SPOTLIGHT pubs.acs.org/crt
ZINC FINGER TARGETS OF ARSENIC In addition to acting as a complete carcinogen, arsenic promotes the carcinogenicity of other genotoxic agents. A possible mechanism by which this occurs is through inhibition of DNA repair enzymes. For example, arsenic binds to the zinc finger domain of poly(ADP-ribose) polymerase-1 (PARP-1) and inhibits its activity. Now, Zhou et al. [(2011) J. Biol. Chem., published online May 5, DOI: 10.1074/jbc.M111.232926] explore the interaction of arsenic with zinc finger proteins in greater detail. PARP-1 contains two zinc fingers, both comprising three cysteine residues in a CCHC motif. Peptides containing either one of these sequences bound As(III) with all three cysteine residues, as indicated by MS analysis. Mutation of the zinc finger to CHHC or CCHH eliminated As(III) binding, whereas binding was retained with a CCCC sequence-containing peptide. Similarly, a peptide constructed from the zinc finger of the DNA repair protein APTX, which contains two cysteine residues (CCHH), did not bind As(III), though binding to this peptide was observed after mutation of the zinc finger to CCHC. All of the test peptides bound Zn(II), confirming the presence of a functional zinc finger. These results suggested that a minimum of three cysteine residues is required for As(III) binding. Incubation of the human keratinocyte cell line (HaCaT) with As(III) resulted in a reduction in the Zn(II) content of immunoprecipitated PARP-1. EMSA analysis revealed reduced binding of PARP-1 to DNA in As(III)-treated cells as compared to controls. In As(III)-treated HaCaT cells, a similar reduction in Zn(II) content occurred for the DNA repair protein XPA, which contains a CCCC zinc finger but not for SP1 or APTX, which contains a CCHH zinc finger. The As(III)-dependent reduction in Zn(II) content was eliminated in the case of PARP-1 containing zinc fingers bearing CCHH or HCHC mutations. Together, the results confirm that arsenic may promote mutagenicity by blocking DNA repair, but only in the cases of repair enzymes containing a C3H or C4 zinc finger structure. Carol A. Rouzer ’ SHEDDING LIGHT ON DNA DAMAGE
will form, yielding a luminescence signal upon addition of substrate. Furman et al. first tested their sensors using a 23-mer oligonucleotide containing one methyl-CpG dinucleotide. Treatment of the oligonucleotide with H2O2/Cu2þ led to a 5-fold increase in the luciferase signal over untreated oligonucleotide upon incubation with the MBD-Nluc/Cluc-OGG1 sensor. The MBD-Nluc/Cluc-DDB2 sensor exhibited a 45-fold increase in response to UV-exposed oligonucleotide as compared to the unexposed control. Greater responses were obtained using the 7667 base pair plasmid pETDuet, following enzymatic methylation and either oxidation or UV exposure. Neither sensor responded to unmethylated DNA. The MBD-Nluc/Cluc-OGG1 sensor exhibited a 32-fold response to 50 ng of human HeLa cell DNA following exposure to 1 mM H2O2/60 μM Cu2þ for 10 min. ELISA indicated that DNA treated in this way contained ∼200 fmol of 8-oxoguanine. A 2 h exposure of HeLa DNA to UV elicited a 100-fold increase in signal in the MBD-Nluc/Cluc-DDB2 sensor. DNA isolated from intact HeLa cells following a 10 min UV exposure produced a 25-fold response using the MBD-Nluc/Cluc-DDB2 sensor. The MBDNluc/Cluc-OGG1 sensor exhibited a 75% higher response for oxidized DNA than for UV-treated DNA, while the MBD-Nluc/ Cluc-DDB2 sensor’s response was 67% higher for UV-treated than oxidized DNA. This cross-specificity was likely due to the ability of either treatment to induce more than one kind of damage or to the ability of each binding protein to recognize multiple lesions. Together, the data support the use of this approach as a rapid and reliable DNA damage detection method requiring minimal DNA processing. Carol A. Rouzer
Reprinted from Furman et al. (2011) J. Am. Chem. Soc., published online April 26, DOI: 10.1021/ja1116606. Copyright 2011 American Chemical Society.
