Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Toward the end of the 20th and beginning of the 21st centuries, clever in vitro biochemical complementation experiments and genetic screens from the laboratories of Xiaodong Wang, Shigekazu Nagata, and Ding Xue led to the discovery of two major apoptotic nucleases, termed DNA fragmentation factor (DFF) or caspase-activated DNase (CAD) and endonuclease G (Endo G). Both endonucleases attack chromatin to yield 3'-hydroxyl groups and 5'-phosphate residues, first at the level of 50-300 kb cleavage products and next at the level of internucleosomal DNA fragmentation, but these nucleases possess completely different cellular locations in normal cells and are regulated in vastly different ways. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45) [also called inhibitor of CAD (ICAD-L)] and a 40 kD latent nuclease subunit (DFF40/CAD). Apoptotic activation of caspase-3 or -7 results in the cleavage of DFF45/ICAD and release of active DFF40/CAD nuclease. DFF40's nuclease activity is further activated by specific chromosomal proteins, such as histone H1, HMGB1/2, and topoisomerase II. DFF is regulated by multiple pre- and post-activation fail-safe steps, which include the requirements for DFF45/ICAD, Hsp70, and Hsp40 proteins to mediate appropriate folding during translation to generate a potentially activatable nuclease, and the synthesis in stoichiometric excess of the inhibitors (DFF45/35; ICAD-S/L). By contrast, Endo G resides in the mitochondrial intermembrane space in normal cells, and is released into the nucleus upon apoptotic disruption of mitochondrial membrane permeability in association with co-activators such as apoptosis-inducing factor (AIF). Understanding further regulatory check-points involved in safeguarding non-apoptotic cells against accidental activation of these nucleases remain as future challenges, as well as designing ways to selectively activate these nucleases in tumor cells.
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PMID:Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. 1572 41

Studies were carried out to address possible cellular mechanisms by which merbarone, a catalytic inhibitor of DNA topoisomerase II, can block tumor cell growth without inducing extensive DNA cleavage. Merbarone induced the release of high molecular weight DNA fragments from the nuclear matrix of HL-60 leukemia cells, which preceded the internucleosomalsize DNA fragmentation characteristic of late-stage apoptosis. The chromatin fragments were enriched in a matrix attachment region (MAR) sequence compared with a non-MAR sequence and were similar in size to DNA loops extracted from nuclear matrices. However, merbarone did not directly induce the excision of high molecular weight DNA fragments from the nuclear matrix by promoting topoisomerase II-catalyzed DNA cleavage, because the drug inhibited topoisomerase II-mediated cleavage in isolated nuclear matrix preparations. Instead, merbarone induced rapid activation of the mitochondrial apoptosis pathway, which included the following temporal sequence of events: dissipation of the mitochondrial transmembrane potential within 30 min, release of mitochondrial cytochrome c, and activation of caspase-activated DNase (CAD) by its inhibitor ICAD. The excision of high molecular weight DNA was inhibited at least 80% in merbarone-treated cells preincubated with the pan-caspase inhibitor z-VAD-fmk [Z-Val-Ala-Asp(OMe)-fluoromethyl ketone] and in caspase-resistant Jurkat cells (ICAD/double-mutated) that express a mutant form of ICAD. These results provide evidence that merbarone can induce rapid disorganization of DNA in tumor cells that have a functional mitochondrial apoptosis pathway without inducing extensive DNA cleavage.
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PMID:Merbarone induces activation of caspase-activated DNase and excision of chromosomal DNA loops from the nuclear matrix. 1643 17

Etoposide-induced treatment-related acute myelogenous leukemia (t-AML) is characterized by rearrangements of the mixed lineage leukemia (MLL) gene with one of its >50 partner genes, most probably as a consequence of etoposide-induced DNA double-strand breaks (DSBs). Recent studies have shown that etoposide-induced DSBs occur predominantly within the breakpoint cluster region (bcr) of the MLL gene. However, bcr-specific DSBs induced by etoposide are not topoisomerase II-linked but the result of apoptotic nuclease-mediated DNA cleavage. Here, we test the involvement of caspase-activated DNase (CAD) and other apoptotic components in etoposide-induced gene rearrangements using two methods. First, we measured the effect of etoposide on the integration frequency of a transfected plasmid. Etoposide strongly stimulated plasmid integration in CAD cDNA-complemented mouse embryonic fibroblasts (MEFs) but not in CAD knockout (KO) MEFs. Consistently, down-regulation of ICAD (inhibitor of CAD, also required for proper folding of CAD) in an HT29-derived cell line, which leads to decreased CAD activity, significantly reduced etoposide-induced plasmid integration. Second, we used long-template inverse PCR to focus on gene rearrangements at the MLL locus. Etoposide stimulated MLL fusion product formation in CAD cDNA-complemented MEFs but not in CAD KO MEFs. Together, these results suggest that CAD and other apoptotic components may play an important role in etoposide-induced t-AML.
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PMID:Role of apoptotic nuclease caspase-activated DNase in etoposide-induced treatment-related acute myelogenous leukemia. 1698 37