Assays and reagents for identifying modulators of cdc25-mediated mitotic activation
OwnerDavid H. Beach, Konstantin Galaktionovinventors
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Abstract
The present invention makes available assays and reagents for identifying agents which can be used to modulate at least one proliferation, differentiation and cell death by apoptosis.
Claims43
An assay for identifying an inhibitor of cdc25-mediated cell cycle progression, comprising
- i. providing a population of test cells comprising a recombinant cdc25 phosphatase gene, which gene is expressible to levels in the cell which cause apoptosis;
- ii. contacting the test cell population with a candidate agent under conditions wherein said recombinant cdc25 phosphatase gene is expressed and causes apoptosis;
- iii. determining the amount of cell death in the test cell population in the presence of the candidate agent;
- and
- iv. comparing the amount of cell death in the presence of the candidate agent to an amount of cell death occurring in the absence of the candidate agent,
- wherein a statistically significant decrease in the amount of cell death in the presence of the candidate agent is indicative of an inhibitor of cdc25-mediated mitotic activation.
An assay for identifying an agent which modulates cdc25-mediated mitotic activation, comprising
- i. providing a population of test cells comprising a recombinant cdc25 phosphatase gene, which gene is expressible to levels in the cell which can cause apoptosis;
- ii. contacting the test cell population with a candidate agent under conditions wherein said recombinant cdc25 phosphatase gene is expressed;
- iii. determining the amount of a diagnostic protein in the test cell population in the presence of the candidate agent, the level of which diagnostic protein is modulated by cdc25-mediated apoptosis;
- and
- iv. comparing the amount of the diagnostic protein in the presence of the candidate agent to an amount occurring in the absence of the candidate agent,
- wherein a statistically significant change in the amount of the diagnostic in the presence of the candidate agent is indicative of a modulator of cdc25-mediated mitotic activation.
An assay for identifying an inhibitor of cdc25-mediated mitotic activation, comprising:
- (i) providing a cell comprising (a) a recombinant cdc25 phosphatase gene, and (b) a reporter gene under transcriptional control of a p53 responsive element;
- (ii) contacting the cell with a candidate agent under conditions wherein said recombinant cdc25 gene is expressed;
- and
- (iii) detecting the level of expression of the reporter gene;
- and
- (iv) comparing the measured level of reporter gene expression in the presence of the candidate agent with a level of expression in the absence of the candidate agent,
- wherein a statistically significant decrease in level of expression of the reporter gene, relative to a level of expression in the absence of the candidate agent, is indicative of cdc25-mediated mitotic activation.
A recombinant cell comprising (i) a recombinant cdc25 phosphatase gene, and (ii) a reporter gene under transcriptional control of a p53 responsive element.
A method of identifying an inhibitor of myc-mediated transcriptional activation, comprising:
- (i) measuring the amount of binding of a myc protein to an isolated nucleic acid which conforms to the consensus binding site CAYGTG;
- (ii) measuring the amount of binding of said myc protein to said nucleic acid in the presence of a test substance;
- and
- (iii) comparing the amount of binding of the myc protein in the presence of said test substance to the amount of binding of the myc protein in the absence of said test substance,
- wherein a statistically significant decrease in the amount of binding indicating an inhibitor of myc-mediated transcriptional activation.
A method of identifying an inhibitor of myc-mediated transcriptional activation, comprising:
- (i) contacting a transfected cell with a test substance, said transfected cell comprising a myc protein and a recombinant reporter gene construct comprising a reporter gene which encodes an assayable product and a sequence which conforms to the myc consensus binding site CAYGTG, wherein said sequence transcriptionally regulates said reporter gene;
- and
- (ii) determining whether the amount of expression of said reporter gene is altered by the test substance.
A nucleic acid construct for use in identifying an inhibitor of myc-mediated transcriptional activation, comprising
- (i) a reporter gene which encodes an assayable product;
- (ii) a myc-responsive transcriptional regulatory sequence which conforms to the myc consensus binding site CAYGTG, which regulatory sequence controls expression of said reporter gene,
- wherein said construct is selected from the group consisting of a recombinant plasmid, a viral vector and an isolated molecule of nucleic acid.
A recombinant cell comprising an expression vector including (i) a reporter gene which encodes an assayable product and (ii) transcriptional regulatory sequence of a cdc25A gene including a myc-responsive transcriptional regulatory sequence, which regulatory sequence are operably linked to and control expression of said reporter gene.
A method for preventing apoptosis of a cell, comprising contacting the cell with an amount of an agent capable of inhibiting myc-induced upregulation of a cdc25 phosphatase.
A method for reducing apoptotic cell death in an animal, comprising administering to the animal an amount of an agent sufficient for inhibiting cdc25-dependent apoptosis, which agent inhibits one of expression of a cdc25 phosphatase, or a phosphatase activity of the cdc25 phosphatase.
A method for determining if a subject is at risk from a disorder characterized by cdc25-mediated apoptosis, comprising
- (i) obtaining a sample of cells from tissue which may undergo apoptosis,
- (ii) determining the level of cdc25 protein or cdc25 mRNA present in cells of the sample,
- (iii) comparing the level of cdc25 protein or cdc25 mRNA present in cells of the sample with the corresponding level in a control sample,
- wherein a statistically significant increase in cdc25 mRNA and/or cdc25 protein in the cells of the sample, relative to the control sample, indicates an increased risk of the tissue undergoing apoptosis.
Dependencies are inferred from the claim text.