author's abstract.) The primary goal of the proposed research is to investigate the molecular mechanism(s) of toxicities consisting of myelosuppression (anemia) and peripheral neuropathy, as a result of the effects of 2',3'-dideoxynucleosides (ddN's) on the expression of specific genes. In completing the two major aims of their project, the Investigator expects that the basic cellular and molecular studies will provide a rational basis for future structural studies leading to the design of second and third generation, highly specific nucleoside analogs active against the human immunodeficiency virus (HIV).
Aim #1 : the elucidation of the mechanism(s) responsible for the anemia induced by AZT, by way of inhibition by AZT and AMT of genes that regulate hemoglobin synthesis. AMT is a newly-identified metabolite of AZT which as been demonstrated by these investigators to be highly toxic to human bone marrow progenitor cells. In the proposed research, the researchers will evaluate the mechanism(s) involved in AMT toxicity for human erythroid cells and its role in AZT-induced anemia observed in patients. They will assess the effects of AZT and AMT on the expression of genes that regulate globin synthesis and heme synthesis (including 5-aminolevulinate synthase, 5-aminolevulinate dehydrase, and phosphobilinogen deaminase) as compared to constitutively-expressed genes. The examination of novel ddN's currently being evaluated in Phase I trials-- such as 3'-fluorothymidine, shown to be very toxic in erythroid progenitor cells--will permit the investigators to evaluate structural factors potentially important for inhibition of globin and """"""""heme"""""""" gene expression. The influence of hemin on the expression of genes regulating the hemoglobin synthesis and affected by AZT and AMT will be examined in an attempt to elucidate the mechanism(s) responsible for hemin protective effects of AZT cytotoxicity in erythroid cells. Elucidation of inhibition of globin gene transcription by AZT will be ascertained in evaluating effects of AZT on the activity and amount of specific erythroid nuclear proteins, such as NF-E1 and NF-E2.
AIM 2 : the examination of toxicity with the elucidation of cellular and molecular mechanism(s) of dideoxycytodine (ddC), dideoxyinosine (ddI), and 2',3'-didhydro-2',3'-dideoxythymidine (d4T) as related to their effects on peripheral neurons. Using a PC-12 cell as a model, the researchers will study the effects of ddC, ddI, and d4T on neurite outgrowth and survival, including the effects of the ddN's on initiation of neurite outgrowth, priming, on neurite degeneration and regeneration, and on biological markers. They will: discriminate the effects of ddN's on electrophysiological function in the soma, neurite, and growth cones of PC-12 cells and multinucleated cells; elucidate the mechanism(s) of toxicity by evaluating effects of ddN's on mitochondrial DNA synthesis and neural-specific protein synthesis; and study the effects of ddN's on the expression of genes that regulate neurofilaments and microtubule-associated proteins.
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