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Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells
Archives of Biochemistry and Biophysics 483 (2009) 90–98
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Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells Hironobu Kouji a,c, Masato Inazu a,b,*, Tomoko Yamada a, Hirohisa Tajima a, Tatsuya Aoki c, Teruhiko Matsumiya a,b a
Department of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan Department of Preventive Medicine (SONOKO), Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan c Third Department of Surgery, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan b
a r t i c l e
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Article history: Received 6 November 2008 and in revised form 12 December 2008 Available online 25 December 2008 Keywords: Choline Transporter Na+/H+ exchanger Colon carcinoma Cell proliferation
a b s t r a c t We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. 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 component of choline uptake under Na+-free conditions was inhibited by a Na+/H+ exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H+ electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H+ gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention. Ó 2008 Elsevier Inc. All rights reserved.
Introduction Choline is an organic cation that plays a critical role in the structure and function of biological membranes in all cells as an essential component of membrane phospholipid, phosphatidylcholine and sphingomyelin. Extensive choline uptake and phosphatidylcholine biosynthesis is necessary for the synthesis of new membranes. Choline also acts as a precursor for synthesis of the neurotransmitter acetylcholine (ACh)1 [1]. Recently, choline kinase (CK) has been proposed to play a role in the onset of human cancer,
* Corresponding author. Address: Department of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. Fax: +81 3 3352 0316. E-mail address: [email protected] (M. Inazu). 1 Abbreviations used: ACh, acetylcholine; CK, choline kinase; NHE1, Na+/H+ exchanger 1; HC-3, hemicholinium-3; CTL1, choline transporter-like protein 1; PCho, phosphocholine; OCTs, organic cation transporters; TBA, tetrabutylammonium chloride; THA, tetrahexylammonium chloride; PAH, p-aminohippuric acid; DMA, 5-(N,Ndimethyl)-amiloride; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; CCCP, carbonyl cyanide-m-chlorophenylhydrazone; FCCP, carbonyl cyanide-p-trifluromethoxyphenylhydrazone; NMDG, N-methyl-D-glucamine. 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.12.008
since it is overexpressed in breast, lung, colon, and prostate tumors [2,3]. CK catalyzes the phosphorylation of choline by ATP in the presence of Mg2+, to give phosphocholine (PCho). Growth factors, chemical carcinogens, and/or ras oncogene transfection have been shown to cause the induction of CK activity, leading to an accumulation of PCho. Uptake studies have shown that enhanced choline transport may play a role in the elevation of PCho levels in cancer cells [4– 6]. The elevation of PCho and total choline is one of the most widely established characteristics of cancer cells. PCho is both a precursor and a breakdown product of phosphatidylcholine, the most abundant phospholipid in biological membranes. Biosynthesis and hydrolysis of phosphatidylcholine are essential processes for mitogenic signal transduction events in cells [7]. Previous studies have demonstrated abnormal choline phospholipid metabolism in cancer cells by positron emission tomography and magnetic resonance spectroscopy [8–10]. The aberrant choline metabolism in cancer cells is strongly correlated with the progression of malignancy [11,12]. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in choline phospholipid metabolism, and a prerequisite for cancer cell proliferation. However, the
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uptake system for choline and the functional expression of choline transporters in human colon cancer are poorly understood. To date, the choline transport system has been categorized into three transporter families according to their affinity for choline. A high-affinity choline transporter (CHT1) has recently been cloned and characterized, and is thought to be unique to cholinergic neurons [13,14]. CHT1 is a Cl–- and Na+-dependent co-transporter that is highly sensitive to the choline analogue HC-3, and is thought to be part of the rate-limiting step in ACh synthesis. As an organic cation, choline is known to be a substrate for carriers of organic cation transporters (OCTs). To date, three different OCTs (OCT1-3) have been cloned and their function, which involves a Na+-independent uptake mechanism, has been characterized [15]. These transporters recognize a multitude of endogenous and exogenous organic cations as substrates and exhibit considerable overlap in substrate specificity. Choline also interacts with these transporters with varying degrees of affinity. OCT1 and OCT2 accept choline as a substrate with comparatively low affinity. However, OCT3 does not recognize choline as a substrate [16–18]. Recently, a choline transporter called choline transporter-like protein (human CTL1-5), has been shown to be present in various human tissues [19]. CTL1 has been cloned from Torpedo marmorata, and was first cloned as a suppressor of a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation [19]. Functional characterization studies with CTL1 have shown that CTL1 is a Na+-independent, intermediate-affinity transporter of choline that can be completely inhibited by a high concentration of HC-3 [20–22]. However, the functions of other transporters in this family, CTL2-5, are completely unknown. In this study, we examined the functional characterization of choline uptake and sought to identify the transporters that mediate choline uptake in the human colon cancer cell line HT-29. We also examined the correlation between choline uptake and cell proliferation.
