An isolated protein having an amino acid sequence of SEQ ID No.
4 and having an activity inducing apoptosis, and a gene encoding
the same are provided. Also, a microarray having a substrate on
which the gene or fragment thereof is immobilized is provided. Also,
a method of diagnosing breast cancer using an antibody specifically
binding to the protein and a method of diagnosing breast cancer
by determining whether the gene is expressed in a cell or not, are
What is claimed is:
1. An isolated protein comprising: an amino acid sequence of SEQ
ID No. 4, wherein the protein is specifically expressed in a breast
2. The isolated protein of claim 1, which is associated with apoptosis.
3. The isolated protein of claim 1, which increases expression
p53, p21, or a combination comprising one or more of the foregoing
proteins in a cell.
4. The isolated protein of claim 1, further comprising a heterologous
polypeptide fused thereto.
5. A method of detecting the presence or absence of a breast cancer
in a test sample, the method comprising: contacting an isolated
anti-BCRP antibody with a polypeptide sample isolated from breast
tissue from a human, wherein the anti-BCRP antibody specifically
interacts with the polypeptide of SEQ ID NO:1; and detecting any
anti-BCRP-antibody-protein complexes that are formed, wherein an
increase in anti-BCRP-antibody-prot- ein complexes compared to a
polypeptide sample isolated from normal breast cells indicates the
presence of the breast cancer in the test sample.
6. The method of claim 4, wherein the increase in anti-BCRP-antibody
protein complexes compared to a polypeptide sample isolated from
normal breast cells is about a 3% increase.
7. A recombinant expression vector comprising: a polynucleotide
encoding a protein of claim 1 operably linked to expression control
8. The recombinant expression vector of claim 7, wherein the polynucleotide
encoding a protein of claim 1 comprises SEQ ID NO. 3.
9. The recombinant expression vector of claim 7, wherein the polynucleotide
is operatively linked to a tag sequence.
10. A polynucleotide or a complementary polynucleotide thereof
for a diagnosis or treatment of breast cancer, comprising: about
10 to about 100 continuous nucleotides derived from a polynucleotide
having a nucleotide sequence of SEQ ID No. 3.
11. The polynucleotide of claim 10, further comprising about 20
to about 100 contiguous nucleotides.
12. A microarray for the diagnosis of breast cancer, comprising:
a substrate, the substrate comprising a plurality of addresses,
wherein at least one address comprises a polynucleotide according
to claim 10.
13. A kit for the diagnosis of breast cancer, comprising: a polynucleotide
of claim 10; a reagent suitable for performing a detection method;
and instructions for use thereof.
14. A method of detecting the presence or absence of breast cancer,
the method comprising obtaining a breast tissue test sample from
a subject; and determining an expression level of a protein of claim
1 in the breast tissue test sample, wherein an elevated expression
level of the protein of claim 1 in the breast tissue test sample
compared to an expression level of the protein of claim 1 in a sample
of normal breast tissue indicates the presence of breast cancer
in the breast tissue test sample.
15. The method of claim 14, wherein the expression level of the
protein of claim 1 in the breast tissue test sample is greater than
or equal to about 2-fold higher than the expression level of the
protein of claim 1 in the sample of normal breast tissue.
16. The method of claim 14, wherein determining the expression
level is determined by northern blotting or electrophoresis.
17. The method of claim 14, wherein determining comprises: isolating
total RNA from the breast tissue test sample; performing reverse-transcription-polymerase
chain reaction on the total RNA with a primer comprising 10 or more
contiguous nucleotides of SEQ ID NO. 3; and quantifying the product
BACKGROUND OF THE INVENTION
 This application claims the benefits of Korean Patent Application
Nos. 10-2004-0011326, filed on Feb. 20, 2004, and 10-2005-0009487,
filed on Feb. 2, 2005, in the Korean Intellectual Property Office,
the disclosures of which are incorporated herein in their entirety
 1. Field of the Invention
 The present invention includes a Breast Cancer Related Protein
(BCRP) having an apoptosis-inducing activity, a gene encoding the
same, and a microarray comprising immobilized fragments of the BCRP
gene. Also, the present invention includes a method of diagnosing
breast cancer with an antibody specifically recognizing the BCRP,
and a method of diagnosing breast cancer by determining whether
the BCRP gene is expressed.
 2. Description of the Related Art
 Breast cancer is diagnosed and occurs most frequently in
women. Breast cancer is next to lung cancer when considering terminal
cancers. The incidence of breast cancer has been steadily increasing
over the past 50 years and, in particular, is surging in Korea.
There are several risk factors that can increase a woman's chance
of developing breast cancer. These factors include age, past breast
cancer history, exposure to radiation, family history for breast
cancer, social and economical class, pregnancy, menarche, menopause,
and first pregnancy after age 30.
 It is known that breast cancer is a heterogeneous disease
and various breast tumors are induced by female sex hormones. There
are many recognized factors and unknown factors. Identified changes
in oncogenes include amplifications of HER-2 and an epithelial growth
factor receptor gene and overexpression of cyclin D1. The overexpression
of an oncogene is associated with considerably slow progress of
breast cancer. Similarly, genetic change or loss of a tumor inhibitory
gene, such as p53, may be associated with slow progress of breast
 Researchers found two genes called BRCA1 and BRCA2, which
are predictors of familial breast cancer before menopause. Early
diagnosis of breast cancer is essential to assure the best treatment
results. Many countries having advanced healthcare systems have
a program for screening for breast cancer. Information employed
in the selection of the treatment and prognosis may include, for
example, measurement of the state of estrogen and progesterone receptors.
 Some objectives in the treatment of breast cancer are to
improve early detection success rate, to find a novel non-invasive
marker capable of tracing the progress of the disease and identifying
recurrence, and to find an improved treatment for progressed disease,
which still has a very low 5-year survival rate. It is desirable
to identify more specific targets for cancerous cells, so as to
attack tumor cells through new prospective methods such as immunotherapy
and targeted toxin therapy, both of which ideally target molecules
expressed on the surface of tumor cells.
SUMMARY OF THE INVENTION
 The present invention provides an isolated Breast Cancer
Related Protein, which is specifically expressed in breast cancer
 The present invention also provides a nucleic acid sequence
encoding the Breast Cancer Related Protein and a microarray on which
the nucleic acid sequence encoding the protein or a fragment thereof
is immobilized. Also included are recombinant expression vectors
comprising a BCRP gene operably linked to expression control sequences.
