Discussion

Invasive urothelial carcinoma (UC) represents a significant proportion of patients diagnosed with bladder cancer and has a poor 5-year survival rate, with resistance to frontline therapies common. Consequently there is a need to understand the molecular mechanisms underlying the evasion of cell cycle control in these cells and how this is associated with the poor prognosis seen with UC. Here we describe a novel phenotypic screening assay that can distinguish the different phases of the cell-cycle including the sub-divisions of mitosis. This assay uses the expression of phase-specific proteins and nuclear morphology to identify cells in G0, G1, S, G2 and M phases as well as apoptotic cells in asynchronous cultures. Here we show assay development and validation, by use of both siRNA and pharmacological inhibitors of specific cell-cycle checkpoints, in U2OS osteosarcoma cells. It is applicable to many cell types and we are currently using it in several UC cell lines, including 5637 and SD cell lines. The assay has been used to identify pro-survival kinases in UC including those with cytostatic effects which are currently undergoing validation. Furthermore we will develop novel protein-binding reagents, termed Adhirons, that block the identified kinases to validate them as drug targets and to aid the identification of small molecule therapeutics.