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medicals Inc. Aurora, OH) and 20 mg/L kanamycin (Gibco BRL, Carlsbad, CA) in non-coated flasks and 24-well plates (BD Biosciences, Bedford, MA). Cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C, and the medium was changed every 3 days. [3H]Choline uptake into HT-29 cells Culture medium was removed from the 24-well culture plates by aspiration and the cells were washed twice with uptake buffer consisting of 125 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl2, 1.2 mM KH2PO4, 5.6 mM glucose, 1.2 mM MgSO4 and 25 mM Hepes adjusted to pH 7.4 with Tris. Uptake was then started by the addition of 250 ll of uptake buffer containing [3H]choline. After incubation, cells were washed twice with ice-cold uptake buffer and then dissolved in 0.1 M NaOH and 0.1% Triton X-100, and aliquots were taken for liquid scintillation counting and protein assay. The radioactivity was measured by a liquid scintillation counter (TriCarbÒ 2100TR, Packard Instrument Company, Meriden, CT, USA). The concentration of Na+ in the uptake buffer was modified by replacing NaCl with an equimolar concentration of N-methyl-Dglucamine chloride. Uptake buffers of varying pH (pH 6.0, 6.5, 7.0, 7.5 and 8.0) were prepared by mixing 25 mM MES (pH 6.0) and 25 mM Tris (pH 8.0). Both buffers contained 125 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl2, 1.2 mM KH2PO4, 5.6 mM glucose and 1.2 mM MgSO4. In experiments dealing with saturation kinetics, the concentration of [3H]choline was kept constant at 10 nM and unlabeled choline was added to give the desired choline concentrations. The nonspecific component of uptake was determined from the radioactivity associated with the cells when incubation was performed with [3H]choline in the presence of 30 mM unlabeled choline. Total uptake at each concentration of choline was adjusted for this nonspecific uptake to calculate the saturable component. Data were analyzed by nonlinear regression and confirmed by linear regression. Protein concentrations were determined using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA).
Materials and methods Materials [Methyl-3H]choline chloride (specific activity: 3182 GBq/mmol) was obtained from PerkinElmer Life Sciences, Inc. (Boston, MA). Hemicholinium-3 (HC-3), choline chloride, acetylcholine, TEA, tetrabutylammonium chloride (TBA), tetrahexylammonium chloride (THA), clonidine, quinine, quinidine, desipramine, diphenhydramine, p-aminohippuric acid (PAH), 5-(N,N-dimethyl)-amiloride (DMA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), carbonyl cyanide-m-chlorophenylhydrazone (CCCP), carbonyl cyanide-p-trifluromethoxyphenylhydrazone (FCCP), NH4Cl, Triton X-100 and Nmethyl-D-glucamine (NMDG) were obtained from Sigma–Aldrich Inc. (St. Louis, MO). QIA shredder and RNeasy Mini Kit were obtained from QIAGEN Inc. (Valencia, CA). TaqManÒ RNA-to-CTTM 1-Step Kit and TaqManÒ Gene Expression Assays were obtained from Applied Biosystems (Foster City, CA). Milk Diluent/Blocking Solution and Wash Solution were purchased from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD). VECTASHIELD mounting medium with DAPI was purchased from Vector Laboratories, Inc. (Burlingame, CA). Alexa Fluor 568 goat anti-rabbit IgG was purchased from Molecular Probes Inc. (Eugene, OR). The human colon carcinoma cell line HT-29 was purchased from the American Type Culture Collection (Manassas, VA). All other reagents were of analytical grade. Cell culture HT-29 cells were grown in MoCoy’s 5A medium (Gibco BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum (ICN Bio-