 The present invention also provides a method of detecting
the presence or absence of breast cancer in a test breast tissue
sample by using an antibody that specifically binds to the Breast
Cancer Related Protein to detect the presence or absence of the
protein. The method includes incubating an anti-BCRP antibody with
a polypeptide test sample isolated from breast tissue from the human,
wherein the anti-BCRP antibody specifically interacts with the polypeptide
of SEQ ID NO:1; and detecting any anti-BCRP-antibody-protein complexes
that are formed, wherein an increase in anti-BCRP-antibody-protein
complexes in the test sample compared to a polypeptide sample isolated
from normal breast cells indicates the presence of the breast cancer
in the test sample.
 The present invention also provides a method of detecting
the presence or absence of breast cancer in a test breast tissue
sample by determining whether gene encoding the Breast Cancer Related
Protein is expressed in the test breast tissue sample.
 According to an aspect of the present invention, there is
provided an isolated protein having an amino acid sequence of SEQ
ID No. 4, wherein the protein is specifically expressed in a breast
 According to another aspect of the present invention, there
is provided a method of detecting the presence or absence of breast
cancer, the method including: reacting an anti-BCRP antibody with
a polypeptide sample derived from human breast tissue, and determining
whether the anti-BCRP antibody interacts with the polypeptide sample.
 According to another aspect of the present invention, there
is provided a polynucleotide encoding the Breast Cancer Related
 According to another aspect of the present invention, there
is provided a polynucleotide or a complementary polynucleotide thereof
for a diagnosis or treatment of breast cancer, including at least
10 continuous nucleotides derived from the Breast Cancer Related
Protein polynucleotide, a microarray on which the polynucleotide
or the complementary polynucleotide thereof is immobilized, and
a kit including the polynucleotide or the complementary polynucleotide
 According to another aspect of the present invention, there
is provided a method of detecting the presence or absence breast
cancer, the method including: obtaining a breast tissue test sample
from a subject; and determining an expression level of the Breast
Cancer Related Protein in the breast tissue test sample and judging
the presence of breast cancer from the results.
BRIEF DESCRIPTION OF THE DRAWINGS
 The above and other features and advantages of the present
invention will become more apparent by describing in detail exemplary
embodiments thereof with reference to the attached drawings in which:
 FIG. 1 illustrates the results of northern blotting of various
normal cells using a breast cancer related protein (BCRP) gene specific
 FIG. 2 illustrates the results of northern blotting of various
cancerous cells using a BCRP gene specific probe;
 FIGS. 3 through 5 illustrate locations of BCRP expressed
in cells, identified through fluorescein isothiocyanate (FITC) fluorescence
observed on each of a colon cancer cell line Clone A (CA), primary
cultured normal kidney cells, and an HEK 293 cell line transfected
with a BCRP-pFLAG vector DNA;
 FIG. 6 illustrates the expression level of BCRP in a CA
cell line transfected with the BCRP gene, and the expression levels
of various apoptosis related genes when the BCRP is overexpressed,
which are identified via reverse transcription-polymerase chain
 FIG. 7 illustrates the effects of overexpression of the
BCRP gene on the proliferation of HEK 293, CA, and CX-1 (referred
to as A, B, and C, respectively) cell lines, which are identified
through a cell proliferation assay (MTT assay);
 FIGS. 8 through 10 illustrate fluorescence activated cell
sorter(FACS) analysis results for HEK 293, CA, and CX-1 transfected
with BCRP-pFLAG vector DNA, which are obtained by conducting an
apoptosis assay to identify the effects of overexpression by transfection
of BCRP on cell lines;
 FIGS. 11 and 12 illustrate the expression level of the BCRP,
which is identified by isolating RNA from breast cancer tissues
of two patients and from a normal tissue and performing a RT-PCR
using oligonucleotides of SEQ ID Nos. 5 and 6 as primers;
 FIG. 13 shows the results of a northern blotting assay for
BCRP using total RNA from breast tumor tissues of three patients
to analyze expression level of BCRP;
 FIG. 14 illustrates changes in the morphology of a cell
when an MDA-MB-231 cell line is treated with an anticancer agent
Taxol to induce apoptosis; and
 FIG. 15 illustrates the effects of Taxol on the expression
of the BCRP gene, which are identified through an RT-PCR assay using
RNA extracted from the cells of FIG. 14.
DETAILED DESCRIPTION OF THE INVENTION
 An isolated protein according to an embodiment of the present
invention comprises a BCRP protein, which is specifically expressed
in breast cancer cells. In one embodiment, the protein has an amino
acid sequence of SEQ ID No. 4. The protein has an apoptosis-inducing
activity and is specifically expressed in breast cancer. The protein
according to an exemplary embodiment of the present invention (hereinafter,
also referred to as breast cancer related protein (BCRP)) is a membrane
protein, which is specifically expressed in normal tissue such as,
for example, heart tissue. Among the various cancer tissues, the
BCRP is specifically expressed in breast cancer tissues. Thus, breast
cancer can be detected by determining whether the BCRP is expressed.
 The BCRP can increase the expression of p53, p21, or both,
when it is overexpressed in cells. The invention includes isolated
or purified BCRP polypeptides. An "isolated" or "purified"
polypeptide or fragment thereof is substantially free of cellular
material or other contaminating polypeptides from the cell or tissue
source from which the protein is derived, or substantially free
of chemical precursors or other chemicals when chemically synthesized.
The language "substantially free of cellular material"
includes preparations of polypeptide in which the polypeptide is
separated from cellular components of the cells from which it is
isolated or recombinantly produced. Thus, a polypeptide that is
substantially free of cellular material includes preparations of
polypeptide having less than about 30%, about 20%, about 10%, or
about 5% (by dry weight) of heterologous polypeptide (also referred
to herein as a "contaminating polypeptide").
 In one embodiment, the preparation is at least about 75%
by weight pure, more specifically at least about 90% by weight pure,
and most specifically at least about 95% by weight pure. A substantially
pure BCRP polypeptide may be obtained, for example, by extraction
from a natural source (e.g., a cancer cell); by expression of a
recombinant nucleic acid encoding a BCRP polypeptide; or by chemically
synthesizing the polypeptide. Purity can be measured by an appropriate
method, e.g., by column chromatography, polyacrylamide gel electrophoresis,
or by high pressure liquid chromatography (HPLC) analysis.
