C-1027, a Radiomimetic Enediyne Anticancer Drug, Preferentially Targets Hypoxic Cells

Research Article

C-1027, A Radiomimetic Enediyne Anticancer Drug, Preferentially Targets Hypoxic Cells

Terry A. Beerman,1 Loretta S. Gawron,1 Seulkih Shin,1 Ben Shen,2 and Mary M. McHugh1

1Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York;and 2Division of Pharmaceutical Sciences, University of Wisconsin National Cooperative Drug Discovery Group, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin

Abstract identified primarily from studies with neocarzinostatin (NCS), a The hypoxic nature of cells within solid tumors limits the holo-form drug, consisting of an apoprotein carrier and an active efficacy of anticancer therapies such as ionizing radiation and chromophore, and was assumed to be representative of all agents conventional radiomimetics because their mechanisms re- in this class (11). The NCS chromophore contains a bicyclic quire oxygen to induce lethal DNA breaks. For example, the enediyne that damages DNA via a Myers-Saito cycloaromatization conventional radiomimetic enediyne neocarzinostatin is 4- reaction, resulting in a 2,6-indacene diradical structure capable of fold less cytotoxic to cells maintained in low oxygen (hypoxic) hydrogen abstractions from deoxyribose (12, 13). Subsequent to compared with normoxic conditions. By contrast, the ene- generation of a sugar radical, reaction with oxygen quickly and diyne C-1027 was nearly 3-fold more cytotoxic to hypoxic than efficiently leads to formation of hydroxyl radicals that induce to normoxic cells. Like other radiomimetics, C-1027 induced DSBs/SSBs at a 1:5 ratio. The more recently discovered holo-form DNA breaks to a lesser extent in cell-free, or cellular hypoxic, enediyne C-1027 (Fig. 1) induces predominately DSBs owing to the compared with normoxic environments. However, the unique efficiency of hydrogen abstraction by its 1,4-benzenoid diradical DNA interstrand cross-linking ability of C-1027 was markedly that results from a similar Bergman cycloaromatization reaction enhanced under the same hypoxic conditions that reduced (14, 15). Although DSBs induced by either NCS or C-1027 are its DNA break induction. Although the unique chemistry of created with a two base pair stagger, variations in alignment of the C-1027 allows it to concurrently generate both DNA breaks diradical element results in different spectrums of preferential and cross-links in normoxic cells, a low oxygen environment cleavage sites (16–18). represses the former and promotes the latter. Thus, treatment In addition to DSBs, a little known aspect of certain enediynes is with C-1027 offers a facile approach for overcoming the that they can form monofunctional and bifunctional adducts with radioresistance associated with poorly oxygenated cells. cell-free oligonucleotides under anoxic conditions (19, 20). [Cancer Res 2009;69(2):593–8] Although the mechanism of adduct formation remains unclear, it is thought that the deoxyribose radical cannot convert to a Introduction hydroxyl radical in the absence of oxygen. Instead, it binds back to the drug, forming a DNA adduct. That adduct