RNA extraction and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay HT-29 cells were washed with sterile Dulbecco’s phosphatebuffered saline (D-PBS) and total RNA was extracted from the cells using QIA shredder and RNeasy Mini Kit (QIAGEN, Valencia, CA). The pairs of primers and the TaqMan probes for the target mRNAs (CHT1, OCT1-3, CTL1-5, NHE1-5 and housekeeping gene GAPDH) were designed based on the human mRNA sequence using TaqManÒ Gene Expression Assays (Applied Biosystems, Assay ID: CHT1, Hs00222367_m1 OCT1, Hs00427554_m1; OCT2, Hs01010723_m1; OCT3, Hs01009568_m1; CTL1, Hs00939357_m1; CTL2, Hs01105938_m1; CTL3, Hs01118290_m1; CTL4, Hs01046784_ml; CTL5, Hs01120485_ml; NHE1, Hs00384604_m1; NHE2, Hs00268166_m1; NHE3, Hs00903842_m1; NHE4, Hs00962495_m1; NHE5, Hs00234441_m1; GAPDH, Hs99999905_ml). For the one-step real-time RT-PCR, 5 lL of total RNA (10 ng) was added to 15 lL of a master mixture by using the TaqManÒ RNA-to-CTTM 1-Step Kit. The final master mixture was contained TaqMan RT Enzyme Mixture, TaqManÒ RT-PCR Mixture and TaqManÒ Gene Expression Assay. The cycling conditions used were 48 °C for 15 min to synthesize cDNA, followed by 1 cycle at 95 °C for 10 min to inactivate the RT and activate the Taq polymerase, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min for amplification. Analysis of the real-time RT-PCR data was performed with the Applied Biosystems StepOne PlusTM Real-Time PCR System. The relative mRNA expression levels of the target genes in HT29 cells were calculated using the comparative cycle time (Ct)
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method. The threshold Ct is the fractional PCR cycle number at which the fluorescent signal reaches the detection threshold. The target PCR Ct value is normalized to the GAPDH PCR Ct value by subtracting the GAPDH Ct value from the target PCR Ct value, which gives the 4Ct value. From this 4Ct value, the relative mRNA expression level to GAPDH for each target PCR can be calculated using the following equation: relative mRNA expression = 2(Ct target–Ct GAPDH) 100%. Antibody production The rabbit polyclonal anti-CTL1 antibody was prepared by immunizing a rabbit with synthetic peptide corresponding to amino acids 261–274 (LYAKQRRSPKETVI), which was coupled to KLH by MBS cross-linking to free sulfhydryl groups. For antibody production, primary immunization with synthetic peptide-KLH conjugate (400 lg) emulsified in complete Freund’s adjuvant was followed by 14, 28 and 42 d of booster injections (200 lg) in incomplete Freund’s adjuvant. Thereafter, monthly boosts were given at 200 lg in incomplete Freund’s adjuvant. Blood was collected, and serum was isolated 2 weeks after boosts and stored at 80 °C. Antisera were tested for approximate antibody titer by ELISA against the immunizing synthetic peptide. Antibody was affinity-purified on a Sepharose-4B column, which was coupled with synthetic peptide. Approximately 2 mg of synthetic peptide was coupled to 3.5 mL of CNBr-activated Sepharose-4B. Antibodies were purified by applying 10 mL of antisera to the column. The column was washed with PBS until