Proc. Nati. Acad. Sci. USAVol. 88, pp. 547-551, January 1991Medical Sciences

Transgenic mice that express the human multidrug-resistance genein bone marrow enable a rapid identification of agents thatreverse drug resistance

[chemosensitizers/daunomycin/taxol/(R)-verapamil/chemotherapy]

GERALD H. MICKISCH*, GLENN T. MERLINO*, HANAN GALSKI*t, MICHAEL M. GOTTESMANt,AND IRA PASTAN*§Laboratories of *Molecular Biology and tCell Biology, Division of Cancer Biology Diagnosis and Centers, National Cancer Institute, National Institutes ofHealth, Bethesda, MD 20892

Contributed by Ira Pastan, October 22, 1990

ABSTRACT The development of preclinical models for therapid testing of agents that circumvent multidrug resistance incancer is a high priority of research on drug resistance. Acommon form of multidrug resistance in human cancer resultsfrom expression oftheMDR] gene, which encodes aM, 170,000glycoprotein that functions as a plasma membrane energy-dependent multidrug efflux pump. We have engineered trans-genic mice that express this multidrug transporter in their bonemarrow and demonstrated that these animals are resistant toleukopenia by a panel of anticancer drugs including anthra-cyclines, vinca alkaloids, etoposide, taxol, and actinomycin D.Differential leukocyte counts indicate that both neutrophils andlymphocytes are protected. Drugs such as cisplatin, methotrex-ate, and 5-fluorouracil, which are not handled by the multidrugtransporter, produce bone marrow suppression in both normaland transgenic mice. The resistance conferred by the MDRIgene can be circumvented in a dose-dependent manner bysimultaneous administration of agents previously shown to beinhibitors of the multidrug transporter in vitro, inclngverapamil isomers, quinidine, and quinine. Verapamil andquinine, both at levels suitable for human trials that producedonly partial sensitization of the MDRl-transgenic mice, werefully sensitizing when used in combination. We conclude thatMDRJ-transgenic mice provide a rapid and reliable system todetermine the bioactivity of agents that reverse mulddrugresistance in animals.

Resistance to chemotherapy poses a major obstacle to thecure of human cancers that are not amenable to definitivesurgical or radiation treatment. Numerous laboratory inves-tigations have uncovered a broad spectrum cross-resistanceto natural product cytotoxic compounds that do not share anyobvious functional or structural similarities (1). This phe-nomenon is termed multidrug resistance (MDR) (2, 3). Theevidence indicates that the presence of a Mr 170,000 plasmamembrane protein, termed P-glycoprotein, which is encodedin humans by the MDR] gene (4, 5) is sufficient to produceMDR in cancer cells (1-5). P-glycoprotein functions as amultidrug transporter that rapidly extrudes many types ofhydrophobic chemotherapeutic agents from the target cancercell before the drugs can exert their cytotoxic effects (6, 7).

Recently, it has become evident that many drug-resistanthuman tumors express the MDRJ gene (8), that MDR] RNAlevels are elevated in many cancers that have not respondedto chemotherapy (2, 9, 10), and, in some cases, the presenceof MDR] gene expression predicts poor results in chemo-therapy with agents affected by the multidrug transporter (11,12). A number of different agents that inhibit the activity of

the multidrug transporter, such as verapamil, have beendescribed (13) and the majority of these appear to be sub-strates for the transporter, which compete with anticancerdrugs for transport (1, 6, 14, 15). An initial report on a limitednumber of patients with drug-resistant multiple myelomasuggests reversal ofMDR by verapamil in a clinical trial (16).However, the inherent side effects of verapamil due to itscardiovascular activities necessitate the search for new andbetter resistance modifiers (17). To confirm the activity oftheMDR] gene in a preclinical in vivo model, this laboratory hasengineered transgenic mice carrying the human MDR] geneand expressing it in their bone marrow, an organ that isnormally very sensitive to anticancer drugs (18). In thesemice, preliminary analysis suggested that the MDRI genewas active since, after treatment with daunomycin, there wasno fall in their peripheral leukocyte count (WBC). Thecurrent studies investigate the protection of bone marrow bythe human MDR] gene from a variety of chemotherapeuticdrugs presently in clinical use and also examine the use ofthese transgenic mice as a suitable model for testing agents,and combinations of agents, that circumvent drug resistance.