formation under It has long been recognized that hypoxic cells associated with anoxic conditions is limited to certain enediynes was indicated by tumor microenvironments, as well as cells grown under conditions the fact that C-1027 can induce ICLs, whereas calicheamicin, which of low oxygen, are more difficult to kill with ionizing radiation (IR), also induces primarily DSBs, produced no detectable adducts (19). which uses oxygen for its DNA strand scission activity (1). In cells, IR and other radiomimetic agents induce cells to Essentially, the deoxyribose radicals generated by radiation respond to DNA DSBs by activation, via the phosphatidylinositol abstraction of hydrogen from DNA require sufficient oxygen levels 3-kinase–like kinase ATM, of cell cycle checkpoint proteins that to allow their conversion along a pathway leading to DNA breaks delay cell cycle progression to allow DNA repair and preserve (2, 3). Radiomimetic enediyne anticancer drugs reportedly suffer chromosomal integrity (21). However, recent studies with C-1027 the same limitation, as break induction occurs via a similar oxygen- and selected analogues revealed that robust activation of cell cycle mediated mechanism (4, 5). However, recent findings identified a checkpoints and cell death hypersensitivity was independent of family of designer enediynes, which can induce an additional type ATM or ATR status (6, 7, 22). In addition to ATM phosphorylation, of DNA damage, interstrand cross-links (ICLs). Because oxygen is these enediynes were able induce activation of the rad3 related not required for ICL formation, these enediynes may be exploited kinase, ATR generally associated with DNA damage that leads to for targeting hypoxic cells (6, 7). replication fork stalling (e.g, ICLs;ref. 23). Although IR induces a plethora of DNA damage events in This sole exception to ATM-dependent responses to DNA DSBs addition to double (DSB) and single-strand breaks (SSB), including was explained by the discovery that C-1027 exhibits a novel a variety of DNA degradation products, enediynes generally induce DNA-damaging activity in cells: an ability to induce concurrently only a subset of these lesions (frank DNA strand breaks;refs. 8–10). DSBs and ICLs in normoxic cells (24). Cellular ICLs induction by The mechanism of action of enediyne DNA break induction was C-1027 was unexpected because deoxyribose radicals should be readily converted to hydroxyl radicals in normoxic cells (25). The presence of both types of cellular DNA lesions explains why either ATM or ATR can regulate DNA damage responses (24). Requests for reprints: Terry Beerman, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY To date, C-1027 is the only drug known to generate DNA damage 14263. Phone: 716-845-3443;Fax: 716-845-1575;E-mail: Terry.Beerman@ that is balanced between DNA breaks and ICLs in a normoxic roswellpark.edu. I2009 American Association for Cancer Research. cellular environment. In a hypoxic environment, C-1027 might be doi:10.1158/0008-5472.CAN-08-2753 expected to induce fewer DSBs, whereas ICL formation, which www.aacrjournals.org 593 Cancer Res 2009; 69: (2). January 15, 2009

