The Interaction of Antitumor Antibiotic CC-1065 with Supercoiled DNA
Dayton, Brian D.
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CC-1065 is a new antitumor antibiotic recently discovered at The Upjohn Company, and, by all experimental indications thus far, it appears to be one of the most cytotoxic antitumor agents. Circular dichroism, ultraviolet spectroscopy, and base-specific alkylation studies suggest that CC-1065 binds to double-stranded DNA in the minor groove at A-T sites. It has been observed that this drug also raises the thermal melting point of DNA greater than 35°C, thus suggesting that CC-1065 stabilizes the DNA helix. In attempting to corroborate these findings, and to suggest a mechanism explaining cytotoxicity of CC-1065, the following findings were made: CC-1065 did not intercalatively bind to DNA, since supercoiled DNA was not unwound when titrated with the drug. In fact, when the CC-1065 DNA complex was then titrated with the known intercalative agent ethidium bromide, CC-1065 stabilized the helix against intercalation. Saturated levels of CC-1065 in DNA increased by 40 times the amount of ethidium needed to unwind the CC-1065 DNA, compared to control DNA. CC-1065 did not induce breaks in DNA under pseudo-physiological conditions, but upon incubation at 70°C, CC-1065 caused breakage in the deoxyribose backbone of the DNA. The breakage rat, was estimated as approximately one break for every 4 drug molecules bound. About half the supercoiled DNA present had been reduced to closed circular DNA via breakage after 4 hours at a drug to nucleotide ratio of one to 2500. Taken together, the previous data and the data from this thesis support the hypothesis that the interaction of CC-1065 with DNA at A-T sites in the minor groove produces a strongly bound adduct that is stable at 37°C, pH 7.2, but yields chain breaks at 70'C. Analysis of these breaks indicate that about 25% of the DNA-bound molecules cause breakage, with half of the DNA being reduced to closed-circular DNA after 4 hours. CC-1065 did not intercalate, and, in fact, stabilizes the helix against ethidium bromide intercalation, thus suggesting that the cytotoxic mechanism of CC-1065 is overstabilization of the DNA molecule such that normal melting and unwinding processes during DNA synthesis cannot occur.