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.

Designer enediynes generate DNA breaks, interstrand cross-links, or both, with concomitant changes in the regulation of DNA damage responses

“…As shown previously (14), 8 nM C-1027 induces significant amounts of linear intracellular SV40 DNA (20%), which increases in a concentration-dependent manner to Ϸ40% (Fig. 3A).…”

“…Although the extent of enediyne induction of cellfree and cellular DSBs is generally well correlated (13), there is no comparable information on cellular ICLs because no such activity has been reported. Thus, we compared the ability of C-1027 and its analogs** to induce both DSBs and ICLs on an intracellular SV40 DNA target (14,15).…”

The Antitumor Enediyne C-1027 Alters Cell Cycle Progression and Induces Chromosomal Aberrations and Telomere Dysfunction

“…When C-1027 was increased from 0.14 to 2.9 nmol/L, the damage score increased dramatically. This was in agreement with earlier pulsed-field gel electrophoresis data from our laboratory that showed minimal detectable damage to genomic DNA with 0.1 nmol/L C-1027 (33). As was evident from the images in Fig.…”

“…Whereas ionizing radiation induces double-strand breaks at random sequences (39), C-1027, as well as neocarzinostatin and bleomycin, can target damage to specific intracellular DNA sequences (33,40,41). If the limited number of strand breaks induced by C-1027 compared with ionizing radiation were directed to more localized regions of the genome as well as to the genome as a whole, clustering of doublestrand breaks would occur and misrejoining likely would be enhanced.…”

Cellular Responses to the DNA Strand-scission Enediyne C-1027 Can Be Independent of ATM, ATR, and DNA-PK Kinases

“…A characteristic feature of C-1027 is its propensity to induce a higher ratio of DNA DSB to SSB as compared with IR. In addition, C-1027 biological effects are similar to IR-induced effects, including reduction of cellular DNA replication, delayed progression through S phase, and G 2 /M cell cycle block (23)(24)(25).…”