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Cancer Research typically is impeded by oxygen, should be robust. This study plate and stirred gently in the hypoxia chamber that had been pre- evaluated the oxygen dependence of C-1027 induction of DNA equilibrated to 0.5% O2 and 5% CO2, unless otherwise indicated. After 1 h, breaks and cross-links and whether C-1027 would overcome the HCT116 cells grown under normoxic conditions were placed in the hypoxia radioresistance associated with hypoxic cells. chamber, and the cell medium was replaced with 2 mL of the pre- equilibrated hypoxic medium. Cells were covered with aluminum foil and incubated on a dry block at 37jC. After 2 h,3 enediynes diluted as described Materials and Methods above in hypoxic water were added, and incubation continued for 1 h. Chemicals. Fermentation, production, isolation, and purification of Normoxic samples were treated similarly, except they were left in a standard j C-1027 from Streptomyces globisporus were carried out as described (26). CO2 incubator at 37 C for an identical time period and treated with NCS was a gift from Bristol-Myers Squibb Co. Stock solutions of all enediynes diluted in normoxic water. compounds were stored at À20jC and diluted in double-distilled water IR treatment of cells. After drug treatment, medium was removed and before use. cells were immediately irradiated on ice under normoxic conditions using a Drug preparations. For dilutions under hypoxic conditions, aliquots of Philips RT 250 Orthovoltage X-ray Unit (GE Healthcare) with a 0.5-mm Cu drugs were placed in a BioSpherix chamber (BioSpherix, Ltd.) pre- filter at 20 Gy, and harvested. Comet analysis. After enediyne incubations with or without IR equilibrated to gas phase O2 at 0.5% unless indicated otherwise, and diluted immediately before use with water pre-equilibrated in the chamber treatment, HCT116 cells were analyzed as described previously (7). Briefly, overnight. Separate dilutions for normoxic samples were prepared at the cells were harvested by Trypsin/EDTA detachment, resuspended in PBS at a Â 7 same time. concentration of 1 10 /mL, diluted into low-melting-temperature agarose, Hypoxic conditions for cell-free assays. Tubes containing DNA and and placed on an agarose-coated slide. The slides were left on ice for 1 mmol/L Tris (pH 7.5) were placed in a hypoxia chamber equilibrated to 30 min, then immersed in alkaline lysis buffer (pH 13) and placed at 4jC overnight. Slides were equilibrated for 20 min in alkaline electrophoresis gas phase O2 at 0.5% unless otherwise indicated. Cell-free DSB and ICL detection. For DNA break detection, 100 ng of buffer before electrophoresis at 27V for 25 min at 4jC. Subsequently, slides supercoiled pBR322 plasmid DNA (New England Biolabs) was incubated were neutralized by washing in 0.4 mol/L Tris (pH 7.5), thrice for 5 min, and with drug in the presence of 1 mmol/L Tris (pH 7.5) at 37jC under hypoxic dried after immersion first in methanol and then in ethanol. Cells were visualized with ethidium bromide and imaged. Because the smaller the DNA (0.5% O2) or normoxic conditions. After incubation for 30 min, tubes were removed from the chamber and loading dye was added. The DNA was fragment the more rapid its migration through agarose, the intensity and electrophoresed on a 1% agarose gel to separate forms, visualized by length of the DNA tail (Comet) extruding from the nucleus was staining with SYBR Gold (Invitrogen Corp.), imaged on the GelDoc XR (Bio- proportional to the level of DNA breaks. Comet signals were quantified Rad Corp.), and quantified using Molecular Dynamics ImageQuant software by tail intensity expressed as a percentage of the total intensity of the comet (GE Healthcare). using Comet Assay IV software (Perceptive Instruments). Typically, 5 fields For ICL detection, EcoR1 restriction enzyme (Fermentas, Inc.) was used were imaged for each treatment condition from which 50 to 60 comets were to linearize pBR322 DNA. DNA (200 ng) and drug were incubated with selected to ensure a representative analysis. 1 mmol/L Tris (pH 7.5) at 37jC under hypoxic conditions for the Immunoblotting. After drug treatment, total cellular extracts were appropriate time. After incubation, denaturation buffer was added to prepared for Western analysis as described previously (6). Essentially, cell achieve a final concentration of 60 mmol/L NaOH, 10 mmol/L NaCl, and lysates were cleared by centrifugation and equal amounts of protein were 0.1 mmol/L EDTA under normoxic conditions for 15 min before the electrophoresed on SDS-PAGE, transferred to a polyvinylidene difluoride addition of loading dye and electrophoresis of DNA as described above. membrane, and probed with the primary antibody Anti–phospho-Histone Cells. HCT116 human colon carcinoma cells (a gift from Dr. B. gH2AX (Ser 139;Millipore) followed by a secondary antibody conjugated Vogelstein, Sidney Kimmel Comprehensive Cancer Center, John Hopkins with horseradish peroxidase (Sigma). Chemiluminescent protein bands University, Baltimore, MD) were grown in McCoy’s 5A, 10% fetal bovine were visualized on X-ray film and quantified using the Gel Doc XR System serum and Pen/strep for 24 h in 6-well dishes, 3 Â 105 cells per well, at 37jC and ImageQuant software. Cytotoxicity. Â 5 and 5% CO2. HCT116 cells were plated in 6-well dishes at 3 10 cells Hypoxic conditions for cell culture. Fifty milliliters of medium in an per well and incubated overnight. Cells were rendered hypoxic (as described uncapped sterile bottle covered with aluminum foil were placed on a stir above), or left normoxic, and enediyne was added. After 1 h, cells were detached using trypsin, counted, diluted, and replated in duplicate at 1,000,

