Effects of 2',3'-dideoxynucleosides on proliferation and differentiation of human pluripotent progenitors in liquid culture and their effects on mitochondrial DNA synthesis

Abstract

2',3'-Dideoxynucleosides (ddNs) including 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-3'-deoxythymidine (FLT), 3'-amino-3'-deoxythymidine (AMT), 2',3'-dideoxycytidine (ddC), and 2',3'-didehydro-3'-deoxythymidine (D4T) were tested for their effects on proliferation and differentiation of pluripotent progenitor cells (CD34+) purified from human bone marrow cells grown in liquid cultures. These highly purified progenitor cells undergo extensive proliferation during 14 days, with a marked differentiation during the last 7 days. These differentiated cells exhibit normal morphological features in response to specific hematopoietic growth factors of both erythroid and granulocyte-macrophage lineages, as demonstrated by flow cytometry cell phenotyping. The potencies of these ddNs in inhibiting proliferation of granulocyte-macrophage lineage cells were in the order FLT > AMT = ddC > AZT >> D4T, and the potencies in inhibiting proliferation of erythroid lineage cultures were in the order FLT > AMT > AZT > ddC >> D4T. The toxic effects of ddNs assessed in these liquid cultures were in agreement with data obtained by using semisolid cultures, demonstrating the consistency of these two in vitro hematopoietic systems toward ddN toxicity. ddC was toxic to CD34+ progenitor cells and/or cells in the early stages of differentiation, whereas the inhibitory effect of AZT on the erythroid lineage was predominantly observed on a more mature population of erythroid progenitors during the differentiation process. Slot blot analysis of granulocyte-macrophage cultures demonstrated that exposure to ddC and FLT was associated with a decrease in total mitochondrial DNA (mtDNA) content, suggesting that these two ddNs inhibit mtDNA synthesis. In contrast, no difference in the ratio of nuclear DNA to mtDNA was observed in cells exposed to toxic concentrations of AZT and AMT is not associated with an inhibition of mtDNA synthesis. This human pluripotent progenitor liquid culture system should permit detailed investigations of the cellular and molecular events involved in ddN-induced hematological toxicity.