Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-02-05
2001-11-06
Ungar, Susan (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S252300, C435S069100
Reexamination Certificate
active
06313277
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the family of proteins known as multidrug resistance proteins. These proteins are xenobiotic transporters which confer resistance to cancer chemotherapeutic drugs. The invention describes a new protein member of this family called Breast Cancer Resistance Protein (BCRP) and the DNA which encodes it.
BACKGROUND OF THE INVENTION
The development of resistance to multiple chemotherapeutic drugs frequently occurs during the treatment of cancer. Two transmembrane xenobiotic transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance protein (NWP) are capable of causing multidrug resistance when transfected into drug-sensitive cells in culture (1,2). Despite this, the role that these transporters play in clinical drug resistance exhibited by human cancers is unclear, and alternate or additional drug resistance mechanisms operative in this disease have been sought.
To address this problem, Chen et. al. (3) selected human breast carcinoma MCF-7 cells for resistance to the anthracycline doxorubicin in the presence of verapamil, an inhibitor of Pgp. The resultant multidrug resistant subline, MCF-7/AdrVp, exhibits marked cross-resistance to other anthracyclines (daunorubicin [DNR], 3′-deamino-3′[3-cyano-4-morpholinyl] doxorubicin, but not idarubicin), and to the anthracenedione mitoxantrone, but remains sensitive to vinca alkaloids, paclitaxel (3,4) and cisplatin. MCF-7/AdrVp cells do not overexpress Pgp or MRP, despite displaying a marked reduction in the intracellular accumulation of the anthracycline daunorubicin and the fluorescent dye rhodamine 123 compared to MCF-7 cells (4,5). MCF-7/AdrVp cells do not display an alteration in the subcellular distribution of drug (4) such as that seen in certain cells that overexpress MRP. Although the decreased accumulation of daunorubicin in MCF-7/AdrVp cells is not reversed by the classical P-glycoprotein antagonist cyclosporin A, depletion of ATP results in complete abrogation of the abnormal efflux of both daunorubicin and rhodamine (4).
The need in the art to elucidate the mechanism of drug resistance is continually present, as chemotherapy remains the primary method for non-invasively treating many types of cancers. There is also a need in the art to counteract the mechanism of drug resistance so to provide a longer and more effective course of chemotherapeutic drug treatment for cancer patients.
SUMMARY OF THE INVENTION
The discovery described in the instant invention fulfills the above needs. The discovery of the BCRP and its corresponding gene greatly advance the knowledge in the art of the drug resistance mechanism by providing a novel xenobiotic transporter which is overexpressed in a variety of drug-resistant human cancer cell lines, and confers resistance to many chemotherapeutic agents.
BCRP is an about 655 amino acid protein and is encoded by a gene which has about 2418 nucleotide cDNA. The protein demonstrates activity and has a sequence homology which places it in the ATP-binding cassette (ABC) superfamily of transporter proteins. The molecular mass is approximately 72.3 kilodaltons (kD) exclusive of any glycoylation. Expression of BCRP in drug-sensitive human cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, and reduces daunorubicin accumulation in the cloned transfected cells.
It is an object of the present invention to provide a mammalian protein that is a multi-drug resistant (MDR) protein and a xenobiotic transporter, and is called Breast Cancer Resistance Protein (BCRP).
It is also an object of the present invention is to provide the gene and/or cDNA which encodes said mammalian MDR protein.
It is another object of the invention to provide antisense fragments of the BCRP gene which inhibit the expression of the BCRP in vivo.
Yet another object of the present invention is to provide a method of using probes derived from the BCRP gene as a diagnostic tool to quantify gene expression or gene amplification in specimens taken from patients with cancer.
It is another object of the invention to provide antibodies to the BCRP.
It is yet another object of the invention to provide a method of reversing the drug resistance of the cancer cells by administering BCRP antibodies.