 The invention also includes homologs of BCRP. "Homolog"
is a generic term used in the art to indicate a polynucleotide or
polypeptide sequence possessing a high degree of sequence relatedness
to a subject sequence. Such relatedness may be quantified by determining
the degree of identity and/or similarity between the sequences being
compared. Falling within this generic term are the terms "ortholog",
meaning a polynucleotide or polypeptide that is the functional equivalent
of a polynucleotide or polypeptide in another species, and "paralog"
meaning a functionally similar sequence when considered within the
same species. Paralogs present in the same species or orthologs
of BCRP genes in other species can readily be identified without
undue experimentation, by molecular biological techniques well known
in the art.
 Related polypeptides are aligned with BCRP by assigning
degrees of homology to various deletions, substitutions and other
modifications. Homology can be determined along the entire polypeptide
or polynucleotide, or along subsets of contiguous residues. The
percent identity is the percentage of amino acids or nucleotides
that are identical when the two sequences are compared. The percent
similarity is the percentage of amino acids or nucleotides that
are chemically similar when the two sequences are compared. Mature
BCRP and homologous polypeptides are preferably greater than or
equal to about 70%, specifically greater than or equal to about
80%, more specifically greater than or equal to about 90%, and most
specifically greater than or equal to about 95% identical.
 Where a particular polypeptide is said to have a specific
percent identity to a reference polypeptide of a defined length,
the percent identity is relative to the reference peptide. Thus,
a polypeptide that is 50% identical to a reference polypeptide that
is 100 amino acids long can be a 50 amino acid polypeptide that
is completely identical to a 50 amino acid long portion of the reference
polypeptide. It might also be a 100 amino acid long polypeptide
that is 50% identical to the reference polypeptide over its entire
length. Of course, many other polypeptides will meet the same criteria.
 By "modification" of the primary amino acid sequence
it is meant to include "deletions" (that is, polypeptides
in which one or more amino acid residues are absent), "additions"
(that is, a polypeptide which has one or more additional amino acid
residues as compared to the specified polypeptide), "substitutions"
(that is, a polypeptide which results from the replacement of one
or more amino acid residues), and "fragments" (that is,
a polypeptide consisting of a primary amino acid sequence which
is identical to a portion of the primary sequence of the specified
polypeptide). By "modification" it is also meant to include
polypeptides that are altered as a result of post-translational
events, which change, for example, the glycosylation, amidation
(e.g., C-terminal amindation), lipidation pattern, or the primary,
secondary, or tertiary structure of the polypeptide. N-terminal
and/or C-terminal modifications are possible.
 Reference herein to either the nucleotide or amino acid
sequence of BCRP also includes reference to naturally occurring
variants of these sequences. Nonnaturally occurring variants that
differ from SEQ ID NO: 4 for the mature polypeptide, and retain
biological function, are also included herein. The variants may
comprise those polypeptides having conservative amino acid changes,
i.e., changes of similarly charged or uncharged amino acids. Genetically
encoded amino acids are generally divided into four families: (1)
acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine);
(3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,
glutamine, cystine, serine, threonine, tyrosine). Phenylalanine,
tryptophan, and tyrosine are sometimes classified jointly as aromatic
amino acids. As each member of a family has similar physical and
chemical properties as the other members of the same family, it
is reasonable to expect that an isolated replacement of a leucine
with an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on
the binding properties of the resulting molecule. Whether an amino
acid change results in a functional polypeptide can readily be determined
by assaying the apoptosis-inducing activity of the BCRP polypeptide
 A method of detecting breast cancer according to another
exemplary embodiment of the present invention comprises contacting
an isolated anti-BCRP antibody that specifically binds to the BCRP
with a polypeptide in a test sample derived from human breast tissue.
As used herein, the term "selectively binds to" refers
to the ability of antibodies of the present invention to preferentially
bind to BCRP and mimetopes thereof including SEQ ID NO. 4. Any protein-antibody
complexes that are formed can be detected using a variety of methods
standard in the art including enzyme immunoassays (e.g., enzyme
linked immunoassays (ELISA)), immunoblot assays, and the like. An
increase in anti-BCRP antibody-protein complexes in the test sample
compared to a control sample isolated from normal (i.e., non-tumor)
breast cells indicates the presence of breast cancer in the test
sample. When the expression level of the BCRP is higher than the
level expressed in normal tissue, specifically about 3%, about 5%,
about 10%, or about 15% higher than its level in a normal tissue,
the sample is judged to be a breast cancer sample. The methods of
producing an antibody to a particular protein antigen are well known
in the art and the anti-BCRP antibody of the present invention may
also be produced by conventional methods.
 Isolated antibodies can include antibodies in serum, or
antibodies that have been purified to varying degrees. Such antibodies
may include polyclonal antibodies, monoclonal antibodies, humanized
or chimeric antibodies, anti-idiotypic antibodies, single chain
antibodies, Fab fragments, fragments produced from a Fab expression
library, epitope-binding fragments of the above, and the like.
 Antibodies that bind to BCRP can be prepared from the intact
polypeptide or fragments containing peptides of interest as the
immunizing agent. The preparation of polyclonal antibodies is well
known in the molecular biology art. A host for preparation and/or
administration of an antibody can mean a human or a vertebrate animal,
including, but not limited to, dog, cat, horse, sheep, pig, goat,
chicken, monkey, rat, mouse, rabbit, guinea pig, and the like.
 A monoclonal antibody composition can be antibodies produced
by clones of a single cell called a hybridoma that secretes or otherwise
produces one kind of antibody molecule. Hybridoma cells can be formed
by fusing an antibody-producing cell and a myeloma cell or other
self-perpetuating cell line. Briefly, monoclonal antibodies can
be obtained by injecting mammals such as mice or rabbits with a
composition comprising an antigen, thereby inducing in the animal
antibodies having specificity for the antigen. A suspension of antibody-producing
cells is then prepared (e.g., by removing the spleen and separating
individual spleen cells by methods known in the art). The antibody-producing
cells are treated with a transforming agent capable of producing
a transformed or "immortalized" cell line. Transforming
agents are known in the art and include such agents as DNA viruses
(e.g., Epstein Bar Virus, SV40), RNA viruses (e.g., Moloney Murine
Leukemia Virus, Rous Sarcoma Virus), myeloma cells (e.g., P3X63-Ag8.653,
Sp2/0-Ag14), and the like. Treatment with the transforming agent
can result in production of a hybridoma by means of fusing the suspended
spleen cells with, for example, mouse myeloma cells. The transformed
cells are then cloned, preferably to monoclonality. The cloning
is preferably performed in a medium that will support transformed
cells, and not support non-transformed cells. The tissue culture
medium of the cloned hybridoma is then assayed to detect the presence
of secreted antibody molecules by antibody screening methods known
in the art. The desired clonal cell lines are then selected.
