Differential effect of GM-CSF pretreatment on intracellular Ara-C metabolism in normal bone marrow mononuclear cells vs acute myeloid leukemia (AML) blasts

Abstract

The effect of recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) on the intracellular metabolism of cytosine arabinoside (Ara-C) was comparatively analyzed in normal bone marrow mononuclear cells (NBMMC) from eight healthy volunteers and in leukemic blasts from 50 patients with acute myeloid leukemia (AML). Pretreatment with GM-CSF (100 U/ml) for 48 h resulted in a significant enhancement of DNA synthesis in both cell types: 21 of 35 AML specimens were found to be responsive to GM-CSF as defined by an increase of ³H-TdR incorporation into the DNA >1.5-fold while NBMMC from normal donors were responsive in all cases. In GM-CSF responsive AML blasts, overall DNA polymerase and DNA polymerase α activity increased from a median of 84.4 to 96.1 and from 3.45 to 5.2 pmol/min × mg as compared to a median of 96.7 to 189.9 and 1.2 to 2.2 pmol/min × mg in NBMMC (P < 0.05). median ara-c-mediated inhibition of dna synthesis was significantly more effective in aml blasts as compared to nbmmc (76.5 vs 55.0% at 0.05 μM and 99.0 vs 96.0% at 5.0 μM Ara-C, P < 0.01) but was not influenced by gm-csf pretreatment. similarly, intracellular ara-ctp levels were higher in aml blasts as compared to nbmmc (median of 46.9 vs 18.7 at 1 μM, 167.8 vs 48.0 at 10 μM and 337.5 vs 59.5 ng/10⁷ cells at 100 μM extracellular Ara-C, P < 0.01) but showed no enhancement in the presence of gm-csf. median deoxycytidine (dck) and thymidine kinase (tk) activity were only slightly increased in aml blasts after gm-csf priming. in contrast, nbmmc revealed a significant increase in tk activity after gm-csf pretreatment (from a median of 1.9 to 3.6 pmol/min × mg, P = 0.039). At low, intermediate and high extracellular Ara-C concentrations GM-CSF pretreatment resulted in a significant enhancement of the ³H-Ara-C incorporation into the DNA in both GM-CSF responsive AML blasts and NBMMC (median of 1.3 to 2.1- and 1.4 to 1.6-fold, P < 0.05). gm-csf non-responsive aml blasts showed no change in ³H-Ara-C incorporation into the DNA in response to GM-CSF at low Ara-C concentrations but significant increases at intermediate and high extracellular Ara-C concentrations (median increases of 1.63-fold at 1.06 μM with P = 0.01 and 1.37-fold at 10 μM extracellular Ara-C with P = 0.0005). NBMMC revealed significantly lower GM-CSF-induced increases of the ³H-Ara-C incorporation into the DNA as compared to the effect of GM-CSF priming on DNA synthesis (median increases of 1.4 to 1.7-fold vs 2.6-fold, P < 0.05). these data reveal a different effect of gm-csf priming on the metabolism of ara-c in normal vs leukemic cells which may cause a preferential increase in the antileukemic cytotoxicity of Ara-C in the presence of GM-CSF.