performed using a FACScan instrument (Becton Dickinson). Results are presented as the number of cells versus the amount of DNAas indicated by fluorescence intensity.

Coculture Experiments. Parental MCF-7 cells were plated in the bottomwells (30 mm in diameter) of Falcon 6-well coculture plates. CYP2B1-expressing cells (MCF-7-P) or CYP2B1-negative cells (MCF-7-Z) were plated

in 25-mm cell culture inserts (0.45 /urn pore size: Falcon 3090). Culturemedium was removed 18-24 h later by aspiration. Culture medium without

drug (1.0 ml) was added to the bottom well, and 1.0 ml medium containingdrug at a 2X concentration was added to the upper cell culture insert. Cellnumbers were determined 6 to 7 days later.

Tumor Growth Delay Measurements. Female homo/ygous (nu+/nu + )nude athymic Swiss mice (Taconic, Germanlown. NY), 20-30 g, were used. Asingle s.c. 17/3-estradiol pellet (1.7 mg. 60-day release pellet; InnovativeResearch. Toledo. OH) was implanted I day prior to s.c. injection of MCF-7,MCF-7-Z. or MCF-7-P tumor cells in the exponential growth phase (2 X IO7

cells/0.2 ml/injection site). Each mouse received two tumor implantations, aCYP2B1-negative tumor (parental MCF-7 or MCF-7-Z) on one flank and a

CYP2B1-expressing tumor (MCF-7-P2 or MCF-7-P9) on the other. Drug

treatment was initiated 4 to 5 weeks later. CPA was given by i.p. injection at150 mg/kg body weight per day for two consecutive days. Tumor size wasdetermined by external caliper measurement, and host toxicity was monitored

by body weight measurement.Tumor Excision Assay. CPA treatment was initiated 6 weeks after tumor

implantation, at which time the tumors were approximately 50-KX) mrrr in

size. The mice were then treated by i.p. injection of CPA at a dose of 150mg/kg body weight, followed by a second drug injection 20 h later. At 4 h afterthe second CPA injection, the mice were sacrificed and soaked briefly in 75%ethanol. The tumors were excised, suspended in DMEM cell culture medium(GIBCO). and minced under sterile conditions. The tumor tissue was thenincubated for 15 min at 37°Cwith shaking in a solution of 500 units/ml of

collagenase (Sigma) containing 0.2 mg/ml of DNusc (Sigma). The sampleswere filtered through a Cell Strainer (Fisher Scientific), washed twice withDMEM. and then suspended in DMEM supplemented with 10% FCS. Thesingle-cell suspensions were counted and plated at densities of 10', IO4, and

IO5 cells/well of a 6-well plate, in duplicate, for a determination of cell

viability by a colony-forming assay. Cells were grown for 10-12 days, and

colonies (>50 cells) were then stained with crystal violet and counted. Resultsare expressed as the surviving cell fraction ±SEM for the drug-treated groups

compared to untreated controls. The untreated tumor cell suspension hada plating efficiency (colony-forming activity) of 5 to 9% in separate

experiments.Recombinant Adenovirus. A recomhinant adenovirus carrying the

CYP2B1 coding sequence was prepared by homologous recombination in thehuman embryonic kidney cell line 293 (27). A shuttle plasmid. pCMV-2Bl.was first constructed by excising the HSV-lk gene from the adenovirus shuttleplasmid pCMV-HSV-tk (28) and then cloning in its place the CYP2B1 codingsequence, excised from pMT2-CYP2Bl. The resulting pCMV-2Bl shuttle

plasmid contains the CYP2B1 gene under the control of the human CMVimmediate-early promoter and enhancer, followed by SV40 polyadenylationsignals, and flanked by type 5 adenovirus map units().()-1.3 and 9.3-17.3. This

shuttle plasmid was transfected by calcium phosphate precipitation into 293cells together with plasmid pJM17, which contains an El region insertion at3.7 map units of the adenoviral genome (Ret. 29; pJM17 kindly provided by

Dr. Frank Graham. McMaster University, Hamilton. Ontario. Canada). Recomhinant adenovirus was isolated from a single plaque and expanded in 293cells. The resulting recomhinant adenovirus, Ad.CMV-2BI, was verified by

PCR and by restriction enzyme digestion. The virus was confirmed to bereplication-defective by the absence of a cytopathic effect in a HeLa cell assay.Ad.CMV-ßGal is a structurally similar replication-deficient recombinunt ad

enovirus (a generous gift of Dr. R. Crystal. New York Hospital. New York.NY), which contains a ßGalreporter gene. Large scale production of recombinunt adenovirus was performed by growth in 293 cells, followed by purifi

cation by double cesium gradient ultracentrifugation as described (27). Thetilers of purified adenovirus were determined in a spectrophotometer at 260 nm

and by plaque assays.Adenovirus-mediated Gene Transduction in Tumor Cells. Cells (0.5-

