Silencing and selective methylation of the normal topoisomerase I gene in camptothecin-resistant CEM/C2 human leukemia cells.
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.
Fujimori
A
A
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Hoki
Y
Y
Popescu
N C
NC
Pommier
Y
Y
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002166:Camptothecin / Q000494:pharmacology*; D019175:DNA Methylation; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis / Q000235:genetics*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000972:Antineoplastic Agents, Phytogenic; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin