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Use of isogenic human cancer cells for high-throughput screening and drug discovery


Cell-based screening for novel tumor-specific drugs has been compromised by the lack of appropriate control cells. We describe a strategy for drug screening based on isogenic human cancer cell lines in which key tumorigenic genes have been deleted by targeted homologous recombination. As a test case, a yellow fluorescent protein (YFP) expression vector was introduced into the colon cancer cell line DLD-1, and a blue fluorescent protein (BFP) expression vector was introduced into an isogenic derivative in which the mutant K-Ras allele had been deleted. Co-culture of both cell lines allowed facile screening for compounds with selective toxicity toward the mutant Ras genotype. Among 30,000 compounds screened, a novel cytidine nucleoside analog was identified that displayed selective activity in vitro and inhibited tumor xenografts containing mutant Ras. The present data demonstrate a broadly applicable approach for mining therapeutic agents targeted to the specific genetic alterations responsible for cancer development.

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Figure 1: Co-culture screening.
Figure 2: Structures of SC-D and TPT.
Figure 3: SC-D and TPT selectivity.
Figure 4: (A) TPT and (B) SC-D dose response.
Figure 5
Figure 6: Selectivity on K-Ras transformed rat kidney epithelial cells.
Figure 7: Effect of SC-D on tumor xenografts.


  1. Druker, B.J. et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344, 1038–1042 (2001).

    Article  CAS  Google Scholar 

  2. Pegram, M.D., Konecny, G. & Slamon, D.J. The molecular and cellular biology of HER2/neu gene amplification/overexpression and the clinical development of herceptin (trastuzumab) therapy for breast cancer. Cancer Treat. Res. 103, 57–75 (2000).

    Article  CAS  Google Scholar 

  3. Bender, A. & Pringle, J.R. Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 11, 1295–1305 (1991).

    Article  CAS  Google Scholar 

  4. Shirasawa, S., Furuse, M., Yokoyama, N. & Sasazuki, T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science 260, 85–88 (1993).

    Article  CAS  Google Scholar 

  5. Cardenas, M.E., Sanfridson, A., Cutler, N.S. & Heitman, J. Signal-transduction cascades as targets for therapeutic intervention by natural products. Trends Biotechnol. 16, 427–331 (1998).

    Article  CAS  Google Scholar 

  6. Campbell, V.W. et al. The G-C specific DNA binding drug, mithramycin, selectively inhibits transcription of the C-MYC and C-HA-Ras genes in regenerating liver. Am. J. Med. Sci. 307, 167–172 (1994).

    Article  CAS  Google Scholar 

  7. Adegboyega, P.A., Adesokan, A., Haque, A.K. & Boor, P.J. Sensitivity and specificity of triphenyl tetrazolium chloride in the gross diagnosis of acute myocardial infarcts. Arch. Pathol. Lab. Med. 121, 1063–1068 (1997).

    CAS  PubMed  Google Scholar 

  8. Otero, A.J., Rodriguez, I. & Falero, G. 2,3,5-Triphenyl tetrazolium chloride (TTC) reduction as exponential growth phase marker for mammalian cells in culture and for myeloma hybridization experiments. Cytotechnology 6, 137–142 (1991).

    Article  CAS  Google Scholar 

  9. Cuenda, A. et al. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364, 229–233 (1995).

    Article  CAS  Google Scholar 

  10. Liverton, N.J. et al. Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinases. Med. Chem. 42, 2180–2190 (1999).

    Article  CAS  Google Scholar 

  11. Geran, R.I., Greenberg, N.H., MacDonald, M.M., Schumaker, A.M. & Abbott, B.J. Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother. Rep. 3, 1–103 (1972).

    Google Scholar 

  12. Kain, S.R. Green fluorescent protein (GFP): applications in cell-based assays for drug discovery. Drug Discov. Today 4, 304–312 (1999).

    Article  CAS  Google Scholar 

  13. Gibbs, J.B. Mechanism-based target identification and drug discovery. Science 287, 1969–1973 (2000).

    Article  CAS  Google Scholar 

  14. Shields, J.M., Pruitt, K., McFall, A., Shaub, A. & Der, C.J. Understanding Ras: 'it ain't over 'til it's over'. Trends Cell Biol. 10, 147–154 (2000).

    Article  CAS  Google Scholar 

  15. West, D.B. et al. Mouse genetics/genomics: an effective approach for drug target discovery and validation. Med. Res. Rev. 20, 216–230 (2000).

    Article  CAS  Google Scholar 

  16. Harris, S. & Foord, S.M. Transgenic gene knock-outs: functional genomics and therapeutic target selection. Pharmacogenomics 1, 433–443 (2000).

    Article  CAS  Google Scholar 

  17. Sedivy, J.M., Vogelstein, B., Liber, H.L., Hendrickson, E.A. & Rosmarin, A. Gene targeting in human cells without isogenic DNA. Science 283, 9 (1999).

    Article  Google Scholar 

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We would like to thank the Developmental Therapeutics Program at the National Cancer Institute (NCI) for providing drug libraries and Dr. S. Shirasawa for providing the K-Ras KO cells. This work was supported by the Clayton Fund, the Miracle Foundation, the National Foundation for Cancer Research, and National Institutes of Health grants CA43460, CA09243, and CA62924.

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Correspondence to Bert Vogelstein.

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Torrance, C., Agrawal, V., Vogelstein, B. et al. Use of isogenic human cancer cells for high-throughput screening and drug discovery. Nat Biotechnol 19, 940–945 (2001).

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