Choline is essential for the synthesis of the major membrane
phospholipid phosphatidylcholine and the
neurotransmitter acetylcholine (ACh). 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 the
small cell lung carcinoma cell line NCI-H69.
Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake
buffer strongly enhanced
choline uptake. This increase in
choline uptake under the Na(+)-free conditions was inhibited by
dimethylamiloride (DMA), a
Na(+)/H(+) exchanger (NHE) inhibitor. Various organic
cations and the
choline analog hemicholinium-3 (HC-3) inhibited the
choline uptake and cell viability. A correlation analysis of the potencies of organic
cations for the inhibition of
choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that
choline transporter-like
protein 1 (CTL1)
mRNA and NHE1 are mainly expressed. HC-3 and CTL1
siRNA inhibited
choline uptake and cell viability, and increased
caspase-3/7 activity. The conversion of
choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that
choline uptake through CTL1 is used for ACh synthesis. Both an
acetylcholinesterase inhibitor (
eserine) and a
butyrylcholinesterase inhibitor (
ethopropazine) increased cell proliferation, and these effects were inhibited by
4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the
choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies
choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine
growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated
choline transport system provides a potential new target for therapeutic intervention.