Selective damage to the active X chromosome by camptothecin and amsacrine as determined by an allele-specific alkaline unwinding assay.
Previous studies with MCF-7 cells demonstrated that several agents induce greater strand breakage in active genes than in nontranscribed centromeric regions. To better assess the effects of gene activity and inactivity, an allele-specific DNA strand break assay was developed, which allowed direct comparison of damage at a specific genetic locus on the active and inactive X chromosomes. The ZP lymphoblastoid cell line is heterozygous at the glucose-6-phosphate dehydrogenase (G6PD) locus, and the unexpressed (A) allele on the inactive X chromosome contains a FokI restriction site that is lacking in the expressed (B) allele on the active X. ZP cells were treated with camptothecin or amsacrine, and subjected to alkaline-induced DNA unwinding. Following detergent lysis and digestion of single-stranded DNA with S1 nuclease, the remaining double-stranded DNA was isolated and subjected to polymerase chain reaction (PCR) with primers that flank the polymorphic FokI site, with [alpha-32P]dCTP being added in the last PCR cycle. The resulting labeled PCR product was cleaved with FokI to assess the A/B allele ratio in the double-stranded DNA fraction. Treatment with camptothecin and amsacrine increased the apparent A/B ratio by factors of 2-3 and 1.5-2 respectively, indicating that the active B allele is preferentially damaged by these agents.
D000483:Alleles; D000677:Amsacrine / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002461:Cell Line, Transformed; D004247:DNA / Q000302:isolation & purification; D004249:DNA Damage; D005821:Genetic Techniques; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000187:drug effects* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D014960:X Chromosome / Q000187:drug effects*
D000972:Antineoplastic Agents, Phytogenic; D000677:Amsacrine; D004247:DNA; D005954:Glucosephosphate Dehydrogenase; D002166:Camptothecin