Wilson J M, Mitchell B S, Daddona P E, Kelley W N
J Clin Invest. 1979 Nov;64(5):1475-84. doi: 10.1172/JCI109606.
Deoxyadenosine and deoxyguanosine are toxic to human lymphoid cells in culture and have been implicated in the pathogenesis of the immunodeficiency states associated with adenosine deaminase and purine nucleoside phosphorylase deficiency, respectively. We have studied the relative incorporation of several labeled nucleosides into DNA and into nucleotide pools to further elucidate the mechanism of deoxyribonucleoside toxicity. In the presence of an inhibitor of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA], 5 muM], deoxyadenosine (1-50 muM) progressively decreased the incorporation of thymidine, uridine, and deoxyuridine into DNA, but did not affect uridine incorporation into RNA. This decrease in DNA synthesis was associated with increasing dATP and decreasing dCTP pools. Likewise, incubation of cells with deoxyguanosine caused an elevation of dGTP, depletion of dCTP, and inhibition of DNA synthesis. To test the hypothesis that dATP and dGTP accumulation inhibit DNA synthesis by inhibiting the enzyme ribonucleotide reductase, simultaneous rates of incorporation of [(3)H]uridine and [(14)C]thymidine into DNA were measured in the presence of deoxyadenosine plus EHNA or deoxyguanosine, and in the presence of hydroxyurea, a known inhibitor of ribonucleotide reductase. Hydroxyurea (100 muM) and deoxyguanosine (10 muM) decreased the incorporation of [(3)H]uridine but not of [(14)C]thymidine into DNA; both compounds also substantially increased [(3)H]cytidine incorporation into the ribonucleotide pool while reducing incorporation into the deoxyribonucleotide pool. In contrast, deoxyadenosine plus EHNA did not show this differential inhibition of [(3)H]uridine incorporation into DNA, and the alteration in [(3)H]cytidine incorporation into nucleotide pools was less impressive. These data show an association between accumulation of dATP or dGTP and a primary inhibition of DNA synthesis, and they provide support for ribonucleotide reductase inhibition as the mechanism responsible for deoxyguanosine toxicity. Deoxyadenosine toxicity, however, appears to result from another, or perhaps a combination of, molecular event(s).
脱氧腺苷和脱氧鸟苷对培养中的人淋巴细胞有毒性作用，分别与腺苷脱氨酶和嘌呤核苷磷酸化酶缺乏相关的免疫缺陷状态的发病机制有关。我们研究了几种标记核苷相对掺入DNA和核苷酸池的情况，以进一步阐明脱氧核糖核苷毒性的机制。在腺苷脱氨酶抑制剂[赤型-9-(2-羟基-3-壬基)腺嘌呤[EHNA]，5 μM]存在的情况下，脱氧腺苷(1-50 μM)逐渐降低胸苷、尿苷和脱氧尿苷掺入DNA的量，但不影响尿苷掺入RNA。DNA合成的这种减少与dATP增加和dCTP池减少有关。同样，用脱氧鸟苷孵育细胞会导致dGTP升高、dCTP耗竭并抑制DNA合成。为了检验dATP和dGTP积累通过抑制核糖核苷酸还原酶来抑制DNA合成这一假说，在存在脱氧腺苷加EHNA或脱氧鸟苷的情况下，以及在存在已知的核糖核苷酸还原酶抑制剂羟基脲的情况下，测量了[(3)H]尿苷和[(14)C]胸苷同时掺入DNA的速率。羟基脲(100 μM)和脱氧鸟苷(10 μM)降低了[(3)H]尿苷而非[(14)C]胸苷掺入DNA的量；这两种化合物还显著增加了[(3)H]胞苷掺入核糖核苷酸池的量，同时减少了其掺入脱氧核糖核苷酸池的量。相比之下，脱氧腺苷加EHNA并未显示出对[(3)H]尿苷掺入DNA的这种差异抑制，并且[(3)H]胞苷掺入核苷酸池的变化不太明显。这些数据表明dATP或dGTP积累与DNA合成的主要抑制之间存在关联，并支持核糖核苷酸还原酶抑制是导致脱氧鸟苷毒性的机制。然而，脱氧腺苷毒性似乎是由另一种或可能是多种分子事件导致的，或者是它们共同作用的结果。