1 X K)'1)were plated on 30-mm diameter tissue culture plates. Recomhinant

adenovirus, Ad.CMV-2B 1 or Ad.CMV-ßGal. was added to the cells at various

multiplicities of infection. The transduced cells were replated and treated withCPA. Six to 7 days after drug treatment, surviving cells remaining attached lothe culture plate were counted, and the results were compared to non-drug-

treated control plates.Western Blot Analysis. Cell lysates prepared from cell lines (stably trans

fected MCF-7 cells, as well as adenovirus-infected cells) were electrophoresedthrough 10% SDS-polyacrylamide gels (10-30 fig/well), transferred to nitrocellulose, and then probed with polyclonal rabbit anti-CYP2Bl antibodies (30,

31 ). Phenobarbital-induced rat liver microsomes ( 1 jug) were used as a positive

control tbrCYP2BI protein.

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P450-BASED CANCER GENE THERAPY

Immunohistochemistry. MCF-7 cells cultured on 30-mm plates were infected with Ad.CMV-2Bl at a multiplicity of infection of 50. After 48 h, the

monoluyers were fixed with Histochoice tissue fixature MB (Amresco. Solon.OH I. The monoluyers were then treated with 5% goat serum, washed withPBS, and reacted with anti-CYP2Bl antibody at room temperature for 1 h.

After washing in PBS. the bound primary antibody was detected by animmunoperoxidase procedure using the Vectastain ABC kit (Vector Laboratories Inc.. Burlingame. CA) and diaminobenzidine tetrahydrochloride as chro-

mogen.

RESULTS

Expression of CYP2B1 Gene in Human Breast Cancer MCF-7Cells. To establish human breast cancer MCF-7 cell lines that stablyexpress the CYP2BI gene. MCF-7 cells were cotransfected withplusmids encoding rat CYP2BI and a neomycin-resistant gene. Cells

resistant to G418 were then selected and clonal lines isolated. Westernblot analysis of cell ly.sates using anti-CYP2BI antibody revealed a

single protein band of approximately 52 kDa, corresponding to themolecular mass of purified CYP2B1, in samples prepared from theclonal cell lines designated MCF-7-P2. -P3. -P5. -P8, -P9. and -P26

(Fig. 1). Each of the cell lines expressed a similar level of CYP2B1protein, which was estimated to be 2-4% of that present in pheno-barbital-induced rat liver microsomes. No CYP2BI protein was detected in parental MCF-7 cells or in the clonal sublines MCF-7-Z andMCF-7-Z2. which stably express the E. coli lacZ gene, encoding ßGal

(Fig. I).Effect of CPA and IFA on Cultured CYP2B1-expressing Cells

and CYP2Bl-negative Cells. To test for the effects of CYP2B1expression on the drug sensitivity of MCF-7 breast cancer cells,CYP2B1-positive cells (MCF-7-P2, -P3.-P9, and -P26) and CYP2B1-negative cells (parental MCF-7 and lacZ-transfected MCF-7-Z cells)

were cultured together with various concentrations of CPA or IFA.The number of viable cells present 6-7 days after drug treatment wasthen determined. CPA and IFA both inhibited the growth of CYP2B1-positive cells in a concentration-dependent manner (ICW, 75-150 /XM;Fig. 2. A and B). By contrast, parental MCF-7 cells and MCF-7-Z

cells both showed little or no sensitivity to millimolar concentrationsof CPA and IFA. CYP2B1-positive and CYP2B1-negative cells were,however, equally sensitive to 4-hydroperoxy-CPA, which spontaneously decomposes to yield the cytotoxic CPA metabolite phosphor-

amide mustard without the need for enzymatic activation (Fig. 2C).

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(MCF-7 and MCF-7-Z) and CYP2B1-positive cells (MCF-7-P2, -P3. -P9, and -P26). Cells(4 X IO4) plated in duplicate in 30-mm tissue culture plates were treated with the indicated

concentrations of CPA, IFA. or 4HC (A-C. respectively). Shown is the number of

surviving cells counted 6 days after drug addition; bars, SE.

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1 23 4 5 6 7 8 9 10 11 12 13 1415Fig. I. Immunodetection ol'CYP2Bl in parental MCF-7 cells and in MCF-7 cells that

stably express CYP2B1. Microsoma] proteins prepared from the indicated cultured celllines (20 ^ig protein/lane) were analyzed on a Western blot probed with pol\clonal rabbitanti-CYP2B 1 antibodies. Phenobarbital-induced rat liver microsomes ( 1 jig) were used asa standard for CYP2B1 (lower hand of doublet in Lane 15). Clonal MCF-7-derived celllines P2. P3. P5. P8. P9. and P26 express CYP2BI protein, whereas parental MCF-7 andlacZ-transfecled MCF-7 cells (Z and Z2| do not.

