DIFFERENTIAL EFFECT OF GM-CSF PRETREATMENT ON INTRACELLULAR ARA-C METABOLISM IN NORMAL BONE-MARROW MONONUCLEAR-CELLS VS ACUTE MYELOID-LEUKEMIA (AML) BLASTS

The effect of recombinant human granulocyte-macrophage colony-stimulat ing factor (GM-CSF) on the intracellular metabolism of cytosine arabin oside (Ara-C) was comparatively analyzed in normal bone marrow mononuc lear cells (NBMMC) from eight healthy volunteers and in leukemic blast s from 50 patients with acute myeloid leukemia (AML). Pretreatment wit h 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 H-3-TdR incorpor ation into the DNA >1.5-fold while NBMMC from normal donors were respo nsive in all cases. In GM-CSF responsive AML blasts, overall DNA polym erase and DNA polymerase alpha activity increased from a median of 84. 4 to 96.1 and from 3.45 to 5.2 pmol/min x mg as compared to a median o f 96.7 to 189.9 and 1.2 to 2.2 pmol/min x mg in NBMMC (P < 0.05). Medi an Ara-C-mediated inhibition of DNA synthesis was significantly more e ffective in AML blasts as compared to NBMMC (76.5 vs 55.0% at 0.05 mu M and 99.0 vs 96.0% at 5.0 mu M Ara-C, P < 0.01) but was not influence d 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 mu M, 167.8 vs 48.0 at 10 mu M and 337.5 vs 59.5 ng/10(7) cells at 1 00 mu M extracellular Ara-C, P < 0.01) but showed no enhancement in th e 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 acti vity after GM-CSF pretreatment (from a median of 1.9 to 3.6 pmol/min x mg, P = 0.039). At low, intermediate and high extracellular Ara-C con centrations GM-CSF pretreatment resulted in a significant enhancement of the H-3-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 H-3-Ara-C i ncorporation into the DNA in response to GM-CSF at low Ara-C concentra tions but significant increases at intermediate and high extracellular Ara-C concentrations (median increases of 1.63-fold at 1.06 mu M with P = 0.01 and 1.37-fold at 10 mu M extracellular Ara-C with P = 0.0005 ). NBMMC revealed significantly lower GM-CSF-induced increases of the H-3-Ara-C incorporation into the DNA as compared to the effect of GM-C SF 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 prim ing on the metabolism of Ara-C in normal vs leukemic cells which may c ause a preferential increase in the antileukemic cytotoxicity of Ara-C in the presence of GM-CSF.