Abstract
Clones resistant to 3-deazaaristeromycin, a potent inhibitor of S-adenosylhomocysteine hydrolase, were selected from a nucleoside kinase-deficient derivative of the WIL-2 human B-lymphoblastoid cell line. The resistant clones took up 3-deazaaristeromycin and showed no alteration in the level of S-adenosylhomocysteine hydrolase activity or in the sensitivity of the enzyme to inhibition by 3-deazaaristeromycin. However, they displayed markedly elevated S-adenosylmethionine content during growth in 3-deazaaristeromycin and, following prolonged selection, enhanced export of S-adenosylhomocysteine. As a result they maintained a high ratio of S-adenosylmethionine to S-adenosylhomocysteine and thus were resistant to the inhibition of S-adenosylmethionine turnover and transmethylation caused by 3-deazaaristeromycin. Expanded S-adenosylmethionine pools declined over several weeks of nonselective growth, suggesting a metabolic adaptation rather than a mutational mechanism. No alterations in S-adenosylmethionine synthetase activity were found in the 3-deazaaristeromycin-resistant clones. S-Adenosylhomocysteine export appeared to be carrier-mediated and largely unidirectional. The resistant clones showed a 5-fold increased rate of S-adenosylhomocysteine export compared with parental cells, but a similar Km for intracellular S-adenosylhomocysteine, estimated to be approximately 1 mM. Our results highlight the opposing effects of S-adenosylmethionine and S-adenosylhomocysteine on transmethylation and suggest that the ability to elevate S-adenosylmethionine pools and to export S-adenosylhomocysteine may provide for homeostatic control of transmethylation in lymphoid cells when S-adenosylhomocysteine hydrolase activity is limited.
Highlights
Clones resistant to 3-deazaaristeromycin, a potent enosylhomocysteine (AdoHcy) as a product and competitive inhibitor of S-adenosylhomocysteine hydrolase, were inhibitor of alltransmethylation reactions are potentially selected from a nucleoside kinase-deficient derivative regulators of methylation-dependent cellular processes (1, 2)
The In mammalian cells, the only metabolic route of AdoHcy resistant clones took up 3-deazaaristeromycinand elimination is hydrolysis to Ado and homocysteine, a reversshowed no alteration in the level of S-adenosylhomo- ible and thermodynamically unfavorable reaction catalyzed cysteine hydrolase activityor in the sensitivity of the enzyme to inhibition by 3-deazaaristeromycin
Impaired catabolism of AdoHcymay transmethylation caused by 3-deazaaristeromycin.Ex- contribute to thefailure of lymphoid development caused by panded S-adenosylmethionine pools declined over sev- inherited deficiency of adenosine deaminase (EC 3.5.4.4)
Summary
Characteristics of Adenosine Analogs and 3-Deazaaristeromycin-resistant (C3Arir) Clones-C3Ari and the related compounds f aristeromycin (Ari) and 3-deazaadenosine (@Ado) all inhibit AdoHcyase, but differ in their ability to undergo deamination, phosphorylation, and AdoHcyase-catalyzed condensation with L-homocysteine We examined these aspects of metabolism in relation to their growth inhibitory effects on WIL-2 lymphoblasts. Compared with K2B, the C3Ari' clones were -25-fold less sensitive to Ari, and -3-fold less sensitive to Ado andto adenine arabinoside, an inactivator of AdoHcyase (10); neither clone was appreciably resistant to C3Ado (Fig. 1,Table 11) These growth characteristics have been stable over a 3-year period of observation and have beendocumented in cells maintained under nonselective conditions for from 10 to 50 doublings.
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