Similar results were obtained with the CYP 2BI-expressing clonalcell lines MCF-7-P5 and -P8 (data not shown). Additional experi

ments established that the cytotoxic effects of CPA and IFA towardCYP2B1-expressing cells are substantially reduced by metyrapone, aheme-ligand and CYP2B1-selective enzyme inhibitor (Ref. 32; Fig.

3). In control experiments, metyrapone alone had no effect on cellviability (data not shown). Moreover, metyrapone did not block thecytotoxic effect of the chemically activated CPA derivative. 4HC(Fig. 3). This metyrapone protection effect demonstrates that thepresence of a catalytically active CYP2B1 enzyme is required for theoxazaphosphorine chemosensiti vity of CYP2BI -expressing cells to be

manifest.Cell Cycle Perturbation by CPA in the CYP2B1 -expressing

Cells but not in CYP2Bl-negative Cells. To elucidate the effect of

intracellular CPA activation on the cell cycle progression of breastcancer cells, we examined the cytokinetic consequences of CPAtreatment in CYP2BI-expressing cells, MCF-7-P2 and MCF-7-P9,and in CYP2B1 -negative MCF-7-Z and parental MCF-7 cell controls.

Cells were harvested and stained with propidium iodine, and thedistribution of cells at each stage of the cell cycle was then determined

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P450-BASED CANCER GENE THERAPY

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Fig. 3. Meiyrapone (MTP) selectively blocks the cylotoxic effects of CPA and IFA onCYP2BI-expressing cells. MCF-7. MCF-7-Z. MCF-7-P2. and MCF-7-P9 cells (4 X IO4)

were treated with either I m\i CPA, 1 m.MIFA, or 25 JAM4HC in the ahsence or presenceof 50 /ÌMmetyrapone, as indicated. Corresponding controls received no drug treatment.Data (mean ±range for duplicate samples) are expressed as final cell number determined6 days after drug treatment.

by flow cytometry. As is seen in the histograms presented in Fig. 4A,the cell cycle profile of the CYP2B1 -negative cells was unaffected byCPA treatment. By contrast, for CYP2B1-expressing cells, there wasa substantial accumulation of S-phase cells, as well as an increase in

the G,-M phase 24 h after CPA treatment. This effect was even more

striking 60 and 84 h after CPA treatment (Fig. 4ß).Further evidencefor a block at the S-phase and the GrM phase was provided by theprogressive decrease in the number of 0,,-G, cells at 60 and 84 h

posttreatment. These findings suggest that the intracellular activationof CPA allows the tumor cells continuing egress out of G,,-G, into theS phase but prevents cells from traversing from G-.-M into G()-G|, a

process that ultimately leads to cell death.CYP2Bl-expressing Cells Mediate a Bystander Killing Effect.

We next examined whether CYP2BI-expressing human breast cancercells mediate a CPA-dependent bystander killing of coculturcdCYP2B1 -negative cells. MCF-7-Z cells and MCF-7-P2 and -P9 cells

were used for these experiments since they have similar doublingtimes in culture (data not shown). MCF-7-Z cells and CYP2B1

transfectants were mixed at ratios of 1:1 and 4:1 and then were treatedwith CPA in cell culture. As shown in Fig. 5A, a significant fractionof the total cell population was eradicated when the mixed culture wasexposed to CPA. even when the ratio of CYP2B1-negative toCYP2B1-positive cells was 4:1. Moreover, the CYP2B1 enzyme

inhibitor, metyrapone. could substantially block this effect (data notshown). Thus, CYP2B1 -positive cells confer a P450 metabolism-dependent bystander killing of adjacent CYP2BI-negative cells.

Fig. 4. CPA interrupts cell cycle progression inCYP2B1-expressing cells. A. representative flow cy-tometric DNA histograms of cells treated with CPA.CYP2BI-negative cells (MCF-7-Z and parentalMCF-7) and CYP2B1-expressing cells (MCF-7-P2and MCF-7-P9) were treated with CPA at 12 h afterplating. Cells were trypsinized, stained with pro-pidium iodide, and analyzed for relative DNA contentby flow cytometry analysis at 24 and 60 h after CPAtreatment (36 and 72 h total culture time, respectively). The scale used for the Y-axis was adjusted to

facilitate comparisons of the effect of drug treatmenton the pattern of cell cycle distribution. B, distributionof cells in Gi-M and in G0-Gt in CPA-treatedCYP2B1-expressing and CYP2BI-negative cells.Data in B are based on experiments similar to thoseshown in A.

