Studies were performed with copper-free culture medium (Fig. 2B) to prevent the complexation and transport of exogenous copper by the cell, but these experiments revealed no changes in the copper uptake or removal in the cells during the period of the study. The intracellular zinc content was examined by atomic absorption spectroscopy but revealed no zinc uptake by cells subjected to similar

DEDTC treatments (Figure S1). To determine the influence of DEDTC in the cell cycle the nuclei were stained with propidium iodide (PI) prior to flow cytometry click here analysis. The cell cycle studies revealed that cells treated with DEDTC exhibited no changes in the cell cycle during the first 24 h of treatment (Fig. 2C) compared with the control cells. However, within 48 h of incubation, the treatment induced an increase in the population of cells in the sub-G1 phase and a slight decrease in the G2/M phase. Approximately 0.7% of the control cells were in the sub-G1 phase, while approximately 10% of the cells treated with 5 μM DEDTC were in this phase (Fig. 2C). To verify if this increase in the sub-G1 population was due to apoptosis, SH-SY5Y cells were labeled with FITC-conjugated Annexin V and PI for flow cytometry Mitomycin C analysis. The results of the flow cytometry study with Annexin V/FITC and PI showed that,

within 12 h of incubation, approximately 7% of the cells treated with 5 μM DEDTC underwent early apoptosis compared to the less than 2% of apoptotic cells observed in the control. During the course of the incubation period 12% of the cells were in early apoptosis and 5% in late apoptosis following 48 h of incubation (Fig. 3B, treatment). The untreated cells maintained a similar percentage of apoptotic cells at all incubation times, with greater than 95% of the cells remaining viable (Fig. 3B, control). Due to the percentage of cells entering apoptosis upon treatment

with 5 μM DEDTC, the apoptotic pathways were investigated to determine a molecular mechanism for this event. The results of the Western blot analysis of cells treated with 5 μM DEDTC showed an approximately 15% increase in caspase 8 protein levels compared with the untreated cells. The same profile was observed after 24 h of incubation with a 28% increase in caspase-8 levels (Fig. 3A). Caspase 3 was also observed to increase upon DEDTC treatment, particularly when the cells were treated for Progesterone 24 h, as this effector caspase is activated after caspase 8. The levels of p53 were also increased at all incubation times compared with their respective controls, with a greater increase in the first 12 h of treatment with 5 μM DEDTC that remained constant until 24 h following the addition of DEDTC (Fig. 3A). Levels of Bcl-2 protein in cells treated with DEDTC remained unchanged and similar to control cells for 24 h (data not shown). To better understand the way in which the apoptotic cascade was activated, we employed immunocytochemistry with colocalization.