E2f reporter melanoma cells

Abstract

A modified melanoma cell line capable of quantification of the effects of MEK inhibitors and CDK4/6 inhibitors in a quantitative, temporal and non-invasive manner both in vitro and in vivo.

What is claimed is: 
     
         1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . A high-throughput in vitro screening method for determining inhibition of E2F comprising:
 a. loading reporter cells comprising an E2F response element-EGFP-firefly luciferase construct onto a screening plate,   b. mixing at least one compound of interest in a test vehicle,   c. administering to at least one cell on said screening plate the compound of interest,   d. administering to at least a second cell, the test vehicle,   e. incubating said cells for a predetermined amount of time,   f. comparing the amount of firefly luciferase activity to the level of actin/tdTomato activity between the compound of interest and of the test vehicle in the incubated cells, and   g. quantifying the level of block for the cells treated with the compound of interest, by normalizing firefly luciferase activity to tdTomato activity/actin levels.   
     
     
         8 . The method of  claim 7  further comprising administering at least two compounds to at least one cell on said screening plate. 
     
     
         9 . The method of  claim 7  further comprising administering at least three compounds to at least one cell on said screening plate. 
     
     
         10 . The method of  claim 7  wherein the cells are incubated for between 12 and 72 hours. 
     
     
         11 . The method of  claim 7  wherein the plate is a 96 well plate. 
     
     
         12 . The method of  claim 7  wherein the plate is a 384 well plate. 
     
     
         13 . The method of  claim 7  wherein the plate is a 1536 well plate. 
     
     
         14 . A kit for testing and determining efficacy of a compound to inhibit E2F activity comprising: (1) E2F response elements-EGFP-Firefly luciferase plasmid/lentivirus, and (2) tdTomato fluorescent protein plasmid/lentivirus, wherein, the firefly luciferase and tdTomato fluorescent protein are transduced into a cancer cell to generate a reporter cell line of interest. 
     
     
         15 . The kit of  claim 14 , wherein a compound of interest is further applied to the reporter cell. 
     
     
         16 . The kit of  claim 14 , wherein at least two compounds of interest are applied to the reporter cell. 
     
     
         17 . The kit of  claim 15 , wherein the reporter cell is incubated after application of the compound of interest. 
     
     
         18 . The kit of  claim 17 , wherein the amount of firefly luciferase activity is compared to the level of tdTomato activity in said reporter cell. 
     
     
         19 . The high-throughput screen of  claim 7  wherein said at least one cell is a melanoma cell harboring a serine/threonine protein kinase B-RAF (BRAF), a melanoma cell harboring a neurblasoma RAS viral oncogene homolog (NRAS) mutation, or a melanoma cell wild type for both BRAF and NRAS.