The anticarcinogenic drug PRIMA-1 (p53 reactivation and induction of massive apoptosis 1) induces suicidal death of tumor cells, an effect in large part attributed to the up-regulation of the proapoptotic transcription factor p53. Erythrocytes are lacking gene transcription but are nevertheless able to enter eryptosis, a suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of

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... s include increase of cytosolic Ca 2+ -activity ([Ca 2+ ] i ) and ceramide formation. The present study tested whether PRIMA-1 stimulates eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca 2+ ] i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and ROS formation from DCFDA fluorescence. Results: A 48 h exposure of human erythrocytes to PRIMA-1 (25 µM) significantly increased the percentage of annexin-V-binding cells without significantly influencing [Ca 2+ ] i or forward scatter. PRIMA-1 (100 µM) induced annexin-V-binding was not significantly blunted by removal of extracellular Ca 2+ or by the caspase-3 inhibitor zVAD. PRIMA-1 (100 µM) further increased the ceramide abundance at the cell surface and ROS formation. Conclusions: PRIMA-1 stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance and ROS formation. C. Faggio and K. Alzoubi contributed equally and share first authorship. 530 Faggio/Alzoubi/Calabrò/Lang: PRIMA-1-Induced Eryptosis Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry Analysis of annexin-V-binding and forward scatter After incubation under the respective experimental condition, 50 µl cell suspension was washed in Ringer solution containing 5 mM CaCl 2 and then stained with Annexin-V-FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37°C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V-FITC fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD, Heidelberg, Germany). Measurement of intracellular Ca 2+ After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Biotium, Hayward, USA) in Ringer solution containing 5 mM CaCl 2 and 5 µM Fluo-3/AM. The cells were incubated at 37°C for 30 min and washed twice in Ringer solution containing 5 mM CaCl 2 . The Fluo-3/ AM-loaded erythrocytes were resuspended in 200 µl Ringer. Then, Ca 2+ -dependent fluorescence intensity 531 Faggio/Alzoubi/Calabrò/Lang: PRIMA-1-Induced Eryptosis Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur. Determination of ceramide formation To determine ceramide abundance, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37°C with 1μg/ml anti-ceramide antibody (clone MID 15B4; Alexis, Grünberg, Germany) in phosphate-buffered saline (PBS) containing 0.1 % bovine serum albumin (BSA) at a dilution of 1:10. After two washing steps with PBS-BSA, cells were stained for 30 min with polyclonal fluoresceinisothiocyanate (FITC)-conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. Samples were then analyzed by flow cytometric analysis at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. 532 Faggio/Alzoubi/Calabrò/Lang: PRIMA-1-Induced Eryptosis Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry Alterations of cytosolic Ca 2+ activity ([Ca 2+ ] i ) were estimated utilizing Fluo3 fluorescence, which was again determined in flow cytometry. As shown in Fig. 3A ,B, a 48 h exposure to PRIMA-1 (10 -100 µM) did not significantly modify Fluo3 fluorescence, indicating that PRIMA-1 did not alter appreciably [Ca 2+ ] i . Further experiments were performed to explore whether the PRIMA-1-induced cell membrane scrambling required entry of extracellular 533 Faggio/Alzoubi/Calabrò/Lang: PRIMA-1-Induced Eryptosis Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry Ca 2+ . To this end, the erythrocytes were exposed for 48 h to 100 µM PRIMA-1 in the presence or nominal absence of extracellular Ca 2+ . As shown in Fig. 3C , removal of extracellular Ca 2+ did not significantly affect the increase of annexin-V-binding following PRIMA-1 treatment.