Designer enediynes generate DNA breaks, interstrand cross-links, or both, with concomitant changes in the regulation of DNA damage responses

Abstract

The ability of the radiomimetic anticancer enediyne C-1027 to induce ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR)-independent damage responses was discovered to reside in its unique ability to concurrently generate robust amounts of double-strand breaks (DSBs) and interstrand cross-links (ICLs) in cellular DNA. Furthermore, a single substitution to the chromophore's benzoxazolinate moiety shifted DNA damage to primarily ICLs and an ATR- but not ATM-dependent damage response. In contrast, single substitutions of the chromophore's beta-amino acid component shifted DNA damage to primarily DSBs, consistent with its induction of conventional ATM-dependent damage responses of the type generated by ionizing radiation and other radiomimetics. Thus, phosphatidylinositol 3-kinase-like protein kinase regulation of DNA damage responses is dictated by the relative proportions of DSBs and ICLs.

Fig. 1.

Structures of the enediyne chromophores of C-1027 and its engineered analogs. (A) Structures of the C-1027 chromophore and its biochemical subunits: benzoxazolinate, deoxy aminosugar, β-amino acid, and enediyne core. (B) Structures of the C-1027 analogs desmethyl, deschloro, and deshydroxy. The rectangles indicate the composition and location of the C-1027 chromophore modifications.

Fig. 2.

C-1027 and desmethyl induce ICLs under cell-free anaerobic conditions. (A) Induction of ICLs produced by C-1027 or analogs. Linearized pBR322 plasmid DNA was incubated with the indicated drug for 4 h at room temperature under anaerobic conditions and examined as described in Materials and Methods. (Upper) Representative gel of C-1027 and desmethyl. (Lower) Representative gel of deschloro and deshydroxy. ND, nondenatured control. (B) Quantitation of ICLs induced by C-1027 and analogs. ICL induction is based on the relative percentage of dsDNA compared with a nondenatured control.

Fig. 3.

C-1027 or analogs can induce DSBs and/or ICLs in intracellular SV40 DNA. (A) Quantitation of DSBs produced by C-1027 and analogs. SV40-infected BSC-1 cells were treated with the indicated drug for 4 h at 37°C, and SV40 DNA was isolated as described in Materials and Methods and examined for topological forms conversion. The percentage of full-length linear DNA represents the drug potency at inducing intracellular SV40 DSBs. (B) DNA from control and drug-treated SV40-infected BSC-1 cells was isolated, converted to a full-length linear form with BamH1, and examined as described in Materials and Methods. Induction of ICLs, as in Fig. 2B, was based on full-length linear DNA that remained after alkaline denaturation. ND, nondenatured control.

Fig. 4.

Desmethyl and C-1027 induce ICLs in genomic DNA. (A) Representative alkaline comets assessing ICLs in HCT116 cells treated with deschloro and desmethyl for 4 h and then mock-treated or exposed to IR as indicated. Induction of ICLs was based on the reduction in IR comet tails by using the procedure described in Materials and Methods. (B) The comet analysis for deschloro and C-1027 is essentially as described in A. (C) Comets from A were scored as described in Materials and Methods, and the black dotted line represents the expected contribution of IR-induced DNA breaks to the comet signal. Bizelesin, an ICL drug, is included as a positive control. (D) The comet tails for deschloro and C-1027 were scored as described in C.

Fig. 5.

Cells require ATR to activate DNA damage response proteins and avoid hypersensitive growth inhibition in response to desmethyl. HCT116 cells were pretreated for 48 h with mock (siRNA buffer) (ATR +) or siATR (ATR −) before treatment with equicytotoxic concentrations of the indicated drug for 1 h at 37°C (A and B) or 3 days (C). (A and B) Cellular extracts were analyzed by Western blotting and probed with an antibody specific for phosphorylated Chk1-Ser-345 (A) or with an antibody specific for phosphorylated Chk2-Thr-68 or p53-Ser-15 (B). (C) Growth inhibition IC₅₀ was calculated based on the concentration of drug required to reduce cell growth by 50%. The fold differences in the IC₅₀ values values were used to determine the ratio of growth inhibition between mock siRNA- or siATR-treated cells.