Induction of apoptosis by lipophilic activators of CTP:phosphocholine cytidylyltransferase alpha (CCTalpha)

Abstract

Farnesol (FOH) inhibits the CDP-choline pathway for PtdCho (phosphatidylcholine) synthesis, an activity that is involved in subsequent induction of apoptosis. Interestingly, the rate-limiting enzyme in this pathway, CCTalpha (CTP:phosphocholine cytidylyltransferase alpha), is rapidly activated, cleaved by caspases and exported from the nucleus during FOH-induced apoptosis. The purpose of the present study was to determine how CCTalpha activity and PtdCho synthesis contributed to induction of apoptosis by FOH and oleyl alcohol. Contrary to previous reports, we show that the initial effect of FOH and oleyl alcohol was a rapid (10-30 min) and transient activation of PtdCho synthesis. During this period, the mass of DAG (diacylglycerol) decreased by 40%, indicating that subsequent CDP-choline accumulation and inhibition of PtdCho synthesis could be due to substrate depletion. At later time points (>1 h), FOH and oleyl alcohol promoted caspase cleavage and nuclear export of CCTalpha, which was prevented by treatment with oleate or DiC8 (dioctanoylglycerol). Protection from FOH-induced apoptosis required CCTalpha activity and PtdCho synthesis since (i) DiC8 and oleate restored PtdCho synthesis, but not endogenous DAG levels, and (ii) partial resistance was conferred by stable overexpression of CCTalpha and increased PtdCho synthesis in CCTalpha-deficient MT58 cells. These results show that DAG depletion by FOH or oleyl alcohol could be involved in inhibition of PtdCho synthesis. However, decreased DAG was not sufficient to induce apoptosis provided nuclear CCTalpha and PtdCho syntheses were sustained.

Figure 1. Transient stimulation of PtdCho synthesis by FOH in CHO-K1 cells

CHO-K1 cells were cultured in medium B for 24 h before the start of experiments. (A) Cells were cultured in choline-free medium B containing [³H]choline (2 μCi/ml) for 30 min prior to the addition of FOH (0–60 μM) for an additional 30 min. Cells were harvested and [³H]choline incorporation into PtdCho (▼), phosphocholine (■) and CDP-choline (▲) was measured. Results are the means±S.D. for triplicate determinations from a representative experiment. Abbreviations: P-choline, phosphocholine. (A–C) Cells were preincubated with [³H]choline (2 μCi/ml) for 30 h prior to the addition of 60 μM FOH (▲) or ethanol (■). At the indicated times, cells were harvested and isotope incorporation into PtdCho (B), phosphocholine (C) and CDP-choline (D) was quantified. Results are the means±S.D. for three independent experiments. *P<0.05 compared with ethanol control.

Figure 2. DAG depletion in FOH-treated CHO-K1 cells

CHO-K1 cells were cultured in medium B for 24 h before the start of experiments. (A) Cells were treated with increasing concentrations of FOH (0–60 μM) in medium B for 30 min and DAG mass was determined in lipid extracts as described in the Experimental section. Results are the means±S.D. for triplicate determinations from a representative experiment. (B) Cells were treated with FOH (60 μM) in medium B for the indicated time periods and masses of DAG (■) and ceramide (▲) were quantified in lipid extracts. Results are the means±S.D. for three independent experiments. *P<0.05 compared with control.

Figure 3. Transient stimulation of PtdCho synthesis by the CCTα activator oleyl alcohol

(A–C) CHO-K1 cells were cultured in medium B for 24 h before the addition choline-free medium B with [³H]choline (1.5 μCi/ml) for 30 min. Cells were then treated with oleyl alcohol (60 μM; ▲) or ethanol vehicle (■) for the indicated time periods and harvested, and [³H]choline incorporation into PtdCho (A), phosphocholine (B) and CDP-choline (C) was measured. Results are the means±S.D. for triplicate determinations from a representative experiment. (D) CHO-K1 cells were cultured in medium B and treated with oleyl alcohol (60 μM) for up to 2 h. Masses of DAG (■) and ceramide (▲) were quantified in lipid extracts. Results are the means±S.D. for three independent experiments. *P<0.005 compared with solvent control.

Figure 4. FOH-induced caspase cleavage of PARP and CCTα is prevented by oleate or DAG

CHO-K1 cells were cultured in medium A (FCS) for 16 h, medium B (LPDS) for 16 h or in medium B for 16 h followed by supplementation with oleate or DiC8 (500 μM) for 30 min prior to the addition of FOH (60 μM). Cells were harvested at the indicated times after FOH addition, total cellular extracts (15 μg of protein) were resolved by SDS/PAGE, and CCTα and PARP were detected by immunoblotting. Similar results were obtained in two independent experiments. Asterisk indicates a protein that cross-reacts with the PARP antibody but is not related to the 85 kDa PARP caspase cleavage product.

