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

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 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).

  2. 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).

  3. 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).

  4. 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).

  5. 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).

  6. 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).

  7. 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).

  8. 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).

  9. 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).

  10. 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).

  11. 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).

  12. 12

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

  13. 13

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

  14. 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).

  15. 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).

  16. 16

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

  17. 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).

Download references

Acknowledgements

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.

Author information

Correspondence to Bert Vogelstein.

Rights and permissions

Reprints and Permissions

About this article

Further reading