Our data show that activating autoantibodies against human beta-adrenergic receptors exist in approximately 25% of patients with dilated cardiomyopathy. Counteraction of such autoantibodies might contribute to the beneficial effects of beta-adrenergic receptor blockade in chronic heart failure.

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (α and β) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIα and IIβ behaved similarly in interphase but differently in mitosis, where only topo IIα was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIα was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.

Topoisomerases are essential for DNA replication in dividing cells, but their genomic targets and function in postmitotic cells remain poorly understood. Here we show that a switch in the expression from Topoisomerases IIα (Top2α) to IIβ (Top2β) occurs during neuronal differentiation in vitro and in vivo. Genome-scale location analysis in stem cell-derived postmitotic neurons reveals Top2β binding to chromosomal sites that are methylated at lysine 4 of histone H3, a feature of regulatory regions. Indeed Top2β-bound sites are preferentially promoters and become targets during the transition from neuronal progenitors to neurons, at a time when cells exit the cell cycle. Absence of Top2β protein or its activity leads to changes in transcription and chromatin accessibility at many target genes. Top2β deficiency does not impair stem cell properties and early steps of neuronal differentiation but causes premature death of postmitotic neurons. This neuronal degeneration is caused by up-regulation of Ngfr p75, a gene bound and repressed by Top2β. These findings suggest a chromatin-based targeting of Top2β to regulatory regions in the genome to govern the transcriptional program associated with neuronal differentiation and longevity.epigenetic regulation | neurogenesis | gene expression | genomewide assays T opoisomerases are essential for solving topological problems arising from DNA-templated processes such as replication, transcription, recombination, chromatin remodeling, chromosome condensation, and segregation (1-5). The type I subfamily of topoisomerases achieves this task by passing one strand of the DNA through a break in the opposing strand; proteins in the type II subfamily pass a region of duplex strands from the same or a different molecule through a double-stranded gap generated in DNA (1-5). Mammalian cells encode two isozymes of type II enzymes that have highly homologous N-terminal ATPase and central core domains but differ at their C-termini (6). These two isozymes, Topoisomerases IIα (Top2α) and IIβ (Top2β), have almost identical enzymatic properties in vitro (7, 8); however, their expression patterns are dissimilar. Top2α is the main isoform expressed in proliferating cells, shows high expression in S/G2/M phases of the cell cycle, and plays important roles in DNA replication and chromosome condensation/segregation during the cell cycle (9-12).The cellular functions of Top2β are much less well understood. It is expressed in all mammalian cells throughout the cell cycle but is up-regulated robustly when cells reach a postmitotic state of terminal differentiation (13-15). For example, the postmitotic granule cells in the external germinal layer of the developing rat cerebellum show a transition from Top2α to Top2β (14), and blocking Top2β catalytic activity affects the expression of about one third of genes induced during differentiation of rat cerebellar granule neurons (16). Genetic deletion of Top2b in mice causes neural defects including aberrant axonal elongation and branching and perinatal death e...

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Alternariol (AOH), a mycotoxin formed by Alternaria alternata, has been reported to possess genotoxic properties. However, the underlying mechanism of action is unclear. Here, we tested the hypothesis that interactions with DNA-topoisomerases play a role in the DNA-damaging properties of AOH. First we compared DNA-damaging properties of AOH with other Alternaria mycotoxins such as AOH monomethyl ether (AME), altenuene and isoaltenuene. AOH and AME significantly increased the rate of DNA strand breaks in human carcinoma cells (HT29, A431) at micromolar concentrations, whereas altenuene and isoaltenuene did not affect DNA integrity up to 100 microM. Next, we selected AOH as the most DNA-damaging Alternaria metabolite for further studies of interactions with DNA topoisomerases. In cell-free assays, AOH potently inhibited DNA relaxation and stimulated DNA cleavage activities of topoisomerase I, IIalpha and IIbeta. Stabilisation of covalent topoisomerase II-DNA intermediates by AOH was also detectable in cell culture, and here, the IIalpha isoform was preferentially targeted. AOH is thus characterised as a poison of topoisomerase I and II with a certain selectivity for the IIalpha isoform. Since topoisomerase poisoning and DNA strand breakage occurred within the same concentration range, poisoning of topoisomerase I and II might at least contribute to the genotoxic properties of AOH.

