Expression of protein-associated DNA damage in the alkaline elution assay in the absence of enzymatic deproteinization.
In the alkaline elution assay, expression of protein-associated DNA damage induced by topoisomerase II antagonists is facilitated by proteinase K digestion of the drug-stabilized topoisomerase-II-DNA complex. In the absence of this enzymatic deproteinization step, drug-induced DNA strand breaks are masked by the binding of the topoisomerase-II-DNA complex to the synthetic filter from which DNA is eluted subsequent to alkaline denaturation. In this manuscript, we report that as the number of cells lysed on the filter is increased, binding of the topoisomerase-II-DNA complex to the filter is compromised, permitting expression of DNA damage in the absence of enzymatic deproteinization.
Gewirtz
D A
DA
Department of Medicine, Medical College of Virginia, Richmond 23298.
Ellis
A L
AL
Randolph
J K
JK
Yanovich
S
S
Swerdlow
P S
PS
Povirk
L F
LF
Yalowich
J C
JC
D000818:Animals; D002460:Cell Line; D004249:DNA Damage; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000302:isolation & purification / Q000528:radiation effects; D019286:Endopeptidase K; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007939:Leukemia L1210; D008114:Liver Neoplasms, Experimental; D051379:Mice; D008942:Mitoxantrone / Q000494:pharmacology*; D011034:Podophyllotoxin / Q000031:analogs & derivatives*; D011485:Protein Binding; D051381:Rats; D012697:Serine Endopeptidases; D013713:Teniposide / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004273:DNA, Neoplasm; D005047:Etoposide; D013713:Teniposide; D008942:Mitoxantrone; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D004250:DNA Topoisomerases, Type II; D011034:Podophyllotoxin