Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells

To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably over-expressed in SIP3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the SIP (sphingosine I-phosphate) receptor S1P(3)]. 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 Cdelta. Overexpression of either PLD1 or PLD2 protected SIP3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cdelta 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-regularted kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD I 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 I-butanol, but not by t-butanol; similar to S I P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in SIP3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways.