U.S. patent application number 11/061694 was filed with the patent office on 2005-08-25 for breast cancer related protein, gene encoding the same, and method of diagnosing breast cancer using the protein and gene.
Invention is credited to Ahn, Tae-jin, Lee, Yeon-su, Park, Jong-hoon, Park, Kyung-hee.
Application Number | 20050186610 11/061694 |
Document ID | / |
Family ID | 34863620 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186610 |
Kind Code |
A1 |
Lee, Yeon-su ; et
al. |
August 25, 2005 |
Breast cancer related protein, gene encoding the same, and method
of diagnosing breast cancer using the protein and gene
Abstract
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 provided.
Inventors: |
Lee, Yeon-su; (Goyang-si,
KR) ; Park, Kyung-hee; (Seoul, KR) ; Ahn,
Tae-jin; (Seoul, KR) ; Park, Jong-hoon;
(Seoul, KR) |
Correspondence
Address: |
CANTOR COLBURN, LLP
55 GRIFFIN ROAD SOUTH
BLOOMFIELD
CT
06002
|
Family ID: |
34863620 |
Appl. No.: |
11/061694 |
Filed: |
February 18, 2005 |
Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.3; 435/7.23; 530/350; 530/388.8;
536/23.5 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C07K 14/47 20130101; C12Q 2600/158 20130101; C07H 21/04 20130101;
G01N 33/57415 20130101 |
Class at
Publication: |
435/006 ;
435/007.23; 435/069.3; 435/320.1; 435/325; 530/350; 530/388.8;
536/023.5 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; C07K 014/82; C07K 016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2004 |
KR |
10-2004-0011326 |
Feb 2, 2005 |
KR |
10-2005-0009487 |
Claims
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
cancer cell.
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
sequences.
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 produced.
Description
BACKGROUND OF THE INVENTION
[0001] 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 by reference.
[0002] 1. Field of the Invention
[0003] 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.
[0004] 2. Description of the Related Art
[0005] 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.
[0006] 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 cancer.
[0007] 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.
[0008] 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
[0009] The present invention provides an isolated Breast Cancer
Related Protein, which is specifically expressed in breast cancer
cells.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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
cancer cell.
[0014] 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.
[0015] According to another aspect of the present invention, there
is provided a polynucleotide encoding the Breast Cancer Related
Protein.
[0016] 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 thereof.
[0017] 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
[0018] 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:
[0019] FIG. 1 illustrates the results of northern blotting of
various normal cells using a breast cancer related protein (BCRP)
gene specific probe;
[0020] FIG. 2 illustrates the results of northern blotting of
various cancerous cells using a BCRP gene specific probe;
[0021] 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;
[0022] 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
reaction (RT-PCR);
[0023] 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);
[0024] 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;
[0025] 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;
[0026] 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;
[0027] 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
[0028] 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
[0029] 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.
[0030] 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").
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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 derivatives.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] "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 (exons).
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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 biology arts.
[0054] 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.
[0055] 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.
[0056] 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
restriction sites.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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 PCR.
[0062] 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.
[0063] 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).
[0064] 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.
[0065] 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.
EXAMPLES
Example 1
Search of BCRP Gene Based on Single Nucleotide Polymorphism (SNP)
Data
[0066] 1. Search of BCRP Gene
[0067] 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.
[0068] 2. Amplification of BCRP Gene Fragment
[0069] 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).
[0070] 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
gene.
[0071] 3. Search of BCRP Full Length Gene Via cDNA Library
[0072] 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.
[0073] 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.
[0074] 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 was obtained.
[0075] 4. Analysis of Base Sequence of the BCRP Full Length Gene
and Prediction of Protein Sequence
[0076] 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.
Example 2
Identification of Expression of BCRP Gene in Cells and Tissues
[0077] 1. Identification of Expression of BCRP Gene in Cells
through Northern Blotting
[0078] 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.
[0079] Specific procedures of the northern blotting were performed
as follows.
[0080] (1) Manufacturing of a Radiolabeled Probe
[0081] 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.
[0082] (2) Prehybridization Using a Hybridization Bottle
[0083] 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.
[0084] (3) Denaturation of the Radiolabeled Probe
[0085] The radiolabeled probe was heated at 95-100.degree. C. for
8-10 minutes, and then quickly placed in ice.
[0086] (4) Hybridization
[0087] 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.
[0088] (5) Washing
[0089] 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.
