Spotlight In Search of DNA Damage DNA glycosylases initiate base excision repair, which is responsible for the removal and replacement of the majority of damaged DNA bases. Most DNA glycosylases are highly specific, but the 3-methyladenine DNA glycosylases, such as AlkA from E. coli, show broad specificity for alkylated bases. To understand how these enzymes recognize such a large number of lesions, Bowman et al. [(2010) J. Biol. Chem., published online Sep 15, DOI: 10.1074/jbc.M110.155663] have obtained the structure of AlkA in complex with undamaged DNA. Bowman et al. engineered a disulfide cross-link between the DNA substrate and AlkA tyr-239 in order to fix the position of the DNA relative to the enzyme’s active site. The resulting structure revealed DNA interacting with AlkA’s HhH DNA binding domain through hydrogen bonds between the DNA’s phosphate backbone and the protein’s main chain. AlkA’s Leu-125, which plays a key role in interrogating the DNA structure for lesions, lay in the minor groove of
Blocking Transcription with Pt The platinum-based drugs cisplatin, carboplatin, and oxaliplatin are widely used and highly effective cancer chemotherapeutic agents. The cytotoxicity of these compounds is dependent upon their ability to form DNA crosslinks. In vitro studies indicate that one outcome of platinum-DNA cross-links is transcription blockade. Now, Ang et al. [(2010) J. Am. Chem. Soc, 132, 7429] have generated reporter plasmids bearing platinum-DNA cross-links in order to study their impact on transcription in intact cells. Ang et al. used the reporter plasmid pGLuc, containing the gene for Gaussia luciferase (GLuc) driven by the CMV promoter. GLuc requires no post-translational modifications and is secreted by the cell, so reliable gene expression data may be obtained by assaying luciferase activity in the culture medium. Incubation of pGLuc with different concentrations of cisplatin produced cross-links at levels of 0 to 8.6 × 10-3 adducts/nucleotide. Ang et al. measured expression levels of these modified plasmids in XPF human fibroblasts (XPF), which are deficient in the XPF protein required for nucleotide excision repair (NER). Transfection of XPF cells with the human XPF gene produced the NER proficient XPFcorr cell line, which was used for comparison. Eight hours following transfection, the expression of platinated pGLuc was markedly suppressed in both XPF and XPFcorr cells. The degree of suppression was directly related to the level of platination. Over time, both cell lines demonstrated increased ability to express the platinated 1860
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the DNA helix, which was straight and showed no disruption of base pairing of the interrogated base. This structure contrasts markedly with that of AlkA in complex with DNA containing a transition state mimic, which shows that the lesion base had extruded into the enzyme active site, Leu-125 had inserted into the helix to take its place, and a sharp bend was present in the helix at the lesion. Modeling studies suggested that close proximity of Leu-125 to the methyl group of 3-methyladenine might explain how this alkylated base is recognized by the enzyme, a hypothesis that was supported by biochemical studies. Together, the data suggest that Leu-125 scans the minor groove of DNA in search of altered bases, either by capturing extrahelical bases or by promoting their extrusion. The rate of removal of the lesion is then likely dependent on the stability of the glycosidic bond, which is often weak in alkylated bases. • Carol A. Rouzer
plasmids; however, the recovery was much greater for XPFcorr than for XPF cells. Next, Ang et al. generated pGLuc containing a sitespecific 1,2-d(G*pG*)-PtA2 or 1,3-d(G*pTpG*)-PtA2 cross-link, where A2 refers to the ammine ligands of cisplatin, or a 1,2d(G*pG*)-Pt(dach) or 1,3-d(G*pTpG*)-Pt(dach) adduct, where dach refers to the R,R-1,2-diaminocyclohexane ligand of oxaliplatin. In each case, the cross-link was inserted between the promoter and the GLuc gene of pGLuc. Transfection into cells confirmed that all four crosslinks caused the inhibition of GLuc expression. Suppression was greater for 1,3 cross-links than for 1,2 cross-links and greater for dach-containing cross-links than A2-containing cross-links. XPF cells exhibited greater suppression of the expression of all plasmids than did XPFcorr cells.
Reprinted from Ang et al. (2010) J. Am. Chem. Soc, 132, 7429. Copyright 2010 American Chemical Society.
Together, the results confirm that platinum-based crosslinks suppress transcription in intact cells and reveal that cross-links generated from oxaliplatin are more inhibitory than those generated from cisplatin. The results also suggest that transcription-coupled NER plays an important role in Published online 12/20/2010 • DOI: 10.1021/tx1003672 © 2010 American Chemical Society
Spotlight the removal of these cross-links, allowing cells to recover from platinum-induced damage. • Carol A. Rouzer
further studies on the physiological role of the AHR in hematopoiesis. • Carol A. Rouzer
AHR’s Link to Hematopoiesis
Protective Chaperones
The aryl hydrocarbon receptor (AHR) is well known to toxicologists as a mediator of the increased expression of key drug and xenobiotic metabolizing enzymes upon exposure to target molecules. However, recent evidence indicates that the AHR also plays a role in developmental processes. Now, Boitano et al. (2010) Science, 329, 1345] demonstrate the influence of AHR signaling on the differentiation of hemopoietic stem cells (HSCs).
