Chk1 and uses thereof

Details Chk1 and uses thereof Chk1 and uses thereof

1999-06-30

2004-04-20

Harris, Alana M. (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C424S009100, C424S009200, C435S325000, C435S006120, C435S007210, C435S007230, C436S063000, C436S064000, C530S300000, C530S350000, C530S333000, C530S333000, C530S333000, C530S333000, C530S333000

Reexamination Certificate

active

06723498

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to chemotherapy and drug resistance.
Cancer chemotherapy commonly involves the administration of one or more cytotoxic or cytostatic drugs to a patient. The goal of chemotherapy is to eradicate a substantially clonal population (tumor) of transformed cells from the body of the individual, or to suppress or to attenuate growth of the tumor. Tumors may occur in solid or liquid form, the latter comprising a cell suspension in blood or other body fluid. A secondary goal of chemotherapy is stabilization (clinical management) of the afflicted individual's health status. Although the tumor may initially respond to chemotherapy, in many instances the initial chemotherapeutic treatment regimen becomes less effective or ceases to impede tumor growth. The selection pressure induced by chemotherapy promotes the development of phenotypic changes that allow tumor cells to resist the cytotoxic effects of a chemotherapeutic drug. Often, exposure to one drug induces resistance to that drug as well as other drugs to which the cells have not been exposed.
Cell cycle checkpoints are regulatory systems that control the order and timing of certain events in the cell cycle. These checkpoints are important for ensuring that cells divide properly. For example, DNA damage leads to activation of a cell cycle checkpoint regulatory system that arrests the cell cycle and activates genes involved in repair of DNA damage. This system prevents progression of the cell cycle until the DNA damage has been repaired.
Chk1, a kinase, is thought to be involved in the DNA damage cell cycle checkpoint. Chk1 is thought to participate in the phosphorylation of Cdc25 in response to DNA damage. Phosphorylation of Cdc25 prevents activation of the Cdc2-cyclin B complex thereby blocking mitotic entry.
SUMMARY OF THE INVENTION
The present invention concerns checkpoint kinase 1 (Chk1; Genbank Accession No. AF016582; Sanchez et al. (1997)
Science
277:1497). Applicants have found that expression of Chk1 is up regulated in certain vinblastin resistant cancer cell lines and in certain adromycin resistant cancer cell lines. Applicants have also found that a ribozyme designed to decrease Chk1 expression can increase drug sensitivity.
Chk1 nucleic acids and polypeptides are useful in diagnostic methods related to identification of drug resistant cells (e.g., cancer cells). Chk1 nucleic acids and polypeptides are also useful in screening methods directed to the identification of compounds that can modulated (increase or decrease) the drug resistance of a particular cell type or multiple cell types.
The invention includes a method for detecting the presence of a Chk1 polypeptide in a sample, e.g., a biological sample. This method features the steps of contacting the sample with a compound which selectively binds to the polypeptide and then determining whether the compound binds to a polypeptide in the sample. In some cases, the compound which binds to the polypeptide is an antibody.
The invention also features methods for detecting the presence of a Chk1 nucleic acid molecule in a sample. This method includes the steps of contacting the sample with a nucleic acid probe or primer which selectively hybridizes to a Chk1 nucleic acid molecule (e.g., an mRNA encoding Chk1); and then determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
Also within the invention are kits that include a compound which selectively binds to a Chk1 polypeptide or nucleic acid and instructions for use. Such kits can be used to determine whether cells within a biological sample, e.g., a sample of patient cells, are drug resistant.
The invention features methods for identifying a compound which binds to a Chk1 polypeptide. These methods include the steps of contacting a Chk1 polypeptide with a test compound and then determining whether the polypeptide binds to the test compound. In various embodiments of these methods, the binding of the test compound to the Chk1 polypeptide is detected using an assay which measures binding of the test compound to the polypeptide or using a competition binding assay.
The invention also includes a method for modulating the activity of a Chk1 polypeptide. This method includes the steps of contacting the polypeptide or a cell expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
In another aspect, the invention provides a method for identifying a compound that modulates the activity of a Chk1 polypeptide (e.g., a Chk1 protein). In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide (e.g., alter the ability of Chk1 to phosphorylated Cdc25). One such method includes the steps of contacting the polypeptide with a test compound and then determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
The invention also features methods for identifying a compound which modulates the expression of a Chk1 nucleic acid or a Chk1 polypeptide by measuring the expression of the nucleic acid or polypeptide in the presence and absence of a compound.
Other aspects of the invention are methods and compositions relating to drug resistance. A “drug-resistant phenotype” refers to a cellular phenotype which is associated with increased survival (compared to a less drug-resistant cell) after exposure to a particular dose of a drug, e.g., a chemotherapeutic drug, compared to a cell that does not have this phenotype. A “drug-resistant cell” refers to a cell that exhibits this phenotype. Drug resistance can be characterized by lower intracellular concentration of a drug compared to a non-resistant cell or a less resistant cell as well as altered ability of a drug to affect its target compared to a non-resistant cell or a less resistant cell. Drug resistance is described in detail by Hochhauser and Harris ((1991)
Brit. Med. Bull
. 47:178-96); Simon and Schindler ((1994)
Proc. Nat'l Acad Sci USA
91: 3497-504); and Harris and Hochhauser ((1992)
Acta Oncologica
31:205-213); Scotto et al. ((1986)
Science
232: 751-55). Multi-drug resistance can be associated with, for example, altered composition of plasma membrane phospholipids; increased drug binding and intracellular accumulation; altered expression or activity of plasma membrane or endomembrane channels, transporters or translocators; altered rates of endocytosis and associated alteration in targeting of endosomes; altered exocytosis; altered intracellular ionic environments; altered expression or activity of proteins involved in drug detoxification; and altered expression or activity of proteins involved in DNA repair or replication.
Also within the invention is a method of determining whether a cell has a drug-resistant phenotype by measuring the expression (or activity) of Chk1 in the cell and comparing this expression to that in a control cell. Increased expression (or activity) of Chk1 in the cell compared to the control cell indicates that the cell has a drug-resistant phenotype. In one embodiment of this method, Chk1 expression is determined by measuring Chk1 protein (e.g., measuring Chk1 protein using an antibody directed against Chk1). In another embodiment, Chk1 expression is measured by quantifying mRNA encoding Chk1 or the copy number of the Chk1 gene. In another embodiment Chk1 activity is measured using any assay which can quantify a biological activity of Chk1.
The invention also includes a method for modulating the drug resistance of a cell by modulating Chk1 expression or activity within the cell. Thus, in one embodiment, the drug-resistance of a cell is reduced by contacting the cell with a molecule (e.g., an antisense nucleic acid molecule) that reduces the expression of Chk1 within the cell.
Another aspect of the present invention is a metho

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