300, or 100 cells per well. After 10 d of growth in a CO2 incubator under normoxic conditions, cells were stained and fixed in Methylene Blue/ methanol. Individual colonies of 50 or more cells were counted, and plating efficiency was calculated relative to that of untreated (control) cells. Evaluation of drug cytotoxicity was based on three or more experiments.

Results Oxygen dependence of C-1027–induced DNA damage under cell-free conditions. The discovery of the ability of C-1027 to induce ICLs was initially observed by treatment of an oligonucle- otide under cell-free anoxic conditions that suppressed DSBs formation (20). To establish whether suppression of DNA breaks occurs under hypoxia, a less stringent oxygen deprivation condition, plasmid DNA was equilibrated to 0.5% gas phase O2 before addition of C-1027. Gel electrophoretic analysis identified

3 Based on optimal suppression of enediyne-induced cellular DNA breaks, it was determined that 2 h was the minimum time necessary for the cells to achieve a Figure 1. The structure of the enediyne chromophore of C-1027. hypoxic state.

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Figure 2. C-1027 induced damage of cell-free plasmid DNA under hypoxic conditions. A, plasmid DNA was treated with varying concentrations of C-1027 for 1 h at 37jC under hypoxic (H) or normoxic (N) conditions and electrophoresed on agarose gels (see Materials and Methods). DNA breaks were shown by decreases in supercoiled form I (FI) and increases in nicked circular form II (FII) or linear form III (FIII). The percent of FI relative to total DNA is indicated for each lane. B, C-1027 cross-link activity was determined by treatment of linearized plasmid DNA with the indicated concentrations of C-1027 for 2 h at 37jC, followed by alkaline denaturation and agarose gel electrophoresis (see Materials and Methods). The upper band represents denaturation-resistant double-strand DNA and the lower band denatured DNA. ND is a nondenatured control lane, and % of ND indicates the relative percentage of nondenatured (i.e., cross-linked) duplex DNA compared with the nondenatured control. C, time-dependant cross-link induction was shown by treatment of linearized plasmid DNA with 150 nmol/L C-1027 for the indicated times at 37jC, followed by alkaline denaturation and electrophoresis as described in B.

DNA breaks based on topological forms conversion of the plasmid. radicals before they can be rendered inactive by interactions with Figure 2A reveals a significant decrease in C-1027 strand scission cellular thiols like glutathiones is limiting (1, 19, 25). To test activity in hypoxic compared with normoxic samples. For example, this prediction, cells were equilibrated to 0.5% gas phase O2, at 250 nmol/L C-1027, the amount of uncut supercoiled plasmid C-1027 treated, and assayed by Western analysis for induction of DNA in the hypoxic sample is 2.2-fold greater than the amount in phosphorylated H2AX, a surrogate marker for DSBs (26). Figure 3A the normoxic sample. A similar strategy was applied to test damage is a representative Western blot that reveals a significant decrease induction at different O2 levels. At 3% O2, full cleavage activity was in phosphorylated H2AX signaling, and, hence, DSBs, in hypoxic obtained, whereas concentrations below 0.5% O2 (e.g., < 0.2%), did versus normoxic cells treated with 0.5 to 4.5 nmol/L C-1027. The not reduce DNA breakage below that observed with 0.5% (data not shown). Recently, cross-linking of plasmid DNA under anoxic conditions was revealed by showing that C-1027–treated DNA was resistant to strand separation under alkaline conditions (24). Using a similar approach, cross-link formation was measured under hypoxic conditions that repress breaks (0.5% O2). The electrophoretic gel analysis in Fig. 2B shows C-1027’s concentration-dependent induction of cross-links after a 1-hour incubation, based on the amount of linearized plasmid DNA that resisted alkaline induced strand separation. At the highest drug level tested, 150 nmol/L, nearly 12% of the DNA failed to denature (i.e., cross-linked). Because enediyne induction of deoxyribose radicals and subsequent DNA breaks is a rapid process (i.e., breaks are detectable within several minutes), ICLs that likely arise from sugar radicals also should form rapidly (25). The time course shown in Fig. 2C was carried out using 150 nmol/L C-1027 and revealed that ICLs are readily detectable (1.5%) after 5 minutes, and continue to increase (8.5%) up to the 60-minute time point. Enediyne induction of cellular H2AX signaling under hypoxic conditions. Unlike a cell-free environment in which DNA strand scission and cross-linking activity predominate under Figure 3. C-1027 induced gH2AX phosphorylation under hypoxic or normoxic normoxic and anoxic/hypoxic conditions, respectively, in cells, conditions. A, hypoxic and normoxic HCT116 cells were each treated with C-1027 for 1 h at 37jC. Cellular extracts were analyzed by Western blotting and C-1027 has a unique ability to induce DSBs and ICLs concurrently probed with an antibody specific for phosphorylated gH2AX (see Materials (24). How formation of these lesions is influenced by a hypoxic and Methods). B, the bar graph represents the average of at least three Western cellular environment was investigated. Based primarily on cell-free blots comparing the percentage of gH2AX phosphorylation induced over a range of concentrations of C-1027 (0.5–4.5 nmol/L), NCS (100–200 nmol/L), studies with IR and radiomimetics, DSB activity should decrease in and calicheamicin (0.5–4.5 nmol/L) under hypoxic conditions relative to normoxic hypoxic cells because the oxygen required to trap deoxyribose treatments. www.aacrjournals.org 595 Cancer Res 2009; 69: (2). January 15, 2009

