Tallimustine Lesions in Cellular DNA are AT Sequence-Specific but Not Region-Specific

Abstract

Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.

AT-rich Islands in Genomic DNA as a Novel Target for AT-specific DNA-reactive Antitumor Drugs

“…The distribution parameters (Table I) remained virtually unchanged after the number of analyzed hits exceeded ϳ10 5 . For the bizelesin cross-linking motif T(A/T) 4 A), this number of hits was reached after analyzing ϳ10 7 bp of DNA sequences. For some sequences, the distribution of drug binding sites was compared with the distribution of known AT elements such as TATA boxes and polyadenylation signals.…”

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Interstrand cross-links at T(A/T) 4 A sites in cellular DNA are associated with hypercytotoxicity of an anticancer drug, bizelesin. Here we evaluated whether these lethal effects reflect targeting critical genomic regions.

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“…Not only are such drugs nonregion-specific (3,4), but they also form most of their adducts with macromolecules other than DNA (5). In contrast, certain minor groove-binding agents (MGBs) 1 combine a high sequence specificity for AT motifs with a lack of reactivity with non-DNA targets.…”

“…Among the small molecules tested, the cyclopropylpyrroleindole drug bizelesin was found to have the greatest potential for producing region-specific damage. In model sequences analyzed, bizelesin adducts were non-randomly distributed and reflected mainly interstrand cross-links at T(A/T) 4 A sites, although some monoadducts at A(A/T) 4 A sites are also observed ( Fig. 1) (8 -10).…”

“…Bizelesin is one of the most cytotoxic compounds ever identified, requiring only a few (Ͻ10 1 ) drug adducts per cell for cell growth inhibition (4,10). Since typical DNA-reactive drugs must form several thousand lesions/cell to achieve similar effects (4), one can infer that bizelesin must damage DNA specifically within regions that are crucial for continued cell growth.…”