MODULATION OF INTRACELLULAR METABOLISM OF CYTOSINE-ARABINOSIDE IN ACUTE MYELOID-LEUKEMIA BY GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR

The current study investigated the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the intracellular metabolism and cytotoxicity of l-beta-D-arabinofuranosylcytosine (araC) in leukemic cells of 45 patients with acute myeloid leukemia (AML). AML blasts from bone marrow (BM) (n = 39) and peripheral blood (PB) (n = 17) were incubated for 48 h with or without GM-CSF (100 U/ml) followed by a concurrent treatment with Increasing concentrations of araC (0.06 100 mu M) for an additional 24 h. After GM-CSF a 1.5-8.4-fold (median 2.3) increase in H-3-araC incorporation into the DNA was observed in ten of 14 peripheral blast specimens and in 23 of 28 bone marrow samples, 18 of whom also showed an enhanced H-3-TdR incorporation (1.5-8.5-fold, median 2.0-fold). Four different types of response were identified when analyzing H-3-araC incorporation into the DNA of bone marrow samples in relation to the applied araC dose: (i) 8/28 cases had increases of the araC incorporation at all araC dose levels applied (0.06-100 mu M), (ii) 12/28 at low araC concentrations only (0.06-1.0 mu M), (iii) 3/28 at high araC concentrations only (10-100 mu M), and (iv) 5/28 showed no increase at any dose level given. Hence, 20 of the 23 responding patients revealed a GMCSF induced enhancement of araC incorporation at low or conventional doses of araC (0.06-1.0 mu M). Fourteen of the 18 cases with concomitant rises of H-3-TdR and H-3-araC incorporation into the DNA after GM-CSF had elevated DNA polymerase cu activity (16-531%, median 72%) and in ten cases overall DNA polymerase activity was enhanced (10-70%, median 22.5%). In contrast, thymidine kinase (TK) and deoxycytidine kinase (dCK) activity were elevated after GM CSF in only ten and five patients, respectively. An increase in the fraction of cells in S phase was found in 11/21 bone marrow specimens and in 5/9 peripheral blast samples. However, no correlation was observed between increases in the proportion of cells in S phase and enhancements in enzyme activities. In 13 cases the cytotoxicity of araC with and without GM-CSF was assessed by means of a blast cell colony assay. Preincubation with GM-CSF increased the araC mediated cytotoxicity in ten of 13 patients by a median of 3.2-fold (range 2.2-229-fold). The respective LD(50) values for araC were reduced from 0.45 to 0.19 mu M on average. These results indicate that GM-CSF pretreatment increases araC incorporation into the DNA and subsequent cytotoxicity of araC mainly by activation of DNA polymerase alpha. The fact that a GM-CSF induced enhancement of araC incorporation into the DNA was observed predominantly at low or conventional doses of araC suggests that it may be more advantageous to apply conventional rather than highdose araC in clinical studies using pretherapeutic GM-CSF for an enhancement of araC cytotoxicity against leukemic cells.