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P450-BASED CANCER GENE THERAPY

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Fig. 5. C YP2B l -expressing cells mediate a bystander cy totoxic effect towardCYP2BI -negative cells in the presence of CPA. A, cytotoxicity of CPA toward mixedcultures of parental MCF-7 and CYP2B1-expressing MCF-7 cells. MCF-7 cells andMCF-7-Z. -P2. or -P9 cells were mixed in the ratios indicated (total initial cell number = 4 X 104/30-mm tissue culture dish). Cells were untreated or were treated with 1 HIMCPA. Cell numbers (mean ± range of duplicates) were determined 6-7 days afterbeginning drug treatment. B. parental MCF-7 cells (2 x IO4) were plated in the bottom

well of 30-mm culture plates. Upper chambers of Falcon culture inserts were seeded withthe CYP2B1-expressing cells. MCF-7-P2 or MCF-7-P9. or with CYP2B1-negative MCF-7-Z cells (2 X IO5), as indicated along the X-axis. The two cell populations were thus

separated by a 0.45 /urn pore size membrane that prevents direct contact between the twocell populations. Cells were treated with 1 mM CPA or received no drug treatment ascontrol. Cell numbers in the bottom chamber were determined 6 days after drug addition(mean ±range of duplicates).

To assess whether direct cell-cell contact is required for this bystander killing effect, parental MCF-7 cells were seeded in the bottomchamber of a Falcon coculture plate, and either CYP2B1 -positive cells(MCF-7-P2 or -P9) or CYP2B1-negative cells (MCF-7-Z) were

placed in the upper chamber of the coculture inserts. In this manner,the two cell populations are physically separated but share the sameculture medium. As shown in Fig. 5B, CPA treatment killed not onlythe CYP2Bl-positive cells in the upper Falcon coculture inserts butalso the parental MCF-7 cells cultured in the bottom wells. The killing

of both cell populations was significantly reduced by the CYP2B1inhibitor metyrapone (data not shown). In control experiments, therewas no killing of either cell population when MCF-7-Z cells werecocultured with parental MCF-7 cells. Thus, cytochrome P450-cata-

lyzed CPA activation leads to the formation of soluble cytotoxicmetabolite(s) that can mediate a significant bystander killing effect.

Iut i af muorili Expression of CYP2B1 Substantially Enhancesthe Antitumor Effect of CPA in Vivo. In view of the positive resultsobtained in the in vitro experiments described above, we carried outan in vivo tumor growth delay study to compare the CPA sensitivityof CYP2Bl-negative MCF-7 tumors to that of CYP2B1-expressingMCF-7 tumors grown s.c. as solid tumors in female nude mice. Sincethe growth of MCF-7 breast cancer is estrogen dependent, a 60-dayß-estradiolrelease pellet was implanted prior to tumor inoculation.CYP2B1-expressing cells formed solid tumors in nude mice thatexhibited growth rates comparable to the CYP2Bl-negative tumors

growing in the same animals (Fig. 6A, upper panel). Moreover, allfour tumors responded to CPA given 35 days after tumor implantation, as demonstrated by the cessation of tumor growth from day 35to day 42 (Fig. 6A, lower panel). However, the CYP2B1 -negative

tumors were only partially responsive, because they resumed theirinitial growth rate after a delay of 7-10 days. This partial response is

most likely due to the activation of CPA by cytochrome P450 enzymes present within the livers of these nude mice. By contrast,substantial tumor regression was observed in the case of the CYP2B1 -positive tumors during a 4-week period following CPA treatment.

This enhanced cytotoxicity of CPA toward MCF-7 tumors express

ing CYP2B1 was achieved without a detectable increase in hosttoxicity. This is indicated by comparing the CPA-induced bodyweight loss over a 1-2-week period after drug treatment in nude micecontaining CYP2B1-negative tumors versus in mice that additionallyhost a CYP2B1-positive tumor (Table 1). A similar degree of modestweight loss (up to 12-15% decrease) was observed in both cases. We

also observed no difference in the antitumor effect of CPA towardCYP2B1-negative tumors grown alone as compared to CYP2B1-negative tumors grown in nude mice that also contained a CYP2B1-

positive tumor at a second s.c. site (data not shown). This indicatesthat the contribution of the CYP2B1-positive tumor to the formation

of circulating, cytotoxic drug metabolites is small when compared tothat of the host liver.

To ascertain whether the enhanced growth delay effect exhibited bythe CYP2B1-expressing tumors reflects enhanced localized drug cy

totoxicity during the period immediately following drug treatment, weused a tumor excision assay to quantitate, by in vitro colony formationactivity, antitumor activity induced in vivo over a 24 h period following CPA treatment. CYP2B1 -expressing tumor cells and CYP2B1 -

negative tumor cells were grown in nude mice and then were treatedwith CPA. The tumors were excised from the mice, dispersed to givea single-cell suspension, and then plated on culture dishes. The

number of surviving tumor cells was then determined by a colonyformation assay. As shown in two separate experiments presented inFig. 65, CPA induced a 15-20-fold greater killing of the CYP2B1-expressing tumor cells as compared to the parental MCF-7 or MCF-7-Z tumor cells. Together, the tumor growth delay and the tumor

excision studies establish that the intratumoral activation of CPA byCYP2B1 renders human breast cancer cells highly susceptible tooxazaphosphorine cytotoxicity in vivo without an increase in hosttoxicity.

