The Breast Cancer Resistance Protein is described, as well as the
cDNA encoding said protein. This protein has been found to confer
resistance to cancer chemotherapeutic drugs.
1) Breast Cancer Resistance Protein which induces resistance to
cancer chemotherapeutic drugs, or fragments or derivatives thereof.
2) The protein of claim 1 which is about 655 amino acids in length.
3) The protein of claim 1 which has a molecular mass of 72.3 kilodaltons.
4) The protein of claim 1 which is substantially identical to the
sequence in SEQ ID No. 1.
5) An antibody which binds to the protein of claim 1.
6) The antibody of claim 5 which is monoclonal.
7) The antibody of claim 5 which is polyclonal.
8) A gene which encodes the protein of claim 1.
9) The gene of claim 8 which is substantially identical to the
sequence in SEQ ID No. 2.
10) An antisense probe which inhibits expression of the protein
of claim 1.
11) The antisense probe of claim 10 which is substantially identical
to the sequence in SEQ ID No. 7.
12) A method of determining the cause of a patient's resistance
to cancer chemotherapy drugs by assaying for expression of the protein
of claim 1, whereby overexpression of the said protein indicates
that it is the cause.
13) A method of inhibiting the activity of the Breast Cancer Resistance
Protein by administering the antibody of claim 5.
14) A method of inhibiting the activity of the Breast Cancer Resistance
Protein by administering the antibody of claim 6.
15) A method of inhibiting the activity of the Breast Cancer Resistance
Protein by administering the antibody of claim 7.
16) A method of inhibiting the activity of the Breast Cancer Resistance
Protein by administering the probe of claim 10.
17) A method of inhibiting the activity of the Breast Cancer Resistance
Protein by administering the probe of claim 11.
18) A method of enhancing a cancer patient's chemotherapy treatment
by administering the antibody of claim 5.
19) A method of enhancing a cancer patient's chemotherapy treatment
by administering the probe of claim 11.
20) A method of enhancing a cancer patient's chemotherapy treatment
by administering Fumitremorgin C.
 This application is based upon U.S. Provisional No. 60/073763,
filed Feb. 5, 1998.
FIELD OF THE INVENTION
 The invention relates to the family of proteins known as
multidrug resistance proteins. These proteins are xenobiotic transporters
which confer resistance to cancer chemotherapeutic drugs. The invention
describes a new protein member of this family called Breast Cancer
Resistance Protein (BCRP) and the DNA which encodes it.
BACKGROUND OF THE INVENTION
 The development of resistance to multiple chemotherapeutic
drugs frequently occurs during the treatment of cancer. Two transmembrane
xenobiotic transporter proteins, P-glycoprotein (Pgp) and the multidrug
resistance protein (MRP) are capable of causing multidrug resistance
when transfected into drug-sensitive cells in culture (1,2). Despite
this, the role that these transporters play in clinical drug resistance
exhibited by human cancers is unclear, and alternate or additional
drug resistance mechanisms operative in this disease have been sought.
 To address this problem, Chen et. al. (3) selected human
breast carcinoma MCF-7 cells for resistance to the anthracycline
doxorubicin in the presence of verapamil, an inhibitor of Pgp. The
resultant multidrug resistant subline, MCF-7/AdrVp, exhibits marked
cross-resistance to other anthracyclines (daunorubicin [DNR], 3'-deamino-3'[3-cyano-4-morpholinyl]d-
oxorubicin, but not idarubicin), and to the anthracenedione mitoxantrone,
but remains sensitive to vinca alkaloids, paclitaxel (3,4) and cisplatin.
MCF-7/AdrVp cells do not overexpress Pgp or MRP, despite displaying
a marked reduction in the intracellular accumulation of the anthracycline
daunorubicin and the fluorescent dye rhodamine 123 compared to MCF-7
cells (4,5). MCF-7/AdrVp cells do not display an alteration in the
subcellular distribution of drug (4) such as that seen in certain
cells that overexpress MRP. Although the decreased accumulation
of daunorubicin in MCF-7/AdrVp cells is not reversed by the classical
P-glycoprotein antagonist cyclosporin A, depletion of ATP results
in complete abrogation of the abnormal efflux of both daunorubicin
and rhodamine (4).
 The need in the art to elucidate the mechanism of drug resistance
is continually present, as chemotherapy remains the primary method
for non-invasively treating many types of cancers. There is also
a need in the art to counteract the mechanism of drug resistance
so to provide a longer and more effective course of chemotherapeutic
drug treatment for cancer patients.
SUMMARY OF THE INVENTION
 The discovery described in the instant invention fulfills
the above needs. The discovery of the BCRP and its corresponding
gene greatly advance the knowledge in the art of the drug resistance
mechanism by providing a novel xenobiotic transporter which is overexpressed
in a variety of drug-resistant human cancer cell lines, and confers
resistance to many chemotherapeutic agents.
 BCRP is an about 655 amino acid protein and is encoded by
a gene which has about 2418 nucleotide cDNA. The protein demonstrates
activity and has a sequence homology which places it in the ATP-binding
cassette (ABC) superfamily of transporter proteins. The molecular
mass is approximately 72.3 Kilodaltons (KD) exclusive of any glycoylation.
Expression of BCRP in drug-sensitive human cancer cells confers
resistance to mitoxantrone, doxorubicin, and daunorubicin, and reduces
daunorubicin accumulation in the cloned transfected cells.
 It is an object of the present invention to provide a mammalian
protein that is a multi-drug resistant (MDR) protein and a xenobiotic
transporter, and is called Breast Cancer Resistance Protein (BCRP).
 It is also an object of the present invention is to provide
the gene and/or cDNA which encodes said mammalian MDR protein.
 It is another object of the invention to provide antisense
fragments of the BCRP gene which inhibit the expression of the BCRP
 Yet another object of the present invention is to provide
a method of using probes derived from the BCRP gene as a diagnostic
tool to quantify gene expression or gene amplification in specimens
taken from patients with cancer.
 It is another object of the invention to provide antibodies
to the BCRP.
 It is yet another object of the invention to provide a method
of reversing the drug resistance of the cancer cells by administering
 It is yet another object of the invention to provide a method
of reversing the drug resistance of the cancer cells by administering
 It is another object of the invention to provide a method
of enhancing a patient's chemotherapy treatment for breast cancer
by administering antibodies to the patient to inhibit the resistance-activity
 These and other objects of the present invention, which
will be apparent from the detailed description of the invention
provided hereinafter, have been met, in one embodiment, by substantially
pure BCRP and the gene encoding BCRP.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1A is an autogradiograph of the RNA fingerprinting
of MCF-7 cells.
