Inhibition of de novo synthesis of phosphatidylcholine (PC) by some anti-cancer drugs such as hexadecylphosphocholine leads to apoptosis in various cell lines. Likewise, in MT58, a mutant Chinese hamster ovary (CHO) cell line containing a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), an important regulatory enzyme in the CDP-choline pathway, inhibition of PC synthesis causes PC depletion. Cellular perturbations like metabolic insults and unfolded proteins can be registered by the endoplasmic reticulum (ER) and result in ER stress responses, which can lead eventually to apoptosis. In this study we investigated the effect of PC depletion on the ER stress response and ER-related proteins. Shifting MT58 cells to the non-permissive temperature of 40°C resulted in PC depletion via an inhibition of CT within 24h. Early apoptotic features appeared in several cells around 30h, and most cells were apoptotic within 48h. The temperature shift in MT58 led to an increase of pro-apoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as GADD153) after 16h, to a maximum at 24h. Incubation of wild-type CHO-K1 or CT-expressing MT58 cells at 40°C did not induce differences in CHOP protein levels in time. In contrast, expression of the ER chaperone BiP/GRP78, induced by an increase in misfolded/unfolded proteins, and caspase 12, a protease specifically involved in apoptosis that results from stress in the ER, did not differ between MT58 and CHO-K1 cells in time when cultured at 40°C. Furthermore, heat-shock protein 70, a protein that is stimulated by accumulation of abnormal proteins and heat stress, displayed similar expression patterns in MT58 and K1 cells. These results suggest that PC depletion in MT58 induces the ER-stress-related protein CHOP, without raising a general ER stress response.
- CHO mutant MT58
- CTP:phosphocholine cytidylyltransferase
Abbreviations used: C/EBP, CCAAT/enhancer-binding protein; CHO, Chinese hamster ovary; CHOP, CCAAT/enhancer-binding protein-homologous protein; CT, CTP:phosphocholine cytidylyltransferase; ER, endoplasmic reticulum; FMK, t-butoxycarbonyl-d-fluoromethylketone; HSP, heat-shock protein; lysoPC, lysophosphatidylcholine; PC, phosphatidylcholine; PERK, double-stranded-RNA-regulated protein kinase-like ER kinase.
- The Biochemical Society, London ©2003