Cell-based assay for determining activity in the retinoblastoma pathway

1. A method of determining the activity of a Rb variant in a cell, comprising the steps of:

a. providing a mammalian cell, said cell comprising a stably integrated first construct, wherein said first construct comprises a first cDNA encoding E2F1 protein which is linked to a first portion of a luciferase gene, and a second cDNA encoding Dp-1 protein;

b. preparing a linear third cDNA of a Rb variant, wherein said Rb variant contains at least one mutation as compared to wild-type Rb, and said third cDNA is linked to a second portion of a luciferase gene to form a second construct, wherein when said first construct is in close proximity with said second construct a complex is formed that emits light;

c. transfecting said second construct into said mammalian cell; and

d. determining the activity of said Rb variant by measuring any generated light emission, wherein an increase in light emission relative to a control mammalian cell is indicative of said Rb variant activity.

2. A method of determining the activity of a p16 variant in a cell comprising the steps of:

a. providing a mammalian cell, said cell having a knockout of endogenous CDK4 and endogenous CDK6, and a first construct stably integrated into said mammalian cell comprising a first cDNA encoding E2F1 protein linked to Dp-1 protein and a first portion of a luciferase gene at its C-terminus;

b. preparing a second construct comprising a second cDNA encoding Rb protein which is linked to a second portion of a luciferase gene at its N-terminus, wherein when said first construct is in close proximity with said second construct a complex is formed that emits light;

c. preparing a third cDNA encoding Cyclin D1 protein and a fourth cDNA encoding CDK4 protein or CDK6 protein;

d. preparing a fifth cDNA of a CDKN2A variant, said CDKN2A variant having at least one mutation as compared to wild-type CDKN2A;

e. preparing a transfection complex, said transfection complex comprising said second construct, said third cDNA, said fourth cDNA, and said fifth cDNA;

f. transfecting said transfection complex into said mammalian cell, wherein said transfection is transient; and

g. determining the activity of said p16 variant by measuring any generated light emission, wherein an increase in light emission relative to a mammalian cell control is indicative of said p16 variant activity.

3. A method of determining the sensitivity of a CDK4 variant or a CDK6 variant to p16 in a cell, comprising the steps of:

a. providing a mammalian cell, said cell having a knockout of endogenous CDK4 and endogenous CDK6, and a first construct stably integrated into said cell, said first construct comprising a first cDNA encoding E2F1 protein linked to Dp-1 protein, wherein said Dp-1 protein is linked to a first portion of a luciferase gene at its C-terminus;

b. preparing a second cDNA of a CDK4 or CDK6 variant from a patient, said CDK4 variant or CDK6 variant containing at least one mutation as compared to wild-type CDK4 or wild-type CDK6;

c. preparing a second construct comprising a third cDNA encoding Rb protein which is linked to a second portion of a luciferase gene at its N-terminus, wherein when said first construct is in close proximity with said second construct a complex is formed that emits light;

d. preparing a fourth cDNA encoding Cyclin D1 protein;

e. preparing a fifth cDNA of wild-type CDKN2A;

f. preparing a transfection complex comprising said second construct, said third cDNA, said fourth cDNA, and said fifth cDNA;

g. transfecting said transfection complex into said mammalian cell, wherein said transfection is transient; and

h. determining whether said CDK4 or CDK6 variant is sensitive to p16 by measuring any generated light emission, wherein an increase in light emission relative to a mammalian cell control is indicative of said CDK4 or CDK6 variant being sensitive to p16.