Cell-based assay for determining activity in the retinoblastoma pathway
Disclosed are methods of determining activity of CDK4 and CDK6 variants upon exposure to CDK inhibitors, methods for determining activity of a Rb variant, methods for determining the activity of a p16 variant in a cell, and methods for determining the sensitivity of a CDK4 variant or a CDK6 variant to p16 in a cell. Stable cell lines for determining activity of CDK4 variants, CDK6 variants, Rb variants, and p16 variants are also disclosed.
1. A method of determining the effect of a CDK inhibitor on a CDK variant, comprising:
a. preparing a first cDNA of a CDK variant, wherein said CDK variant contains at least one mutation as compared to wild-type CDK;
b. providing a mammalian cell, said cell comprising a knockout of endogenous CDK, and a first construct stably integrated into said mammalian cell, wherein said first construct comprises a second cDNA of SEQ. ID. NO: 215 encoding E2F1 protein which is linked to Dp-1 protein and a first portion of a luciferase gene at its C-terminus;
c. transfecting into said mammalian cell said first cDNA from step a);
d. transfecting into said mammalian cell a third cDNA encoding Cyclin D1 and a second construct, comprising a fourth cDNA of SEQ. ID. NO: 216 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;
e. exposing said transfected cell with a CDK inhibitor; and
f. measuring any generated light emission, wherein an increase in light emission relative to said CDK inhibitor is indicative of said CDK variant being susceptible to treatment with said CDK inhibitor.
2. The method of claim 1 , wherein the CDK variant is selected from the group consisting of CDK4 variants and CDK6 variants.
3. The method of claim 1 , wherein the wild-type CDK is selected from the group consisting of wild-type CDK4 and wild-type CDK6.
4. The method of claim 1 , wherein the first cDNA of said CDK variant is prepared from the DNA of a patient.