Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065.
CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand breaks [Reynolds, R. L., Molineux, I. J., Kaplan, D.J., Swenson, D.H., & Hurley, L.H. (1985) Biochemistry 24, 6228-6237]. We have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, we observe thermally induced breakage of [14C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [3H]thymidine-labeled untreated cells with the [14C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10(6) base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The induction of heat-labile sites in cellular DNA was confirmed by gel electrophoretic analyses of the damage to intracellular simian virus 40 (SV40) DNA in SV40-infected BSC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Zsido
T J
TJ
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263.
Woynarowski
J M
JM
Baker
R M
RM
Gawron
L S
LS
Beerman
T A
TA
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D004247:DNA / Q000187:drug effects* / Q000494:pharmacology*; D018736:DNA Adducts; D004249:DNA Damage; D004279:DNA, Viral / Q000187:drug effects; D000080890:Duocarmycins; D006358:Hot Temperature; D007211:Indoles; D007933:Leucomycins / Q000494:pharmacology*; D013539:Simian virus 40 / Q000235:genetics
C055641:CC-1065-(N(3)-adenine)-DNA; D018736:DNA Adducts; D004279:DNA, Viral; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; D004247:DNA; D000225:Adenine