Adenovirus-mediated Transduction of the CYP2B1 Gene toBreast Cancer Cells Confers Chemosensitivity to CPA. Recombinant adenovirus has been widely used as a highly efficient vector todeliver therapeutic genes to a variety of cell types, both in vitro andin vivo (33). To assess whether transduction of CYP2B1 into othertumor cell lines can sensitize them to CPA, we constructed a replication-defective recombinant adenovirus that carries the CYP2BI

gene. This was accomplished by homologous recombination of apCMV-2Bl shuttle plasmid with the adenovirus 5 genome plasmidpJM17 in 293 cells (see "Materials and Methods"). The resultant

recombinant adenovirus, Ad.CMV-2Bl, contains an insertion of the

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CYP2BÃŒgene coding sequence in the adenovirus El region under thecontrol of the CMV promoter. Adenovirus infection of MCF-7 cells,followed by Western blot analysis using anti-CYP2B 1 antibody, dem

onstrated the expression of CYP2B1 protein in the cultured tumorcells 48 h after infection with Ad.CMV-2Bl, but not after infection

o

E

co<D

O

100-

80-

60-

40-

20-

CPA

20 30 40 50 60 70

Days After Tumor Implantation

100 200 300

CPA (mg/kg)

Fig. 6. Evaluation of growth-inhihitory and cytotoxic effects of CPA toward CYP2BI-expressing and CYP2BI-negative tumors grown in a nude mouse model. A, growth delayexperiment. Female athymic nude mice were inoculated with either of two CYP2BI-expressing cells, MCF-7-P2 and MCF-7-P9, or with the CYP2BI-negative tumor cellsMCF-7-Z and parental MCF-7 cells, by s.c. injection of 2 X IO7 cells/0.2 ml in the flanksof the animals as described in "Materials and Methods." About 5 weeks after tumor

implantation, each mouse received CPA ( 150 mg/kg body weight, daily for 2 days. i.p.).Tumor sizes were measured for 2 months, at which time the animals were sacrificed toavoid complications relating to expiration of the 17ß-estradiolpellets (see "Materials andMethods"). Results are presented as the means of four to six individual animals; hurs. SE.

fl. tumor excision assay shown for two independent experiments. Mice bearing parentalMCF-7 (D). MCF-7-Z (O), MCF-7-P2 (•).and MCF-7-P9 (•)tumors were treated withCPA at 150 mg/kg body weight two times (see "Materials and Methods") or saline as

control. Tumors were excised 4 h after the second dose, and single-cell suspensions wereprepared lor assay of colony formation activity measured IO to 12 days later. Results arepresented as the survival fractions as compared to control groups (means of n = 4

mice/group for each experiment: burs. SE).

Table I Influence iif CYP2BI-expressing tumor an CPA-induced hoily weight /<>.\s

Nude mice carrying CYP2B1-positive or CYP2B1-negative MCF-7 tumors weretreated with two single injections of CPA 4-5 weeks after tumor implantation, asdescribed in Fig. 6/1. The presence of a CYP2B1-positive tumor is shown to have nosignificant impact on CPA-induced host toxicity associated with body weight loss. Bodyweights generally stabili/ed within 2 weeks of drug treatment and then began to increase.Data shown are mean ±SD for the indicated number of mice. The percentage of weightvalues relative to / = 0 time point are shown in italics.

Weeks afterCFAtreatment0I2Mice/groupBody

weight (g) of mice hearing Body weight (g) of mice hearingCYP2BI -positive tumors CYP2BI -negativetumors-CPA29.3

±0.610030.3

±0.9103.430.8

±0.8105.1II+

CPA30.3

±1.4I(X)26.5

±1.387.fi25.9

±1.085.517+CPA31.2

±2.2im27.3

±2.487.627.3

±1.587.fi5

with the ßGalconstruct Ad.CMV-ßGal (data not shown). Moreover,as shown in Fig. 1A, at least 70-75% of MCF-7 cells infected withAd.CMV-2Bl express a high level of CYP2B1, as revealed by im-munohistochemical staining with anti-CYP 2B1 antibody. By contrast, uninfected cells (Fig. IB) and Ad.CMV-ßGal-infected cells(data not shown) did not stain with anti-CYP2Bl antibody. To test the

functional activity of this adenoviral vector, the human breast cancercell lines MCF-7 and ZR-75-1 were infected with Ad.CMV-2BI at