 FIG. 1B is an autoradiograph of a Northern blot hybridization
of mRNA from MCF-7/W, MCF-7/AdrVp, and MCF-7/AdrVpPR cells.
 FIG. 1C is an autoradiograph of a genomic Southern blot
hybridization of DNA from MCF-7/AdrVp, MCF-7/W and MCF-7/AdrVpPR
 FIG. 2A is the deduced amino acid sequence of BCRP with
 FIG. 2B shows the relative similarity of BCRP to selected
members of the ABC transporter superfamily.
 FIG. 2C is the cDNA sequence which encodes the BCRP.
 FIG. 2D is a graph of a phylogram showing the evolution
of the amino acid seqeunce of BCRP in relation to certain other
members of the ABC family of transport proteins.
 FIG. 3 shows an autoradiograph of a multiple tissue Northern
 FIG. 4A is an autoradiograph of a Northern blot of subclones
of BCRP transfectants.
 FIG. 4B is a graph of Daunorubicin (DNR) accumulation and
retention in the pcDNA3 vector control cells and BCRP-transfected
clones 6 and 8.
 FIG. 4C shows the relative resistance factors-MCF-7, vector
control, clones 19, 6, and 8.
 FIG. 4D are graphs showing the effect of various chemotherapeutic
drugs' concentrations on BCRP-transfected MCF-7 clone 8 cell survival.
 FIG. 4E shows a graph of the effects of ATP deletion of
the retention of rhodamine 123 by transfectant MCF-7/pcDNA3 (empty
vector control) or MCF-7/BCRP clone 8 cells.
 FIG. 5 is a table showing the effect of various chemotherapeutic
drugs on BCRP-transfected MCF-7 cells.
 FIG. 6 is an autoradiograph showing the expression of Human
.omega. gene in MCF-7 cells detected by the Reverse Transcription-Polymerase
chain reaction (RT-PCR).
 FIG. 7 is an autoradiograph showing the expression of BCRP
in samples of blast cells from patients with acute myelogenous leukemia.
 FIG. 8A, 8B, and 8C are autoradiographs showing the results
of Northern blot hybridizations of mRNA from various drug resistant
cell lines probed with a BCRP probe.
 FIG. 9 is an autoradiograph of a Southern blot hybridization
from various MCF-7 cell lines.
 FIG. 10 is a graph showing the results of administration
of FTC to BCRP transfected cells.
DETAILED DESCRIPTION OF THE INVENTION
 A novel gene and the protein encoded by said gene, called
the Breast Cancer Resistance-associated Protein (BCRP) are described
in the instant invention. The BCRP is shown to be overexpressed
in human multi-drug resistant (MDR) breast carcinoma cells, colon
carcinoma, gastric carcinoma, fibrosarcoma, and myeloma origin.
The BCRP is a xenobiotic transporter which confers resistance to
multiple chemotherapeutic drugs, and belongs to the ABC transporter
superfamily. The BCRP appears to be responsible for the alteration
in drug transport and drug resistance manifested by various cancer
 The present invention pertains partially to the BCRP, to
fragments of this factor, as well as to functional derivatives,
agonists and antagonists, and metabolic breakdown products of this
factor. The BCRP amino acid sequence is depicted in SEQ ID No. 1
and FIG. 2A. The invention especially concerns agents which are
capable of inhibiting BCRP, preferably antibodies to BCRP or antisense
probes to the BCRP gene. The invention further encompasses chemical
agents which inhibit expression of the BCRP gene or mRNA, including
Fumitremorgin C (FTC). The invention also concerns methods of inhibiting
activity of BCRP or expression of the BCRP gene by administering
 A "functional derivative" of BCRP is a compound
which possesses a biological activity (either functional or structural)
that is substantially similar to a biological activity of BCRP.
The term "functional derivatives" is intended to include
the "fragments," "variants," "analogues,"
or "chemical derivatives" of a molecule. A "fragment"
of a molecule such as BCRP, is meant to refer to any polypeptide
subset of the molecule. A functional fragment means that a molecule
with a similar, but not identical, amino acid sequence, but has
the same function of the full length BCRP. A "variant"
of a molecule such as BCRP is meant to refer to a molecule substantially
similar in structure and function to either the entire molecule,
or to a fragment thereof. A molecule is said to be "substantially
similar" to another molecule if both molecules have substantially
similar structures or if both molecules possess a similar biological
 Thus, provided that two molecules possess a similar activity,
they are considered variants as that term is used herein even if
the structure of one of the molecules is not found in the other,
or if the sequence of amino acid residues is not identical. An "analogue"
or agent which mimics the function of a molecule such as BCRP is
meant to refer to a molecule substantially similar in function but
not in structure to either the entire molecule or to a fragment
thereof. As used herein, a molecule is said to be a "chemical
derivative" of another molecule when it contains additional
chemical moieties not normally a part of the molecule. Such moieties
may improve the molecule's solubility, absorption, biological half
life, etc. The moieties may alternatively decrease the toxicity
of the molecule, eliminate or attenuate any undesirable side effect
of the molecule, etc. Moieties capable of mediating such effects
are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures
for coupling such moieties to a molecule are well known in the art.
More specifically, the scope of the present invention is intended
to include functional derivatives of BCRP which lack one, two, or
more amino acid residues, or which contain altered amino acid residues,
so long as such derivatives exhibit the capacity to influence cell
resistance to chemotherapy.