 Other types of antibodies include humanized monoclonal antibodies,
chimeric antibodies, anti-idiotypic monoclonal antibodies, and recombinant
antibodies. Humanized monoclonal antibodies are produced by transferring
mouse complementarity determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
then substituting human residues into the framework regions of the
murine counterparts. Chimeric antibodies can be obtained by splicing
the genes from a mouse antibody molecule with appropriate antigen
specificity together with genes from a human antibody molecule of
appropriate biological specificity. An anti-idiotypic monoclonal
antibody made to a first monoclonal antibody will have a binding
domain in the hypervariable region that is the "image"
of the epitope bound by the first monoclonal antibody. Recombinant
antibodies can be prepared by recombinant DNA techniques as is known
in the art.
 A suitable method to produce anti-BCRP antibodies includes
(a) administering to an animal an effective amount of BCRP (ranging
in size from a polypeptide fragment to a full-length protein) or
mimetope thereof to produce the antibodies and (b) recovering the
antibodies. Antibodies can be recovered and/or purified by methods
known in the art. Suitable methods for antibody purification include
purification on Protein A or Protein G beads, protein chromatography
methods (e.g., diethyl-amino-ethyl (DEAE) ion exchange chromatography,
ammonium sulfate precipitation), antigen affinity chromatography,
and the like.
 An isolated polynucleotide according to another exemplary
embodiment of the present invention encodes an isolated protein
having an amino acid sequence of SEQ ID No. 4 wherein the protein
has an apoptosis-inducing activity. The polynucleotide may have
the nucleotide sequence of SEQ ID No. 3. The polynucleotide may
be employed for expressing the BCRP and for judging the presence
of breast cancer by investigating whether the BCRP is expressed.
 The term "isolated polynucleotide" includes polynucleotides
that are separated from other nucleic acid molecules present in
the natural source of the nucleic acid. For example, with regard
to genomic DNA, the term "isolated" includes polynucleotides
that are separated from the chromosome with which the genomic DNA
is naturally associated. An "isolated" polynucleotide
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and/or 3' ends of the nucleic acid)
in the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated polynucleotide
can contain less than about 5 kb, about 4 kb, about 3 kb, about
2 kb, about 1 kb, about 0.5 kb, or about 0.1 kb of 5' and/or 3'
nucleotide sequences which naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
Moreover, an "isolated" polynucleotide, such as a cDNA
molecule, can be substantially free of other cellular material,
or culture medium when produced by recombinant techniques, or substantially
free of chemical precursors or other chemicals when chemically synthesized.
By free of other cellular material, it is meant that an isolated
polynucleotide is greater than or equal to about 70%, about 75%,
about 80%, about 85%, about 90%, about 95%, or about 99% pure.
 "Polynucleotide" or "nucleic acid" refers
to a polymeric form of nucleotides at least 5 bases in length. The
nucleotides can be ribonucleotides, deoxyribonucleotides, or modified
forms of either nucleotide. Modifications include but are not limited
to known substitutions of a naturally-occurring base, sugar or internucleoside
(backbone) linkage with a modified base such as 5-methylcytosine,
a modified sugar such as 2'-methoxy and 2'-fluoro sugars, and modified
backbones such as phosphorothioate and methyl phosphonate. As used
herein, the term "gene" means the segment of DNA involved
in producing a polypeptide chain; it includes regions preceding
and following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
 The polynucleotide can be a DNA molecule, a cDNA molecule,
genomic DNA molecule, or an RNA molecule. The polynucleotide as
DNA or RNA comprises a sequence wherein T can also be U. The polynucleotide
can be complementary to a polynucleotide encoding a BCRP polypeptide
(e.g., SEQ ID NO:3), wherein complementary refers to the capacity
for precise pairing between two nucleotides. For example, if a nucleotide
at a certain position of a polynucleotide is capable of hydrogen
bonding with a nucleotide at the same position in a DNA or RNA molecule,
then the polynucleotide and the DNA or RNA molecule are complementary
to each other at that position. The polynucleotide and the DNA or
RNA molecule are substantially complementary to each other when
a sufficient number of corresponding positions in each molecule
are occupied by nucleotides that can hybridize with each other in
order to effect the desired process. As used herein, hybridization
means hydrogen bonding, which may be Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary nucleoside
or nucleotide bases.
 In addition, polynucleotides that are substantially identical
to a polynucleotide encoding a BCRP polypeptide (e.g., SEQ ID NO:3)
or which encode proteins substantially identical to SEQ ID NO:4
are included. By "substantially identical" is meant a
polypeptide or polynucleotide having a sequence that is at least
about 85%, specifically about 90%, and more specifically about 95%
or more identical to the sequence of the reference amino acid or
nucleic acid sequence. For polypeptides, the length of the reference
polypeptide sequence will generally be at least about 16 amino acids,
or specifically at least about 20 amino acids, more specifically
at least about 25 amino acids, and most specifically at least about
35 amino acids. For nucleic acids, the length of the reference nucleic
acid sequence will generally be at least about 50 nucleotides, specifically
at least about 60 nucleotides, more specifically at least about
75 nucleotides, and most specifically about 110 nucleotides.
 Typically, homologous sequences can be confirmed by hybridization,
wherein hybridization under stringent conditions as described, for
example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,
2d ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) is preferred.
Using the stringent hybridization outlined in Sambrook et al., (i.e.,
washing the nucleic acid fragments twice where each wash is at room
temperature for 30 minutes with 2.times. sodium chloride and sodium
citrate (SCC) and 0.1% sodium dodecyl sulfate (SDS); followed by
washing one time at 50.degree. C. for 30 minutes with 2.times.SCC
and 0.1% SDS; and then washing two times where each wash is at room
temperature for 10 minutes with 2.times.SCC), homologous sequences
can be identified comprising at most about 25 to about 30% base
pair mismatches, or about 15 to about 25% base pair mismatches,
or about 5 to about 15% base pair mismatches.