DNA strand separation. For example, when cells were treated with the non cross-linking member of the C-1027 family, deschloro- C-1027, followed by IR, a significant increase in the IR-induced Comet signal was observed due to the DNA breaks induced by 5nmol/L deschloro-C-1027 (e.g., 148% of IR alone). However, treatment with C-1027 produced a modest decrease in the expected C-1027 plus IR Comet signal (24). To test whether hypoxic conditions alter C-1027 cross-linking potential, cells equilibrated with 0.5% gas phase O2 were treated with C-1027 for 1 h, removed from the chamber, and irradiated before assessing DNA breaks via Comet. Figure 5A shows representative Comet images that reveal the ability of C-1027 to reduce the size of IR-generated Comets. A smaller C-1027–induced Comet was observed under hypoxic compared with normoxic conditions. Also, irradiated cells pretreated with C-1027 formed Comets that were dramatically reduced in size. Based on measurements of the Comet tail moment, increasing concentrations of C-1027 resulted in correspondingly more severe reductions in the combined drug plus IR Comet signal consistent with substantial increases in ICLs (Fig. 5B). Notably, the higher concentrations of C-1027, (i.e., 5.0 and 10 nmol/L) dramatically reduced the combined drug plus IR Comet to 10% or less of the IR Figure 4. Oxygen dependence of C-1027–induced cellular DNA breaks. A, representative alkaline Comet (see Materials and Methods) images are alone value (Fig. 5C). In striking contrast, in a previous study under shown of HCT116 cells incubated with C-1027 for 1 h under hypoxic or normoxic normoxic conditions, C-1027 plus IR Comets were 90% the conditions. B, the extent of DNA breaks is reflected by quantitation of Comet intensity of those induced by IR alone (24). For comparison, NCS tail intensity as described in Materials and Methods. The bar graph represents the average of 2 to 3experiments that measured the oxygen-dependent break activity of C-1027, NCS, and calicheamicin expressed as a percentage of break activity in hypoxic relative to normoxic cells. oxygen sensitivity of DSB repression in cells is not unexpected and was similar to that observed with other enediynes, including NCS and calicheamicin (Fig. 3B). Oxygen dependence of C-1027 induction of cellular DNA breaks. DNA damage response pathways including repair proteins are effected by hypoxia and could influence H2AX signaling (27, 28). The influence of oxygen on C-1027 break activity also was evaluated using a more direct assessment of DNA breaks. Alkaline Comet electrophoretic analysis of single cells can be used to show the extent of DNA breaks, which are proportional to the length of the DNA tail extruding from the nucleus (29). Figure 4A reveals a significant reduction in the Comet signals of cells treated under hypoxic versus normoxic conditions. Quantitation of the extent of DNA break repression as a function of oxygen is in general agreement with that identified by Western analysis. Like the cell- free experiments, significant repression and full restoration of cellular DNA damage induction were observed at 0.5% O2 and 3% O2, respectively (Fig. 4B). C-1027 induction of cellular ICLs under hypoxic conditions. DSB suppression under low-oxygen conditions suggests that deoxyribose radicals are not efficiently converted to hydroxyl radicals/DNA breaks. If so, the formation of ICLs rather than DSBs may be expected under hypoxic but not normoxic conditions. In a prior study, Comet analysis was used to detect not only C-1027 induced DNA breaks but also cross-links (24). In those experiments, cells were treated sequentially with C-1027 and IR. If C-1027 Figure 5. Induction of ICLs by C-1027 in hypoxic cells. A, representative caused only DSBs, the Comet score resulting from the combination alkaline Comet images are shown that assess ICLs induction in hypoxic HCT116 of C-1027 and IR treatment should equal the sum of the scores cells treated with C-1027 or NCS for 1 h and then mock treated or exposed to 20 Gy IR as indicated. B, comets were scored as described in Materials and obtained with each agent individually. If C-1027 induced not only Methods. Black dashed line, the Comet signal of IR alone. Induction of ICLs strand breaks but also ICLs, the Comet signal would be noticeably is indicated by the extent of reduction in the IR Comet tail signal. NCS, an enediyne that does not induce ICLs, is included as a negative control. C, the less than the sum of the individual scores because cross-links bar graphs represent averages from two to three experiments and indicate the would render IR-fragmented DNA resistant to alkaline-induced percent of the IR tail intensity signal in the presence of C-1027 or NCS.

