Site specificity of bleomycin cleavage in purified and intracellular simian virus 40 DNA

Grimwade, Julia E.; Cason, E B; Beerman, Terry A.

doi:10.1093/nar/15.16.6315

Cited by 19 publications

(9 citation statements)

References 26 publications

(32 reference statements)

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“…African green monkey kidney BSC-1 cells (from Dr. Terry Beerman, Roswell Park Cancer Inst., Buffalo, NY) were cultured as described previously (24,27). When drug effects on intracellular SV40 DNA were examined, cells were infected with SV40 virus as described (24,27,28) and at 40 h postinfection treated with drug for 4 h at 37 °C. SV40 DNA was then isolated as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

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Tallimustine Lesions in Cellular DNA are AT Sequence-Specific but Not Region-Specific

Herzig¹,

Treviño

Arnett

et al. 1999

Biochemistry

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Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.

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Section: Methodsmentioning

confidence: 99%

“…When drug effects on intracellular SV40 DNA were examined, cells were infected with SV40 virus as described (24,27,28) and at 40 h postinfection treated with drug for 4 h at 37 °C. SV40 DNA was then isolated as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

Tallimustine Lesions in Cellular DNA are AT Sequence-Specific but Not Region-Specific

Herzig¹,

Treviño

Arnett

et al. 1999

Biochemistry

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“…(African green monkey kidney) cells was in Minimal Essential Medium plus 10% calf serum (MEM), and infection with 5-15 plaque-forming units of SV40 virus per 0.6 x 106 cells was as described elsewhere (Grimwade et al, 1987).…”

Section: Methodsmentioning

confidence: 99%

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

McHugh

Woynarowski²,

Gawron³

et al. 1995

Biochemistry

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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.

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“…Maintenance of BSC-I (African green monkey kidney) cells was in Minimal Essential Medium, 10% calf serum (MEM). Infection with 5-15 plaque-forming units of SV40 virus per 6 X 10 5 cells was as described elsewhere [14]. BSC-1 cells at 6 X 10 5 per 35 mm plate were infected as described above with SV40 virus.…”

Section: Cleurlage Of Intracellular Dna B! the Holoatrtibioticmentioning

confidence: 99%

Kinetics of cleavage of intra- and extracellular simian virus 40 DNA with the enediyne anticancer drug C-1027

Kirk

Goodisman

Beerman

et al. 1997

Biophysical Chemistry

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A kinetic analysis of cleavage of simian virus DNA (SV40 DNA) inside and outside green monkey BSC-1 cells by the enediyne-protein antibiotic C-1027 and its free chromophore is described. Information on rate constants was obtained by fitting populations of forms I (closed circular DNA), II (nicked circular DNA) and III (linear DNA) SV40 DNA as a function of drug concentration to a kinetic model which includes: cutting of form I to give form II with rate constant k1, cutting of form I to give form III with rate constant K4, and cutting of form II to give form III with rate constant k2. The ratio of single-strand (ss) to double-strand (ds) cutting for the holoantibiotic and the free chromophore, k1/k4, is approximately 1.8 for extracellular SV40 DNA. For intracellular DNA and extracellular DNA which has been post-treated with putrescine, ds cutting is much more probable, with k4 about four times as large as k1. This observation suggests that amine groups present in the cell are able to convert abasic sites opposite an ss break into a ds break in SV40 chromatin. The overall rate of cleavage of form-I DNA inside the cell is much larger than the rate outside, the sum k1 + k4 being about three times as large for intracellular DNA as for extracellular DNA.

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Site specificity of bleomycin cleavage in purified and intracellular simian virus 40 DNA

Cited by 19 publications

References 26 publications

Tallimustine Lesions in Cellular DNA are AT Sequence-Specific but Not Region-Specific

Tallimustine Lesions in Cellular DNA are AT Sequence-Specific but Not Region-Specific

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

Kinetics of cleavage of intra- and extracellular simian virus 40 DNA with the enediyne anticancer drug C-1027

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