All-trans-retinoic acid increases cytosine arabinoside cytotoxicity in HL-60 human leukemia cells in spite of decreased cellular ara-CTP accumulation

Background: Accumulation of the cytosine arabinoside (ara-C) metabolite ara-C-triphosphate (ara-CTP) in leukemic blast cells is considered to be the main determinant of ara-C cytotoxicity in vitro and in vivo. Retinoids such as all-trans-retinoic acid (ATRA) have been shown to increase the sensitivity of acute myelogenous leukemic (AML) blast cells to ara-C. To investigate the mechanism of this sensitisation, the hypothesis was tested that ATRA augments cellular ara-CTP levels in human-derived myelogenous leukemia HL-60 cells. Materials and methods. The effect of ATRA and 13-cis-retinoic acid on ara-CTP accumulation and ara-C-induced apoptosis was studied. Ara-CTP levels were measured by highperformance liquid chromatography (HPLC), cytotoxicity by the tetrazolium (MTT) assay, and apoptosis by occurrence of DNA fragmentation (gel electrophoresis), cell shrinkage and DNA loss (flow cytometry). Results. Pretreatment of HL-60 cells with ATRA (0.01-1 mu M) caused a significant decrease in intracellular ara-CTP levels; e.g., incubation for 72 hours with ATRA 1 mu M prior to one hour ara-C 10 mu M reduced ara-CTP levels to 41% +/- 4% of control. Similar results were obtained after preincubation with 13-cis-retinoic acid. In spite of decreased ara-CTP levels, the cytotoxicity of the combination was supraadditive and ATRA augmented ara-C-induced apoptosis. Conclusions: At therapeutically relevant concentrations ATRA. increased ara-C cytotoxicity and ara-C induced apoptosis but this augmentation is not the corollary of elevated ara-CTP levels. The feasibility of ara-C treatment optimisation via strategies other than those involving elevation of ara-CTP levels should be investigated further.