Characterization of covalent Adriamycin-DNA adducts

Abstract

Adriamycin is a popular antineoplastic agent whose ability to form covalent adducts with DNA has been correlated to cellular apoptosis (programmed cell death) in tumor models. We have isolated and purified this adduct formed under oxido-reductive (Fenton) conditions in Tris buffer. We show by homo-and heteronuclear NMR spectroscopy that the covalent Adriamycin-DNA adduct is structurally equivalent to that resulting from direct reaction with formaldehyde. Covalent linkage of the drug to one of the DNA strands confers remarkable stability to the duplex, indicated by a 162-fold reduction in the rate of strand displacement compared with the complex with noncovalently bound drug. Glyceraldehyde also engenders covalent Adriamycin-DNA complexes, providing a possible relevant biological context for in vivo adduct formation.

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

“…Doxorubicin adducts with cellular DNA have been shown to be formed from reduction of the drug in vitro and to involve the C7 position of the reductively activated quinone methide and guanine-base position of DNA (Cullinane et al, 1994). It was also evidenced that the covalent linkage of the drug to one of the DNA strand increases remarkably the stability of the duplex in comparison with the complex with noncovalently bound DOX (Zeman et al, 1998). The relevance of reductive activation of DOX by CPR for increasing cytotoxic activity of this drug towards sensitive human breast cell line MCF-7 has been also demonstrated using purified rat CPR (Bartoszek and Wolf, 1992).…”

Analysis of covalent ellipticine- and doxorubicin-derived adducts in DNA of neuroblastoma cells by the ³²P-postlabeling technique

“…To answer this question, development of methods suitable for detecting and quantifying covalent DNA adducts formed by ellipticine and doxorubicin is the first and key target of the research. Using 14 C-labeled doxorubicin, doxorubicin-DNA adduct formation has been detected in several cancer cells 17,20,21,25 . Moreover, accelerator mass spectrometry has recently also been shown suitable for detecting the covalent 14 C-labeled doxorubicin-DNA adducts in cancer cells exposed to this drug [26][27][28] .…”

“…The resulting drug-DNA monoadduct is further stabilized through in- tercalation and hydrogen bonding with the second strand of DNA (ref. 21 ) (Fig. 2B).…”

“…The adducts are formed predominantly at 50-GC-30 sites in DNA (ref. 18 ), where the doxorubicin sugar group (daunosamine) is covalently linked to the N-2 amino group of guanine via an aminal (N-C-N) bond [19][20][21][22] . The central carbon atom in the aminal bond is derived from formaldehyde, hence formaldehyde is an absolute requirement for adduct formation 20,21 .…”

“…18 ), where the doxorubicin sugar group (daunosamine) is covalently linked to the N-2 amino group of guanine via an aminal (N-C-N) bond [19][20][21][22] . The central carbon atom in the aminal bond is derived from formaldehyde, hence formaldehyde is an absolute requirement for adduct formation 20,21 . This compound is present in cancer cells, at levels which are often even higher in tumor cells (1.5-4.0 mM) than normal cells 23,24 .…”