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(as well as calicheamicin;data not shown), which lacks an ability to cross-link, produced a corresponding increase in the drug plus IR signal to 160% of the IR alone signal under hypoxic conditions (Fig. 5C). Cytotoxicity of enediynes under normoxic and hypoxic conditions. Treatment with IR or radiomimetics such as esperamicin or NCS results in diminished cytotoxicity in hypoxic compared with normoxic cells, presumably due to reduced DSBs formation (1, 30). To corroborate these findings, we treated HCT116 cells with NCS under hypoxic conditions that we determined would suppress break induction (data not shown) and then measured cytotoxicity based on a colony formation assay carried out under normoxic conditions. Figure 6B shows that survival was increased when cells were treated with NCS under hypoxic compared with normoxic conditions. By contrast, a similar experiment showed that survival was decreased when cells were treated with C-1027 under hypoxic compared with normoxic conditions (Fig. 6A). Overall, hypoxic cells were nearly 4-fold more resistant to NCS, but 3-fold more sensitive to C-1027 cytotoxicity, than were normoxic cells. Thus, killing of hypoxic cells by C-1027 is enhanced about an order of magnitude over that of the conventional radiomimetic drug NCS.

Discussion Figure 6. Oxygen dependence of enediyne cytotoxicity. HCT116 cells were The paradigm for the interaction of aromatized enediyne core incubated under hypoxic or normoxic conditions for 1 h with either (A) C-1027 or diradicals with DNA depicts deoxyribose radicals generated from (B) NCS. Cells were collected, replated, and grown for 10 d under normoxic hydrogen abstraction reactions culminating in DNA breaks through conditions. Cells were stained, and cell colonies were counted. Colony formation was expressed as plating efficiency relative to control cell samples (see Materials an oxygen mediated mechanism (31). The unprecedented ability and Methods). A and B compare C-1027 and NCS cytotoxicity, respectively, of C-1027 to induce both DSBs and ICLs in cells, an apparent under hypoxic and normoxic treatment conditions. contradiction to the current model, prompted an investigation of the oxygen dependence of its DNA-damaging activities. Implicitly, the fate of C-1027 generated sugar radicals seems balanced induction in hypoxic cells is increased and is well above levels between completing the reactions required to induce DNA breaks detected in earlier studies carried out in normoxic cells (Fig. 5;24). or binding back to the drug to form a cross-link. If the DNA In both cell-free and cellular environments, hypoxia results in damage shifts toward ICLs in low-oxygen environments, then C- decreased oxidation of deoxyribose radicals, which preserves their 1027 unlike IR has the potential to target cells within hypoxic potential to rebind the drug and form ICLs (19, 25). It had been microenvironments associated with many solid tumors (1). presumed that even in cellular environments with available The inverse relationship between the DSB and ICL activity of deoxyribose radicals, the presence of hydrogen donors such as C-1027 is apparent from cell-free experiments in which breaks are glutathiones would readily lead to quenching. Although conditions suppressed under the hypoxic conditions (0.5% O2) required for promoting deoxyribose radicals rebinding to the drug are not well- cross-link formation (Fig. 2A). The findings are consistent with understood, positioning of the drug within the DNA minor groove, observations from the Goldberg laboratory that under anoxic availability of oxygen, as well as the presence of reducing factors conditions, C-1027 generated deoxyribose radicals can rebind to are considered critical (19). The oxidizing environment within portions of the aromatized enediyne core positioned within the normoxic cells seems compatible with generation of both C-1027– DNA minor groove leading to ICL formation (19, 20). Although induced breaks and cross-links (24). The repressed oxidative state the 50% reduction of DSBs under hypoxic conditions is less than in hypoxic cells apparently promotes the ICL activity of the drug at the decreases (>80%) reported for anoxia, it is apparently sufficient the expense of DNA breaks. Although C-1027 provides a novel to allow ICLs to form (20, 31). Also, like DNA breaks, ICLs form agent for targeting DNA in low-oxygen environments, the basis for rapidly (Fig. 2C), which supports the notion that both are generated its unique DNA-damaging ability in normoxic and hypoxic cells is from unstable deoxyribose radicals, which need to commit to unclear. All enediynes, including C-1027, trigger a DNA breakage either type of DNA damage to avoid inactivation (25). reaction by going through a cycloaromatization process via a Similar to cell-free experiments, cells treated with C-1027 show Myers-Saito or Bergman-type rearrangement resulting in biradicals substantial decreases in DSBs under hypoxic conditions based on capable of position-specific hydrogen abstraction from the either gH2AX activation or tail Comet intensity (i.e., 45% and 60% deoxyribose backbone of DNA (32). At present, predicting whether decreases, respectively;Figs. 3 and 4). 4 At the same time, ICL an enediyne has the potential to induce DSBs, ICLs, or both is not possible. Although all known enediynes (with the exception of the engineered C-1027 analogue, desmethyl-C-1027) induce robust amounts of DSBs, an accompanying ICL activity may depend on 4 Because C-1027 induces breaks and cross-links, the more robust reduction in comet signal could be due in part to increased ICL activity, which would also tend to the reactivity as well as the proximity and steric availability of the depress the comet score. aromatized enediyne chromophore to the deoxyribose radicals (7, www.aacrjournals.org 597 Cancer Res 2009; 69: (2). January 15, 2009