MATERIALS AND METHODSMDR Transgenic Mice. The construction of the original

plasmid carrying the full-length cDNA encoding MDR] (19)and the injection of this cDNA under control of a chicken3-actin promoter using standard techniques (20) have beendescribed (18). The cDNA construct (1-2 ng) was injectedinto fertilized mouse embryos of single-cell stage, and thesetransgenic embryos were implanted in foster mice. Afterestablishing a homozygous line (MDR-39) of mice carryingthe transgene, homozygous males were backcrossed toMDR-negative females of the progenitor line (C57BL/6 XSJL)F1. The resultingMDR] heterozygous descendants wereanalyzed for expression of the human MDR] gene using tailsamples (6, 20) and hybridized with the MDRJ-specific probeMDR5A generated in this laboratory (21). In these studies,only 6- to 8-week-old sex-matched littermates were investi-gated.

Test Conditions. Cisplatin and etoposide were a gift ofBristol-Myers Squibb (Syracuse, NY). Verapamil and (R)-verapamil were provided by courtesy of BASF Bioresearch(Cambridge, MA). Taxol was from the Developmental Ther-apeutics Branch, National Cancer Institute (Bethesda, MD).All other drugs were purchased from Sigma. The drugs were

Abbreviations: MDR, multidrug resistance; WBC, leukocyte count.tPresent address: Institute of Life Science, Hebrew University,Jerusalem 91904, Israel.§To whom reprint requests should be addressed at: Laboratory ofMolecular Biology, Building 37, Room 4E 16, National CancerInstitute, National Institutes of Health, Bethesda, MD 20892.

547

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Bone marrow toxicity of chemotherapeutic agents fornormal and MDR transgenic mice. Drugs were administered i.p. ina single dose on day 0. Values are expressed as the ratio of WBCson day 5 to those on day 0. Experiments included a minimum of threeanimals per group and were repeated at least once. Dosage wasselected to ensure a significant (>50o) reduction ofWBCs in normalmice without causing generalized toxicity. DOX, doxorubicin; VBL,vinblastine; TAX, taxol; DAU, daunomycin; ETO, etoposide; ActD,actinomycin D; 5FU, 5-fluorouracil; MTX, methotrexate.

administered by a single intraperitoneal injection in the lowerright quadrant of the abdominal cavity. Drug concentrationswere adjusted so that a maximum vol of 400 ILI was injectedper experiment. Experiments included a minimum of threeanimals per group and were repeated at least once. Theresults were highly reproducible with

Proc. NatL. Acad. Sci. USA 88 (1991) 549

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FIG. 3. Dose-dependent reversal of bone marrow protectionagainst daunomycin effected by the human MDR] gene. (A) Dauno-mycin (DAU) (10 mg/kg) combined with verapamil. (B) Daunomycin(10 mg/kg) combined with quinidine. (C) Daunomyin (10 mg/kg)combined with quinine. Experiments were conducted as described inMaterials and Methods and in the legend to Fig. 1.

MDR drugs, the MDR mice were treated with appropriateamounts of seven different commonly used anticancer drugsbelonging to the MDR group. As shown in Fig. 1, there wasno fall in the WBC of transgenic animals after treatment withdoxorubicin, vinblastine, vincristine, taxol, daunomycin,etoposide, or actinomycin D, whereas the WBC fell by >50%in the normal animals. Three non-MDR drugs were alsostudied at concentrations that lowered the WBC in normalmice. There was no difference in the response of the MDRand non-MDR animals to 5-fluorouracil, methotrexate, orcisplatin (Fig. 1). These experiments show that the MDRtransgenic mice are specifically resistant to drugs in the MDRfamily and not to other cytotoxic agents that suppress bonemarrow.

IC To determine which cells in the bone marrow were pro-] tected in the MDR mice, animals were challenged with

daunomycin (10 mg/kg) and differential WBC performed on100 day 0 (before treatment) and on day 5. In the control mice,

there was a fall in the total WBC, which was due to a fall in80 both neutrophils and lymphocytes. There was no significant

change in the hematocrit or in the number of platelets as60 estimated from a blood smear, and none of the animals

exhibited bleeding during the experiments. Basophil and40 eosinophil counts were too low to evaluate the effects of

chemotherapy. In the MDR mice there was no change in the20 granulocyte or lymphocyte counts, indicating that both major

types of leukocytes were protected (Table 1).0 The bone marrow of the MDR mice was found to be

completely resistant to the action of several different cyto-toxic drugs that had a profound effect on normal bone

C marrow. One example is shown in Fig. 2, where the effect ofincreasing amounts of taxol (4, 6, and 10 mg/kg) on the WBCof normal mice is depicted. There was a clear dose-responserelationship, with all concentrations producing a marked

10 decrease in the WBC, which reached a nadir on day 5 andthen began to increase (Fig. 2A). In the MDR mice, even the

