Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-03-26
2002-03-26
Caputa, Anthony C. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S018700, C536S023100, C536S023500, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06362321
ABSTRACT:
BACKGROUND OF THE INVENTION
Acquired drug resistance to currently available chemotherapeutic drugs is a major cause of failure of cancer treatment (Gottesman et al., 1993, Annu Rev. Biochem 62:385-427; Van Der Zee et al., 1995, Gynecologic Oncol. 58:165-178; Casazza et al., 1996, Cancer Treat. Res. 87:1-171). Many of the most common carcinomas, including breast and ovarian cancer, are initially relatively sensitive to a wide variety chemotherapy agents. However, acquired drug resistance phenotype typically occurs after months or years of exposure to chemotherapy. Determining the molecular basis of drug resistance may offer opportunities for improved diagnostic and therapeutic strategies.
Taxol is a natural product derived from the bark of
Taxus brevafolio
(Pacific yew). Taxol inhibits microtubule depolymerization during mitosis and results in subsequent cell death. Taxol displays a broad spectrum of tumorcidal activity including against breast, ovary and lung cancer (McGuire et al., 1996
, N. Engld. J. Med
. 334:1-6; and Johnson et al., 1996
, J. Clin. Ocol
. 14:2054-2060). While taxol is often effective in treatment of these malignancies, it is usually not curative because of eventual development of taxol resistance. Cellular resistance to taxol may include mechanisms such as enhanced expression of P-glycoprotein and alterations in tubulin structure through gene mutations in the &bgr; chain or changes in the ratio of tubulin isomers within the polymerized microtubule (Wahl et al., 1996
, Nature Medicine
2:72-79; Horwitz et al., 1993
, Natl. Cancer Inst
. 15:55-61; Haber et al., 1995
, J. Biol. Chem
. 270:31269-31275; and Giannakakou et al., 1997
, J. Biol. Chem
. 272:17118-17125). Some tumors acquires taxol resistance through unknown mechanisms.
SUMMARY OF THE INVENTION
A gene that is overexpressed in taxol-resistant cancer cell lines has now been discovered and characterized. The gene is designated Taxol Resistance Associated Gene-3 (“TRAG-3”). At least two alternatively spliced forms of TRAG-3, referred to herein as TRAG-3&agr; and TRAG-3&bgr;, exist. The TRAG-3&agr; cDNA sequence shown in
FIG. 2
encodes a 110 amino acid protein. The TRAG-3&bgr; cDNA shown in
FIG. 3
encodes a 127 amino acid protein (FIG.
4
). TRAG-3 is expressed predominantly in chemotherapy resistant cell lines and the majority of melanoma cell lines and melanoma malignant tissues. It is minimally, if at all, expressed in normal cells and tissues. As used herein, the term TRAG-3 refers to any and all TRAG-3 gene products. The TRAG-3 gene product, i.e., the TRAG-3 polypeptide, modulates expression of several genes involved in taxol resistance displayed by mammalian cells.
Based on the discoveries, the invention features an isolated nucleic acid containing a nucleotide sequence that encodes a polypeptide that shares at least 80% sequence identity with SEQ ID NO:2 or SEQ ID NO:4. Preferably, it shares at least 90% sequence identity with SEQ ID NO:2 or SEQ ID NO:4, and more preferably, it shares at least 95% sequence identity with SEQ ID NO:2 or SEQ ID NO:4. Alternatively, the nucleotide sequence defines a DNA molecule whose complement hybridizes under stringent hybridization conditions to a DNA molecule having a sequence consisting of SEQ ID NO:1 or SEQ ID NO:3.
The invention also includes an isolated nucleic acid (antisense) that hybridizes under stringent hybridizaton conditions to a nucleic acid whose nucleotide sequence is the complement of SEQ ID NO:1 or SEQ ID NO:3.
The invention also includes an isolated nucleic acid containing a nucleotide sequence that encodes: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:2 with one or more conservative amino acid substitutions; or SEQ ID NO:4 with one or more conservative amino acid substitutions. In some embodiments, the nucleotide sequence is SEQ ID NO:1 or SEQ ID NO:3.
