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Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA
Baylor College of Medicine, Houston, TX 77030, USA
Michael E. DeBakey Veterans Affairs Medical Center, 2002 Holcombe Boulevard, Research Building 109, Room 204, Houston, TX 77030, USA
* Author to whom correspondence should be addressed.
Received: 28 April 2011; in revised form: 20 May 2011 / Accepted: 24 May 2011 / Published: 3 June 2011
Abstract: In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous DNA sequences. Although the phagocytic phase is critical for the efficient execution of apoptosis, there are currently few methods specifically adapted for its detailed visualization in the fixed tissue section format. To resolve this we developed new fluorescent probes for in situ research. The probes selectively visualize active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells) which engulf and digest apoptotic cell DNA. These fluorescent probes are the covalently-bound enzyme-DNA intermediates produced in a topoisomerase reaction with specific “starting” oligonucleotides. They detect a specific marker of DNase II cleavage activity, which occurs exclusively in phagolysosomes of the cells that engulfed apoptotic nuclei. The probes provide snap-shot images of the digestion process occurring in cellular organelles responsible for the actual execution of phagocytic degradation of apoptotic cell corpses. We applied the probes for visualization of the phagocytic reaction in tissue sections of normal thymus and in several human lymphomas. We also discuss the nature, stability and properties of DNase II-type breaks as a marker of phagocytic activity. This development provides a useful fluorescent tool for studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders.
Keywords: fluorescent protein-DNA probes; clearance of apoptotic cells; fluorescence labeling of phagocytosis; vaccinia topoisomerase I; phagolysosomes; DNase II-type breaks
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Minchew, C.L.; Didenko, V.V. Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA. Molecules 2011, 16, 4599-4614.
Minchew CL, Didenko VV. Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA. Molecules. 2011; 16(6):4599-4614.
Minchew, Candace L.; Didenko, Vladimir V. 2011. "Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA." Molecules 16, no. 6: 4599-4614.