[0090] (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.
[0091] 2. Identification of Expression of BCRP Gene in Tissues
[0092] 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 breast tissue
[0093] 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.
[0094] 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.
Example 3
Identification of Location of Expression of BCRP Gene in Cells
[0095] 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
above.
[0096] 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.
[0097] Based on these results, it is believed that the BCRP, in
some embodiments, is specifically expressed in the cell
membrane.
Example 4
Effects of the Expression of BCRP Gene on Expression of other Genes
in a Cell Line
[0098] 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 cell line.
[0099] 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
[0100] 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.
Example 5
Effects of Overexpression of the BCRP Gene on the Amplification of
a Cell Line and Apoptosis
[0101] 1. MTT Assay
[0102] 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.
[0103] 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.
[0104] 2. Apoptosis Assay
[0105] 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.
[0106] 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.
Example 6
Effects of Apoptosis in Human Breast Cancer Cells on Expression of
BCRP Gene
[0107] It was conventionally known that when human breast cancer
cells were treated with an anticancer agent Taxol, apoptosis was
induced. In the present Example, to investigate the expression
level of the BCRP gene when apoptosis was induced, an MDA-MB-231
cell line was treated with Taxol to induce apoptosis and then
RT-PCR was performed in the same manner as described above.
[0108] FIG. 14 illustrates changes in the form of cell when the
MDA-MB-231 cell line was treated with Taxol. It can be seen from
panel B of FIG. 14 that apoptosis is induced by Taxol.
[0109] FIG. 15 illustrates the effects of Taxol on the expression
of the BCRP gene, identified through RT-PCR. Referring to FIG. 15,
the expression of the BCRP gene was increased by Taxol.
[0110] As is apparent from the above results, cell proliferation is
inhibited by the BCRP gene according to an embodiment of the
present invention. Further, although apoptosis is not strongly
induced by the BCRP, it is slightly induced compared to controls.
In addition, considering the results of expression of BCRP in the
Taxol treated cell line as an indirect evidence, it is believed
that BCRP is associated with apoptosis. Also, as is demonstrated in
Example 4, expression of the p53 and p21 genes were increased by
the BCRP gene. Consequently, it is believed that overexpression of
the BCRP gene increases the expression level of p53 and the
activation of p53 increases the expression level of p21.
[0111] An isolated protein and a nucleic acid encoding the same
according to embodiments of the present invention can be used for
diagnosing breast cancer and for developing medicaments targeting
the protein.
[0112] A method of diagnosing breast cancer using an antibody
specifically binding to the BCRP according to an embodiment of the
present invention can be used for effectively diagnosing breast
cancer.
[0113] A method of diagnosing breast cancer by measuring the
expression level of the BCRP gene in cells according to an
embodiment of the present invention can be used for effectively
diagnosing breast cancer.
[0114] A microarray according to an embodiment of the present
invention can be used in various analysis methods such as an
analysis for detecting the presence of breast cancer.
[0115] While the present invention has been particularly shown and
described with reference to exemplary embodiments thereof, it will
be understood by those of ordinary skill in the art that various
changes in form and details may be made therein without departing
from the spirit and scope of the present invention as defined by
the following claims.