HSC transplants are used in the treatment of diseases of the blood or bone marrow as well as some forms of cancer; however, the availability of this form of therapy is often limited by the difficulty of finding an appropriately matched donor. Banked cord blood (CB) would be a potential source of HSCs for many patients if the cells could be grown under culture conditions that selectively increase the number of HSCs, which are identified by the surface expression of the marker CD34. Culture conditions that are currently used for HSC expansion lead to an increased number of cells but a loss of CD34 expression. A screen of 100,000 compounds led to the discovery of SR1 as a compound that conserves CD34 expression during the culture of HSCs from CB. SR1 did not directly induce proliferation or change division time in CB-derived CD34+ cell cultures, suggesting that it increases the number of HSCs by preventing differentiation. Incubation of CB CD34+ cells with SR1 led to an increased proportion of cells capable of forming colonies of all blood cell lineages and to an increased success rate in a mouse model of HSC transplantation. Transcriptional profiling of SR1-treated CD34+ cells indicated that the compound inhibited the expression of the AHR-dependent genes CYP1B1 and AHRR. Further studies showed that SR1 directly binds to the AHR and that it blocks AHR-dependent transcription responses to the agonist dioxin. The structurally similar compound LGC006, which had no effect on CD34+ cell expansion, showed poor activity in AHR binding and functional assays. Boitano et al. went on to show that two other AHR antagonists and RNAi knockdown of AHR expression promote retention and expansion of CD34+ cells in culture. Furthermore, SR1 failed to induce CD34+ cell expansion in cultures of cells expressing a constitutively active AHR. Together, the results suggest that AHR suppression promotes the retention of HSCs in an undifferentiated state. The exact mechanism by which this occurs remains unknown, but the data provide a strong foundation for Published online 12/20/2010 • DOI: 10.1021/tx1003672 © 2010 American Chemical Society
Nonsteriodal anti-inflammatory drugs (NSAIDs) are among the most widely used pharmaceutical agents, available both over the counter (e.g., aspirin, ibuprofen, and naproxen) and by prescription. The most serious clinical toxicity resulting from NSAID use is gastric ulceration, which has been attributed to the drugs’ primary mode of action, the inhibition of cyclooxygenase enzymes. Recent work suggests, however, that the mechanism of gastric ulceration may be more complex, involving the induction of apoptosis through activation of the endoplasmic reticulum (ER) stress response pathway. This mechanism was explored in greater detail by Namba et al. [(2010) J. Biol. Chem., published online Sep. 22, DOI:10.1074/jbc.M110.148882] who also evaluated the interaction between Helicobacter pylori infection and NSAID-induced ulceration. Incubation of AGS gastric cells with live H. pylori resulted in a decreased mRNA and protein expression of the ER chaperones ORP150 and GRP78, which protect against protein damage during the ER stress response. This reduction in chaperone expression was attributed to a decrease in levels of the ER stress-activated transcription factor p90ATF6. Studies using selective inhibitors indicated that H. pylori-induced decreases in p90-ATF6 levels were due primarily to proteasomal- and lysosomal-mediated degradation of the protein. In contrast to the decreased levels of ORP150 and GRP78, incubation of H. pylori with AGS cells induced the expression of CHOP, an ER stress-activated proapoptotic protein. Prior studies had indicated that increased expression of ORP150 and GRP78 in the presence of NSAIDs helps to protect gastric cells against NSAID-mediated apoptosis. Namba et al. showed that H. pylori blocks this increase in chaperone expression and actually results in decreased expression in the presence of NSAIDs. Thus, H. pylori exposure eliminates a possible mode of cellular protection against NSAID toxicity. The importance of ER chaperones was confirmed in vivo. Mice inoculated with H. pylori exhibited decreased gastric levels of the mRNA for ORP150 and GRP78, and H. Pylori reversed the increased expression of these chaperones induced by the NSAID indomethacin. H. pylori inoculation alone did not produce gastric ulcers in mice, but the bacterial exposure increased the number of apoptotic cells in gastric mucosa and gastric ulceration in mice treated with indomethacin. Similarly, mice genetically deficient in ORP150 exhibited an increased susceptibility to indomethacin-induced gastric ulcers. Namba et al. conclude that ER chaperones play a significant role in protecting gastric cells against NSAIDinduced apoptosis and consequent ulceration. H. pylori exacerbates ulcer formation by blocking the transcription of these protective proteins. • Carol A. Rouzer TX1003672 Vol. 23,
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