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

Biochemistry. 1991 Apr 16;30(15):3733-8. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine / analogs & derivatives*
  • Adenine / pharmacology
  • Animals
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Chlorocebus aethiops
  • DNA / drug effects*
  • DNA / pharmacology*
  • DNA Adducts*
  • DNA Damage*
  • DNA, Viral / drug effects
  • Duocarmycins
  • Hot Temperature
  • Indoles*
  • Leucomycins / pharmacology*
  • Simian virus 40 / genetics


  • CC-1065-(N(3)-adenine)-DNA
  • DNA Adducts
  • DNA, Viral
  • Duocarmycins
  • Indoles
  • Leucomycins
  • DNA
  • Adenine