various multiplicities of infection, and their sensitivity to CPA wasthen determined. As shown in Fig. 8, A and B, Ad.CMV-2B I-infectedMCF-7 and ZR-75-1 cells were rendered highly sensitive to CPA ina dose-dependent manner. These effects were obtained at a multiplic

ity of infection as low as 10. A similar sensiti/.ation to CPA wasobtained following Ad.CMV-2B 1 infection of several other humantumor cell lines, including MDA-MB231, a poorly differentiated

breast cancer cell line. T98G, a glioblastoma cell line, and DU 145, aprostate cancer cell line (Fig. 8C). In control experiments, MCF-7cells infected with Ad.CMV-ßGal failed to show any sensitivity toCPA, despite a high degree of infectivity, demonstrated by X-gal(5-bromo-4-chloro-3-indolyl ß-D-galactoside) staining (data not

shown).

DISCUSSION

Previously, rat gliosarcoma 9L and C6 cells were used to investigate the impact of cytochrome P450 gene transfer on the responsiveness of rodent tumors to CPA (24, 25). In the present study, humanbreast cancer MCF-7 cells were used to test whether the paradigm of

cytochrome P450 gene transfer/oxazaphosphorine chcmoscnsitizationcan also be applied to human cancers, as well as to tumors ofnon-central nervous system origin. Our primary goals were: (a) to

establish whether the expression of a cytochrome P450 gene,CYP2B1, sensitizes human breast cancer cells to the prodrugs CPAand IFA; (b) to determine the impact of intracellular CPA activationon cell cycle progression; (c) to evaluate the therapeutic potential ofintratumoral CPA activation in vivo using human breast cancer cellsgrown as solid tumors in a nude mouse model; and (cl) to developrecombinant adenovirus that can efficiently deliver the CYP2BI geneinto human tumor cells for additional preclinical studies of cytochrome P450-based cancer gene therapy.

We found human breast cancer MCF-7 cells to be well suited as a

recipient cell line for cytochrome P450 gene transfer studies, sincethese cells contain little or no endogenous CYP enzyme activity butexpress significant amounts of NADPH cytochrome P450 reducíase(34), which transfers electrons required for all microsomal cyto-

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P450-BASF.D CANCER GENE THERAPY

B

Fig. 7. Immunohistochemical staining of Ad.CMV-2B I-infected and uninfected MCF-7 breast cancer cells with polyclonal anti-CYP2Bi antibody. A, shown are Ad.CMV-2Bl-infected MCF-7 cells exhibiling strong positive cellular CYP2B1 immunostaining in about 25% of cells (large arrows) and more moderate immunostaining in 45-50% of the cells(small arrowhead) 48 h after infection at a multiplicity of 50. X200. B. uninfected MCF-7 cells exhibited no CYP2B1-positive staining. X200.

chrome P450-dependent enzyme reactions. Transfer of the oxazaphos-phorine-activating CYP2B1 gene rendered MCF-7 tumor cells highly

susceptible to oxazaphosphorine cytotoxicity, thus confirming andextending our previous findings in a rat glioma cell model (24). Thefact that CYP2B! gene transfer also confers a striking chemosensiti-zation of MCF-7 tumors to CPA treatment in vivo, in the context of anintact mouse liver which contains a high level of CPA-activating

cytochrome P450 (35), lends strong support to our earlier conclusionthat there is a very substantial proximity effect in the case of intratu-moral CPA activation in CYP2B1-expressing tumor cells. This effect

is not achieved when CPA is activated within the liver during conventional CPA therapy, and this undoubtedly contributes to the striking positive impact that intratumoral CYP2BI gene transfer has on theresponsiveness of MCF-7 tumors to systemic CPA treatment.

The establishment of stable, CYP2BI-expressing MCF-7 cell lines

enabled us to investigate the cytokinetic effects of CPA following itsdirect activation within breast cancer target cells, thus extendingearlier cell cycle studies carried out with mafosfamide. a chemicallyactivated CPA derivative (36). Direct, intratumoral CPA activationled to a delayed S-phase progression and G,-M arrest that likely is aconsequence of the DNA cross-links and single-strand breaks that areinduced by P450-activated CPA (37-39). Conceivably. P450-acti-

vated CPA may induce apoptosis/programmed cell death subsequentto the initial DNA alkylation and cell cycle disruption events. Otheranticancer drugs, including etoposide (40) and cisplatin (41), induceG2-phase cell cycle arrest, which in turn leads to activation of an

apoptotic cascade. Indeed, the chemically activated CPA derivativesmafosfamide and 4HC cause DNA fragmentation and chromatincondensation, two characteristics of apoptosis (36, 42). In agreement

with these findings, Hoechst dye 33528 staining revealed severalmorphological characteristics of apoptosis, including nuclear condensation, cell shrinkage, and the presence of apoptotic bodies followingCPA treatment of CYP2B1 -expressing but not parental MCF-7 cells

