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.
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