 The BCRP polynucleotides can be inserted into a recombinant
expression vector or vectors. The term "recombinant expression
vector" refers to a plasmid, virus, or other means known in
the art that has been manipulated by insertion or incorporation
of the BCRP genetic sequence. The term "plasmids" generally
is designated herein by a lower case p preceded and/or followed
by capital letters and/or numbers, in accordance with standard naming
conventions that are familiar to those of skill in the art. Plasmids
disclosed herein are either commercially available, publicly available
on an unrestricted basis, or can be constructed from available plasmids
by routine application of well-known, published procedures. Many
plasmids and other cloning and expression vectors are well known
and readily available, or those of ordinary skill in the art may
readily construct any number of other plasmids suitable for use.
These vectors may be transformed into a suitable host cell to form
a host cell vector system for the production of a polypeptide.
 The BCRP polynucleotides can be inserted into a vector adapted
for expression in a bacterial, plant, yeast, insect, amphibian,
or mammalian cell that further comprises the regulatory elements
necessary for expression of the nucleic acid molecule in the bacterial,
yeast, insect, amphibian, or mammalian cell operatively linked to
the nucleic acid molecule encoding BCRP. "Operatively linked"
refers to a juxtaposition wherein the components so described are
in a relationship permitting them to function in their intended
manner. An expression control sequence operatively linked to a coding
sequence is ligated such that expression of the coding sequence
is achieved under conditions compatible with the expression control
sequences. As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of
a nucleic acid sequence to which it is operatively linked. Expression
control sequences are operatively linked to a nucleic acid sequence
when the expression control sequences control and regulate the transcription
and, as appropriate, translation of the nucleic acid sequence.
 Thus, expression control sequences can include appropriate
promoters, enhancers, transcription terminators, a start codon (i.e.,
atg) in front of a protein-encoding gene, splicing signals for introns
(if introns are present), maintenance of the correct reading frame
of that gene to permit proper translation of the mRNA, and stop
codons. The term "control sequences" is intended to include,
at a minimum, components whose presence can influence expression,
and can also include additional components whose presence is advantageous,
for example, leader sequences and fusion partner sequences. Expression
control sequences can include a promoter. By "promoter"
is meant minimal sequence sufficient to direct transcription. Also
included are those promoter elements which are sufficient to render
promoter-dependent gene expression controllable for cell-type specific,
tissue-specific, or inducible by external signals or agents; such
elements may be located in the 5' or 3' regions of the gene. Both
constitutive and inducible promoters are included.
 Transformation of a host cell with an expression vector
or other DNA may be carried out by techniques well known to those
skilled in the art. By "transformation" is meant a permanent
or transient genetic change induced in a cell following incorporation
of new DNA (i.e., DNA exogenous to the cell). Where the cell is
a mammalian cell, a permanent genetic change is generally achieved
by introduction of the DNA into the genome of the cell. By "transformed
cell" or "host cell" is meant a cell (e.g., prokaryotic
or eukaryotic) into which (or into an ancestor of which) has been
introduced, by means of recombinant DNA techniques, a DNA molecule
encoding a polypeptide of the invention (i.e., a BCRP polypeptide),
or fragment thereof. When the host is a eukaryote, such methods
of transfection with DNA include calcium phosphate co-precipitates,
mechanical procedures such as microinjection, electroporation, insertion
of a plasmid encased in liposomes, or virus vectors, as well as
others known in the art, may be used.
 The BCRP polynucleotides can also be designed to provide
additional sequences, such as, for example, the addition of coding
sequences for added C-terminal or N-terminal amino acids that would
facilitate purification by trapping on columns or use of antibodies.
Such tags include, for example, histidine-rich tags that allow purification
of polypeptides on nickel columns. Such gene modification techniques
and suitable additional sequences are well known in the molecular
 A BCRP fusion polypeptide is also provided, comprising a
BCRP polypeptide covalently joined to a heterologous polypeptide
to which it would not be joined in nature. Fusion polypeptides are
useful for use in various assay systems. Therefore, fusion polypeptides
may be used, for example, to detect BCRP expression and to provide
a defense mechanism for BCRP expression when desired. For example,
BCRP fusion polypeptides can be used to identify proteins that interact
with the BCRP protein and influence its function. This interaction
may impart specificity to the ability of BCRP to regulate other
proteins, or it may increase or decrease the effect of BCRP function.
Physical methods, such as protein affinity chromatography, or library-based
assays for protein-protein interactions, such as the yeast two-hybrid
or phage display systems, can be used for this purpose. Such methods
are well known in the art.
 A fusion polypeptide comprises at least two heterologous
polypeptide segments fused together by means of a peptide bond.
The first polypeptide segment can comprise in whole or in part the
contiguous amino acids of a BCRP polypeptide. Where in part, at
least about 8 contiguous amino acids of the BCRP polypeptides are
used, specifically at least about 10 may be employed, more specifically
about 15, and most specifically at least about 20. The first polypeptide
segment can also be a full-length BCRP protein. The second polypeptide
segment can comprise an enzyme which will generate a detectable
product, such as beta-galactosidase or other enzymes that are known
in the art. Alternatively, the second polypeptide segment can include
a fluorescent protein such as green fluorescent protein, HcRed (Clontech)
or other fluorescent proteins known in the art. Additionally, the
fusion protein can be labeled with a detectable marker, such as
a radioactive maker, a fluorescent marker, a chemiluminescent marker,
a biotinylated marker, and the like. Techniques for making fusion
polypeptides, either recombinantly or by covalently linking two
polypeptide segments are well known.