30 highest dose of 10 mg/kg had no effect.Effect of MDR Reversing Agents. The clear-cut difference

in the response of normal and transgenic mice made it,,0 possible to test the effect of standard drugs that reverse

MDR, such as verapamil, quinine, and quinidine. None of!0 these agents administered by themselves had any effect on

the WBC ofnormal mice (Fig. 2A) or oftransgenic mice (data0 not shown). However, when administered with a cytotoxic

agent such as taxol (Fig. 2 B-D), the chemosensitizing agentshad a large effect on the WBC. Fig. 2 (B-D) shows thatquinine (40 mg/kg), quinidine (150 mg/kg), and verapamil (30mg/kg) all caused a profound fall in the WBC of the trans-genic mice when given with taxol. Furthermore, these agentsacted in a dose-dependent manner (Fig. 3). In the examplesshown in Fig. 3, groups of transgenic animals received asingle dose of daunomycin (10 mg/kg) and increasingamounts of verapamil (Fig. 3A), quinidine (Fig. 3B), orquinine (Fig. 3C). At the highest doses ofreversing agent, thefall in WBC induced by the daunomycin was equivalent to thedrop seen in non-MDR mice given daunomycin alone.

(R)-verapamil is a verapamil stereoisomer that can be givenin large amounts in vivo because it has less cardiovascularactivity than the (L)-isomer (22) but exhibits similar potencyto overcome MDR compared to racemic verapamil in vitro(23). It is currently in phase 1 clinical trials as a MDR-reversing agent of potential clinical interest. Some of theeffects of (R)-verapamil with or without daunomycin areshown in Fig. 4. By itself, (R)-verapamil even at 150 mg/kghad no effect on the WBC (Fig. 4A). With daunomycin it didhave a small but significant effect on the WBC in non-MDRmice (Fig. 4B). The basis of this effect is currently unknown.However, in the MDR mice its effects are much moredramatic. Daunomycin at 8 mg/kg had no effect by itself (Fig.4C), but with (R)-verapamil the WBC fell by 80% (Fig. 4D).Because the reversing agents currently in use all have

specific pharmacological actions of their own, as well as acommon action on the multidrug transporter, it has beensuggested that they can be used in combination at lower doselevels to overcome drug resistance without producing unde-sirable side effects (24). Since intrinsic cardiovascular effectsof prototype reversing agents such as verapamil proved to bea limitation in experimental therapeutic trials to inactivateMDR (16, 17), we chose to combine verapamil with theantimalarial quinine (25) rather than its optical isomer quini-dine, which has potent anti-arrhythmic effects. To examinethe efficacy of this approach, MDR transgenic animals weretested with daunomycin (8 mg/kg) with either verapamil (0.5mg/kg) or quinine (20 mg/kg) alone (Fig. SA) or together (Fig.

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1 3 5 7Days

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FIG. 4. Toxicity of daunomycin alone or incombination with (R)-verapamil to the bone mar-row of mice. (A) Normal mice, daunomycin or(R)-verapamil (150 mg/kg). (B) Normal mice,daunomycin and (R)-verapamil (150 mg/kg). (C)MDR transgenic mice, daunomycin or (R)-verapamil (150 mg/kg). (D) MDR transgenic mice,daunomycin and (R)-verapamil (150 mg/kg). Ex-periments were conducted as described in Materi-als and Methods and in the legend to Fig. 1.Daunomycin: solid symbols, 4 mg/kg; open sym-bols, 6 mg/kg; hatched symbols, 8 mg/kg. (R)-Verapamil: stippled circles, 150 mg/kg.

5B). The data show that the combination of verapamil andquinine, at levels that are clinically achievable in humans,gives a much larger effect than either alone.

DISCUSSION

Function of the MDR Transgene. These data show that thebone marrow of transgenic mice expressing a human MDR]cDNA is resistant to doxorubicin, daunomycin, vinblastine,vincristine, taxol, etoposide, and actinomycin D. Thesedrugs have very different structures but have been defined bytissue culture studies to belong to the family of drugs trans-ported by the multidrug transporter. The data presented hereplus the finding that the bone marrow of the MDR mice wasnot resistant to 5-fluorouracil, methotrexate, or cisplatinclearly shows that expression of a functional DNA elementencoding a single multidrug transport protein is sufficient tocause resistance to drugs with widely different structures inanimals. Because many drug-resistant cancers such as renalcell carcinoma express the MDR] gene at levels comparableto those found in the marrow of the MDR transgenic mice(18), it is reasonable to ascribe their resistance to certainchemotherapeutic drugs to the ability of the product of this

1 3 5 7Days

gene to pump the cytotoxic drugs out of the drug-resistantcancer cells. In addition, these transgenic mice can serve asa simple test system to determine whether a drug that appearson the basis of tissue culture samples to be useful in over-coming MDR will act in a similar manner in an animal.