 An "antagonist" of BCRP is a compound which inhibits
the function of BCRP. Such antagonists can be immunoglobulin (such
as, for example, monoclonal or polyclonal antibody, or active fragments
of such antibody). The antagonists of the present invention may
also include non-immunoglobulin compounds (such as polypeptides,
organic compounds, etc.), and substrates of BCRP transport that
may modulate or inhibit the transport of cytotoxic drugs. Antagonists,
or inhibitors of BCRP are one embodiment of the invention. These
antagonists or inhibitors are useful for inhibiting the drug resistance
effect caused by BCRP on cancer cells. The preferred inhibitor is
an antibody raised to the BCRP, an antigenic fragment thereof, or
a drug which blocks BCRP transporter activity. A preferred inhibitor
which is a drug is fumitremorgin C (FTC), a mycotoxin. FTC was obtained
from Dr. Lee Greenberg at Wyeth-Ayerst Laboratories in Pearl River,
 A polyclonal antibody capable of binding to BCRP can be
prepared by immunizing a mammal with a preparation of BCRP or functional
derivative of BCRP. Methods for accomplishing such immunizations
are well known in the art. Monoclonal antibodies or fragments thereof
can also be employed to assay for the presence or amount or BCRP
in a particular biological sample. Such antibodies can be produced
by immunizing splenocytes with activated BCRP (7). The BCRP-binding
antibodies of the present invention can be administered to patients
to reduce resistance to chemotherapy drugs, and hence enhance their
treatment. Methods of administration will depend on the particular
circumstances of each individual patient and are within the skill
of those skilled in the art.
 The BCRP of the present invention may be obtained by natural
processes (such as, for example, by inducing the production of BCRP
from a human or animal cell); by synthetic methods (such as, for
example, by using the Merrifield method for synthesizing polypeptides
to synthesize BCRP, functional derivatives of BCRP, or agonists
or antagonists of BCRP (either immunoglobulin or non-immunoglobulin);
or by the application of recombinant technology (such as, for example,
to produce the BCRP of the present invention in diverse hosts, e.g.,
yeast, bacterial, fungi, cultured mammalian cells, to name a few,
or from recombinant plasmids or viral vectors). The compounds of
the present invention are said to be "substantially free of
natural contaminants" if preparations which contain them are
substantially free of materials with which these products are normally
and naturally found.
 The choice of which method to employ will depend upon factors
such as convenience, desired yield, etc. It is not necessary to
employ only one of the above-described methods, processes, or technologies
to produce BCRP; the above-described processes, methods, and technologies
may be combined in order to obtain BCRP. It is most preferable to
prepare BCRP by expressing the gene or cDNA sequence which encodes
the BCRP protein. Such gene or cDNA sequence hereinafter termed
the "BCRP gene" or "BCRP cDNA sequence".
 The technique of RNA fingerprinting was employed to clone
the BCRP cDNA. RNA fingerprinting uses the polymerase chain reaction
(PCR) and degenerate primer pairs to amplify cellular mRNA. This
technique is based on modifications of the technique of "Differential
Display of mRNA" developed by Liang and Pardee (6). We used
these techniques as a means to discover genes that are differentially
expressed in drug-selected cell lines compared to parental cells.
The major difference between RNA Fingerprinting and Differential
Display is that the mRNA fingerprinting protocol uses a single cDNA
synthesis reaction, followed by amplification with upstream and
downstream primers. Differential Display uses 9 to 12 cDNA syntheses
for each RNA sample with an anchored oligo(dT) primer, followed
by amplification with an upstream primer.
 The cloned BCRP gene, obtained through the methods described
above and in the examples, may be operably linked to an expression
vector, and introduced into bacterial, or eukaryotic cells to produce
BCRP protein. Techniques for such manipulations are disclosed in
Maniatis, T. et al. supra, and are well known in the art (8).
 The BCRP cDNA sequence is about 2418 nucleotides long. The
BCRP cDNA is depicted in SEQ ID No. 2 or FIG. 2C. The BCRP cDNA
can be used to express the BCRP. Also, the BCRP cDNA sequence, or
a portion thereof, can be used as a probe in a Northern blot assay
or for selection of probes in a RT-PCR assay to measure BCRP mRNA
in various tissue samples. Measurement of expression of BCRP by
Northern blot or RT-PCR assay can be determinative of drug response
to chemotherapeutic drugs over time. The techniques for these assays
are described in the examples and are well-known in the art (8).
Therefore, such an assay could be used to determine if a patient's
failure to respond to chemotherapy is due to overexpression of BCRP,
and hence resistance to the drugs. Also, antisense probes could
be developed based on the cDNA sequence depicted in SEQ ID 2 and
FIG. 2C. These probes can be administered to patients to bind to
the BCRP cDNA endogenously and hence inhibit the expression of the
BCRP. Such a therapy could be used to halt or slow a patient's propensity
to become resistant to the chemotherapy drugs and hence render treatment
more effective. Techniques for the production and administration
of antisense probes are well known in the art. Techniques of nucleic
acid hybridization and cloning are well known in the art (8).
 The data presented in the examples and corresponding figures
strongly support the conclusion that the novel ABC family member
BCRP reported here is a xenobiotic transporter that is primarily
responsible for the drug resistance phenotype of MCF-7/AdrVp cells.
 The overexpression of BCRP in several cancer cell lines
is also shown in the present invention. These cell lines include
colon carcinoma cells S1, HT29, gastric carcinoma cells EPG85-257,
fibrosarcoma cells EPR86-079, and myeloma 8226 cells. The overexpression
of BCRP mRNA in each of these cell lines, and the amplification
of the BCRP gene in the drug-resistant cells demonstrate an important
role for BCRP in resistance to cytotoxic agents. Furthermore, the
enforced overexpression of BCRP in MCF-7 cells diminished daunorubicin
cellular accumulation and imparted a pattern of drug cross-resistance
to the transfected cells that was virtually identical to that of
MCF-7/AdrVp cells. The degree of overexpression of BCRP in transfectant
clones 6 and 8 correlates with the alterations in the intracellular
steady state level of daunorubicin and their degree of resistance
to mitoxantrone, daunorubicin and doxorubicin.
 A major difference between the BCRP-overexpressing transfectant
clones and the original MCF-7/AdrVp subline is that the degree of
drug resistance in the latter is greater than in the transfected
cells, while the steady state BCRP mRNA levels in each are comparable
(FIG. 4A). A number of possibilities may contribute to this difference.
Differences in protein stability and/or localization may contribute
to the full drug-resistant phenotype, or the expression of other
proteins may be required. Recently, we reported that members of
the carcinoembryonic antigen (CEA) family, primarily the non-specific
cross reacting antigen (NCA) and CEA itself, are markedly overexpressed
on the cell surface of MCF-7/AdrVp and MCF-7/AdrVpPR cells compared
to drug-sensitive MCF-7 cells (15). A high density of these acidic
glycoproteins on the cell surface may protonate drugs such as mitoxantrone,
daunorubicin or doxorubicin which prevents entry into the cell.
