Institute of Medical Science, Tokyo Medical University, Japan
Abstract:
Choline is essential for the synthesis of major membrane phospholipid phosphatidylcholine. Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism, and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable, and mediated by a single transport system. Choline uptake was inhibited by unlabeled choline and the choline analogue hemicholinium-3 (HC-3). Various organic cations also interacted with the choline transport system. Interestingly, removal of Na+ from uptake buffer strongly enhanced choline uptake. Increase component of choline uptake under the Na+-free condition was inhibited by dimethylamiloride, a Na+/H+ exchanger (NHE) inhibitor. RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed in NCI-H69. Furthermore, the conversion of choline to ACh was confirmed with NCI-H69 cells, and was enhanced under Na+-free conditions, which is sensitive to HC-3. These results indicate that choline uptake through CTL1 was used for ACh synthesis. CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. These results demonstrate that functional inhibition of CTL1 could promote apoptotic cell death. We conclude that NCI-H69 express choline transporter CTL1. These choline transporters utilized a directed H+ gradient as a driving force, and its transport functions co-operatively with NHE1. This system primarily supplies choline for the synthesis of ACh and secrete ACh to act as an autocrine growth factor. Identification of this new CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.