Advantages of UsingMDR Mice to Test Reversing Agents. Awide variety of drugs that are themselves not cytotoxic havebeen found to overcome MDR by competitively or noncom-petitively inhibiting the multidrug transporter (13). Unfortu-nately, the reversing agents currently in clinical use arepotent pharmacologic agents that have undesirable side ef-fects at levels that must be given to substantially reverseMDR. Agents with fewer side effects remain to be developed.To determine whether an agent is capable of reversing drugresistance in vitro by using cultured cells is relatively easy;this is done by comparing its activity on a matched set ofdrug-sensitive and drug-resistant cell lines. However, todetermine whether a reversing agent has the appropriatebioactivity and useful pharmacokinetic properties in an ani-mal is more difficult. Until the development of this transgenicmodel for testing agents that reverse drug resistance, it hasbeen necessary to show that these agents enhance the anti-tumor activity of a cytotoxic drug against a drug-resistant

FIG. 5. Additive effects of verapamil plus qui-nine combined with daunomycin in MDR trans-genic mice. (A) Daunomycin alone or combinedwith verapamil (0.5 mg/kg) or quinine (20 mg/kg).(B) Daunomycin (8 mg/kg) combined with vera-pamil (0.5 mg/kg) and quinine. Experiments wereconducted as described in Materials and Methodsand in the legend to Fig. 1. Daunomycin, 8 mg/kg(hatched boxes); verapamil, 0.5 mg/kg (boxes withhorizontal lines); quinine, 5 mg/kg (solid symbols),10 mg/kg (open circles), and 20 mg/kg (hatchedcircles).

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Proc. Natl. Acad. Sci. USA 88 (1991) 551

tumor. These assays require many animals, take weeks tomonths to perform, and are not entirely reproducible becauseof variability in the growth of tumor cells.

In the current study, we have used both taxol and dauno-mycin as chemotherapeutic agents because they have knownbone marrow suppressive effects (26, 27). The finding thatperipheral WBCs drop gradually over several days withnadirs on day 5 (data not shown) supports the published dataindicating that they are suppressing bone marrow activityrather than having a direct toxic effect on peripheral WBCs.This assay allows a rapid, quantitative estimate ofthe activityof reversing agents by directly comparing the WBC on dayso and 5. Because the assay is highly reproducible, only a fewanimals are needed to evaluate each dose ofdrug. In addition,several different doses of a cytotoxic drug (Figs. 2 and 4) ora reversing agent can be given in the same experiment (Figs.3 and 5). Furthermore, combinations of reversing agents canbe tested together (Fig. 5). Because different reversing agentsmay bind to serum proteins (28, 29) or to different tissue sites,a functional assay of their activity in animals is necessarybefore taking such drugs into clinical studies.The transgenic animal model also allows this rapid deter-

mination of the bioactivity of reversing agents without theneed for expensive and time-consuming pharmacokineticmeasurements. Because the toxicity of even very high dosesofa drug such as taxol is blocked by bone marrow expressionof the MDR] gene in the MDR transgenic mice (Fig. 2B vs.Fig. 2A), it seems unlikely that the human MDR] gene has aprimary effect on pharmacokinetics in these animals. Simi-larly, the reversing effect of drugs such as verapamil cannotbe attributed to altered pharmacodynamics of the chemo-therapeutic drug, since chemosensitization occurs even forlow doses ofdaunomycin (Fig. 4D) or taxol (data not shown)in the MDR animals. Since this assay simply asks if the drugworks as a reversing agent in an animal, without requiringcomplex pharmacologic testing, it will speed the screeningprocess and shorten the development time ofMDR-reversingagents for use in human trials.

It has not escaped our attention that these MDR transgenicanimals prove that bone marrow cells expressing a drug-resistance gene have a selective advantage in the presence ofanticancer drugs. This is the kind of selective system that isneeded for the development of gene therapy in which anunselected gene can be introduced into bone marrow, or anyother organ, by selecting for expression of the drug-resistance gene. Thus, the MDR transgenic animals shouldserve as a useful model not only for development of antican-cer therapies but for establishing new strategies for genetherapy.

The authors wish to thank Dr. T. Licht for his valuable help withthe hematological procedures in these studies and Jennie Evans forsecretarial assistance. G.H.M. is supported by Deutsche Forschungs-gemeinschaft (DFG-Mi 334/1-1) and by Boehringer Mannheim Foun-dation.

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