 Polynucleotides encoding BCRP sequences allow for the preparation
of relatively short DNA (or RNA) sequences having the ability to
specifically hybridize to such gene sequences. A polynucleotide
or a complementary polynucleotide thereof for diagnosis or treatment
of breast cancer according to another embodiment of the present
invention includes at least about 10, about 20, about 30, about
40, about 50, about 60, about 70, about 80, about 90, and about
100 continuous nucleotides derived from a polynucleotide having
a nucleotide sequence of SEQ ID No. 3. The polynucleotide may be
about 10 to about 100, specifically about 10 to about 50, more specifically
about 20 to about 50 contiguous nucleotides in length. Such a polynucleotide
may be used as a primer or a probe. Such fragments may be prepared
by, for example, directly synthesizing the fragment by chemical
means, by application of nucleic acid reproduction technology, such
as PCR technology, or by excising selected nucleic acid fragments
form recombinant plasmids containing appropriate inserts and suitable
 A microarray for diagnosing breast cancer according to another
embodiment of the present invention comprises a substrate on which
an isolated polynucleotide or a complementary polynucleotide thereof
for the diagnosis or treatment of breast cancer including at least
10 continuous nucleotides derived from a polynucleotide having a
nucleotide sequence of SEQ ID No. 3 is immobilized. The polynucleotide
may be about 10 to about 100, specifically about 10 to about 50,
more specifically about 20 to about 50 contiguous nucleotides in
length, but is not limited thereto.
 A microarray array includes a substrate having a plurality
of addresses. At least one address of the plurality includes a BCRP
polynucleotide or complement thereof that binds specifically to
a BCRP polynucleotide. The capture probe may include the sense and/or
anti-sense strand. The array can have a density of about 10, 50,
100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm.sup.2,
and ranges between. The plurality of addresses can include 10, 100,
500, 1,000, 5,000, 10,000, 50,000 addresses. The substrate can be
a two-dimensional substrate such as a glass slide, a wafer (e.g.,
silica or plastic), a mass spectroscopy plate, or a three-dimensional
substrate such as a gel pad. Addresses in addition to address of
the first plurality can be disposed on the array. An array can be
generated by various methods, e.g., by photolithographic methods,
mechanical methods, and bead-based techniques.
 A kit for the diagnosis or treatment of breast cancer according
to another embodiment of the present invention includes a polynucleotide
comprising at least 10 continuous nucleotides derived from a polynucleotide
having a nucleotide sequence of SEQ ID No. 3. A kit may also comprise
a reagent suitable for performing a detection method such as a hybridization
reaction, an immunological reaction, and the like. A kit also suitably
comprises instructions for use thereof.
 A method of detecting the presence or absence of breast
cancer in a test sample according to another embodiment of the present
invention includes obtaining a polynucleotide sample derived from
breast tissue from a subject and determining an expression level
of a protein having the amino acid sequence of SEQ ID No. 4 in the
breast tissue test sample, and then determining the presence of
breast cancer from the results. The expression level of BCRP can
be determined, for example, by quantifying the level of BCRP mRNA
in the cell. The presence of breast cancer in the test sample can
be used to diagnose breast cancer in the subject.
 In the method, the expression level of the protein may be
measured using various methods known in the art. In the method,
the expression level of the BCRP gene may be determined by northern
blotting using a BCRP gene specific probe to quantify the level
of mRNA. Alternatively, the expression level of the BCRP gene may
be determined by extracting total RNA containing mRNA, performing
RT-PCR using a BCRP gene specific primer, and quantifying the product
produced. However, methods of determining the expression level of
BCRP are not limited thereto. The nucleic acid sample derived from
breast tissue does not necessarily mean only a purely purified nucleic
acid sample and only the presence of a nucleic acid capable of being
used in the analysis is required in any analysis method. For example,
a sample having disrupted cells may be used as it is without isolating
the nucleic acids when identifying the expression of BCRP gene using
 When the expression level of the BCRP in the test sample
is higher than the level expressed in normal breast tissue cells,
it may be judged to be breast cancer. The expression level of BCRP
may be greater than or equal to about 2-fold higher in breast cancer
cells than in control cells.
 Nucleic acids or proteins specifically expressed in breast
cancer cells were searched with respect to various nucleic acid
sequences selected from a database of commercially available nucleic
acids or proteins. As a result, a nucleic acid identified to be
specifically expressed only breast cancer cells or a protein presumed
therefrom, a BCRP nucleic acid or protein was selected. Next, a
BCRP full-length gene was identified by searching the cDNA library
and the nucleotide sequence thereof was analysed. It was identified
through northern blotting assay that the gene was specifically expressed
in a breast cancer cell line and breast cancer cells of patients
(FIGS. 11 and 12). Also, It was identified through repeated assay
that the expression level in breast cancer cells is twice higher
than in normal cells (FIG. 12). Immunocytochemistry methods were
used to localize BCRP gene expression in cells. As one localization
method, a BCRP polynucleotide was cloned into a pFLAG vector using
recombinant DNA techniques to produce a construct for the production
of BCRP protein with a pFLAG tag. The clone thus obtained was transfected
to an animal cell line, and then, the expression of the BCRP protein
was identified using a pFLAG fluorescence detection system (FIGS.
3, 4 and 5).
 In another method, the BCRP was overexpressed in cells and
an effect of the overexpressed BCRP on cells was investigated in
order to identify function of the BCRP. For this method, a gene
encoding the BCRP was transfected to an animal cell line, total
RNA containing mRNA was extracted therefrom, and RT-PCR was performed
using the total RNA as a template and primers suitable to specifically
amplify the gene to be detected. The expression levels of the BCRP
gene and other genes associated with apoptosis such as p53 and p21
were monitored by the RT-PCR (FIG. 6). A structure of the promoter
of the BCRP gene was also identified.
 The present invention will be described in greater detail
with reference to the following examples. The following examples
are for illustrative purposes and are not intended to limit the
scope of the invention.
Search of BCRP Gene Based on Single Nucleotide Polymorphism (SNP)
 1. Search of BCRP Gene
 The nucleotide sequence of the site, at which the SNP was
found was searched through a database (NCBI) search and analysis.
The association of the SNP with breast cancer was accomplished in
a separate study. The information suitable for fabricating a primer
for amplifying a gene was obtained therefrom.
 2. Amplification of BCRP Gene Fragment
 A primer for amplifying DNA around the searched SNP was
designed using the sequence information obtained through the database
(SEQ ID Nos. 1 and 2).
 Next, PCR was performed using genomic DNA as a template
and the designed primer set as a primer so that BCRP gene fragment
of 239 bp was amplified. In the PCR, 10 pmol of each of forward
and reverse primers (SEQ ID Nos. 1 and 2) and 200 pg-1 .mu.g of
genomic DNA template were used and the reaction was 35 times repeated
at 95.degree. C. for 40 seconds, at 57.degree. C. for 40 seconds,
and 72.degree. C. for 1 minute, respectively. The result was identified
via 1% agarose gel electrophoresis and amplification of expected
DNA fragment of 239 bp was identified. The 239 base pair amplified
DNA fragment was used as a probe for searching a BCRP full length
 3. Search of BCRP Full Length Gene Via cDNA Library
 A human fetal brain cDNA library (.lambda. triplrEx library,
available from Clontech Corp.) was used as a cDNA library and a
search procedure followed the experimental guidelines of the manufacturer
(PT3003-1). The search procedure was briefly as follows.
