ALCOHOL EFFECT OF AZT PHOSPHORYLATION REQUIRED FOR ACTIVATION OF AZT

3 '-叠氮基-2'，3 '-双脱氧胸苷（AZT），第一个实施方案的化疗剂， 艾滋病患者的选择，需要细胞活化 由一系列细胞激酶催化成三磷酸形式（AZT- TP）的抗病毒活性。级联反应中的两个限速步骤 已经鉴定了以下激活：1）胸苷激酶， 将AZT转化为单磷酸（AZT-MP），和2）胸苷酸激酶， 将AZT转化为二磷酸盐（AZT-DP）的酶。早期的研究 显示AZT的抗病毒活性可以通过缺陷而降低， 任何一种激酶的表达。此外，AZT治疗是 经常伴有严重的造血毒性，导致贫血 和白细胞减少症。目前还没有关于酒精如何 服用AZT可能会改变AZT向其活性形式的转化， 该药物对HIV-1感染者的毒性作用。有 有证据表明酒精能够下调 胸苷激酶以及损害造血祖细胞的生长 在文化中。我们的假设是，在HIV-1存在的情况下， 蛋白质将1）下调胸苷-胸苷酸的表达 激酶并抑制AZT活化成其活性形式;和2） 增强AZT对造血祖细胞的毒性作用。这 这项提案将在表达完整基因的转基因小鼠中检验这一假设， 长度达特（达特/86）蛋白，并且在小鼠（HIV小鼠）中表达大量 HIV编码的蛋白质，并有6个特定的目的。具体目标1，测试 酒精抑制胸苷酸活性预测 小鼠骨髓、胸腺和肝组织中的激酶。由于AZT是 已知减少其自身的磷酸化，在具体目标2中，我们将 评估酒精对AZT治疗小鼠的影响。为了测试 预测抑制胸苷-胸苷酸激酶与 降低AZT磷酸化。在具体目标3中，我们将测量 体外试验中小鼠组织磷酸化AZT的能力。在 具体目标4，我们将测试的预测，抑制 目的1和2中观察到的胸苷激酶活性是可比较的 减少mRNA的合成。在具体目标5和6中，我们将测试 预测酒精消费对 增殖的骨髓祖细胞，并进一步增强 AZT的抑制作用拟议研究的结果将 为酒精在艾滋病毒中的辅助因素作用提供了新的见解 通过降低抗病毒活性和增加 AZT及相关药物的造血毒性。

3'-Azido-2',3'-dideoxythymidine (AZT), the chemotherapeutic agent of first choice for patients with HIV disease, requires cellular activation catalyzed by a series of cellular kinases to the triphosphate form (AZT- TP) for antiviral activity. Two rate-limiting steps in the cascade of activation have been identified: 1) thymidine kinase, the enzyme that converts AZT to its monophosphate (AZT-MP), and 2) thymidylate kinase, the enzyme that converts AZT to its diphosphate (AZT-DP). Earlier studies have shown that the antiviral activity of AZT can be reduced through defect in the expression of any one of these kinases. In addition, AZT therapy is regularly accompanied by severe hematopoietic toxicity leading to anemia and leukopenia. Currently, there is no information on how alcohol consumption might alter the conversion of AZT to its active form or modify the toxic effects of the drug in HIV-1 affected individuals. There is evidence that alcohol has the ability to down-regulate the expression of thymidine kinase as well as impair the growth of hematopoietic progenitors in culture. It is our hypothesis that alcohol in the presence of HIV-1 protein(s) will 1) down-regulate the expression of thymidine-thymidylate kinases and inhibit the activation of AZT to its active form; and 2) enhance toxic effects of AZT on hematopoietic progenitor cells. This proposal will test the hypothesis in transgenic mice that express full- length Tat (Tat/86) protein, and in mice (HIV mice) that express a number of HIV-encoded proteins, and has 6 Specific Aims. Specific Aim 1, to test the prediction that alcohol suppresses activities of thymidine-thymidylate kinases in bone marrow, thymus and liver tissues of mice. Since AZT is known to diminish its own phosphorylation, in Specific Aim 2, we will assess the effects of alcohol in AZT-treated mice. In order to test the prediction that suppression of thymidine-thymidylate kinases is linked to decreased AZT phosphorylation. In Specific Aim 3, we will measure the capacity of the mouse tissues to phosphorylate AZT in in vitro assays. In Specific Aim 4, we will test the prediction that the suppression of thymidine kinase activity seen in Aims 1 and 2 is the result of comparable decreases in its mRNA synthesis. In Specific Aims 5 and 6, we will test the prediction that alcohol consumption has suppressive effect on the proliferation of bone marrow progenitors and further enhances the suppressive effects of AZT. The results of the proposed studied will provide new insights into the co-factor role of alcohol in HIV pathogenesis through decreased antiviral activity and increased hematopoietic toxicity of AZT and related drugs.