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 associat ed with a topoisomerase I (top1) mutation: Asn722Ser (Fujimori, A. et al. Cancer Res. 55:1339-1346; 1995). The corresponding DNA point mutat ion generates a novel site for the restriction endonuclease DdeL. We f ound 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 norma l top1 genes were present in CEM/C2 cells. The mechanism of normal top 1 allele silencing was further investigated. Cytogenetic analysis with a human chromosome 20 specific probe and restriction mapping by South ern 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 met hylcytosine-sensitive restriction endonuclease (HpaII) indicated diffe rential top1 methylation in CEM/C2 cells. Global cytosine methylation, however, appeared similar in CEM/C2 and wild-type CEM cells. These re sults indicate that gene-specific DNA methylation can play a role in d ownregulating top1 gene(s) and in the cellular resistance to camptothe cins. Copyright (C) 1996 Elsevier Science Inc.