The overall long-term objective of this proposal is to investigate the mechanisms associated with drug-induced toxicities in AIDS therapy. The hematological toxicity of zidovudine (AZT) remains a limiting factor in clinical management of AIDS and is the most important complication requiring cessation of therapy.
The specific aims of this proposal are therefore directed to investigate the mechanisms of bone marrow toxicity induced by AZT and other anti-AIDS drugs. Since high plasma levels of erythropoietin (Epo) in certain patients have been ineffective in ameliorating AZT-induced anemia, we hypothesize that AZT interferes with Epo receptor expression and function. Preliminary results from our laboratory have indeed demonstrated that AZT causes a dose-dependent down regulation of Epo receptor expression which correlated with inhibition of CFU-E derived colonies. Incubation of bone marrow progenitor cells with Epo prevented AZT mediated down regulation of Epo receptor but did not completely restore the proliferative capacity suggesting that AZT interferes with both Epo receptor expression and signalling pathways. We therefore, propose to quantitate the Epo receptor numbers with 125I-Epo and/or biotinylated Epo after exposure of the human bone marrow erythroid progenitor cells with various doses of AZT with or without Epo. We will further examine the steady state levels of Epo receptor mRNA, half life and turn over rates in erythroid progenitor cells upon exposure to AZT and other ddNs. Since transmembrane signalling by the Epo ligand-- receptor complex involves protein kinase C (PKC) activation and our preliminary data have demonstrated significant inhibition of PKC activity by AZT, we propose to further investigate the inhibitory effect of AZT on this enzyme and to delineate the specific isoforms which may be inhibited. Since Epo-induced expression of protooncogenes (c-myc and c-fos) has been shown to be an early cellular event in erythroid proliferation and differentiation and is linked to PKC activation, we will also investigate the effects of AZT and other ddNs on the levels of mRNA transcribed by c-myc, c-fos and raf-1. We have shown in preliminary experiments that AZT down regulated c-fos mRNA whereas levels of c-myc mRNA were unaffected. The proposed studies will provide (a) an in-depth understanding of the mechanisms associated with hematological toxicity of AZT and other ddNs and (b) a basis for effective therapeutic approaches to overcome AZT-induced bone marrow toxicity.
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