Topoisomerases are involved in many aspects of DNA metabolism such as replication and transcription reactions. Camptothecins, which stabilize the covalent intermediate of topoisomerase I and DNA are effective, though toxic, drugs for cancer therapy. In this study, a new class of topoisomerase I inhibitors was identified, and their mode of action was characterized using recombinant human topoisomerase I preparations and human HL-60 leukemic cells. Quercetin and the related natural flavones, acacetin, apigenin, kaempferol, and morin, inhibit topoisomerase I-catalyzed DNA religation. In contrast to camptothecin, these compounds do not act directly on the catalytic intermediate and also do not interfere with DNA cleavage. However, formation of a ternary complex with topoisomerase I and DNA during the cleavage reaction inhibits the following DNA religation step. 3,3,4,7-Tetrahydroxy-substituted flavones stabilize the covalent topoisomerase I-DNA intermediate most efficiently. Enhanced formation of covalent topoisomerase I-DNA complexes was also demonstrated in human HL-60 cells. In contrast, synthetic 3,5-dibromo-4-hydroxy-3-methylflavones bind selectively to topoisomerase I in its non-DNA-bound form and block the following DNA binding step. As a consequence, these synthetic flavonoids are capable of counteracting topoisomerase I-directed effects of camptothecin. Inhibition of DNA binding is obtained by voluminous hydrophobic substituents in 6-position of the flavone structure. Our data show that selective inhibitors of both half-reactions of topoisomerase I can be derived from the flavone structure.

We visualized DNA topoisomerases in A431 cells and isolated chromosomes by isoenzyme-selective immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10–15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted (α) or reticular (β) nuclear patterns throughout interphase. In contrast to topoisomerase IIα, topoisomerase IIβ was completely excluded from nucleoli. In mitosis, topoisomerase IIβ diffused completely into the cytosol, whereas topoisomerases I and IIα remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIα accumulated in the long axes of the chromosome arms and in the centriols. Topoisomerase antigens were 2–3-fold higher in mitosis than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by etoposide or camptothecin, whereas in mitosis, 50% of topoisomerase IIα escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIα, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIβ remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIβ is released from the heterochromatin, whereas topoisomerase I and IIα remain chromosome bound. Scaffold-associated topoisomerase IIα appears not to be involved in catalytic DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M phase.

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3-phosphate of DNA breaks. When expressed in human cells as biofluorescent chimera, TDP1 appeared more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound at early mitosis. Upon exposure to camptothecin both proteins were cleared from nucleoli and rendered less mobile in the nucleoplasm. However, with TDP1 this happened much more slowly reflecting most likely the redistribution of nucleolar structures upon inhibition of rDNA transcription. Thus, a steady association of TDP1 with topoisomerase I seems unlikely, whereas its integration into repair complexes assembled subsequently to the stabilization of DNA⅐topoisomerase I intermediates is supported. Cells expressing GFP-tagged TDP1 > 100-fold in excess of endogenous TDP1 exhibited a significant reduction of DNA damage induced by the topoisomerase I poison camptothecin and could be selected by that drug. Surprisingly, DNA damage induced by the topoisomerase II poison VP-16 was also diminished to a similar extent, whereas DNA damage independent of topoisomerase I or II was not affected. Overexpression of the inactive mutant GFP-TDP1 H263A at similar levels did not reduce DNA damage by camptothecin or VP-16. These observations confirm a requirement of active TDP1 for the repair of topoisomerase I-mediated DNA damage. Our data also suggest a role of TDP1 in the repair of DNA damage mediated by topoisomerase II, which is less clear. Since overexpression of TDP1 did not compromise cell proliferation, it could be a pleiotropic resistance mechanism in cancer therapy.Tyrosyl DNA phosphodiesterase 1 (TDP1) 1 is an enzyme capable of hydrolyzing phosphodiester bonds between tyrosine and the 3Ј-phosphate of DNA (1, 2), which are typically generated in a transient manner by DNA topoisomerase I (topo I) (3). In keeping with this, yeast deletion mutations of TDP1 are deficient in the repair of DNA damage induced by camptothecin, a drug that stabilizes the transient topo I⅐DNA intermediate (2, 4 -6). More precisely, TDP1 has been characterized in these studies as a non-exclusive effector upstream of Rad52 that removes structurally modified topo I adducts (7, 8) as well as oxidative adducts (9) from the 3Ј-phosphate of a DNA break prior to homologous recombination repair. In mammals, TDP1 is (in addition or instead?) involved in an XRCC-dependent single-stranded DNA repair pathway also directed at topo I⅐DNA adducts (10 -12). Despite all the evidence of yeast deletion studies implying TDP1 in DNA repair, a familial disease caused by a mutation in the active site of the human ortholog of the enzyme exhibits a phenotype not at all typical for inadequate DNA repair, namely a slow onset of neuronal degeneration (13). This unexpected finding has prompted speculations that at least in mammals TDP1 could serve a much broader scope of functions, some of which may not even depend on catalytic activity. To further clarify the importance of TDP1 for...