TY - JOUR
T1 - SR compartment calcium and cell apoptosis in SERCA overexpression
AU - Ma, T. S.
AU - Mann, D. L.
AU - Lee, J. H.
AU - Gallinghouse, G. J.
N1 - Funding Information:
G.J.G. and J.R.L. were Cardiology Fellows at the Bugher Foundation of Molecular Cardiology. We appreciate the gift of the adenovirus shuttle vectors and adenoviral LacZ reporter from Dr Frank L. Graham of McMaster University. We are indebted to Drs Andrew Schafer and Robert Roberts for their constant encouragement. We wish to acknowledge help with flow cytometry from Dr Dorothy Lewis and Mr Jeff Scott of the Department of Immunology, Baylor College of Medicine. The editorial assistance from Ms Pat Salis is appreciated. This research was supported by Veterans Administration Merit Review funds and by a Grant-in-Aid from the American Heart Foundation.
PY - 1999/7
Y1 - 1999/7
N2 - The relationship between SR Ca2+ ATPase (SERCA) activities, cell calcium level, SR calcium store and cell cycle events is not clearly understood. We studied SERCA overexpression in Cos cells using an adenovirus vector. Twofold increases in SERCA mRNA and in protein were correlated with a 2.3-fold and a 1.6-fold paralleled increase in SR calcium pump activity (R = 0.97 and R = 0.99 respectively). Dose-related apoptotic cell death was associated with SERCA overexpression (R = 0.92). When serum was reduced to 4%, cell apoptosis further increased from 20.7 ± 4.8% to 47.5 ± 12.9% (M ± SD; P < 0.05; n = 3). Flow cytometry identified cell cycle arrest at the G2/M phase. The interleukin-1 converting enzyme (ICE) inhibitor z-VAD-fmk reduced apoptosis for low-, medium- and high-expressing constructs, whereas the CPP-32 inhibitor z-DEVD-fmk had no effect. Flow cytometry using Fluo-3 and Fura-Red revealed a 1.5-fold higher basal calcium and a 10-fold SR calcium overload. ICE inhibitor z-VAD-fmk did not alter calcium loading. An epitope-tagged SERCA mutant, which has no intrinsic Ca2+-pump activities, had a much smaller effect on the SR calcium. These findings suggest that SERCA2A overexpression has an intrinsic role in altering cell-cycle progression, augmenting cellular and SR calcium loading, and precipitating ICE protease-mediated apoptosis; this represents as a novel model for primary SR calcium overload and associated cell apoptosis.
AB - The relationship between SR Ca2+ ATPase (SERCA) activities, cell calcium level, SR calcium store and cell cycle events is not clearly understood. We studied SERCA overexpression in Cos cells using an adenovirus vector. Twofold increases in SERCA mRNA and in protein were correlated with a 2.3-fold and a 1.6-fold paralleled increase in SR calcium pump activity (R = 0.97 and R = 0.99 respectively). Dose-related apoptotic cell death was associated with SERCA overexpression (R = 0.92). When serum was reduced to 4%, cell apoptosis further increased from 20.7 ± 4.8% to 47.5 ± 12.9% (M ± SD; P < 0.05; n = 3). Flow cytometry identified cell cycle arrest at the G2/M phase. The interleukin-1 converting enzyme (ICE) inhibitor z-VAD-fmk reduced apoptosis for low-, medium- and high-expressing constructs, whereas the CPP-32 inhibitor z-DEVD-fmk had no effect. Flow cytometry using Fluo-3 and Fura-Red revealed a 1.5-fold higher basal calcium and a 10-fold SR calcium overload. ICE inhibitor z-VAD-fmk did not alter calcium loading. An epitope-tagged SERCA mutant, which has no intrinsic Ca2+-pump activities, had a much smaller effect on the SR calcium. These findings suggest that SERCA2A overexpression has an intrinsic role in altering cell-cycle progression, augmenting cellular and SR calcium loading, and precipitating ICE protease-mediated apoptosis; this represents as a novel model for primary SR calcium overload and associated cell apoptosis.
UR - http://www.scopus.com/inward/record.url?scp=0032701794&partnerID=8YFLogxK
U2 - 10.1054/ceca.1999.0049
DO - 10.1054/ceca.1999.0049
M3 - Article
C2 - 10892568
AN - SCOPUS:0032701794
SN - 0143-4160
VL - 26
SP - 25
EP - 36
JO - Cell Calcium
JF - Cell Calcium
IS - 1-2
ER -