Sequence CWU 1
1
20 1 20 DNA Artificial Sequence forward primer 1 acggacgagg
gtgacaatag 20 2 20 DNA Artificial Sequence reverse primer 2
aggtaaaaga agggcatggg 20 3 672 DNA Homo sapiens 3 atgaggctcc
aaagaccccg acaggccccg gcgggtggga ggcgcgcgcc ccggggcggg 60
cggggctccc cctaccggcc agacccgggg agaggcgcgc ggaggctgcg aaggttccag
120 aagggcgggg agggggcgcc gcgcgctgac cctccctggg caccgctggg
gacgatggcg 180 ctgctcgcct tgctgctggt cgtggcccta ccgcgggtgt
ggacagacgc caacctgact 240 gcgagacaac gagatccaga ggactcccag
cgaacggacg agggtgacaa tagagtgtgg 300 tgtcatgttt gtgagagaga
aaacactttc gagtgccaga acccaaggag gtgcaaatgg 360 acagagccat
actgcgttat agcggccgtg aaaatatttc cacgtttttt catggttgcg 420
aagcagtgct ccgctggttg tgcagcgatg gagagaccca agccagagga gaagcggttt
480 ctcctggaag agcccatgcc cttcttttac ctcaagtgtt gtaaaattcg
ctactgcaat 540 ttagaggggc cacctatcaa ctcatcagtg ttcaaagaat
atgctgggag catgggtgag 600 agctgtggtg ggctgtggct ggccatcctc
ctgctgctgg cctccattgc agccggcctc 660 agcctgtctt ga 672 4 223 PRT
Homo sapiens 4 Met Arg Leu Gln Arg Pro Arg Gln Ala Pro Ala Gly Gly
Arg Arg Ala 1 5 10 15 Pro Arg Gly Gly Arg Gly Ser Pro Tyr Arg Pro
Asp Pro Gly Arg Gly 20 25 30 Ala Arg Arg Leu Arg Arg Phe Gln Lys
Gly Gly Glu Gly Ala Pro Arg 35 40 45 Ala Asp Pro Pro Trp Ala Pro
Leu Gly Thr Met Ala Leu Leu Ala Leu 50 55 60 Leu Leu Val Val Ala
Leu Pro Arg Val Trp Thr Asp Ala Asn Leu Thr 65 70 75 80 Ala Arg Gln
Arg Asp Pro Glu Asp Ser Gln Arg Thr Asp Glu Gly Asp 85 90 95 Asn
Arg Val Trp Cys His Val Cys Glu Arg Glu Asn Thr Phe Glu Cys 100 105
110 Gln Asn Pro Arg Arg Cys Lys Trp Thr Glu Pro Tyr Cys Val Ile Ala
115 120 125 Ala Val Lys Ile Phe Pro Arg Phe Phe Met Val Ala Lys Gln
Cys Ser 130 135 140 Ala Gly Cys Ala Ala Met Glu Arg Pro Lys Pro Glu
Glu Lys Arg Phe 145 150 155 160 Leu Leu Glu Glu Pro Met Pro Phe Phe
Tyr Leu Lys Cys Cys Lys Ile 165 170 175 Arg Tyr Cys Asn Leu Glu Gly
Pro Pro Ile Asn Ser Ser Val Phe Lys 180 185 190 Glu Tyr Ala Gly Ser
Met Gly Glu Ser Cys Gly Gly Leu Trp Leu Ala 195 200 205 Ile Leu Leu
Leu Leu Ala Ser Ile Ala Ala Gly Leu Ser Leu Ser 210 215 220 5 19
DNA Artificial Sequence forward primer of BCRP amplification 5
cggacgaggg tgacaatag 19 6 20 DNA Artificial Sequence reverse primer
for BCRP amplification 6 aggtaaaaga agggcatggg 20 7 26 DNA
Artificial Sequence forward primer for beta-actin amplification 7
aggactttga ttgcacattg ttgttt 26 8 26 DNA Artificial Sequence
reverse primer for beta actin amplification 8 gagaccaaaa gccttcatac
atctca 26 9 20 DNA Artificial Sequence forward primer for p53
amplification 9 atttgcgtgt ggagtatttg 20 10 20 DNA Artificial
Sequence reverse primer for p53 amplification 10 ggaacaagaa
gtggagaatg 20 11 22 DNA Artificial Sequence forward primer for p21
amplification 11 gtgagcgatg gaacttcgac tt 22 12 22 DNA Artificial
Sequence reverse primer for p21 amplification 12 ggcgtttgga
gtggtagaaa tc 22 13 25 DNA Artificial Sequence forward primer for
CytC amplification 13 tttggatcca atgggtgatg ttgag 25 14 30 DNA
Artificial Sequence reverse primer for CytC amplification 14
tttgaattcc tcattagtag cttttttgag 30 15 19 DNA Artificial Sequence
forward primer for caspase 5 amplification 15 ctgacattga aggaagagg
19 16 19 DNA Artificial Sequence reverse primer for caspase 5
amplification 16 gccaggtgat caaactttg 19 17 20 DNA Artificial
Sequence forward primer for caspase 3 amplification 17 tggaattgat
gcgtgatgtt 20 18 20 DNA Artificial Sequence reverse primer for
caspase 3 amplification 18 ggcaggcctg aataatgaaa 20 19 20 DNA
Artificial Sequence forward primer for Apaf 1 amplification 19
gggtttcagt tgggaaacaa 20 20 20 DNA Artificial Sequence reverse
primer for Apaf 1 amplification 20 cacccaagag tcccaaacat 20
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