Q L Zhao1, T Kondo, A Noda, Y Fujiwara. 1. Department of Radiation Biophysics and Genetics, Kobe University School of Medicine, Japan.
Abstract
PURPOSE: To investigate the mechanisms and pathways of X-ray apoptosis in Molt-4 cells, focusing on mitochondrial and cytosolic Ca2+ ([Ca2+]i) regulation. MATERIALS AND METHODS: X-irradiated Molt-4 cells and cell extract (CE) were used to analyse: (1) induced apoptosis (Giemsa stain), (2) p53, Bcl-2 and Bax expressions (immunoblot), (3) mitochondrial potential deltapsi(m) and (4) [Ca2+]i (flow cytometry), (5) caspase-3 activity, and (6) roles of [Ca2+]- and caspase-3-mediated pathways by inhibiting either or both pathways for induced apoptosis. RESULTS: Molt-4 cells were sensitive to apoptosis since 5 Gy induced 57 and 94% apoptosis at 6 and 24 h. After 5Gy, p53 was accumulated that upregulated Bax but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6 h. Predominant Bax reduced deltapsi(m), and low-deltapsi(m) cells increased 45 min earlier than apoptosis after 5 Gy. Caspase-3 was activated in apoptotic CE. The caspase-3 inhibitor Ac-DEVD-CHO inhibited apoptosis and DNA-ladder formation by approximately 50%, suggesting a approximately 50% role of caspase-3-activated DNase (CAD). [Ca2+]i was increased after 5 Gy. [Ca2+]i-chelating BAPTA-AM (5 microM) and/or DNase gamma-inhibiting Zn2+ (0.5 mM) inhibited approximately 50% of induced apoptosis and DNA-laddering, indicating a 50% participation of Ca2+/Mg2+-dependent DNase gamma. CONCLUSIONS: The p53-Bax-mitochondria-caspase-3-CAD pathway and the [Ca+2]i-mediated DNase gamma pathway were involved in the regulation of X-ray apoptosis in sensitive Molt-4 cells.
PURPOSE: To investigate the mechanisms and pathways of X-ray apoptosis in Molt-4 cells, focusing on mitochondrial and cytosolic Ca2+ ([Ca2+]i) regulation. MATERIALS AND METHODS: X-irradiated Molt-4 cells and cell extract (CE) were used to analyse: (1) induced apoptosis (Giemsa stain), (2) p53, Bcl-2 and Bax expressions (immunoblot), (3) mitochondrial potential deltapsi(m) and (4) [Ca2+]i (flow cytometry), (5) caspase-3 activity, and (6) roles of [Ca2+]- and caspase-3-mediated pathways by inhibiting either or both pathways for induced apoptosis. RESULTS: Molt-4 cells were sensitive to apoptosis since 5 Gy induced 57 and 94% apoptosis at 6 and 24 h. After 5Gy, p53 was accumulated that upregulated Bax but which repressed Bcl-2 with time, resulting in a 7-fold increase in Bax/Bxl-2 at 6 h. Predominant Bax reduced deltapsi(m), and low-deltapsi(m) cells increased 45 min earlier than apoptosis after 5 Gy. Caspase-3 was activated in apoptotic CE. The caspase-3 inhibitor Ac-DEVD-CHO inhibited apoptosis and DNA-ladder formation by approximately 50%, suggesting a approximately 50% role of caspase-3-activated DNase (CAD). [Ca2+]i was increased after 5 Gy. [Ca2+]i-chelating BAPTA-AM (5 microM) and/or DNase gamma-inhibiting Zn2+ (0.5 mM) inhibited approximately 50% of induced apoptosis and DNA-laddering, indicating a 50% participation of Ca2+/Mg2+-dependent DNase gamma. CONCLUSIONS: The p53-Bax-mitochondria-caspase-3-CAD pathway and the [Ca+2]i-mediated DNase gamma pathway were involved in the regulation of X-ray apoptosis in sensitive Molt-4 cells.
Authors: Patrick Voos; Sebastian Fuck; Fabian Weipert; Laura Babel; Dominique Tandl; Tobias Meckel; Stephanie Hehlgans; Claudia Fournier; Anna Moroni; Franz Rödel; Gerhard Thiel Journal: Front Immunol Date: 2018-04-30 Impact factor: 7.561