Indeed, Kawaharata, et.al. (16) reported that the enforced expression
of CEA in transfected NIH3T3 cells results in both diminished accumulation
of and resistance to doxorubicin in the transfected cells. Hence,
the relative overexpression of CEA family members on the MCF-7/AdrVp
cell surface could act in concert with BCRP to cause greater resistance
to mitoxantrone, doxorubicin and daunorubicin than that caused by
BCRP alone. This hypothesis could be tested by co-transfecting the
MCF-7/BCRP-clone 8 subline with an expression vector containing
NCA or CEA.
 Another possible explanation for the greater degree of resistance
of MCF-7/AdrVp cells compared to the transfectants is that BCRP
is part of a multiprotein transporter complex. The translocation
pathway of typical ABC transporters consists of two ATP-binding
domains and two highly hydrophobic domains which contain membrane-spanning
regions. This can be accomplished in a single molecule, as is the
case of MRP or Pgp, which are twice the size of BCRP (approximately
1,300 compared to 655 amino acids). Alternatively, the active complex
of certain ABC transporters can be formed by the heterodimerization
of two non-identical proteins, each of which contains a single ATP-binding
and hydrophobic region. The .omega. and brown (b) proteins of Drosophila
and the Tap-1 and Tap-2 proteins that transport major histocompatibility
class I proteins are examples of ABC family members that exhibit
such a cooperative interaction. The presence of the phosphopantetheine
attachment site on BCRP suggests that BCRP may be a part of a multiprotein
complex. Thus, it is possible that BCRP has a protein cofactor(s)
which makes it a much more efficient transporter in a heteromeric
state. The activation or overexpression of this cofactor in MCF-7/AdrVp
relative to MCF-7 cells could explain the increased drug transport
in the MCF-7/AdrVp subline relative to the BCRP transfectants.
 The finding of elevated expression of BCRP mRNA in the human
colon carcinoma S1M1-3.2 cells suggests that BCRP is the "non-Pgp,
non-MRP" drug transporter manifested by this multi-resistant
cell line. This is of particular importance because of the recent
report (25) of a specific inhibitor of the transporter identified
in S1M1-3.2 cells. This inhibitor, fumitrimorgin C (FTC), does not
reverse resistance in cells that overexpress Pgp or MRP. FIG. 10
shows that FTC is able to enhance the accumulation and inhibit the
efflux of BBR 3390 (an aza-anthrapyrazole drug that is effluxed
by BCRP) in BCRP-transfected MCF-7 cells.
 The following examples are provided for illustrative purposes
only and are in no way intended to limit the scope of the present
invention. All references cited are incorporated by reference.
 Cell lines. MCF-7 breast carcinoma cells, their drug-resistant
subline MCF-7/AdrVp, and a partially drug-sensitive revertant subline
(MCF-7/AdrVpPR, obtained from Dr. Antonio Fojo, Medicine Branch,
National Cancer Institute), were maintained in culture as described
previously (5). The MCF-7/AdrVp subline was continuously maintained
in the presence of 100 ng/ml doxorubicin (Pharmacia Adria, Dublin,
Ohio) and 5 .mu.g/ml verapamil (Sigma Chemicals, St. Louis, Mo.).
 Growth conditions for the cell lines used in the Northern
blot studies are contained in the references listed in Table 1.
The S1M1-3.2 colon carcinoma cells were derived from S1 cells (a
subclone of human colon carcinoma cell line LS174T) by selection
for growth in increasing concentrations of mitoxantrone until a
final concentration of 3.2 .mu.M was acheived. HL-60/MX2 cells were
purchased from the American Type Culture Collection (Manassas, Va.),
and maintained in culture as described previously (17).
 Synthesis of cDNA by Reverse Transcription of mRNA
 Purified total cellular RNA (2 .mu.g) from MCF-7/W, MCF-7/AdrVp
or MCF-7/AdrVpPR cells which have partially reverted to drug sensitivity
by culture in the absence of the selecting agents were reverse transcribed
with 200 units of Moloney murine leukemia virus reverse transcriptase
in the presence of an oligo(dT) primer (0.1 .mu.M), and 1 mM dNTP
at 42.degree. C. for 1 hour. The reactions were terminated by heating
at 75.degree. C. for 10 minutes. The cDNAs thus produced were stored
at-20.degree. C. until further use.
 RNA Fingerprinting
 RNA fingerprinting was performed using the Delta.TM. RNA
fingerprinting kit (Clontech Laboratories, Palo Alto, Calif.), with
minor modifications. RNA fingerprinting is accomplished by amplification
of the cDNA by the polymerase chain reaction (PCR), using random
 For each fingerprinting reaction, cDNA diluted 1:10 (dilution
A) or 1:40 (dilution B) from each cell line was amplified with one
upstream (P) and one downstream (T) primer in the presence of 50
.mu.M dNTP, 50 nM [.sup.33P]dATP, and the "Advantage KlenTaq
Polymerase Mix" supplied with the Clontech kit. The upstream
P primers were arbitrary 25-mers. The downstream T primers were
30-mer anchored oligo(dT)primers whose 3' terminal contained the
sequence 5'-T.sub.9N.sub.1N.sub.1-3', where N.sub.1 is A, C or G.
The P primer binds to the cDNA based on chance homology. We paired
ten P primers and nine T primers to give 90 possible combinations.
 The first three PCR cycles were performed at a relatively
low stringency (annealing temperature 40.degree. C.). Because of
this, the P primer bound imperfectly, which increased the number
of amplified products. The products of these early cycles were then
amplified by 24 PCR cycles at high stringency (annealing temperature
60.degree. C.). Control PCR reactions were prepared containing sterile
water instead of cDNA (water control), or 0.02 .mu.g of total cellular
RNA (RNA control). The RNA controls were prepared to assess whether
the RNA was contaminated with genomic DNA.
 Following the PCR reaction, a small amount of each reaction
mixture was loaded onto a 5% polyacrylamide gel, after which the
gels were dried, then autoradiographs made (FIG. 1A). These autoradiographs
demonstrated a characteristic "RNA Fingerprint" pattern
of 50 to 100 PCR product bands of 100 to 2000 nucleotides in length.
