Silencing and selective methylation of the normal topoisomerase I gene in camptothecin-resistant CEM/C2 human leukemia cells

Oncol Res. 1996;8(7-8):295-301.


Camptothecin resistance of the human leukemia CEM/C2 cells is associated with a topoisomerase I (top1) mutation: Asn722Ser (Fujimori, A. et al. Cancer Res. 55:1339-1346; 1995). The corresponding DNA point mutation generates a novel site for the restriction endonuclease DdeI. We found that only the mutated top1 transcript was detectable in CEM/C2 by reverse transcriptase-polymerase chain reaction. Genomic DNA analysis by Southern blotting with DdeI showed that both the mutated and normal top1 genes were present in CEM/C2 cells. The mechanism of normal top1 allele silencing was further investigated. Cytogenetic analysis with a human chromosome 20 specific probe and restriction mapping by Southern blotting showed that both cell lines had a similar copy number of chromosome 20, with the predominant population containing 5-6 copies, and no detectable top1 gene rearrangement. Southern blotting using methylcytosine-sensitive restriction endonuclease (HpaII) indicated differential top1 methylation in CEM/C2 cells. Global cytosine methylation, however, appeared similar in CEM/C2 and wild-type CEM cells. These results indicate that gene-specific DNA methylation can play a role in downregulating top1 gene(s) and in the cellular resistance to camptothecins.

MeSH terms

  • Antineoplastic Agents, Phytogenic / pharmacology*
  • Camptothecin / pharmacology*
  • DNA Methylation
  • DNA Topoisomerases, Type I / biosynthesis
  • DNA Topoisomerases, Type I / genetics*
  • DNA, Neoplasm / chemistry
  • DNA, Neoplasm / genetics*
  • Enzyme Induction
  • Enzyme Inhibitors / pharmacology*
  • Gene Expression Regulation, Leukemic*
  • Humans
  • In Situ Hybridization, Fluorescence
  • Neoplasm Proteins / biosynthesis
  • Neoplasm Proteins / genetics*
  • Precursor Cell Lymphoblastic Leukemia-Lymphoma / pathology*
  • Tumor Cells, Cultured / drug effects


  • Antineoplastic Agents, Phytogenic
  • DNA, Neoplasm
  • Enzyme Inhibitors
  • Neoplasm Proteins
  • DNA Topoisomerases, Type I
  • Camptothecin