the optical density at 280 nm was below 0.05. The antibodies were then eluted with 0.1 M glycine– HCl buffer (pH 2.5). The eluate was neutralized and dialyzed against PBS. The antibody was aliquoted and stored at 80 °C. Western blotting analysis HT-29 cells were washed three times with D-PBS, harvested in D-PBS, and recovered by centrifugation. Cell pellets were extracted on ice in a RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris–HCl) containing a protease inhibitor cocktail and centrifuged (14,000g) for 15 min at 4 C. The supernatant was incubated for 30 min at room temperature in a 1: 1 ratio (v:v) of gel-loading buffer (20 mM Tris–HCl, 2 mM EDTA, 10% ß-mercaptoethanol, 2% SDS, 20% glycerol and 0.2% bromophenol blue, pH 6.8). The samples were then stored at 80 °C until electrophoresis. The proteins in the samples were resolved by one-dimensional gel electrophoresis on 7.5% SDS gel with molecular weight standards. Gels, Whatman filter paper, and PVDF membranes were soaked in electroblotting buffer (25 mM Tris–HCl, 193 mM glycine, 20% methanol) for 15 min prior to transfer. Proteins that had been separated on 7.5% SDS–PAGE were transferred to PVDF membranes by electroblotting in 25 mM Tris–HCl, 192 mM glycine, and 20% methanol at 15 V for 60 min. Following protein transfer, the membrane was blocked with casein solution overnight at 4 °C and incubated with 2 lg/mL rabbit anti-CTL1 polyclonal antibody in new casein solution overnight at 4 °C. The membrane was then washed three times in wash solution. After being washed with wash buffer, membrane was incubated with an EnVision systems-alkaline phosphatase (Dako, Glostrup, Denmark). The alkaline phosphatase reaction was performed with BCIP/NBT substrate. Molecular weight standards were used to determine the molecular weight of immunoreactive species. Immunocytochemistry HT-29 cells grown on glass bottom culture dishes (MatTek Corporation, Ashland, MA) were washed twice with D-PBS and fixed with 100% methanol for 10 min at room temperature. Cells were
permeabilized with D-PBS containing 0.2% Triton X-100 for 5 min and then washed three times with D-PBS. Fixed cells were incubated with non-fat dry milk blocking solution overnight at 4 °C. After being washed with wash solution, cells were incubated with 4 lg/mL rabbit anti-CTL1 polyclonal antibody in new blocking solution overnight at 4 °C. After being washed with wash solution, the cells were incubated with 10 lg/mL Alexa Fluor 568 goat antirabbit IgG for 1 h at room temperature in an area protected against light. After excess antibody was washed out three times with wash solution, the specimens were mounted using VECTASHIELD mounting medium with DAPI. Fluorescence images were collected by a BZ-8000 fluorescence microscope (KEYENCE, Osaka, Japan). Cell proliferation assay Cells were plated at 25,000 cells per well in 24-well plates. Choline uptake inhibitors (THA and HC-3) were added immediately after cell plating, and the final medium volume of each well was 1.0 mL. Inhibitors were added every day for 4 days, and cell numbers were measured using an ATPLiteTM (PerkinElmer Life and Analytical Sciences, Boston, MA). A luminescence ATP detection assay was carried out as described in the manufacturer’s instructions. Luminescence was measured on a GloMaxTM 20/20n Luminometer (Promega Corporation, Madison, WI).
