Human T-cell leukemia (Jurkat) cells were obtained from ATCC, Manassas, VA. These cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 10 μM gentamycin (Life Technologies, Mississauga, ON) in an incubator set at 37°C, 5% CO2 and 95% humidity.
Human nucleated blood cells were isolated from whole blood taken from a healthy non-smoking male according to the University of Windsor REB #04-147. The whole blood sample was collected in a BD Vacutainer™ CPT (Cell Preparation Tube) obtained from Becton Dickinson, Franklin Lakes, NJ, and spun at 2900 rpm for 30 min at 25°C. The upper layer consisting of mononuclear cells, platelets and plasma was collected and maintained with RPMI 1640 media supplemented with 10% FBS and 10 μM gentamycin (Life Technologies) in the same incubator as the Jurkat cells.
Cells were grown and treated with either AMD4 or AMD5 alkaloids at various concentrations and time-points. AMD4 and AMD5 (99.5% pure) were derived as detailed by Pettit et al., 1993 .
Cellular viability assay
Cells were grown to 70% confluence and treated with AMD4 and AMD5 at a final concentration of 10 μM. The cells were then stained with the cell permeable dye Hoechst 33342 (Molecular Probes, Eugene, OR) at 10 μM final concentration and incubated for 10 min at 25°C. Brightly stained, condensed nuclei are characteristic features of apoptotic cells, as visualized with a fluorescent microscope (Leica DM IRB, Germany). Images were captured at 10× and 40× objectives; the percentage of apoptotic cells was calculated from the total number of cells with Microsoft® Excel 6.0 software and pictures were compiled using Adobe® Photoshop 7.0 software. A minimum of 5 fields of at least 100 cells per field was counted. Statistical significance was determined using STATISTICA® software.
Annexin-V binding assay
Jurkat cells were treated with 10 μM AMD4 and AMD5 for 24 hours. The Annexin-V binding assay was conducted according to the manufacturer's protocol using a kit purchased from Molecular Probes, Eugene, OR. Briefly, cells were washed with phosphate-buffered saline (PBS) and re-suspended in Annexin-V binding buffer (10 mM HEPES/NaOH pH 7.5, 140 mM NaCl, 2.5 mM CaCl2), containing Annexin-V Alexa Fluor® 488 conjugate (1:50) for 15 min at 25°C; pictures were taken with a fluorescent microscope (Leica DM IRB, Germany) at 40× objective and compiled using Adobe® Photoshop 7.0 software.
After treating Jurkat cells with AMD4 and AMD5 at indicated times the TUNEL Assay was performed as per manufacturer's protocol (Molecular Probes, Eugene, OR) and a previously published method , to detect DNA damage. Cells were fixed by suspending them in 70% (v/v) ethanol and stored at -20°C overnight. The sample was then incubated with DNA-labeling solution (10 μL reaction buffer, 0.75 μL TdT enzyme, 8 μL BrdUTP, 31.25 μL of dH2O) for 1 hr at 25°C. Each sample was then exposed to an antibody solution consisting of 5 μL Alexa Fluor® 488 labeled anti-BrdU antibody with 95 μL rinse solution and allowed to react for 20 min; pictures were taken at 20× objective using a fluorescent microscope (Leica DM IRB, Germany).
The caspase-3 assay was carried out using a previously published method . Briefly, Jurkat or normal lymphocyte cellular lysates were collected and incubated with the fluorogenic substrate DEVD-AFC (MP Biomedicals, Aurora, OH) in DEVD buffer (0.1 M HEPES, pH 7.4, 2 mM DTT, 0.1% CHAPS, 1% sucrose) and allowed to incubate at 37°C for 45 min. Fluorescence was measured at 400 nm excitation and 505 nm emission using the Spectra Max Gemini XS (Molecular Devices, Sunnyvale, CA). Caspase-3 activity was calculated per microgram of protein, and expressed as a percentage of control activity. Protein concentration was determined utilizing the BioRad protein assay reagent (BioRad, Mississauga, ON, Canada) with bovine serum albumin as a standard. Microsoft® Excel 6.0 software was used for data representation; statistical significance was determined using STATISTICA® software.
The Mito-Casp assay was performed as per manufacturer's protocol using a kit purchased from Cell Technology Inc., Mountain View, CA. Briefly, cells were treated with either alkaloid at 10 μM concentration for the desired times and washed twice in PBS. Cells were re-suspended in PBS according to protocol and incubated with both MMP dye and caspase reagent at a 1:30 dilution for 60 min at 37°C in darkness. Following incubation, the cell pellet was collected and re-suspended in wash buffer; fluorescence was measured from a 96-well micro-titre plate at 549 nm excitation and 574 nm emission using the Spectra Max Gemini XS (Molecular Devices, Sunnyvale, CA). Loss of MMP was presented as a loss in relative fluorescence units per 10,000 cells. Microsoft® Excel 6.0 software was used for data representation; statistical significance was determined using STATISTICA® software.