Effects of the DNA-Damaging Enediyne C-1027 on Intracellular SV40 and Genomic DNA in Green Monkey Kidney BSC-1 Cells

Abstract

This study describes the selective ability of C-1027 to induce limited double-strand damage in a viral DNA target. The effect of the cellular environment on C-1027 activity was examined by assaying the extent, as well as the specificity, of damage to simian virus 40 (SV40) DNA in lytically infected mammalian BSC-1 cells and in purified SV40 DNA preparations. C-1027 damage to intracellular SV40 DNA was quantitated by topological forms conversion analysis. A gradual decrease in intracellular supercoiled form I accompanied by an increase in form III was observed with C-1027 concentrations from 2 to 100 nM, with a 50% reduction in form I observed at 50 nM. Damage to purified SV40 DNA also was most pronounced between 10 and 100 nM C-1027. When concentrations were expressed as r values (drug/DNA molar ratio), the amount of C-1027 necessary to effect a 50% reduction in form I was lower for intracellular (r = 0.002) than for purified SV40 DNA (r = 0.0035). Double-strand damage was more likely to occur with C-1027 treatment of intracellular compared to purified SV40 DNA. However, with both purified and intracellular DNA, restriction enzyme digestion analysis revealed double-strand damage at a number of specific sites throughout the genome, particularly within the early region of the SV40 genome (e.g., within the coding sequence for large T-antigen). No significant damage was observed in either the origin (ORI) or the termination (TER) regions of SV40 replication. The extent of C-1027 damage to uninfected BSC-1 cell DNA was also quantitated using pulsed-field gel electrophoresis. At 0.1 nM (r = 2.8 x 10(-5), where incorporation of [3H]thymidine was reduced by 80%, 600 rad equiv of damage was detected in uninfected BSC-1 cells. At C-1027 r values from 1 x 10(-4) to 40 x 10(-4), double-strand breaks were from 80- to 40-fold more frequent in SV40 than in BSC-1 cell genomic DNA. By contrast, 50-fold more drug was necessary to inhibit intracellular SV40 DNA accumulation compared to [3H]thymidine incorporation into uninfected BSC-1 cells. Thus, SV40 DNA synthesis appeared to be less sensitive to C-1027-induced lesions than replication in uninfected BSC-1 cells.