DNA alkylating agents, including nitrogen mustards (NM) such as cyclophosphamide (CPA), remain in heavy use as anti-cancer agents. They are employed in adjuvant chemotherapy regimens, such as the AC (anthracycline-CPA) therapy frequently administered to breast cancer patients. The primary site of alkylation in DNA is the N7-guanine position. This Program focuses upon chemistry and biology that happens after N7-dG alkylation: the NM N7-alkylated guanines undergo interstrand cross-link formation (ICL), base-induced ring-opening, yielding stable alkyl-formamidopyrimidine (N5-substituted-Fapy) lesions, and depurination, yielding apurinic (AP) sites. These types of complex DNA damage are may contribute substantially to the mechanisms of action of NM agents, including cytoxicity. Knowledge of the spectrum of complex DNA damage created by NM agents during therapy and the biological processing of this damage is critical for the design of effective treatment regimens. We have shown that the anthracyline antibiotic doxorubicin (Adriamycin), a component of AC therapy, forms covalent conjugates with AP sites, a previously unrecognized activity for this drug. Thus, AP sites formed as a consequence of NM treatment or as base excision repair (BER) intermediates provide targets for adjuvant therapies designed to increase cytotoxicity. Complex DNA damage arising from rearrangement of N7-dG alkylation sites has until now been understudied, in part, due to an inability to rigorously prepare DNAs containing such damage for biological, biochemical, and structural studies. Insights gained from our Program will yield fundamental and applied understanding of complex DNA damage arising from DNA alkylation and its detection in cellular DNA, innovative syntheses for the production and characterization of DNAs containing site-specific complex damage, structural understanding of how complex damage and AP site conjugation products alters DNA structure, and modulates repair and replication, effective design of anthracycline analogs targeted to AP sites, new biomarkers and analytical methods with which to monitor real time therapeutic efficacy in patients undergoing chemotherapy, and improved adjuvant therapy regimens designed to maximize cytotoxic response in cancer vs normal cells.
The alkylation of DNA by clinically used chemotherapeutic agents such as nitrogen mustards (NM) and thioTEPA results in complex forms of DNA damage. These may be genotoxic or cytotoxic, and in addition, may react further with other chemotherapeutic agents, such as the anthracyline antibiotics. Delineating the chemistry and biology of these complex damage sites, individually or in combination with other agents, and how they are processed in humans can serve as the basis for novel strategies to improve therapeutic treatments of cancers.
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