Adenosine metabolism and its effect on methylation potential in cultured cells: methodological considerations.
Cell culture models are frequently used to study the role of adenosine in several physiological and pathological processes. In the present study, we have shown that adenosine deaminase activity in medium supplemented with calf serum significantly reduces adenosine concentration in culture medium. In the presence of HepG2 cells, the adenosine concentration in culture medium is decreased much faster, because a large amount of exogenous adenosine is metabolized by cellular enzyme. In order to measure intracellular adenosine, inosine, adenine nucleotides, S-adenosylhomocysteine (AdoHcy) and Sadenosylmethionine (AdoMet) contents, two methods for cell harvesting were compared. First, cells were removed with trypsin/EDTA, second, cells were lysed in cell culture dishes immediately after removing culture medium. Our results show that exact determination of adenosine metabolites requires immediate inactivation of metabolism by cell lysis in culture dishes. Application of adenosine (1mM) resulted in a time-dependent increase in intracellular adenosine, inosine, AMP, ATP, AdoHcy and AdoMet concentration. Since AdoHcy levels increased to a larger extent than AdoMet, the methylation potential, expressed as the ratio of AdoMet/AdoHcy, was reduced from 51.8 (control) to 2.9 (adenosine 1 mM, 2 hrs), suggesting that AdoMet-dependent methylation reactions might be impaired. In conclusion our data demonstrate that extracellular adenosine concentration and intracellular metabolite concentration strongly depend on the methods used to culture and harvest the cells.
Hermes
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Department of Pharmacology and Toxicology, Faculty of Medicine, University of Tuebingen, Wilhelmstrasse 56, D-72074, Tuebingen, Germany. marina.hermes@uni-tuebingen.de
Osswald
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Kloor
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D000227:Adenine Nucleotides / Q000032:analysis; D000241:Adenosine / Q000032:analysis / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007288:Inosine / Q000032:analysis; D007700:Kinetics; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008745:Methylation; D012436:S-Adenosylmethionine / Q000032:analysis / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D000227:Adenine Nucleotides; D002614:Chelating Agents; D003470:Culture Media; D007288:Inosine; D012436:S-Adenosylmethionine; D004492:Edetic Acid; D014357:Trypsin; D000241:Adenosine