Results Time-course and kinetics of [3H]choline uptake in HT-29 cells We first examined the time-course of [3H]choline uptake at a concentration of 10 nM in the presence and absence of Na+ in HT-29 cells for 60 min (Fig. 1a). [3H]Choline uptake in HT-29 cells increased in a time-dependent manner; it was linear with time at up to 30 min (r2 = 0.9921, p = 0.004 in the presence of Na+, r2 = 0.9959, p = 0.002 in the absence of Na+). Based on these findings, subsequent experiments were performed using an uptake period of 10 min. When NaCl in the uptake buffer was replaced by NMDG, the uptake of [3H]choline under Na+-free conditions was strongly enhanced as compared with control uptake under normal conditions. The characteristics of the kinetics of [3H]choline uptake by HT29 cells were determined (Fig. 1b). HT-29 cells were incubated for 10 min with [3H]choline at a concentration of from 0.78 to 50 lM. Kinetic analysis of [3H]choline uptake data (specific uptake), as computed by a non-linear regression analysis, yielded a Michaelis–Menten constant (Km) of 12.1 ± 1.2 lM and a maximal velocity (Vmax) of 387 ± 15 pmol/mg protein/min. The Eadie–Hofstee plot (Fig. 1b, inset) shows a single straight line (r2 = 0.9857, p < 0.0001), suggesting that [3H]choline uptake by HT-29 cells is mediated by a single transport system. Effects of various compounds on [3H]choline uptake in HT-29 cells An inhibition study was performed to determine the substrateselectivity of the choline transport system in HT-29 cells (Fig. 2). The Ki values for the inhibition of [3H]choline uptake were calculated from the corresponding inhibition curves and are shown in Table 1. [3H]Choline uptake was strongly inhibited by THA, unlabeled choline, HC-3, desipramine, quinidine and quinine, in a concentration-dependent manner. Moderate inhibition was seen with diphenhydramine and acetylcholine and only weak inhibition was seen with TEA, corticosterone and TBA. In addition, the Ki values for the inhibition of [3H]choline uptake by MPP+, carnitine and betaine were over 1 mM, and PAH did not cause any significant inhibition at up to 10 mM.
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Fig. 1. Time-course of 10 nM [3H]choline uptake in the presence (d) and absence (s) of Na+ in HT-29 cells for 60 min. (a) Na+-free buffer was modified by replacing NaCl with an equimolar concentration of N-methyl-D-glucamine chloride. Each point represents the mean ± S.D. of three determinations. The time-course of [3H]choline uptake was fitted to the experimental data by a non-linear regression analysis. The kinetic characteristics of [3H]choline uptake in HT-29 cells. (b) HT-29 cells were incubated for 10 min with [3H]choline at concentrations from 0.78 to 50 lM. Each point represents the mean ± S.D. of four determinations. Specific [3H]choline uptake is saturable with a Km of 12.1 ± 1.2 lM and a Vmax of 387 ± 15 pmol/mg protein/min. Inset: Eadie–Hofstee plots of [3H]choline uptake. The correlation is highly significant (r2 = 0.9920, p < 0.0001).
Fig. 2. Effects of various organic cation compounds and PAH on [3H]choline uptake in HT-29 cells. Cells were pre-incubated with test compounds for 20 min and the uptake of 10 nM [3H]choline was measured for 10 min. Results are given as a percentage of control uptake measured in the presence of vehicle. Each point represents the mean ± S.D. of four determinations.
Effects of extra- and intracellular pH on [3H]choline uptake in HT-29 cells The effect of extracellular pH on the uptake of [3H]choline in HT-29 cells was examined by varying the pH of the pre-incubation and incubation media between pH 6.0 and 8.0 (Fig. 3a). [3H]Choline uptake significantly decreased when the extracellular pH was changed from a physiological value of 7.5–6.0. We further investigated the effect of intracellular pH on [3H]choline uptake in HT-29 cells. It is well known that exposure to NH4Cl in Na+-free conditions cause rapid cytosolic alkalinization [23]. Cells were pre-incubated in Na+-free uptake buffer containing various concentrations of NH4Cl for 5 min, and [3H]choline uptake was measured for 10 min. The increase in [3H]choline uptake under the Na+-free condition was inhibited by the extracellular application of NH4Cl in a concentration-dependent manner (Fig. 3b). These results indicate
