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 ind uce greater strand breakage in active genes than in nontranscribed cen tromeric regions. To better assess the effects of gene activity and in activity, an allele-specific DNA strand break assay was developed, whi ch 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 o n the active X. ZP cells were treated with camptothecin or amsacrine, and subjected to alkaline-induced DNA unwinding. Following detergent l ysis and digestion of single-stranded DNA with S1 nuclease, the remain ing double-stranded DNA was isolated and subjected to polymerase chain reaction (PCR) with primers that flank the polymorphic FokI site, wit h [alpha-P-32]dCTP being added in the last PCR cycle. The resulting la beled 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 d amaged by these agents.