The anticancer
anthracycline compound
Adriamycin is a known
topoisomerase II inhibitor but is also capable of exerting other cellular consequences. After intercalation,
Adriamycin can form covalent adducts with
DNA, and the magnitude of these adducts appears to be limited by the cellular availability of
formaldehyde. Adducts produced by
Adriamycin in the presence of
formaldehyde have been well characterized in cell-free systems but not in cells. In this study, we show that when
Adriamycin is used in conjunction with the
formaldehyde-releasing
prodrug AN-9 in IMR-32
tumor cells, this allows the formation of sufficiently high levels of adducts in genomic
DNA to enable detection of their DNA sequence specificity for the first time. The 340-bp alpha-satellite EcoRI repeat sequence was isolated from
drug-treated cells and digested with lambda-
exonuclease to determine adduct sites at which
exonuclease digestion was blocked. The
Adriamycin adducts were formed predominantly at 5'-GC and GG sequences and unstable with respect to elevated temperatures and extended times at 37 degrees C. The use of three
anthracycline derivatives lacking a 3'amino group demonstrated that this amino portion is critical for the formation of
anthracycline adducts in cells. The structure of these
drug-
DNA adducts can therefore be considered to be identical to the
Adriamycin adducts, which have been characterized rigorously in cell-free systems by X-ray crystallography, two-dimensional nuclear magnetic resonance, and mass spectrometry.