Lanes 1, 3, and 5 are reaction mixes where cDNA diluted 1:10 (dilution
A) was added; lanes 2, 4, and 6 represent reaction mixtures where
cDNA diluted 1:40 (dilution B) was added. Lanes 7 and 8 are "H.sub.2O
controls", where sterile water was added to the PCR reaction
mixture instead of cDNA. Lanes 9, 10 and 11 are "RNA controls",
where 0.02 .mu.g of cellular RNA from MCF-7/W, MCF-7/AdrVp, or MCF-7/AdrVpPR
cellular is added instead of cDNA. These "RNA controls"
serve to indicate contamination of the RNA with genomic DNA. The
autoradiographs were inspected for PCR products that were produced
in greater abundance in reactions that used reverse transcribed
RNA from MCF-7/AdrVp cells, compared to those that used RNA from
MCF-7/W or MCF-7/AdrVpPR cells (FIG. 1A). The ARROW indicates a
PCR product that represents a mRNA species that is overexpressed
in MCF-7/AdrVp cells, compared to MCF-7/W or MCF-7/AdrVpPR cells.
This is the PCR product that was cut out of the gel and amplified
and cloned using the "TA Cloning" method, the desired
clone of which was called Clone 8 (see below).
 Amplification of the Target cDNA by TA Cloning
 The PCR product overexpressed in MCF-7/AdrVp cells was excised
from the dried gel and eluted by boiling in 40 ml ddH.sub.2O for
5 min, then amplified by PCR for 20 cycles using the original primers
and separated on 2% agarose/ethidium bromide gels. These PCR products
were then ligated into a "TA Cloning Vector" plasmid,
pCR.RTM.2.1, which was then cloned using standard techniques for
PCR products (Original TA Cloning.RTM. Kit, Invitrogen Corporation,
San Diego, Calif.).
 The pCR.RTM.2.1 plasmids containing the PCR product were
used to transform the TOP 10F strain of E. coli. Individual bacterial
colonies were picked and plasmid DNA was isolated by minipreps (Wizard.TM.
Miniprep, Promega, Madison, Wis.). Plasmid DNA was amplified by
PCR with the original "P" and "T" primers, then
subjected to gel electrophoresis. The original sized band was cut
out, and the DNA was isolated by boiling in 100 .mu.l ddH.sub.2O
at 100.degree. C. for 5 min. An aliquot of the DNA was reamplified
by PCR with the original primers for 20 cycles. A single band was
visualized on ethidium bromide gels which was cut out, electroeluted
 Isolation of the BCRP Clone
 The "reverse" Northern blot method was used to
screen the TA vector clones. Briefly, a "reverse" Northern
analysis was performed as follows. The PCR product isolated from
12 different colonies of E. coli that was transformed by the pCR2.1
plasmid were fixed in duplicate to Zeta Probe (BioRad, Richmond,
Calif.) membranes in a slot blot apparatus. One of the duplicate
membranes was probed with the [.sup.33P]-labeled PCR reaction mixture
that amplified MCF-7 cDNA using the original "P" and "T"
primers in the RNA Fingerprinting kit. The other membrane was probed
with the original [.sup.33P]-labeled parallel PCR reaction mixture
that amplified the cDNA produced from MCF-7/AdrVp cells, using standard
Northern blot conditions of hybridization, after which the binding
of probe was assessed by autoradiography. A single TA clone (Clone
8-SEQ ID No. 7) was thus identified whose PCR product insert identified
a 2.4 kb mRNA species that was markedly overexpressed in MCF-7/AdrVp
cells, compared to MCF-7 cells (FIG. 1B, top panel). The partially
revertant MCF-7 AdrVpPR subline had intermediate expression of the
2.4 kb mRNA species (FIG. 1B, top panel). To control for equivalence
in lane loading, the blot was stripped then reprobed with radiolabeled
18S RNA (FIG. 1B, bottom panel).
 Southern blots were performed using the Clone-8 PCR product.
Briefly, DNA was isolated, digested with EcoR1, subjected to agarose
gel electrophoresis, transferred and fixed to a nitrocellulose filter.
The filter was probed with the Clone-8 PCR product that was end-labeled
with [.sup.32P]-dCTP, then the radioautograph shown was made (FIG.
1C, top panel). This demonstrated that the cognate gene for BCRP
was amplified in both MCF-7/AdrVp and MCF-7/AdrVpPR cells, compared
to parental MCF-7 cells (FIG. 1C, top panel). The lower panel in
FIG. 1C shows the ethidium bromide-stained agarose gel electrophoretogram
of the corresponding genomic DNA after digestion with EcoR1, to
demonstrate approximate equivalence of gel loading.
 Sequencing of the BCRP Clone
 Sequencing of the cDNAs was performed with an automated
DNA sequencer (Perkin Elmer, Inc., Foster City, Calif.). All DNA
sequences were confirmed by sequencing in the reverse direction.
The differentially expressed PCR product in the TA Clone 8 was sequenced
and found to be a 795 bp cDNA (SEQ ID No. 7). Protein database searches
of the deduced amino acid sequence revealed a high degree of homology
to members of the ABC superfamily of transporter proteins.
 Isolation of the Full-length BCRP cDNA
 An MCF-7/AdrVp cDNA library was constructed using the CapFinder.TM.
PCR cDNA library construction kit (Clontech) according to the manufacturer's
protocol. The CapFinder.TM. technique is designed specifically to
produce full-length double stranded cDNA. The 795 bp Clone 8 cDNA
fragment was radiolabeled and used as a probe to screen the cDNA
library prepared from MCF-7/AdrVp cells. Positive clones isolated
were subjected to secondary and tertiary screening, then tested
by Northern blot hybridization using RNA obtained from MCF-7, MCF-7/AdrVp
and MCF-7/AdrVpPR cells. Multiple clones were found to have 2.4
kb inserts, the approximate size of the BCRP mRNA suggested by Northern
 Four of the 2.4 kb inserts were ligated into the pCR2.1
plasmid, then these TA vectors were cloned in E. coli (as described
above). One TA vector clone containing a 2.4 kb cDNA fragment insert
was identified and isolated. Sequencing of the 2.4 kb cDNA insert
was performed with an automated DNA sequencer (Perkin Elmer Inc.,
Foster City, Calif.). All DNA sequences were confirmed by sequencing
in the reverse direction. After sequencing, the cDNA insert was
found to be 2418 bp in length as in FIG. 2C or SEQ ID No. 2. Analysis
of the cDNA for open reading frames (ORF) using the program "FRAMES"
contained in the Genetics Computer Group (GCG) software package
indicated the presence of a long ORF that began at position 239,
and ended with the stop codon TAA at position 2204-6. The deduced
amino acid sequence of this ORF is shown in FIG. 2A, and SEQ ID
No. 1. The protein has 655 amino acids and a approximate molecular
weight of about 72.3 kilodaltons. The protein encoded by this sequence
has been designated Breast Cancer Resistance Protein, or BCRP (FIG.
