To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably overexpressed in S1P3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the S1P (sphingosine 1-phosphate) receptor S1P3]. Treatment of S1P3-CHO cells with ActD (actinomycin D) induced apoptosis, as shown by the occurrence of nuclear fragmentation and the caspase-dependent proteolytic cleavage of PARP [poly(ADP-ribose) polymerase] and protein kinase Cδ. Overexpression of either PLD1 or PLD2 protected S1P3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cδ cleavage. However, in the early phase of apoptosis, ActD induced an increase in PLD activity and activation of key factors in the cell-survival signalling pathways, such as PI3K (phosphoinositide 3-kinase), Akt, p70S6K (p70 S6 kinase) and ERK (extracellular-signal-regulated kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD1 or PLD2. The PI3K inhibitor LY294002 inhibited the ActD-induced activation of Akt and p70S6K, and completely abolished the effects of PLD1 or PLD2, whereas inhibition of ERK activity by the MEK inhibitor U0126 had a milder effect. The ActD-induced activation of p70S6K and ERKs was blocked by 1-butanol, but not by t-butanol; similar to S1P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in S1P3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways.
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Research Article|
March 01 2004
Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells
Momoko YAMADA;
Momoko YAMADA
*Department of Biochemistry, Gifu Pharmaceutical University, Mitahora, Gifu, Japan
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Yoshiko BANNO;
Yoshiko BANNO
1
†Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu, Japan
1To whom correspondence should be addressed (e-mail banno@cc.gifu-u.ac.jp).
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Yoh TAKUWA;
Yoh TAKUWA
‡Department of Physiology, Kanazawa University Graduate School of Medicine, Takaramachi, Kanazawa, Japan
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Masahiro KODA;
Masahiro KODA
†Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu, Japan
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Akira HARA;
Akira HARA
*Department of Biochemistry, Gifu Pharmaceutical University, Mitahora, Gifu, Japan
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Yoshinori NOZAWA
Yoshinori NOZAWA
§Gifu International Institute of Biotechnology, Kakamigahara, Japan
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Publisher: Portland Press Ltd
Received:
September 15 2003
Revision Received:
November 17 2003
Accepted:
November 25 2003
Accepted Manuscript online:
November 25 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 378 (2): 649–656.
Article history
Received:
September 15 2003
Revision Received:
November 17 2003
Accepted:
November 25 2003
Accepted Manuscript online:
November 25 2003
Citation
Momoko YAMADA, Yoshiko BANNO, Yoh TAKUWA, Masahiro KODA, Akira HARA, Yoshinori NOZAWA; Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells. Biochem J 1 March 2004; 378 (2): 649–656. doi: https://doi.org/10.1042/bj20031398
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