A novel mammalian cell-based approach for the discovery of anticancer drugs with reduced cytotoxicity on non-dividing cells.
A key asset of cytotoxic drugs in cancer therapeutics is their ability to discriminate between proliferating and mitotically inert cells and eliminate preferentially neoplastic ones. We have designed a high throughput-compatible mammalian cell-based assay for the discovery of cytotoxic drugs, which selectively kill proliferation-competent target cells. This cytotoxic drug discovery assay is based on a transgenic CHO-K1-derived cell line engineered for a conditional G1-specific growth arrest following tetracycline-responsive overexpression of the human cyclin-dependent kinase inhibitor p27(Kip1). The CHO-derived cell line CHO-p27(Kip1) shows wild type proliferation rates and can be expanded in the presence of tetracycline antibiotics when p27(Kip1) expression is repressed. Upon withdrawal of regulating antibiotics CHO-p27(Kip1) differentiates into a 1:1 mixed population consisting of two different proliferation phenotypes: (i) a G1-arrested cell population induced by heterologous expression of p27(Kip1) which mimics mitotically inactive terminally differentiated cells and (ii) a proliferation-competent cell population which eliminated the p27(Kip1) expression unit and imitates neoplastic cell characteristics. Addition of chemical or metabolic libraries to CHO-p27(Kip1) populations cultivated in tetracycline-free medium followed by scoring for cell viability will reveal cytotoxic drug candidates associated with a high viability ratio of proliferation-competent/arrested populations. We have validated the cell-based cytotoxic drug discovery assay using the clinically licensed cancer drugs mitomycin C, doxorubicin, etoposide and 5-fluorouracil. Comparative proof-of-concept studies showed that these top-prescribed cancer therapeutics preferentially eliminate proliferating cells while showing less interference with the viability of G1-arrested cell populations. These results demonstrate the CHO-p27(Kip1)-based cytotoxic drug finder technology is ready-to-apply for high throughput screenings of chemical as well as metabolic libraries to discover novel cancer therapeutics which show reduced cytotoxicity on terminally differentiated cells.
Gonzalez-Nicolini
Valeria
V
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland.
Fux
Cornelia
C
Fussenegger
Martin
M
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000096:biosynthesis; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004317:Doxorubicin / Q000494:pharmacology; D015195:Drug Design; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005047:Etoposide / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005472:Fluorouracil / Q000494:pharmacology; D016685:Mitomycin / Q000494:pharmacology; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016685:Mitomycin; D005047:Etoposide; D004317:Doxorubicin; D005472:Fluorouracil