Preferably, the polypeptide modulates expression of at least one gene involved in taxol resistance displayed by a mammalian cell. Examples of genes involved in taxol resistance include: annexin I, interleukin 6 (IL-6), interleukin 8 (IL-8), macrophage inflammatory protein 2&agr;, and natural killer cell enhancing factor B (NKEFB).
The invention also includes a vector containing an above-described nucleic acid. In the vector, the nucleic acid can be operably linked to at least one expression control sequence. The invention also includes a cell comprising the vector.
The invention also includes a substantially pure polypeptide that has an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO:2 or SEQ ID NO:4. The invention also includes a substantially pure polypeptide whose amino acid sequence is: SEQ ID NO:2; SEQ ID NO:4, SEQ ID NO:2 with one or more conservative amino acid substitutions; or SEQ ID NO:2 with one or more conservative amino acid substitutions. Preferably, the polypeptide modulates expression of at least one gene involved in taxol resistance displayed by a mammalian cell, e.g., annexin I, IL-6, IL-8, macrophage inflammatory protein 2&agr;, and NKEFB.
The invention also includes an antibody that binds to a TRAG-3 polypeptide. Preferably, the antibody binds specifically to a TRAG-3 polypeptide, or a biologically active portion thereof. The antibody can be a monoclonal, polyclonal, or engineered antibody. The antibody is useful in detecting a TRAG-3 polypeptide, e.g., in a biological sample, or to alter the activity of TRAG-3.
The invention also includes a method for modulating expression of annexin I, IL-6, IL-8, macrophage inflammatory protein 2&agr;, or NKEFB, in a mammalian cell. The method includes introducing into the cell a vector containing a TRAG-3 coding sequence operably linked to expression control sequences.
The invention also includes a screening method for identifying a compound that inhibits expression of a TRAG-3 gene in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which express endogenous TRAG-3, (b) contacting the treatment test cell with a candidate compound, and (c) detecting a decrease in the level of TRAG-3 gene expression in the treatment test cell, compared to the level of TRAG-3 gene expression in the control test cell. The test cell can be a taxol-resistant mammalian cancer cell that overexpresses endogenous TRAG-3 as compared to a non-taxol-resistant cancer cell from a parental cell line. The decrease in the expression level of TRAG-3 gene expression can be detected through measurement of TRAG-3 RNA level in the cell, or through measurement of TRAG-3 polypeptide level in the cell. TRAG-3 polypeptide level can be measured by means of a TRAG-3 antibody.
The invention also includes a screening method for identifying a compound that inhibits the biological activity of a TRAG-3 polypeptide in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which: (1) contain a TRAG-3 coding sequence operably linked to expression control sequences, and (2) express an endogenous gene whose expression is upregulated by a TRAG-3 polypeptide; (b) contacting the treatment test cell with a candidate compound; and (c) detecting a decrease in the expression level of the endogenous gene in the treatment test cell, compared to the expression level of the endogenous gene in the control test cell. The level of TRAG-3 -modulated gene expression can be measured according to TRAG-3 RNA level or TRAG-3 polypeptide level. The endogenous gene whose expression is upregulated by a TRAG-3 polypeptide can be an annexin I gene, IL-6 gene, IL-8 gene, or macrophage inflammatory protein 2&agr; gene.
The invention also includes a screening method for identifying a compound that inhibits the biological activity of a TRAG-3 polypeptide in a cell. The method includes: (a) providing a treatment test cell and a control test cell, both of which contain: (1) a TRAG-3 coding sequence operably linked to expression control sequences, and (2) a reporter gene operably linked to a positive expression control sequence naturally associated with an endogenous gene whose expression is upregulated by a TRA
Duan Zhenfeng
Feller Aynn
Seiden Michael V.
Caputa Anthony C.
Fish & Richardson P.C.
Harris Alana M.
The General Hospital Corporation
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