It is yet another object of the invention to provide a method of reversing the drug resistance of the cancer cells by administering Fumitremorgin C.
It is another object of the invention to provide a method of enhancing a patient's chemotherapy treatment for breast cancer by administering antibodies to the patient to inhibit the resistance-activity of BCRP.
These and other objects of the present invention, which will be apparent from the detailed description of the invention provided hereinafter, have been met, in one embodiment, by substantially pure BCRP and the gene encoding BCRP.
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A multidrug resistance transporter form human MCF-7 breast cancer cells, L. Austin Doyle, Weidong Yang, Lynne V. Abruzzo, Tammy Krogmann, Yongming Gao, Arun K. Rishi, and Douglas D. Ross. Proc. Natl. Acad. Sci. USA, vol. 95, pp. 15665-15670, Dec. 1998, Medical Sciences.
A Human Placenta-Specific ATP-Binding Cassette Gene (ABCP) on Chromosome 4q22 That Is Involved in Multidrug Resistance. Rando Allikmets, Lynn M. Schriml, Amy Hutchinson, Vincenzo Ramano-Spica, and Michael Dean, Cancer 5337-5339, Dec. 1, 1998.
Molecular Cloning of cDNAs Which Are Highly Overexpressed in Mitoxantrone resistant Cells: Demonstration of Homology to ABC Transport Genes. Keisuke Miyake, Lyn Mickley, Thomas Litman, Zhirong Zhan, Robert Robey, Barbara Cristensen, Marifiorella Brangi, Lee Green Berger, Michael Dean, Tito Fojo, and Susan E. Bates, Cancer Research 59, 8-13, Jan. 1, 1999.
A Human Canalicular Multispecific Organic Anion Transporter (cMOAT) Gene Is Overexpressed in Cisplantin-resistant Human Cancer Cell Lines with Decreased Drug Accumulation. Ken Taniguchi, Morimase Wada, Kimitoshi Kohno, Takanori Nakamura, Takeshi Kawabe, Mina Kawakami, Kazuhiro Kagotani, Katsuzumi Okumura, Shin-ichi Akiyama, and Michihiko Kuano, Cancer Research 56 4124-4129, Sep. 15, 1996.
Cloning of the cDNA for a Human Homoloromosome 21q22.3gue of the Drosophila White Gene and Mapping to Chromosome 21q22.3. Haiming Chen, Colette Rossier, Maria D. Lalioti, Audrey, Lynn, Aravinda Chakravarti, Caelle Perrin, and Stylianos E. Antonarakis, Am. J. Genet. 59:66-75, 1996.
Atypical Multidrug Resistance: Breast Cancer Resistance Protein Messenger RNA Expression in Mitaxantrone-Selected Cell Lines. Douglas D. Ross, Weidong Yang, Lynne V. Abruzzo, William S. Dalton, Erasmus Schneider, Hermann Lage, Munfred Dietel, Lee Greenberger, Susan P.C. Cole, L. Austin Doyle. JNCI, vol. 91, No. 5, Jan. 28, 1999, pp. 1-11.
Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the Expressed Sequence Tags database. Rando Allikmets, Bernard Gerrard, Amy Hutchinson and Michael Dean, Human Molecular Genetics, 1996, vol. 5, No. 1, pp. 1649-1655.
Overexpression of a Transporter Gene in a Multidrug-Resistant Human Lung Cancer Cell Line. S.P.C. Cole, G. Bhardwaj, J.H. Gerlach, J.E. Mackie, C.E. Grant, K.C. Almquist, A.J. Stewart, E.U. Kurz, A.M.V. Duncan, R.G. Deeley, vol. 258, Dec. 4, 1992, pp. 1650-1654.
Reduced Intracellular Drug Accumulation in the Absence of P-Glycor
Abruzzo Lynne V.
Doyle L. Austin
Ross Douglas D.
Morgan, Lewis & Bockius, L.L.P.
Ungar Susan
University of Maryland Baltimore
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