The human genome is subject to numerous chemical and physical insults, leading to >104 lesions per cell per day. Because of the toxic potential of these lesions, the ability to measure DNA damage rapidly and accurately is an ongoing concern. Now, Furman et al. [(2011) J. Am. Chem. Soc., published online April 26, DOI: 10.1021/ja1116606] address that concern by introducing DNA damage sensors based on a split luciferase platform. Furman et al. used a cell free system to express the N-terminal fragment of firefly luciferase attached to a methyl-CpG binding domain (MBD-Nluc) and the C-terminal fragment of the luciferase attached to a DNA damage binding domain. The DNA damage binding domains were derived from oxoguanine glycosylase 1 (Cluc-OGG1), which binds to 8-oxoguanine, a product of oxidative damage, and damaged DNA binding protein 2 (Cluc-DDB2), which recognizes photoproducts arising from UV exposure. Since 5080% of CpG sites in DNA are methylated, the MBD-Nluc fragment binds at numerous locations in the genome. The DNA damage binding domain directs the ClucOGG1 or Cluc-DDB2 fragment to a site of damage. If the two fragments are sufficiently close together, a functional luciferase r 2011 American Chemical Society
Published: July 18, 2011 990
dx.doi.org/10.1021/tx200227x | Chem. Res. Toxicol. 2011, 24, 990–991
Chemical Research in Toxicology
’ GNF531: A COMPLETE AHR ANTAGONIST
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor well known to toxicologists for its regulation of the expression of some xenobiotic metabolizing enzymes. However, the AHR also plays a role in the immune response, reproduction, and development. Many AHR-controlled processes occur through its ligand-dependent binding to dioxinresponse elements (DREs) in the promotor regions of target genes. However, the recent discovery of selective AHR modulators (SAhRMs), which mediate some AHR-dependent but DRE-independent processes, suggests multiple mechanisms of action for this transcription factor. To better understand AHR function, Smith et al. [(2011) J. Pharmacol. Exp. Ther., published online April 20, DOI:10.1124/pet.110.178392] have discovered and characterized GNF531, a highly potent pure AHR antagonist. Smith et al. started with a previously known AHR antagonist, SR1, which was potent only against the human receptor. Chemical modification led to GNF531, a structurally similar compound that showed equal potency to SR1 in a binding assay based on displacement of a photoaffinity AHR ligand. Using a mixture of assays based on DRE-driven luciferase expression and determination of AHR-dependent gene expression by PCR, Smith et al. showed that GNF531 has no DRE-dependent agonist activity and that it antagonizes the action of the highly potent xenobiotic agonist TCDD (see figure) and the endogenous agonist 3-indoxyl sulfate. Unlike SR1, GNF531 displayed roughly equal affinity to both the human and the mouse AHR. Using repression of IL-1β- and IL-6-stimulated expression of the acute phase gene SAA1 as an assay for SAhRM activity, Smith et al.. showed that GNF531 lacks this activity and antagonizes the action of the known SAhRM, SGA360 (see figure). These findings were confirmed in vivo in a murine model of 12-Otetradecanoylphorbol 13-acetate-induced ear edema, which is suppressed by SAhRMs. The potency and efficacy of GNF531 compared favorably to those of other known AHR ligands, and time course studies revealed that its antagonism of TCDD-mediated AHR activation remained effective for up to 12 h. Molecular modeling indicated high affinity binding of GNF531 to both the human and murine AHR ligand binding pocket. As a result of this work, Smith et al. define three kinds of AHR ligands, full agonists, such as TCDD that mediate both DRE-dependent and SAhRM responses, SAhRMs, which mediate non-DRE-dependent responses only, and complete antagonists. To date, GNF531 is one of the best characterized and potent complete antagonists. This compound will serve as a useful probe for future studies of AHR function. Carol A. Rouzer
SPOTLIGHT
’ IMPORTANCE OF γH2AX IN DNA REPAIR The histone H2AX plays a well-characterized role in the DNA damage response to double strand breaks (DSBs). Phosphorylation at serine-139 generates γH2AX, which becomes the focal point for assembly of the DNA repair protein complex. So well understood is this pathway that staining for γH2AX has become a familiar way to identify and quantify sites of DNA damage. However, it is not clear to what degree H2AX plays a role in the repair of damage that does not involve DSBs. Now, Revet et al. [(2011) Proc. Natl. Acad. Sci. U.S.A., published online May 9, DOI: 10.1073/pnas.1105866108] provide the answer. Revet et al. constructed a set of isogenic mouse cell lines in which the H2AX gene was deleted and replaced with nothing (knockout), the wild-type H2AX gene (wild-type), or an S139A mutant H2AX gene (mutant) incapable of being converted to γH2AX. They then exposed these cells to DNA damaging agents, including γ-rays, etoposide (topoisomerase inhibitor), rotenone (reactive oxygen species generator), temozolamide (alkylating agent), cisplatin (intrastrand cross-linking agent), mitomycin C (interstrand cross-linking agent), and UV (photoproduct generator). They discovered that, compared to wild-type, both mutant and knockout cells showed increased sensitivity to γ-rays, etoposide, rotenone, and temozolamide. All of these agents can directly induce DSBs or may form them indirectly during the process of DNA repair. The sensitivity of mutant and knockout cells to mitomycin C and cisplatin was no different from that of wild-type cells, while the knockout but not the mutant cells showed increased sensitivity to UV. These results clearly indicated that for some forms of DNA damage, such as those inflicted by cisplatin and mitomycin C, neither H2AX nor γH2AX play a critical role in repair or survival. In the case of UV, H2AX appears to play a role, but via a mechanism other than phosphorylation to γH2AX. Revet et al. next exposed their wild-type cells to LD50 doses of each DNA damaging agent and measured the level of γH2AX formation. The remarkable outcome of this experiment was the demonstration that γH2AX formation did not correlate with its role in cell survival. Thus, the highest levels of γH2AX were observed in UVtreated cells, for which γH2AX was not required. In contrast, γH2AX formation did not rise above the level observed in untreated cells in the case of the γ-ray, rotenone, or temozolamide treatments, and only small increases in γH2AX were observed in etoposide-treated cells. The results indicate that γH2AX is not involved in the repair of all kinds of DNA damage. Indeed, H2AX itself is not required in some cases. They also demonstrate that there is no clear correlation between the level of γH2AX formed in response to DNA damage and the importance of its role in DNA repair or survival. The authors consequently caution that γH2AX alone cannot be used as a priori evidence for DSBs, and its uncritical use is potentially misleading. Carol A. Rouzer
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dx.doi.org/10.1021/tx200227x |Chem. Res. Toxicol. 2011, 24, 990–991