 Analysis of the sequence of BCRP with the GCG program "MOTIFS"
demonstrated a single Walker "A" ATP/GTP binding region
(11) at amino acids 80-87 and a phosphopantetheine attachment site
at amino acids 213-228 (FIG. 2A). Phosphopantetheine (or pantetheine
4' phosphate) is the prosthetic group of acyl carrier proteins in
some multienzyme complexes where it serves in the attachment of
activated fatty acid and amino-acid groups (12).
 Examination of BCRP structure with GCG programs "PEPPLOT"
and "PLOTSTRUCTURE" revealed a relatively hydrophilic
amino-terminal domain (amino acids 1-400) that contains the ATP-binding
sequence and a relatively hydrophobic carboxy-terminal domain (amino
acids 401-655), containing at least three putative transmembrane
domains (TM1, TM2, and TM3), and four potential N-glycosylation
sites (Glyc) (FIG. 2A). The transmembrane domains were estimated
by the use of a program to predict helices in integral membrane
proteins (13). Analysis of the BCRP sequence by the GCG program
"DOTPLOT" demonstrates that the peptide is homologous
with one-half of the duplicated Pgp or MRP molecule, except that
Pgp or MRP have the configuration NH.sub.2-[transmembrane domains]-[ATP
binding 1]-[transmembrane domains]-[ATP binding 2]-COOH, whereas
that of BCRP is NH.sub.2-[ATP binding]-[transmembrane domains]-COOH.
The relative similarity of BCRP to other members of the ABC transporter
superfamily was determined using the "PILEUP" program
of GCG. This analysis demonstrated that the peptide sequence of
BCRP is only distantly related to P-glycoprotein (PgP or Mdr1) or
MRP (FIG. 2B).
 Comparison of BCRP Sequence to the .omega. Sequence
 Analyses of cDNA and deduced protein sequences were accomplished
using protein and nucleotide sequence databases that were accessed
using the Wisconsin Sequence Analysis Package Version 8 (Genetics
Computer Group [GCG], Madison, Wis.) which are available through
the Frederick Cancer Research Center's Supercomputing Facility (Frederick,
 A "FASTA" comparison of the BCRP amino acid sequence
revealed a high degree of homology to at least 50 ATP-binding cassette
transport proteins. The highest match was PIR2:G02068, the human
homologue of the Drosophila white (.omega.) gene, which has 638
amino acids, and is 29.3% identical to BCRP. The .omega. gene in
Drosophila functions in the cellular transport of guanine and tryptophan,
which are retinal pigment precursors (9). We found that the human
homologue of .omega. is not overexpressed in MCF-7/AdrVp cells compared
to MCF-7 cells, as detected by a reverse-transcription PCR assay
 The program "Oligo" (Version 5.0, National Biosciences,
Inc., Plymouth, Minn.) was used to help determine suitable primers
for detection of the human homologue of .omega. by reverse transcription-PCR.
These assays were done using a modification of those described previously
for beta actin and MRP (10), except that primers specific for the
.omega. gene were used instead of MRP. The upper primer began at
5' position 2136 of human .omega. mRNA, and had the sequence 5'-CGA
CCG ACG ACA CAG A-3) (SEQ ID No. 3); The lower primer began at 3'
position 2590, and had the sequence 5'-CTT AAA ATG AAT GCG ATT GAT-3')
(SEQ ID No. 4). To assure uniformity of gel loading, a reverse transcription-PCR
assay for beta-actin was also performed. The final concentrations
of primers used was 200 nM. Twenty-five cycles of denaturation (94.degree.
C., 1 minute), annealing (50.degree. C., 1 minute) and elongation
(72.degree. C., 2 minutes) were carried out. FIG. 6 shows an agarose
gel electrophoresis of an aliquot of the PCR reaction mixtures that
used RNA from MCF-7 or MCF-7/AdrVp cells demonstrating that both
human .omega. and beta-actin are expressed approximately equally
in these cell lines.
 Northern Blots of Various Human Tissue with BCRP Probe (Clone
 Northern blotting with a .sup.32P-labeled Clone 8 cDNA probe
was performed. Pre-blotted agarose gel-electrophoresed RNA from
multiple tissues was purchased from Clontech, for use in multiple
tissue Northern blot assays (FIG. 3). The greatest expression of
BCRP was seen in placental tissue, with lower amounts of expression
demonstrable in brain, prostate, small intestine, testis, ovary,
colon and liver. BCRP transcripts were below the level of detection
in heart, lung, skeletal muscle, kidney, pancreas, spleen, thymus
and peripheral blood leukocytes.
 Expression of BCRP in MCF-7 Cells-functional Studies
 The full-length BCRP cDNA was inserted into the multiple
to cloning site of expression vector pcDNA3 (Invitrogen). Following
subcloning of the pcDNA3-BCRP construct, DNA sequence analysis was
performed to confirm that the insert in the clone that was chosen
was in a sense orientation to the CMV promoter of the pcDNA3 vector.
MCF-7 cells were transfected with pcDNA3-BCRP, using the calcium
phosphate precipitation method (17), selected by culture with geneticin
(G418, 1 mg/ml), then subclone by limiting dilution in 96 well flat-bottomed
culture plates. Subclones were tested for expression of BCRP mRNA
by Northern blot analysis, using radiolabeled Clone 8 cDNA as a
probe (FIG. 4A). As a control, MCF-7 cells were also transfected
with the empty pcDNA3 vector, then selected by growth in medium
containing 1 mg/ml G418 (FIG. 4A). Two clones of MCF-7 cells transfected
with pcDNA3-BCRP that were found to overexpress BCRP (clones 6 and
8) were selected and expanded for further studies (FIG. 4A). A third
clone of pcDNA3-BCRP transfected cells, clone 19, did not overexpress
BCRP, and was selected for study as a control.
