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

Nat Biotechnol. 2001 Oct;19(10):940-5. doi: 10.1038/nbt1001-940.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Androstenes / pharmacology
  • Animals
  • Antineoplastic Agents / pharmacology*
  • Bacterial Proteins
  • Drug Screening Assays, Antitumor / methods*
  • Female
  • Gene Deletion
  • Genes, ras
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins
  • Mice
  • Mice, Knockout
  • Mice, Nude
  • Mutagenesis
  • Plicamycin / pharmacology
  • Recombination, Genetic
  • Sulfur Compounds / pharmacology
  • Tetrazolium Salts / pharmacology
  • Tumor Cells, Cultured


  • Androstenes
  • Antineoplastic Agents
  • Bacterial Proteins
  • Luminescent Proteins
  • Sulfur Compounds
  • Tetrazolium Salts
  • blue fluorescent protein, Aequorea victoria
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins
  • demethoxyviridin
  • triphenyltetrazolium
  • Plicamycin