Manfred Wiessler

Epigenetic Reactivation of Tumor Suppressor Genes by a Novel Small-Molecule Inhibitor of Human DNA Methyltransferases

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.

The anticancer agent ellipticine on activation by cytochrome P450 forms covalent DNA adducts.

Ellipticine is a potent antitumor agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Using [3H]-labeled ellipticine, we observed substantial microsome (cytochrome P450)-dependent binding of ellipticine to DNA. In rat, rabbit, minipig, and human microsomes, in reconstituted systems with isolated cytochromes P450 and in Supersomes containing recombinantly expressed human cytochromes P450, we could show that ellipticine forms a covalent DNA adduct detected by [32P]-postlabeling. The most potent human enzyme is CYP3A4, followed by CYP1A1, CYP1A2, CYP1B1, and CYP2C9. Another minor adduct is formed independent of enzymatic activation. The [32P]-postlabeling analysis of DNA modified by activated ellipticine confirms the covalent binding to DNA as an important type of DNA modification. The DNA adduct formation we describe is a novel mechanism for the ellipticine action and might in part explain its tumor specificity.

The Anticancer Drug Ellipticine Forms Covalent DNA Adducts, Mediated by Human Cytochromes P450, through Metabolism to 13-Hydroxyellipticine and Ellipticine N2-Oxide

Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N2-oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N2-oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N2-oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N2-oxide.

Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid-DNA adducts found in patients with urothelial cancer.

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individuals susceptibility to this plant carcinogen. Using the 32P postlabeling assay, we examined the ability of microsomal samples from 8 human livers and from 1 human kidney to activate AAI, the major component of the plant extract AA, to metabolites forming adducts in DNA. Microsomes of both organs generated DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7‐(deoxyadenosin‐N6‐yl)aristolactam I, 7‐(deoxyguanosin‐N2‐yl)aristolactam I and 7‐(deoxyadenosin‐N6‐yl)aristolactam II were identified as AA‐DNA adducts formed from AAI by all human hepatic and renal microsomes. To define the role of human microsomal enzymes in the activation of AAI, we investigated the modulation of AAI‐DNA adduct formation by cofactors and selective inhibitors of microsomal reductases, cytochrome P450 (CYP) enzymes, NADPH:CYP reductase and NADH:cytochrome b5 reductase. We also determined whether the activities of CYP and NADPH:CYP reductase in different human hepatic microsomal samples correlated with the levels of AAI‐DNA adducts formed by the same microsomal samples. On the basis of these studies, we attribute most of the activation of AAI in human hepatic microsomes to CYP1A2. In contrast to human hepatic microsomes, in human renal microsomes NADPH:CYP reductase is more effective in AAI activation. In addition, prostaglandin H synthase is another enzyme activating AAI in renal microsomes. The results demonstrate for the first time the potential of microsomal enzymes in human liver and kidney to activate AAI by nitroreduction.

DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by 32P-postlabeling

Diesel exhaust is known to induce tumors in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust and in airborne particulate matter, 3‐nitrobenzanthrone (3‐NBA), is a particularly powerful mutagen. We investigated the capacity of 3‐NBA to form DNA adducts in vivo that could be used as agent‐specific biomarkers of exposure. Female Sprague‐Dawley rats were treated orally with 2 mg/kg body weight of 3‐NBA, and DNA from various organs was analyzed by 32P‐postlabeling. High levels of 3‐NBA‐specific adducts were detectable in all organs. Both enrichment versions nuclease P1 digestion and n‐butanol extraction resulted in patterns consisting of either 3 or 4 adducts remarkably similar in all tissues examined. The highest level of DNA adducts was found in the small intestine (38 adducts per 108 nucleotides) followed by forestomach, glandular stomach, kidney, liver, lung and bladder. To provide information on the nature of the adducts formed in vivo in rats, DNA adducts were cochromatographed in 2 independent systems with standardized deoxyguanosine adducts and deoxyadenosine adducts produced by reaction of 3‐NBA in the presence of xanthine oxidase with deoxyribonucleoside 3′‐monophosphates in vitro. In both systems, each of the rat adducts comigrated either with a deoxyguanosine or a deoxyadenosine‐derived 3‐NBA adduct. Our results demonstrate that 3‐NBA binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vivo, all of which are products derived from reductive metabolites bound to the purine bases (deoxyguanosine 60% and deoxyadenosine 40%).

