title: Methods to monitor and compare mitochondrial and glycolytic ATP production.
abstract: ATP is commonly considered as the main energy unit of the cell and participates in a variety of cellular processes. Thus, intracellular ATP concentrations rapidly vary in response to a wide variety of stimuli, including nutrients, hormones, cytotoxic agents, and hypoxia. Such alterations not necessarily affect cytosolic and mitochondrial ATP to similar extents. From an oncological perspective, this is particularly relevant in the course of tumor progression as well as in the response of cancer cells to therapy. In normal cells, mitochondrial oxidative phosphorylation (OXPHOS) is the predominant source of ATP. Conversely, many cancer cells exhibit an increased flux through glycolysis irrespective of oxygen tension. Assessing the relative contribution of glycolysis and OXPHOS to intracellular ATP production is fundamental not only for obtaining further insights into the peculiarities and complexities of oncometabolism but also for developing therapeutic and diagnostic tools. Several techniques have been developed to measure intracellular ATP levels including enzymatic methods based on hexokinase, glucose-6-phosphate dehydrogenase, and firefly luciferase. Here, we summarize conventional methods for measuring intracellular ATP levels and we provide a detailed protocol based on cytosol- and mitochondrion-targeted variants of firefly luciferase to determine the relative contribution of glycolysis and OXPHOS to ATP synthesis.
authors.affiliation: Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Ferrara, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Patergnani
authors.affiliation: Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Ferrara, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Baldassari
authors.affiliation: Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Ferrara, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: De Marchi
authors.affiliation: Department of Pathology, The Children's Memorial Health Institute, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Karkucinska-Wieckowska
authors.affiliation: Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
authors.forename: Mariusz R
authors.initials: MR
authors.lastname: Wieckowski
authors.affiliation: Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Ferrara, Italy. Electronic address: pnp@unife.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism*; D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D004867:Equipment Design; D049454:Firefly Luciferin / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D006019:Glycolysis; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D005954:Glucosephosphate Dehydrogenase; D008156:Luciferases
keywords: ATP; Glucose-6-phosphate dehydrogenase; Hexokinase; Luciferase/luciferin; Mitochondria; Respiratory chain