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

Abstract

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 high-performance 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 μM) caused a significant decrease in intracellular ara-CTP levels; e.g., incubation for 72 hours with ATRA 1 μM prior to one hour ara-C 10 μM 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.