MPR-related ABC transporter encoding nucleic acids and...

Details MPR-related ABC transporter encoding nucleic acids and... MPR-related ABC transporter encoding nucleic acids and...

2000-09-27

2004-10-12

Chen, Shin-Lin (Department: 1632)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S320100, C435S325000, C435S455000, C536S023100, C536S023500, C424S093210

Reexamination Certificate

active

06803184

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the fields of medicine and molecular biology. More specifically, the invention provides nucleic acid molecules and proteins encoded thereby which are involved in the development of resistance to pharmacological and chemotherapeutic agents in tumor cells.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
P-glycoprotein, the product of the MDR1 gene, was the first ABC transporter shown to confer resistance to cytotoxic agents. Pgp functions as an ATP-dependent efflux pump that reduces the intracellular concentration of a variety of chemotherapeutic agents by transporting them across the plasma membrane (1). The multidrug resistance phenotype associated with overexpression of Pgp is of considerable clinical interest because natural product drugs are second only to alkylating agents in clinical utility, and many effective chemotherapeutic regimens contain more than one natural product agent. More recently, we and others have reported transfection studies indicating that MRP, another ABC family transporter, confers a multidrug resistance phenotype that includes many natural product drugs, but is distinct from the resistance phenotype associated with Pgp (2-6). MRP shares only limited amino acid identity with Pgp, and this is reflected in the different substrate specificities of the two transporters. In contrast to Pgp, MRP can transport a wide range of anionic organic conjugates, including glutathione S-conjugates (7). In addition to Pgp and MRP there may be other transporters that are involved in cytotoxic drug resistance. In the case of natural product drugs, resistant cell lines have been described that display a multidrug resistant phenotype associated with a drug accumulation deficit, but do not overexpress Pgp or MRP (8). ABC transporters have also been linked to cisplatin resistance, and several lines of evidence suggest the possibility that pumps specific for organic anions may be involved: 1) decreased cisplatin accumulation is consistently observed in cisplatin resistant cell lines (9); 2) cisplatin is conjugated to glutathione in the cell, and this anionic conjugate is toxic in an in vitro biochemical assay (10); and 3) biochemical studies using membrane vesicle preparations have shown that cisplatin resistant cells lines have enhanced expression of an ATP-dependent transporter of CDDP-glutathione and other glutathione S-conjugates such as the cystinyl leukotriene LTC, (11, 12). These data thus suggest that an organic anion transporter may contribute to cisplatin resistance by exporting CDDP-glutathione. While MRP is an organic anion transporter, the reported drug resistance profile of MRP-transfected cells does not extend to this agent (5, 6), and to date only one cisplatin-resistant cell line has been reported to overexpress MRP (13). This suggests that organic anion transporters other than MRP may contribute to cisplatin resistance. Consistent with this possibility, the canalicular multispecific organic anion transporter, cMOAT, an MRP-related transporter that functions as the major organic anion transporter in liver, has been reported to be overexpressed in cisplatin resistant cell lines (14, 15). A more direct link between cMOAT and cytotoxic drug resistance is suggested by a recent report in which transfection of a cMOAT antisense construct into a liver cancer cell line resulted in sensitization to cisplatin, daunorubicin and other cytotoxic agents (16).
Clearly, a need exists for identifying the essential components and mechanisms giving rise to drug resistance and the transport of anticancer agents out of the tumor cell. The elucidation of these mechanisms may be used to advantage for the design of efficacious chemotherapeutic agents.
SUMMARY OF THE INVENTION
This invention provides novel, biological molecules useful for identification, detection, and/or molecular characterization of components involved in the acquisition of drug resistance in tumor cells. According to one aspect of the invention, an isolated nucleic acid molecule is provided which includes a sequence encoding a protein transporter of a size between about 1300 and 1350 amino acids in length. The encoded protein, referred to herein as MOAT-B, comprises a multi-domain structure including a tandem repeat of nucleotide binding folds appended C-terminal to a hydrophobic domain that contains several potential membrane spanning helices. Conserved Walker A and B ATP binding sites are present in each of the nucleotide binding folds.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human MOAT-B protein. In a particularly preferred embodiment, the human MOAT-B protein has an amino acid sequence the same as Sequence I.D. No. 2. An exemplary MOAT-B nucleic acid molecule of the invention comprises Sequence I.D. No. 1.
According to another aspect of the invention, a second isolated nucleic acid molecule is provided which includes a sequence encoding a transporter between about 1400 and 1450 amino acids. The encoded protein, referred to herein as MOAT-C contains a multi-domain structure including a tandem repeat of nucleotide binding folds appended C-terminal to a hydrophobic domain that contains several potential membrane spanning helices. Conserved Walker A and B ATP binding sites are present in each of the nucleotide binding folds. While similar in structure to MOAT-B described above, MOAT-C contains distinct sequence differences.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human MOAT-C protein. In a particularly preferred embodiment, the human MOAT-C protein has an amino acid sequence the same as Sequence I.D. No. 4. An exemplary MOAT-C nucleic acid molecule of the invention comprises Sequence I.D. No. 3.
According to yet another aspect of the invention, an isolated nucleic acid molecule is provided which includes a sequence encoding a protein of a size between about 1500 and 1550 amino acids in length. The encoded protein, referred to herein as MOAT-D, contains a multidomain structure including an N-terminal hydrophobic extension which harbors five transmembrane spanning helices.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a MOAT-D protein. In a particularly preferred embodiment, the human MOAT-D protein has an amino acid sequence the same as Sequence I.D. No. 6. An exemplary MOAT-D nucleic acid molecule of the invention comprises Sequence I.D. No. 5.
According to yet another aspect of the invention, an isolated nucleic acid molecule is provided which includes a sequence encoding a protein of a size between about 1480 and 1530 amino acids in length. The encoded protein, referred to herein as MOAT-E, contains a multidomain structure including an N-terminal hydrophobic extension which harbors several transmembrane spanning helices. While similar in structure to MOAT-D described above, MOAT-E contains distinct sequence differences.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a MOAT-E protein. In a particularly preferred embodiment, the human MOAT-E protein has an amino acid sequence the same as Sequence I.D. No. 8. An exemplary MOAT-E nucleic acid molecule of the invention comprises Sequence I.D. No. 7.
According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) Sequence I.D. No. 1; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 1 comprising nucleic acids encoding amino acids 1-1154 of Sequence ID No. 2; (3) a sequence encoding preselected portions of Sequence I.D. N

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