Effects of Arsenic Trioxide on Megakaryocytic Cell Lines: Apoptosis, Cell Cycle Dysfunction and Signaling Pathways.

Despite progress made in the elucidation of the actions of arsenic trioxide (ATO) in acute promyelocytic leukemia, the molecular mechanisms leading to apoptosis in other malignancies remain unclear. In particular, the effects of ATO on the megakaryocytic (MK) lineage have not been well characterized. In this study, we focused on two MK cell lines CHRF-288-11 (CHRF) and MEG-01, which were derived from an infant and adult acute megakaryocytic leukemia (AMKL), respectively. Our data showed that these cells underwent apoptosis within 24 – 48 h post-ATO (6 μM) treatment, as demonstrated by the Annexin V assay (Table 1). By flow cytometry, significant activation of caspase-3 was detected in the MK cells at 24 h, and was preceded by the loss of mitochondrial membrane potential (8 h) as determined by the fluorescent dye JC-1. Western blotting experiments showed that ATO induced Bax expression and down-regulated Bcl-2, which led to an increase in Bax/Bcl-2 ratio. ATO exerted immediate and significant interference on the cell cycle by delaying S-phase progression and the subsequent accumulation of cells in the G2/M phase (43.2% vs 13.6%, p < 0.01). By multivariate analysis (BrdU and 7-AAD), active caspase-3 was detected in all phases of the cell cycle. The responses of CHRF and MEG-01 cells to ATO were similar, except that the latter appeared more resistant, in terms of the dosage of ATO and the slight delayed onset of apoptosis. We screened the expression levels of 96 genes involved in apoptosis using the GEArray Q Series Human Apoptosis Gene Array at 0, 4, 8 and 16 h (each n = 2) post-ATO treatment. We identified the up-regulation of mRNA of two extrinsic components of apoptosis. Fas was progressively increased in both cell lines (up to 6.14-fold) and caspase-8 was elevated in MEG-01 (3.58-fold). The protein expressions of Fas and activated caspase-8 were demonstrated in both cell lines by flow cytometry. Increased mRNA expressions of caspase-1 (2.30-fold) and CD137 (2.33-fold) were also noted, but their significance in apoptosis of our system remained to be investigated. To demonstrate the direct effect of ATO on gene expressions in AMKL cells, a more comprehensive microarray (Human 19K Array, Ontario Cancer Institute Microarray Centre) was used. Treatment with ATO for 4 h (n = 3) prompted an elevation in the mRNA levels of stress-associated proteins, such as metallothioneins (MT1G: 6.31-fold; MT2A: 3.64-fold), Hsp72 (5.81-fold), Hsp73 (3.77-fold), Hsp90 (2.11-fold), ferritin (2.02-fold) and ubiquitin (2.76-fold). Interestingly, WT1, a cell cycle regulatory gene elevated in many types of leukemia, was induced by ATO (2.44-fold). In conclusion, our results suggested that apoptosis in AMKL cells mediated by ATO involved a switch from pro-survival in the early phase to the activation of multiple death machineries, consisting of the intrinsic (mitochondrial, Bax, Bcl-2) and the extrinsic (Fas, caspase-8) compartments. Table 1: Signals regulated by ATO in CHRF cells 0 h 24 h 48 h Mean ± SEM; * p < 0.05 compared to 0 h; # n = 2, others n = 3–5. Annexin V +/PI − (%) 4.56±0.28 8.28±0.53* 9.83±0.73* Active caspase-3 (%) 2.28±0.13 4.58±0.87* 14.7±1.16* JC-1 greenhi/redlo (%) 4.18±0.52 8.05±0.60* 20.76±8.69* Bax/Bcl-2 (Fold)# 0.63±0.08 2.65±0.68 - Fas (Fold) 1 1.73±0.17* 1.96±0.20* CD137 (Fold) 1 1.55±0.08* 1.76±0.03*