Site-Specific One-Pot Dual Labeling of DNA by Orthogonal Cycloaddition Chemistry

Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.

Inverse-electron-demand Diels-Alder reaction as a highly efficient chemoselective ligation procedure: synthesis and function of a BioShuttle for temozolomide transport into prostate cancer cells.

Hormone‐refractory prostate cancer (HRPC), insensitive to most cytostatic interventions, features low response rates and bad prognosis. Studies with HRPC treated with temozolomide (TMZ) showed a poor response and the results were discouraging. Therefore, TMZ has been considered to be ineffective for the treatment of patients with symptomatic and progressive HRPC. A solution to this problem is demonstrated in this study by combining proper solid‐phase peptide synthesis and a chemoselective new ‘click’ chemistry based on the Diels–Alder reaction with ‘inverse‐electron‐demand’ (DARinv) for the construction of a highly efficient TMZ‐BioShuttle in which TMZ is ligated to transporter and subcellular address molecules. The transport to the targeted nuclei resulted in much higher efficiency and better pharmacological effects. The reformulation of TMZ to TMZ‐BioShuttle achieved higher in vitro killing of prostate cancer cells. Accordingly, the potential of TMZ for the treatment of prostate tumors was dramatically enhanced even in a tenfold lower concentration than applied normally. This TMZ‐BioShuttle may be well suited for combining chemotherapy with other cytostatic agents or radiation therapy. Copyright

DNA adduct formation by the anticancer drug ellipticine in rats determined by 32P postlabeling

Ellipticine is a potent antineoplastic agent whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts in vitro and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). Here, we investigated the capacity of ellipticine to form DNA adducts in vivo. Male Wistar rats were treated with ellipticine, and DNA from various organs was analyzed by 32P postlabeling. Ellipticine‐specific DNA adduct patterns, similar to those found in vitro, were detected in most test organs. Only DNA of testes was free of the ellipticine‐DNA adducts. The highest level of DNA adducts was found in liver (19.7 adducts per 107 nucleotides), followed by spleen, lung, kidney, heart and brain. One major and one minor ellipticine‐DNA adducts were found in DNA of all these organs of rats exposed to ellipticine. Besides these, 2 or 3 additional adducts were detected in DNA of liver, kidney, lung and heart. The predominant adduct formed in rat tissues in vivo was identical to the deoxyguanosine adduct generated in DNA by ellipticine in vitro as shown by cochromatography in 2 independent systems. Correlation studies showed that the formation of this major DNA adduct in vivo is mediated by CYP3A1‐ and CYP1A‐dependent reactions. The results presented here are the first report showing the formation of CYP‐mediated covalent DNA adducts by ellipticine in vivo and confirm the formation of covalent DNA adducts as a new mode of ellipticine action.

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase - 32P-postlabeling analysis of DNA adduct formation

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the (32)P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (alpha-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.

Mutagenicity of the two main components of commercially available carcinogenic aristolochic acid in Salmonella typhimurium

One of the 2 main components of the commercially available carcinogenic aristolochic acid (AA) was isolated, the other was enriched. Three different aristolochic acid samples (AAI 99% pure; AAI 65% + AAII 35%; AAI 32% + AAII 68%) were assayed for mutagenic activity in Salmonella typhimurium TA1537, TA100 and TA100 NR with and without the addition of a metabolizing mixture. The two main components (AAI and AAII) were direct mutagens in Salmonella strains TA1537 and TA100 with almost equal mutagenic potency. In TA100 NR the aristolochic acid samples showed no or only a very low level of biological activity, indicating the necessity of nitroreduction for the bioactivation of the samples. These findings suggest that both AAI as well as AAII can be used in further studies to elucidate the metabolism of aristolochic acid.