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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Proc. NatL. Acad. Sci. USA 88 (1991) 549
A WBC[%j
100
80 -
60 -
40 -
20 -
0-0 0.5 5 10 20 30 40Verapamil (mg/kg)
40 0NO DAU Non.%IDR
WBC[%]
100 -
80 -
60 -
40 -
20 -
O-
WB(r%J
100
80
60 -
40 -
20 -
0-
0 25 50 75 100 125 150Quinidine (mg/kg)
.I- -~~~~~
0 5 10 20 30 40 50Quinine (mg/kg)
50 0NODAU Non MDR
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.
B
c
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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.
1. Gottesman, M. M. & Pastan, I. (1988) J. Biol. Chem.
263,12163-12166.
2. Pastan, I. & Gottesman, M. M. (1987) N. Engl. J. Med.
316,1388-1393.
3. Endicott, J. A. & Ling, V. (1989) Annu. Rev. Biochem.
58,137-171.
4. Chen, C.-J., Chin, J., Ueda, K., Clark, D., Pastan, I.,
Gottes-man, M. M. & Roninson, I. (1986) Cell 47, 381-389.
5. Roninson, I. B., Chin, J. E., Choi, K., Gros, P., Housman,D.
E., Fojo, A., Shen, D.-W., Gottesman, M. M. & Pastan, I.(1986)
Proc. Natl. Acad. Sci. USA 83, 4538-4542.
6. Horio, M., Gottesman, M. M. & Pastan, I. (1988) Proc.
Nat!.Acad. Sci. USA 85, 3580-3584.
7. Willingham, M. C., Cornwell, M. M., Cardarelli, C.
O.,Gottesman, M. M. & Pastan, I. (1986) Cancer Res. 46,
5941-5946.
8. Goldstein, L. J., Galski, H., Fojo, A., Willingham, M. C.,
Lai,S.-L., Gazdar, A., Pirker, R., Green, A., Crist, W.,
Brodeur,G., Grant, C., Lieber, M., Cossman, J., Gottesman, M. M.
&Pastan, I. (1989) J. Natl. Cancer Inst. 81, 116-124.
9. Bourhis, J., Goldstein, L. J., Riou, G., Pastan, I.,
Gottesman,M. M. & Benard, J. (1989) Cancer Res. 49,
5062-5065.
10. Goldstein, L. J., Fojo, A. T., Ueda, K., Crist, W., Green,
A.,Brodeur, G., Pastan, I. & Gottesman, M. M. (1990) J.
Clin.Oncol. 8, 128-136.
11. Sato, H., Gottesman, M. M., Goldstein, L. J., Pastan,
I.,Block, A. M., Sandberg, A. A. & Preisler, H. D. (1990)
Leu-kemia Res. 14, 11-22.
12. Chan, H. S. L., Thorner, P. S., Haddad, G. & Ling, V.
(1990)J. Clin. Oncol. 8, 689-704.
13. Tsuruo, T. (1988) Jpn. J. Cancer Res. 79, 285-296.14.
Akiyama, S., Cornwell, M. M., Kuwano, M., Pastan, I. &
Gottesman, M. M. (1987) Mol. Pharmacol. 33, 144-147.15.
Cornwell, M. M., Pastan, I. & Gottesmann, M. M. (1987) J.
Biol. Chem. 262, 2166-2170.16. Dalton, W. S., Grogan, T. M.,
Meltzer, P. S., Scheper, R. J.,
Durie, B. G. M., Taylor, C. W., Miller, T. P. & Salmon, S.
E.(1989) J. Clin. Oncol. 7, 415-424.
17. Gottesman, M. M. & Pastan, I. (1989) J. Clin. Oncol.
7,409-411.
18. Galski, H., Sullivan, M., Willingham, M. C., Khew-Voon,
C.,Gottesman, M. M., Pastan, I. & Merlino, G. T. (1989)
Mol.Cell. Biol. 9, 4357-4363.
19. Ueda, K., Cardarelli, C., Gottesman, M. M. & Pastan, I.
(1987)Proc. Natl. Acad. Sci. USA 84, 3004-3008.
20. Hogan, B., Constantini, F. & Lacy, E. (1986)
Manipulation ofMouse Embryo (Cold Spring Harbor Lab., Cold Spring
Harbor,NY), pp. 1-322.
21. Pastan, I., Gottesman, M. M., Ueda, K., Lovelace, E.,
Ruth-erford, A. V. & Willingham, M. C. (1988) Proc. Nat!.
Acad.Sci. USA 85, 4486-4490.
22. Echizen, H., Brecht, T., Niedergesaess, S., Vogelgesang, B.
&Eichelbaum, M. (1985) Am. Heart J. 109, 210-217.
23. Mickisch, G. H., Kossig, J., Keilhauer, G., Schlick,
E.,Tschada, R. K. & Alken, P. M. (1990) Cancer Res. 50,
3670-3674.
24. Lehnert, M., Dalton, W. S., Roe, D., Emerson, S. &
Salmon,S. E. (1990) Blood, in press.
25. White, N. J., Looareesuwan, S., Warrell, D. A., Warrell,M.
J., Bunnang, D. & Harinasuta, T. (1982) Am. J. Med.
73,564-567.
26. Maral, R. J. & Jouanne, M. (1981) Cancer Treat. Rep.
65,Suppl. 4, 9-18.
27. Rowinsky, E. K., Cazenave, L. A. & Donehower, R. C.
(1990)J. Natl. Cancer Inst. 82, 1247-1259.
28. Lehnert, M., Kunke, K., Dalton, W. S., Roe, D., Dorr, R.
T.& Salmon, S. E. (1990) Proc. Am. Assoc. CancerRes. 31,
2250.
29. Garfinkel, D., Mamelok, R. D. & Blaschke, T. F. (1987)
Ann.Intern. Med. 107, 48-50.
Medical Sciences: Mickisch et aL
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