 Effect of Chemotherapeutic Drugs on BCRP-transfected MCF-7
 Daunorubicin accumulation and retention was examined in
the transfected cells by means of flow cytometry. The BCRP-overexpressing
clones 6 and 8 displayed diminished accumulation and retention of
daunorubicin, compared to the vector-transfected controls (FIG.
4B), with intracellular steady-state concentrations of drug in clones
8 and 6 respectively approximately 30% or 50% of that attained in
the vector control cells. This difference was not due to differences
in cell volume, since the volumes of the BCRP-overexpressing sublines
tested was not less than that of the empty vector-transfected control
cells. The cell volumes, measured by Coulter Channelyzer.TM. are
2515.+-.56, 3074.+-.112 and 2459.+-.56 um.sup.3 for MCF-7/BCRP-clone
6, MCF-7/BCRP-clone 8 and MCF-7/pcDNA3 vector control cells, respectively.
These values are comparable to our previous measurements of MCF-7
cell volumes (5).
 The sensitivities of the various transfected sublines to
chemotherapeutic agents were tested by the sulforhodamine-B (SRB)
cytotoxicity assay (14). The LC.sub.50, defined as the concentration
of drug that caused lethality to 50% of the cells, was calculated.
From this, the "Fold of Resistance" (RF) was calculated
by dividing the LC.sub.50 for a given drug against a transfected
cell line by the LC.sub.50 of that drug against non-transfected
MCF-7 cells. The BCRP-overexpressing clones 6 and 8 displayed resistance
to mitoxantrone, daunorubicin and doxorubicin, compared to non-BCRP-overexpressing
clone 19 cells, MCF-7 cells, or the empty vector-transfected controls
(FIGS. 4C, 4D, 5). FIG. 5 contains the median LC.sub.50 values for
multiple cytotoxicity experiments for all cell lines and drugs tested.
FIG. 4D shows typical LC.sub.50 studies for the six drugs tested
for MCF-7/W and MCF-7/pcDNA3-BCRP clone 8 cells to illustrate the
data from which the LC.sub.50 values were derived, and the accuracy
of the measurements. The asterisk and solid line in FIG. 4D indicate
MCF-7/W cells, the closed squares and dotted lines represent MCF-7/pcDNA3-BCRP
clone 8 cells. The vertical bars in the figure represent the standard
deviation of six replicate determinations.
 Like MCF-7/AdrVp cells, the MCF-7/BCRP transfectant clones
6 and 8 displayed the greatest degree of resistance to mitoxantrone.
The pattern of cross-resistance displayed by the BCRP-overexpressing
transfected cells is very similar to that displayed by MCF-7/AdrVp
cells, except that MCF-7/AdrVp cells have greater relative resistance
to all cytotoxic drugs within the phenotype. The BCRP-transfected
clones 6 and 8 remained relatively sensitive to idarubicin, cisplatin
and paclitaxel (taxol), as are MCF-7/AdrVp cells (FIGS. 4C, 4D and
 To determine the effects of ATP depletion on the retention
of rhodamine 123 by the BCRP transfected cells compared to controls,
cells were incubated in complete medium or under ATP-depleting conditions.
MCF-7 cells were depleted of ATP by incubation in glucose-free DMEM
containing 50 mM 2-deoxy-D glucose and 15 mM sodium azide for 20
minutes (37.degree. C.). Rhodamine 123 was added (0.5 .mu.g/ml final
concentration) for an additional 30 minutes. The cells were placed
on ice, washed free of rhodamine, and incubated under ATP-depleting
conditions for an additional 30 minutes, and rhodamine retention
was determined by flow cytometry (excitation 488 nm, emission 520
nm). This demonstrates that the transport function of BCRP appears
to depend on ATP.
 Expression of BCRP in Blast Cells from Patients with Acute
Myelogenous Leukemia (AML) as Detected by a Reverse-transcription
Polymerase Chain Reaction (RT-PCR) Assay
 The RT-PCR assays were performed using a modification of
those described previously for beta actin and MRP (10), except that
primers specific for BCRP were used instead of MRP. For BCRP, the
primers used were (sense) 5'-TTA GGA TTG AAG CCA AAG G-3' (SEQ ID
No. 5), and (antisense) 5'-TAG GCA ATT GTG AGG AAA ATA-3' (SEQ ID
No. 6). The 5' end of the sense primer begins at nucleotide position
1727 of the BCRP cDNA (SEQ ID No. 2 and FIG. 2C); the 3' end of
the antisense probe corresponds to position 2152 of the BCRP cDNA
(FIG. 2C). The final concentrations of primers used was 200 nM.
The final magnesium concentration used for PCR was 700 uM. Thirty-five
cycles of denaturation (94.degree. C., 1 minute), annealing (50.degree.
C., 1 minute) and elongation (72.degree. C., 2 minutes) were carried
out. Following agarose gel electrophoresis of an aliquot of the
PCR reaction mixture, the gels were transferred to nitrocellulose
and Southern blotting was done as described previously (12), using
the 795 bp Clone 8 PCR product (5' end labeled with .sup.32P-dCTP)
as a probe for BCRP. The expected PCR product length is 446 bp.
 Total cellular RNA was obtained from the blast cells of
fourteen patients with AML. Controls were done using varying volumes
of the PCR reaction mixture that was run with reverse-transcribed
MCF-7/W RNA. The results of these controls and of the RT-PCR assays
of the patient blast cell samples are depicted in FIG. 7. These
controls using MCF-7/W RNA indicate the RT-PCR assay we developed
is quantitative. Note in FIG. 7 that some patients have very low
levels of expression of BCRP, while others (patients 3, 4, 5 and
7) have levels of expression comparable to or greater than that
of MCF-7/W cells. This variation in expression of BCRP amongst blast
cell samples from AML patients holds open the possibility that those
patients who have relatively high expression of BCRP are more resistant
to treatment with the anti-neoplastic drugs which are susceptible
to the resistance caused by BCRP (anthracyclines and mitoxantrone).
Mitoxantrone and the anthracycline daunorubicin are important drugs
used in the treatment of AML.
 Northern Blot Hybridization in Various Cancer Cell Lines
 Total cellular RNA was used for Northern analysis in all
cases except for H209 or H69 cells, where poly A.sup.+ RNA was used.