(data not shown).Rats implanted with 9L gliomas that express CYP2B1 exhibit a

striking growth inhibition following CPA treatment that far exceedsthe modest tumor growth delay effect obtained with non-CYP2Bl-

expressing 9L tumors (24). However, a recent report has indicated that9L tumor cells engineered to express foreign genes can be potentiallyimmunogenic in a syngeneic host (43), raising the question of whetherhost immune response, rather than intratumoral drug activation per se,is the key factor contributing to the CPA-induced growth inhibition

that we had observed. To address this question, we used in the presentstudy an immune-incompetent nude mouse model to evaluate theresponsiveness of CYP2B1-expressing tumor cells to CPA treatment

in vivo. Using this model, we were able to demonstrate a strikingtherapeutic benefit of intratumoral CYP expression in the form of asubstantial tumor growth delay effect following CPA treatment; nosuch effect was obtained with MCF-7 tumors engineered to express

ßGal.Moreover, using an independent experimental approach, CPAinduced a 15-20-fold greater cytotoxic response in CYP2B1-expressing tumors compared to control MCF-7 tumors when measured by a

tumor excision assay 24 h after initiation of drug treatment. Thus,using two distinct assay systems, we demonstrate that expression ofcytochrome P450 gene 2BI by human tumor cells does indeed translate into a marked therapeutic advantage in vivo.

Despite the striking enhancement in CPA-dependent antitumor

activity, no additional host toxicity was associated with the presence

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100

SO-

£100

O

--*-

•¿�-T-

No Infection

Ad.CMV-ßgal (MOI = 50)

Ad.CMV-2B1(MOI = 10)

Ad.CMV-2B1(MOI = 50)

Ad.CMV-2B1(MOI = 100)

100

oü

50-

50-

0.5

CPA (mM)

Fig. 8. Adenoviru.s-medialed transfer oí*CYl'2Hl gene to breast tumor cells sensiti/es cells to CPA and IFA. A, human MCF-7 breast cancer cells. li. human ZR-75-1 breast cancer

cells. Cells were infected with Ad.CMV-2BI at multiplicities of infection (MOI) ranging from K)-KM), as indicated. Uninfected cells or cells infected with Ad.CMV-ßGal were usedas control. About 48 h later, cells were replated n\ 4 x IO4 cells/well on 6-well plates. CPA was then added at concentrations ranging from 0 to I m\i. After 7 days incubation, viablecells were counted; hurs. SF. (', human tumor cells MDA-MB231 (a poorly differentiated breast cancer cell line). T98G (a gliohlastoma cell line), and DIJI45 (a prostate cancer cell

line) were infected with Ad.CM V-2BI at multiplicities of infection of 50 and treated with I HIMCPA 48 h after infection. The number of surviving cells was determined using an XTTcolorimetrie assay. The effects of CPA on cell survival are expressed as growth ralio (%). i.e.. cell number (or XTT activity) in piales containing CPA as a percentage of thecorresponding drug-free controls: hars, SE.

of CYP2BI within the MCF-7 tumors, as indicated by the similardegree of CPA-induced weight loss over a I-2 week period followingdrug treatment in nude mice bearing CYP2BI-negative tumors versusmice that additionally contain a CYP2BI-positive tumor. Further

evidence that the effects of intratumoral CYP2BI are primarily localized, rather than systemic, is provided by our observation that themodest growth-inhibitory effect of CPA on non-P450-containingMCF-7 tumors is not enhanced by the presence in the same animals ofa CYP2BI-positive MCF-7 tumor at a second distal s.c. site. Thesefindings are consistent with the fact that the CYP2BI-positive tumor

is small and contains a very low specific content of P4SO protein ascompared to that of the host liver. Accordingly, intratumoral transferof the CYP2B1 gene under conditions that are sufficient to enhancethe localized antitumor effect of CPA is unlikely to significantlyincrease host toxicity associated with an increased formation of systemic, cytotoxic drug metabolites. This suggests that the antitumoreffect of CPA, in the case of CYP2BI-expressing tumors, may largely

be independent of liver drug activation, in which case a decrease inhost toxicity without compromising antitumor effect may be achiev

able by combining P450 gene transfer/CPA therapy with the selectiveinhibition of liver P450-catalyzed drug activation. Further preclinical

model studies will be required to evaluate the therapeutic potential ofthis combined modulator approach.

