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Many chemotherapeutic agents preferentially act on proliferating more than on arrested cells. However, no cellular system existed until recently to determine the proliferation-dependent cytotoxicity of existing and investigational compounds on an isogenic background. A cellular system has been now developed which allows to evaluate the cytotoxic effect of a test compound on proliferating as well as resting cells on the same cellular background. The resulting assay is suitable for screening of large compound libraries to identify cell cycle interfering chemotherapeutics targeting preferentially proliferating cells. It is also useful to characterize the mode of action of single lead compounds.
The test system is based on genetically engineered human colon adenocarcinoma cells (RKO) which inducibly express the CDK inhibitor protein p27Kip1. After stimulation with an inducer p27Kip1 is expressed and cells are arrested in the G1 phase of the cell cycle. Comparing the effect of your test compounds on induced/arrested cells and uninduced/proliferating cells we can find out whether your compounds are just cytotoxic in general or whether they preferentially kill proliferating cells a feature which is very desirable.
The figure below shows the results of differential cytotoxicity assays with Taxol, a tubulin-stabilizing agent which acts during mitosis and does not affect resting cells, and Cisplatin which causes crosslinking of DNA and subsequent apoptosis in a cell cycle-independent manner.
Cell cycle-dependent (Taxol) and cell-cycle independent (Cisplatin) cytotoxicity demonstrated in the RKOp27 cell system. Arrested and proliferating RKOp27 cells were incubated for 72 hours with Taxol/Cisplatin. Subsequently cell viability was determined and calculated in comparison to untreated control cells.
The service we offer in collaboration with a very experienced partner includes IC50 determination with 10 semi-logarithmic dilutions of your compounds, tested on proliferating versus arrested RKOp27 cells. Single concentration experiments are also possible. Read-out is the percentage of viable cells, measured through conversion of Alamar Blue into a fluorescent product by metabolically active cells. The assay is performed in 96-well plates and thus suitable for high throughput screening.
Fact Sheet - Differential Cytotoxicity Assay
Cellular Phosphorylation Assays
Spheroid-Based Cellular Angiogenesis Assay
Please inquire for a specific quote.
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