Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065

• Published in: Biochemistry (Easton) 1991-04, Vol.30 (15), p.3733-3738
• Main Authors: Zsido, Tunde J, Woynarowski, Jan M, Baker, Raymond M, Gawron, Loretta S, Beerman, Terry A
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; ADDUCTS; Adenine - analogs & derivatives; Adenine - pharmacology; ALKYLATING AGENTS; ANIMAL CELLS; ANIMALS; ANTINEOPLASTIC DRUGS; AZINES; BACTERIA; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; BIOLOGICAL EFFECTS; BODY; CARBON 14 COMPOUNDS; CARBON COMPOUNDS; Cell physiology; Cells, Cultured; Centrifugation, Density Gradient; Cercopithecus aethiops; DNA; DNA - drug effects; DNA - pharmacology; DNA ADDUCTS; DNA Damage; DNA, Viral - drug effects; DRUGS; Fundamental and applied biological sciences. Psychology; HETEROCYCLIC COMPOUNDS; Hot Temperature; Indoles; KIDNEYS; LABELLED COMPOUNDS; Leucomycins - pharmacology; MAMMALS; MICROORGANISMS; Molecular and cellular biology; MOLECULAR STRUCTURE; MONKEYS; NUCLEIC ACIDS; NUCLEOSIDES; NUCLEOTIDES; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PRIMATES; PYRIMIDINES; Responses to growth factors, tumor promotors, other factors; RIBOSIDES; SEDIMENTATION; Simian virus 40 - genetics; STREPTOMYCES; THYMIDINE; TIME DEPENDENCE; VERTEBRATES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00229a021

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.