 PCR was performed to obtain mRNA, and simultaneously a search
of a cDNA library was performed using the previously obtained PCR
products as a probe. Cells used in the search were E. coli XL-1
blue cells generally used in the art.
 First, upon assay of titer of the library, both sets were
2.0.times.10.sup.9 pfu/ml. Then, the cDNA library was smeared on
an E. coli XL-1 blue plate. Generally, 2.about.5.times.10.sup.4
pfu/150 mm of the cDNA library was smeared. Next, .lambda. pharge
was transferred to a positively charged nylon film. Filter hybridisation
was performed using a probe labelled by random primed DNA labelling
with a radioactive isotope labelled dCTP ([.alpha.-.sup.32P]dCTP,
3000 Ci/mmol) and the hybridisation result was measured by detecting
the signals for the labelled probe. As a result, a positive clone
 4. Analysis of Base Sequence of the BCRP Full Length Gene
and Prediction of Protein Sequence
 The nucleotide sequence of the BCRP full length gene obtained
from the clone was identified by analysing with an automated sequence
analyzer (ABI 3700). Further, a deduced protein sequence of the
gene was identified using NCBI and the GENSCANW web program. A nucleotide
sequence of the BCRP gene was identical with SEQ ID No. 3, and thus,
an amino acid sequence of the protein encoded was identical with
SEQ ID No. 4. The SNP used for searching the BCRP is located in
the promoter region, and BCRP gene is located in the genome between
16032 and 96546 and is composed of 3 introns and 4 exons.
Identification of Expression of BCRP Gene in Cells and Tissues
 1. Identification of Expression of BCRP Gene in Cells through
 A northern blotting analysis was performed on a plurality
of human normal tissues and tumor tissues (available from Clontech
Corp.) using the PCR product obtained from Example 1 as a probe.
The results are illustrated in FIGS. 1 and 2. Referring to FIGS.
1 and 2, the BCRP gene was specifically expressed only in heart
tissue among normal tissues and in breast cancer tissue among tumor
tissues. Consequently, it is apparent that the expression of the
BCRP gene can be used to detect the presence of breast cancer. In
FIG. 1, tissues used in the northern blotting were brain heart,
heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small
intestine, placenta, lung, and peripheral blood leukocyte cell.
In FIG. 2, cancer tissues used in the northern blotting were cell
lines of breast, ovary, uterine, lung, kidney, stomach, colon, and
rectum (multiple tissue northern blot manufactured by Clontech Corp.).
1.00 kb and 1.2 kb in the top of FIGS. 1 and 2, respectively, represented
size markers and blotting results were shown at 1.37 kb.
 Specific procedures of the northern blotting were performed
 (1) Manufacturing of a Radiolabeled Probe
 A probe was manufactured using random primed DNA labeling
(Roche Corp. Random primed DNA labelling kit, #1004760). About 25
ng of a purified BCRP PCR product and an isotope [.alpha.-.sup.32P]dCTP,
250 .mu.Ci (available from BMS Corp.) were used.
 (2) Prehybridization Using a Hybridization Bottle
 The nylon film was prehybridized for 30 minutes in 7 ml
of ExpressHyb solution (#8015-1, available from BD Clontech Corp.)
previously heated at 68.degree. C.
 (3) Denaturation of the Radiolabeled Probe
 The radiolabeled probe was heated at 95-100.degree. C. for
8-10 minutes, and then quickly placed in ice.
 (4) Hybridization
 100 ml of a fresh ExpressHyb solution was mixed with the
radiolabeled probe. The prehybridized solution was removed from
the hybridisation bottle containing the nylon film and the fresh
ExpressHyb solution mixed with the radiolabeled probe was poured
thereto. Then, incubation was performed at 68.degree. C. for 1 hour
while shaking the bottle.
 (5) Washing
 The nylon film in the bottle was washed with a wash solution
1 at room temperature for 30-40 minutes, and then washed again with
a wash solution 2 at 50.degree. C. for 40 minutes. Then, the nylon
film was removed from the bottle and dried to the extent of maintaining
some moisture, and then wrapped with plastic.
 (6) The nylon film was placed in X-ray film and exposed
at -70.degree. C. After 1-2 days, the nylon film was removed and
band was identified.
 2. Identification of Expression of BCRP Gene in Tissues
 Expression of the BCRP gene in a lesional part and a nonlesional
part of breast cancer patients was identified. The lesional part
indicates tumor tissue, and the nonlesiongal part indicates normal
 FIGS. 11 and 12 illustrate the results of an RT-PCR experiment
performed using RNA isolated from cells, which are derived from
breast cancer tissues of two patients, and using the oligonucleotides
of SEQ ID Nos. 5 and 6 as primers. Referring to FIGS. 11 and 12,
the expression of the BCRP gene in breast cancer tissues increased
compared to in the normal tissue and expression of p53, which was
found to be associated with the breast cancer and selected as a
comparative gene, was also significantly increased.
 FIG. 13 illustrates the results of a northern blotting assay
of RNA of breast tumor tissues and normal breast tissues using the
PCR product obtained from Example 1 as a probe. Referring to FIG.
13, expression of the BCRP gene increased (about 1.8 kb). In FIG.
13, Lanes 1 to 3 are the results for breast tumor tissues of different
donors and Lane 4 is the result for a normal breast tissue. Comparing
the mean expression level of Lanes 1 to 3 with the expression level
in the normal breast tissue, it can be seen that the expression
level of the BCRP gene in breast cancer tissues was about 2.05 times
higher than the expression level in the normal tissue.