that choline uptake in HT-29 cells was decreased by acidification of the extracellular medium and by intracellular alkalinization. In addition, we also examined the effects of protonophores, CCCP and FCCP, which known to dissipate transmembrane H+ gradients, on [3H]choline uptake in HT-29 cells. Upon collapse of the plasmamembrane H+ electrochemical gradient with 50 lM CCCP and 50 lM FCCP, the uptake of [3H]choline was significantly reduced to 52.3 ± 5.7% and 56.1 ± 4.9%, respectively (Fig. 3c). Effects of NHE1 inhibitors on [3H]choline uptake in HT-29 cells We demonstrated that choline transport activity was enhanced by the removal of Na+ ion from uptake buffer and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization, suggesting that the activity of NHE1 might affect choline transport activity. Therefore, we investigated
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Table 1 Ki values of various organic cation compounds and PAH on [3H]choline uptake in HT29 cells. Compound
Ki value (lM)
Tetrahexylammonium chloride (THA) Choline Hemicholinium-3 (HC-3) Desipramine Quinidine Diphenhydramine Quinine Acetylcholine Tetraethylammonium chloride (TEA) Corticosterone Tetrabutylammonium chloride (TBA) 1-Methyl-4-phenylpyridinium (MPP+) Carnitine Betaine PAH (p-aminohippric acid)
6.6 7.9 15.4 50.5 64.5 149.1 216 533 1813 3350 5618 >1000 >1000 >1000 >10000
the effects of NHE1 inhibitors, such as DMA and EIPA, on [3H]choline uptake into HT-29 cells (Fig. 4). The increase in [3H]choline uptake under Na+-free conditions was inhibited by 10 lM DMA and EIPA. However, DMA and EIPA did not cause any significant inhibition of [3H]choline uptake in the normal condition. Expression of mRNA for CHT1, CTLs, OCTs and NHEs in HT-29 cells The expression of mRNA for CHT1, CTL1-5, OCT1-3 and NHE15 in HT-29 cells was investigated by Real-time RT-PCR analysis.
The amplified PCR products of transporters were analyzed to determine the expression profiles of mRNA for choline transporters, such as CHT1 and CTLs, in HT-29 cells. CTL1, CTL2 and CTL4 mRNAs are mainly expressed in HT-29. In addition, CHT1, CTL3 and CTL5 mRNAs are expressed at low levels (Fig. 5a). As an organic cation, choline is a substrate for the OCT family. Therefore, we investigated the expression of mRNAs for OCTs. OCT1-3 mRNAs were expressed, albeit OCT1 and OCT2 mRNAs were expressed at low levels (Fig. 5b). We demonstrated that the increase in choline uptake under the Na+-free condition was inhibited by NHE1 inhibitors. Therefore, we determined the expression profile of NHEs in HT-29 cells. As shown in Fig. 5c, NHE1 mRNA is mainly expressed, and NHE2, NHE3 and NHE5 mRNAs are expressed at low levels. Under similar conditions, NHE4 was not detectable in HT-29 cells. Detection of CTL1 protein expression in HT-29 cells by Western blotting analysis and immunocytochemistry Extracts of HT-29 cells were immunoblotted with anti-CTL1 polyclonal antibody. Western blotting analysis indicated that anti-CTL1 polyclonal antibody recognized a band of 80 kDa (Fig. 6A). In addition, CTL1 immunoreactivity in HT-29 cells was detected by immunocytochemical staining using anti-CTL1 antibody (Fig. 6B), which was consistent with the results of RT-PCR and Western blotting. Immunocytochemical staining using anti-CTL1 antibody in HT-29 cells revealed that the plasma membrane showed a considerable level of immunoreactivity.
Fig. 3. Effect of pH on [3H]choline uptake in HT-29 cells. (a) Effect of extracellular pH on [3H]choline uptake. The uptake of 10 nM [3H]choline was measured for 10 min under different pH conditions. Each column represents the mean ± S.D. of four determinations; **p < 0.01 compared with pH 7.5. (b) Effect of intracellular pH on [3H]choline uptake. Cells were pre-incubated in Na+-free uptake buffer containing various concentrations of NH4Cl for 5 min, and then [3H]choline uptake was measured for 10 min. Each column represents the mean ± S.D. of three determinations. ***p < 0.001 compared with the [3H]choline uptake in the normal condition, #p < 0.05, ##p < 0.01 and ###p < 0.001 compared with 0 mM NH4Cl in the Na+-free condition. (c) Effects of protonophore on [3H]choline uptake in HT-29 cells. The plasma-membrane H+ electrochemical gradient was collapsed by 50 lM CCCP and 50 lM FCCP for 20 min and [3H]choline uptake was then measured for 10 min. Each column represents the mean ± S.D. of three determinations. ***p < 0.001 compared with the vehicle control.