The application to human cancer treatment of a gene therapystrategy using drug susceptibility genes, such as cytochrome P450.imposes two essential requirements: that gene transfer to the tumor beselective and that it be efficient. Retroviruses have been used totransduce the HSV-tk gene into tumor cells, both in vitro and in vivo

(3, 4, 44, 45). Retroviral vector systems are limited, however, by thedifficulty in producing high viral titers for efficient gene transduction,by the restriction that only actively dividing tumor cells becomeinfected, and by the potential risks of insertional mutagenesis as aconsequence of the random integration of retrovirus. In this regard,recombinant adenovirus presents a promising alternative vector system. Adenovirus can infect a broad spectrum of dividing and nondi-

viding cells and has little or no significant potential for viral integration and insertional mutagenesis (33, 46). The El gene-disrupted

adenovirus. used in the present study, is replication defective but can

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P450-BASED CANCER GENE THERAPY

be prepared to a high titer in 293 cells, which provide the Eia and Elbgene products required for complementation of the replication defectin trans (47, 48). Adenovirus-based in vivo gene therapy is effectivefor in vivo trunsduction of the HSV-tk gene for treatment of gliomas

and head and neck tumors in rodent models (49, 50). Moreover, asshown in the present study, the replication-defective recombinantadenovirus Ad.CMV-2Bl can sensitize a variety of cancer cell types

to CPA, including breast, glioma, and prostate cancer cells, suggestingthat adenovirus may be widely useful as an efficient and therapeuti-cally effective vector for transfer of drug-activating cytochrome P450genes. Recombinant adenovirus constructed with other oxazaphos-phorine-activating P450 genes, such as human CYP2B6 (17), may be

useful in testing whether they provide any added pharmacological ortherapeutic benefit beyond that of CYP2B1. These recombinant adenovirus may also serve as useful tools for in vivo studies to exploreapproaches to control the tumor cell targeting specificity that will berequired for clinical applications of CPA/P450-based cancer gene

therapy. In one potentially useful approach to gene targeting specificity, tumor tissue-specific DNA regulatory sequences could be used

to direct the selective expression of cytochrome P450 enzymes intumor cells. For example, the oncogene ERBB2 (51, 52) and themucin-like glycoprotein DF3/MUC1 (53) are both overexpressed inhuman breast carcinomas under the influence of well-defined DNAregulatory elements (54-57). Construction of recombinant adenovi-

ruses containing the CYP2BI gene under the control of these or othertumor tissue-specific enhancer/promoter elements may be a useful

approach in this regard.The present studies indicate that the CPA/cytochrome P450 system

has several distinct advantages as compared to other prodrug/enzymeactivation systems, such as ganciclovir/HSV-tk and 5-fluorocytosine/CD: (a) the P450-based approach uses a class of widely used alky-

lating agent prodrugs whose chemotherapeutic potential, mechanismof action, and spectrum of host toxicity are well established. Bycontrast, although ganciclovir/HSV-tk and 5-fluorocytosine/CD have

shown promise in model systems, the ultimate effectiveness of thesedrugs for treatment of human cancer patients remains unproven; (b)the HSV-tk and CD genes are derived from nonmammalian sources

and could potentially elicit a host immune response that interfereswith prodrug activation. This problem can be avoided by use of anendogenous liver cytochrome P450 enzyme, such as rat CYP2B1 in arat model (24) or the human counterpart CYP2B6 (which contributesto the activation of CPA in human liver; Ret. 17) in human genetherapy trials; (<•)in contrast to ganciclovir/HSV-tk (58, 59), CPA/

P450 exhibits a bystander cytotoxic effect that does not require directcell-cell contact and is mediated by diffusible substances, most likely

the activated metabolites of CPA. Since this bystander effect ismanifest even when as little as 20% of a tumor cell populationexpresses CYP2BI (Fig. 5), a significant therapeutic response may beanticipated, even if in vivo gene transfer to tumor cells is less than100% complete, owing to the extensive bystander tumor killing; and(d) a fourth distinct advantage of the CPA/P450 system as comparedto ganciclovir/HSV-tk and 5-fluorocytosine/CD relates to the cellcycle dependence of each of these drugs. Ganciclovir and 5-fluoro

cytosine are metabolized to activated nucleoside analogues that preferentially kill cells in the S-phase of the cell cycle by inhibiting DNA

polymerase, thereby interfering with DNA synthesis (60). By contrast,the DNA-alkylating metabolites of CPA and IFA kill tumor cells, ina cell cycle-independent manner, by inducing DNA cross-links whose

cytotoxic potential becomes manifest at whichever point the cellsbegin to replicate. Tumor cells that have entered the quiescent (G0)state are thus more susceptible to the cytotoxic effects of CPA/P450than to those of ganciclovir/HSV-tk or 5-fluorocytosine/CD, resulting

in a higher fractional tumor killing in the case of CPA/P450. Com

bination of the cell cycle nonspecific CPA/P450 system with theS-phase-specific ganciclovir/HSV-tk or 5-fluorocytosine/CD system

may be one possible way to take advantage of the distinct actions ofthese alternative drug/suicide gene combinations in a manner thatcould lead to an additive or perhaps a synergistic tumor killing effect,as well as decrease the potential for tumor cells to develop resistanceto these cancer gene therapy regimens.

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