RNA extraction and Northern blotting were performed by standard
techniques, and as described in Example 4. A 795 bp fragment (clone
8, SEQ ID No. 7) of the 3' end of the 2418 bp BCRP cDNA was used
as the hybridization probe after labeling with [.sup.32P]-dCTP ("Prime-a-Gene"
labeling kit, Promega, Madison, Wis.). To control for variations
in sample loading, the blots were stripped, then re-hybridized with
.sup.32P-labeled .beta.-actin or 18S RNA probes.
 FIG. 8A shows the results of the Northern blot hybidization
of mRNA from MCF-7 cells (lane 1), MCF-7/MITOX (lane 2), 8226/W
cells (lane 3), and 8226/MR20 (lane 4). The blot was probed for
BCRP with a 795-bp cDNA (Clone 8, SEQ ID No. 7) after labeling with
.sup.32P-dCTP (top panel). To control for equivalence in sample
loading, the blot was stripped and reprobed for .beta.-actin (bottom
 FIG. 8B shows the results of a Northern blot hybridization
of mRNA from S1/M1-3.2 cells (lane 1), S1/W cells (lane 2), MCF-7/W
cells (lane 3), MCF-7/MX.sub.PR cells (lane 4), MCF-7/MX.sub.RS250
cells (lane 5), MCF-7/MX.sub.RS600 cells (lane 6), MCF-7/VP (MRP+)
cells (lane 7), MCF-7/Adr (Pgp+) cells (lane 8), MCF-7/MTX (DHFR+)
cells (lanes 9), MCF-7/AdrVp1000 (BCRP+) cells (lane 10). The blot
was probed as described for FIG. 8A.
 FIG. 8C shows a Northern blot hybridization of mRNA from
human colon carcinoma HT29 cells (lane 1), HT29RNOV cells (lane
2), human breast carcinoma MDA-MB-231 cells (lane 3), MDA-MB-231RNOV
cells (lane 4), human fibrosarcoma EPF86-079 cells (lane 5), EPF86-079RNOV
cells (lane 6), human gastric carcinoma EPG85-257 cells (lane 7),
EPG85-257RNOV cells (lane 8), EPG85-257RDB (Pgp+) cells (lane 9),
human pancreatic carcinoma EPP85-181 cells (lane 10), EPP85-181RNOV
cells (lane 11), and EPP85-181RDB (Pgp+) cells (lane 12). The blots
were probed as described above for FIG. 8A.
 Southern Blot Hybridization
 Genomic DNA was isolated using standard techniques (8) from
the parental drug sensitive MCF-7/W cells (lanes 1, 7), MCF-7/MX.sub.PR
cells (lanes 2, 8), MCF-7/MX.sub.RS250 cells (lanes 3, 9), MCF-7/MX.sub.RS600
cells (lanes 4, 10), MCF-7/VP cells (overexpress MRP, lanes 5, 11)
and MCF-7/MTX cells (derive resistance by overexpression of DHFR,
lanes 6, 12), digested with EcoR1 or BamH1, separated by 0.8% agarose
gel electrophoresis, stained with ethidium bromide, transferred,
and fixed to a nitrocellulose filter, using standard techniques
(8). The filter was hybridized with the [.sup.32P]-labeled 795 bp
BCRP probe as described above for FIG. 8 (FIG. 9, top panel). Ethidium
bromide stained 0.8% agarose gel electrophoresis of genomic DNA
after digestion with the restriction endonucleases, and prior to
nitrocellulose filter transfer, demonstrated approximate equivalency
of sample loading (FIG. 9, bottom panel).
 Fumitremorgin C (FTC) Effects on BCRP Transfected Cells
 MCF-7 cells transfected with either the pcDNA3 empty vector
or pcDNA3 containing the full-length BCRP cDNA (transfectant clone
8) were cultured as monolayers in tissue culture flasks. The effects
of FTC on the accumulation of the aza-anthrapyrazole BBR3390 were
measured by exposing these cells to the fluorescent aza-anthrapyrazole
BBR3390 (5 uM) in the presence or absence of 10 uM FTC for 60 minutes.
Then, the cells were removed from the flasks by trypsinization,
and intracellular BBR3390 content was measured by flow cytometry.
The effects of FTC on BBR3390 retention were measured by exposing
another set of cells (vector control and transfectant clone 8) to
5 uM BBR3390 with and without 10 uM FTC for 60 minutes, washing
the cells free of drug, then reincubating the cells for an additional
30 minutes in fresh medium with and without FTC. Intracellular BBR3390
content was measured by flow cytometry. (See FIG. 10).
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1TABLE 1 Characteristics of Selected Multidrug Resistant Human
Cancer Cell Lines Fold Expression levels resistance relative to
Tumor to selecting Selecting parental cell line Cell line origin
agent agent Pgp MRP BCRP Reference MCF-7/Mitcox breast 1,206 mitcarantrone
- - ++ 18 MCF-7/MX.sub.pr " 270 " - +/- ++ 19 MCF-7/MX.sub.azaco
" 4,600 " - +/- +++ 20 MCF-7/MX.sub.azaco " 7,450
" - - +++ 20 MCF-7/AdrVp1000 " 4000 dox verap - - + 3,4
MCF-7/AdrVp3000 " -7000 " - - ++ 3,4 MCF-7/VP " 26
etoposide - ++ - 21 MCF-7/Adr " 192 doxorubicin ++ - - 22 MCF-7/MTX
" 1,000 methotrecate - - - 23 MDA-MD-231RNOV " 83 milcoxantrone
- - +/- H. Lage, unpublished 8225/MR20 mylaloma 36 mitoxantrone
- - + 24 S1M1-3.2 colon 1,435 mitoxantrone - - + 25 HT29RNOV "
100 mitoxantrone +/- - + H. Lage, unpublished H209/MX2 small cell
lung 2 mitoxantrone - - - S. Cole, unpublished H209/MX4 " 4
" - - - " H209/V6 " 22 etoposide - - - 26 H69/AR
" 100 doxorubicin - +++ - 2 EPG36-267RNOV gastric 457 mitoxantrone
- - ++ 27, 28 EPG36-267RCB " 1,857 deunorubicin ++ - - 27,
28 EPP85-181RNOV pancreatic 27 mitoxantrone - - - H. Lage, unpublished
EPP85-181RDS " 845 deunorubicin ++ - - 29 EPP86-079RNOV florosarcoma
7 mitoxantrone - - ++ H. Lage, unpublished HL-60/MX2 leukemia 35
mitoxantrone - - - 30