title: Selection and characterization of a murine lymphoid cell line partially deficient in S-adenosylhomocysteine hydrolase.
abstract: The exact role of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) in mediating the toxic effects of adenosine toward mammalian cells has not been ascertained. The selection and characterization of S-adenosylhomocysteine hydrolase-deficient cell lines offers a biochemical genetic approach to this problem. In the present experiments, a mutant clone (Sahn 12) with 11-13% of wild-type S-adenosylhomocysteine hydrolase activity was selected from the murine T lymphoma cell line R 1.1 after mutagenesis and culture in adenosine, deoxycoformycin, uridine and homocysteine thiolactone-supplemented medium. In the presence of 0.5 mM homocysteine thiolactone and 10-200 microM adenosine, wild-type and mutant cells synthesized S-adenosylhomocysteine intracellularly at markedly different rates, and excreted the compound extracellularly. Thus, at time points up to 10 h, the S-adenosylhomocysteine hydrolase-deficient lymphoblasts required 5-10-fold higher concentrations of adenosine in the medium to achieve the same intracellular S-adenosylhomocysteine levels as wild-type cells. Similarly, the Sahn 12 lymphoblasts were 5-10-fold more resistant than R 1.1 cells to the toxic effects of adenosine plus homocysteine thiolactone. These results establish that (i) 11-13% of wild-type S-adenosylhomocysteine hydrolase activity is compatible with normal growth, (ii) in medium supplemented with both adenosine and homocysteine thiolactone, intracellular S-adenosylhomocysteine is synthesized by S-adenosylhomocysteine hydrolase, (iii) the net intracellular level of S-adenosylhomocysteine is determined by both the rate of S-adenosylhomocysteine synthesis and its rate of excretion, (iv) under such conditions the accumulation of S-adenosylhomocysteine is related to cytotoxicity, (v) in the absence of an exogenous homocysteine source, S-adenosylhomocysteine derives from endogenous sources, and the accumulation of S-adenosylhomocysteine is not the primary cause of adenosine induced cytotoxicity.
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D002999:Clone Cells / Q000201:enzymology; D006710:Homocysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D006867:Hydrolases / Q000172:deficiency*; D008223:Lymphoma / Q000201:enzymology*; D051379:Mice; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology
chemical_list: D006710:Homocysteine; D012435:S-Adenosylhomocysteine; C007957:homocysteine thiolactone; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine