Metabolism of deoxynucleosides by lymphocytes in long-term culture deficient in different purine enzymes.
The metabolism of 8-14C-labelled 2'-deoxyadenosine (dAR) and 2'-deoxyguanosine (dGR) has been investigated using lymphocytes in long-term culture transformed by Epstein-Barr (EB) virus (B-cells) from eight patients with different inherited purine enzyme defects. The use of such lines enabled accurate mapping of the route of metabolism by acting as a 'trap' for the radiolabel at specific points. With either substrate (25 microM) most of the label was recovered in the medium. Using dAR, less than 30% of the radiolabel was incorporated into cellular nucleotides. For dGR, values were less than 18%. Studies with dAR alone confirmed the principal route of metabolism was to hypoxanthine, with further metabolism (by lines with intact salvage pathways) to ATP and GTP in the ratio of approximately 4:1. Lack of accumulation of deoxyinosine in the purine nucleoside phosphorylase (PNP) deficient line, or hypoxanthine in the hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient line, using dAR together with the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin (dCF) at 10 microM, confirmed the effectiveness of ADA inhibition. Nevertheless, some ATP was still formed by all lines in the presence of dCF by a route as yet unknown. Only the ADA deficient lines formed dATP with dAR alone. However, some dATP was formed by all lines in the presence of dCF. A partially HGPRT deficient line formed extremely high dATP levels, well in excess of those formed by the T-cell line CEM. Studies with dGR revealed some interesting differences, a large proportion of the substrate being metabolized predominantly to xanthine by most enzyme deficient lines. In the PNP deficient line most of the substrate remained unmetabolized, but some dGTP was formed. No other enzyme deficient line formed any dGTP--with or without the PNP inhibitor 8-aminoguanosine (8-NH2GR)--with one exception. Again this was the partially HGPRT deficient line, which with the inhibitor again formed more dGTP than the T-cell line. Within the cells most of the substrate was metabolized to GTP, except in the PNP, and totally HGPRT deficient lines. Levels of GTP formed were not altered by the inhibitor, reflecting the lack of effective PNP inhibition by 8-NH2GR. Some counts were also found in ATP and IMP, confirming the existence of this route in mammalian cells of lymphoid origin. The results also support previous studies by us using cell lines with intact purine pathways, which demonstrated that, contrary to current beliefs, some B-cell lines are capable of accumulating high levels of deoxynucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)
D058892:Adenosine Deaminase Inhibitors; D002478:Cells, Cultured; D003839:Deoxyadenosines / Q000097:blood*; D003849:Deoxyguanosine / Q000097:blood*; D006151:Guanosine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency; D007042:Hypoxanthines / Q000097:blood; D008214:Lymphocytes / Q000378:metabolism*; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000097:blood*
D058892:Adenosine Deaminase Inhibitors; D003839:Deoxyadenosines; D007042:Hypoxanthines; D006151:Guanosine; D019271:Hypoxanthine; C032533:8-aminoguanosine; D011683:Purine-Nucleoside Phosphorylase; D007041:Hypoxanthine Phosphoribosyltransferase; D003849:Deoxyguanosine