Figure 5. Oleyl alcohol induces DAG-dependent apoptosis

CHO cells were cultured in medium B for 24 h before the start of experiments. (A) Cells (in medium B) were incubated for 4 h with increasing concentrations of oleyl alcohol (0–89 μM) or preincubated for 30 min with DiC8 (500 μM) prior to the addition of oleyl alcohol (60 μM). Total cellular extracts (15 μg of protein) were separated by SDS/PAGE and immunoblotted for CCTα. (B) Cytoplasmic nucleosomes were measured in cells cultured in medium B and treated with oleyl alcohol (60 μM) for 4 h in the absence or presence of a 30 min preincubation with DiC8 (500 μM). Results are expressed as fold increase in apoptosis relative to ethanol-treated control cells and are the averages of duplicate determinations from a representative experiment. Abbreviation: OIOH, oleyl alcohol.

Figure 6. Oleate and DiC8 prevent FOH- and oleyl alcohol-mediated export of CCTα from the nucleus

(A) CHO-K1 cells cultured on glass coverslips were incubated in medium A (FCS) or medium B (LPDS) for 24 h, or cultured in medium B supplemented with oleate (500 μM), DiC8 (500 μM) or TPA (200 nM) for 30 min prior to addition of FOH (60 μM) or ethanol (no addition, NA) for 2 h. CCTα was localized by immunofluorescence using an Alexa-488 conjugated secondary antibody and chromatin was visualized with Hoechst 33258 as described in the Experimental section. (B) CHO-K1 cells cultured in medium B for 24 h were treated with oleyl alcohol (60 μM) in medium B for the indicated time periods. One set of cells was preincubated for 30 min with DiC8 (500 μM) prior to addition of oleyl alcohol for 4 h. Cells were processed for immunofluorescence localization of CCTα and chromatin as described above.

Figure 7. Triacsin C prevents oleate-mediated rescue from FOH-induced apoptosis

CHO-K1 cells were cultured in medium B for 24 h prior to the start of experiments. (A, B) Cells (in medium B) were preincubated for 30 min with oleate (500 μM) or DiC8 (500 μM) in the absence or presence of triacsin C (9.6 μM) prior to treatment with FOH (60 μM) for 2 h. Total cellular extracts (15 μg of protein) were resolved by SDS/PAGE, and CCTα and PARP were detected by immunoblotting. (C, D) Levels of cytoplasmic nucleosomes were measured in cells treated as described for (A, B) using the Cell Death ELISA, and results are expressed relative to ethanol-treated controls. Results are the averages of duplicate determinations from a representative experiment.

Figure 8. Effects of oleate and DiC8 on PtdCho synthesis and DAG mass in FOH-treated CHO-K1 cells

(A) CHO-K1 cells were cultured for 16 h in medium B followed by the addition of medium B supplemented with oleate (250 μM), DiC8 (200 μM) or solvent control (no addition; ‘NA’). After 30 min, cells received FOH (60 μM) (black bars) or no treatment (white bars). After FOH treatment for 2 h, cells were harvested and total lipids were extracted for DAG quantification. Results are the means±S.D. for 3–6 independent experiments. *P<0.001 compared with untreated or oleate-treated cells; ns, not significant compared with DiC8-treated cells. (B) PtdCho synthesis was measured by pulse-labelling cells (treated as described for A) with [³H]choline for the final 1 h of FOH treatment (60 μM). Results are the means±S.D. for three independent experiments.

Figure 9. Overexpression of CCTα in MT58 cells partially prevents apoptosis in response to FOH

MT58 or MT58-CCTα cells were cultured in medium B for 16 h prior to the addition of medium B containing FOH (60 μM). (A) Cells were incubated with FOH (0–6 h) or solvent (zero time control), and cytoplasmic nucleosomes were measured as an index of apoptosis. Results are presented as fold increase in nucleosome release relative to solvent-treated control, and are the means±S.D. for five independent experiments. (B) Cells received choline-deficient medium B for 6 h followed by staggered addition of FOH for the indicated time periods. For the final hour of each treatment, cells were pulse-labelled with [³H]choline (2 μCi/ml) and incorporation into PtdCho was quantified. Results are the means±S.D. for three independent experiments. *P<0.005.