Identification of Location of Expression of BCRP Gene in Cells
 The localization of the expression of the BCRP gene in cells
was identified using an immunocytochemistry method. For this experiment,
the BCRP gene was first cloned to pFLAG vector (Sigma, Amherst,
N.Y.) using recombinant DNA techniques. The cloning procedure was
as follows. The BCRP gene of SEQ ID No. 3 was digested with Not
I enzyme and Sal I enzyme, the pFLAG vector was digested with the
same enzymes, and then the digested BCRP gene and the pFLAG vector
were ligated. The cloned BCRP-pFLAG vector DNA was transfected to
various cell lines using Lipofection 2000. The transfected cells
were incubated in 5% CO.sub.2 at 37.degree. C. for 48 hours such
that the BCRP gene could be expressed. Then, the incubated cells
were fixed on a plate using 3.5% paraformaldehyde. To dye the inside
of the cell, the cell was changed to be permeable by 0.1% Triton
X-100. A blank space was blocked with 1% BSA blocking solution.
A flag specific antibody (anti-FLAG M2) was incubated with the plate
on which the cells were fixed so as to specifically bind to the
BCRP-Flag. Finally, an FITC conjugated second antibody (anti-mouse
IgG-FITC) was reacted with the BCRP-Flag-primary antibody conjugate.
The location of the expression of the BCRP was identified through
fluorescence generated from FITC using a fluorescence microscope.
For comparison, an experiment was performed on a non-transfected
cell line using a Flag specific antibody in the same method as described
 The results were illustrated in FIGS. 3 through 5. FIGS.
3 through 5 illustrate FITC fluorescence observed for each of the
BCRP-pFLAG vector DNA transfected colon cancer cell line Clone A
(CA), the primary cultured normal kidney cell, and the HEK 293 cell
line. As shown in FIGS. 3 through 5, the BCRP was observed in cell
membranes in all three cell lines.
 Based on these results, it is believed that the BCRP, in
some embodiments, is specifically expressed in the cell membrane.
Effects of the Expression of BCRP Gene on Expression of other Genes
in a Cell Line
 The effects of the expression of the BCRP gene on the expression
of the others genes in a cell line were identified. For this assay,
the presence of mRNA of the BCRP gene was first identified through
northern blotting assay as described above. Then, the effect of
the expression of the BCRP gene on the expression of genes associated
with known cancers and apoptosis in cells was investigated in the
 First, BCRP-pFLAG vector DNA manufactured as above was transfected
into a CA (colon cancer Colon A) cell line using Lipofection 2000
and the transfected cell line was incubated in 5% CO.sub.2 at 37.degree.
C. for 48 hours. Then, total RNA containing mRNA was extracted and
RT-PCR was performed. .beta.-actin was used as a control. Primer
sets capable of amplifying BCRP, .beta.-actin, p53, p21, CytC, caspase
5, caspase 3, and Apaf 1 genes, respectively, were used as primers
(Table 1). The expression levels of each gene were identified by
monitoring the amounts of the PCR product obtained from the RT-PCR.
1TABLE 1 Primer sequence used in the amplification of each gene
Gene Primer sequence BCRP F: SEQ ID No. 5 R: SEQ ID No. 6 .beta.-actin
F: SEQ ID No. 7 R: SEQ ID No. 8 p53 F: SEQ ID No. 9 R: SEQ ID No.
10 p21 F: SEQ ID No. 11 R: SEQ ID No. 12 CytC F: SEQ ID No. 13 R:
SEQ ID No. 14 Caspase 5 F: SEQ ID No. 15 R: SEQ ID No. 16 Caspase
3 F: SEQ ID No. 17 R: SEQ ID No. 18 Apaf 1 F: SEQ ID No. 19 R: SEQ
ID No. 20
 RT-PCR was performed as follows: Total RNA containing mRNA
extracted from the cell line in which the BCRP gene was overexpressed
was reverse-transcribed using Superecript II reverse transcriptase
(available from Invitrogen), thereby obtaining cDNA. PCR was performed
on 5 ng of the obtained cDNA using Taq polymerase as a template
and using 10 pmol of each of the forward and reverse primers (referred
to Table 1). In the PCR, the reaction was repeated 30 times at 95.degree.
C. for 40 seconds, at different annealing temperatures depending
on genes to be amplified for 40 seconds, and at 72.degree. C. for
1 minute, respectively. The annealing temperature was 58.degree.
C. for .beta.-actin, caspase 5, and BCRP, and 52.degree. C. for
p53, p21, Cyt C, caspase 3, and Apaf 1. The results were identified
through 1% agarose gel electrophoresis and were illustrated in FIG.
6. Referring to FIG. 6, the expression of p53 increased and as did
the expression of p21, which was known as a temporary mediator of
p53-dependent growth arrest. However, other genes did not show specific
changes in expression.
Effects of Overexpression of the BCRP Gene on the Amplification
of a Cell Line and Apoptosis
 1. MTT Assay
 The effects of overexpression of the BCRP gene in cells
on amplification of the cell line and apoptosis were identified
through an MTT assay. The MTT assay was a method of measuring absorbance
of formazan generated by reducing MTT with mitochondrial dehydrogenase
in a living cell. The measured absorbance reflected a concentration
of a metabolically vigorous cell. The cell lines used in the assay
were Clone A and CX-1 (colon cancer cell lines), and a normal kidney
cell line, HEK 293. BCRP-pFLAG vector DNA was transfected into the
cells with Lipofection 2000 as in Example 4. Controls used in the
assay were a vector control to which only pFLAG was transfected,
a nothing control which was not transfected but encountered equivalent
stress, and a blank control having no cell.
 The results were obtained from twice-repeated experiments.
Cell amplification occurred less in CA and CX-1 cell lines than
in controls. In other words, cell growth in CA and CX-1 cell lines
was inhibited compared to controls. Also, cell amplification in
the HEK 293 cell line was relatively less as in colon cancer cell
lines (CA and CX-1) compared to other two controls. The results
are illustrated in FIG. 7, which shows the evaluation results of
the effects of overexpression of BCRP in the HEK 293, CA, and CX-1
(represented as A, B, and C, respectively) cell lines on amplification
of the cell line.
 2. Apoptosis Assay
 The effect of the expression of the BCRP gene on apoptosis
was investigated using the HEK 293, CA, and CX-1 cell lines through
flow cytometry. A vector control in which only the pFLAG vector
was transfected was used as a control.
 As a result of the twice-repeated assay, CA and CX-1 showed
less apoptosis than the control in both assays, and HEK 293 showed
little or no apoptosis. The assay results are illustrated FIGS.
8 through 10. FIGS. 8 through 10, respectively, illustrate FACS
assay results for HEK 293, CA, and CX-1.