19, 24). The precise mechanistic reasons for the differing activities ICLs under hypoxic conditions that approximate a radioresistant of C-1027 and its designer analogues also are unclear. However, environment (1% O2 or lower) for many types of human tumors that the desmethyl-C-1027 analogue predominately induces ICLs suggests a potential for using C-1027 based enediynes to target indicates the potential for fine tuning the DNA-damaging activity hypoxic cells within tumors (1, 33). These findings also highlight of enediynes by introducing minimal structural perturbation. the potential for rational engineering of C-1027 as well as other Similarly, other C-1027 analogues with modifications of the enediyne biosynthetic pathways based on SAR studies to yield enediyne core, such as deschloro-C-1027, induce primarily cellular new generations of enediyne analogues that could be explored as DSBs and not ICLs (24). Interestingly, the degree to which C-1027 antitumor (34). and its analogues induce DSBs, ICLs, or both correlates with whether DNA damage responses are regulated by ATM, ATR, or both, respectively (6, 7, 24). Disclosure of Potential Conflicts of Interest Treatment of hypoxic cells with NCS, as with IR or other No potential conflicts of interest were disclosed. radiomimetics, decreased DNA breaks, and a 4-fold drop in cytotoxicity was observed (Fig. 6B). Although it is difficult to Acknowledgments judge the cytotoxic potential of DSBs versus ICLs, the DNA Received 7/18/2008;accepted 11/6/2008. damage induced by C-1027 revealed decreased breaks and Grant support: National Cancer Institute research grant CA106312 & National elevated cross-links and a corresponding 2.7-fold increase in Cancer Institute Center Support grant CA16056 and Roswell Park Alliance Foundation cytotoxicity in hypoxic compared with normoxic cells (Fig. 6A). (T.A. Beerman) and NIH grants CA078747 & CA113297 (B. Shen). The costs of publication of this article were defrayed in part by the payment of page Thus, increased ICLs production seemed to correlate with charges. This article must therefore be hereby marked advertisement in accordance increased cytotoxicity. That C-1027 DNA damage shifts toward with 18 U.S.C. Section 1734 solely to indicate this fact.

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Terry A. Beerman, Loretta S. Gawron, Seulkih Shin, et al.

Cancer Res 2009;69:593-598.

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