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Fig. 4. Effects of Na+/H+ exchange (NHE) inhibitor, DMA (a) and EIPA (b) on [3H]choline uptake in HT-29 cells. HT-29 cells were pre-incubated with test compounds and vehicle for 20 min in the presence and absence of Na+, and [3H]choline uptake was then measured for 10 min. Results are given as a percentage of vehicle control uptake measured under the normal condition. Each column represents the mean ± S.D. of three determinations. *p < 0.05 and **p < 0.01 compared with choline uptake under the normal condition. N.S.; not significant.
Effects of choline uptake inhibition by THA and HC-3 on cell proliferation To explore the possible correlation between choline uptake and cell growth, the effect of transporter inhibitors on cell proliferation was examined by luminescence ATP detection assays. As shown in Fig. 7, potent choline uptake inhibitors such as THA and HC-3 both inhibited HT-29 cell proliferation in a concentration-dependent and time-dependent manner, with relative inhibitory potencies as follows: THA > HC-3. The inhibitory potencies of these inhibitors on cell growth were consistent with the inhibitory potencies on choline uptake inhibition. Discussion Colorectal carcinoma is the most common cancer in both men and women worldwide. Despite the use of aggressive surgical resection and chemotherapy, nearly 50% of patients with colorectal carcinoma develop recurrent disease. Thus, novel approaches to the medical treatment of human colon cancer are required. Intracellular choline accumulation through choline transporters appears to be a prerequisite for cancer cell proliferation based on CK activity [6,24,25]. In this sense, the identification and characterization of choline transporters in cancer may offer a new target for the design of anti-tumor strategies. Therefore, it is important to identify choline transporters in cancer cells. In this study, we first found that the human colon carcinoma cell line HT-29 takes up [3H]choline by a saturable process that is mediated by a single transport system. The Km value was 12.1 lM, which is similar to the blood concentration of choline (10–30 lM). The kinetic parameters are very close to those of the choline transport system in endothelial cells of the blood–brain barrier [26], keratinocytes [27] and astrocytes [20], and these reports have shown conclusively that choline is taken up into these cells by a specific transporter that is not OCT1-3 or CHT1. In astrocytes, the inhibition of CTL1 mRNA expression using siRNA inhibited Na+-independent choline uptake, suggesting that CTL1 is responsible for Na+-independent choline uptake in these cells. The Km value of choline uptake in CTL1-expressing cells was approximately 30 lM [21], which is very close to the Km value in this study, in contrast to those for CHT1 (Km = 0.5 2 lM) and the OCT family of transporters (Km = 100 450 lM). The kinetics data indicate that choline transport in HT-29 cells does not occur via CHT1 or the OCT family. Unfortunately, the functions of CTL25 are unclear. CHT1 is a choline transporter that has been reported to be found at cholinergic neuronal membranes; it is Na+-dependent and can be blocked by HC-3 at a very low concentration (Ki value = 50– 100 nM). Recently, CHT1 has also been identified in non-neuronal cholinergic cells, i.e., keratinocytes [28], and in ciliated cells of
the tracheal epithelium [29]. CHT1 is a novel component of the intrinsic non-neuronal cholinergic system. We found that CHT1 mRNA is expressed in HT-29 cells at low levels. However, the Ki value of HC-3 in HT-29 cells (Ki value = 15.4 lM) is much higher than the values in the literature (Ki value = 50–100 nM), and [3H]choline uptake could also be observed under Na+-free conditions. These findings strongly suggest that CHT1 plays no role in the uptake of choline by HT-29 cells. As an organic cation, choline is a substrate for the OCT family. These transporters, known as OCT1, OCT2 and OCT3, exhibit a broad and overlapping substrate-specificity toward organic cations [16,18,30]. OCT1 and OCT2 accept choline as a substrate with comparatively low affinity. The transport process is Na+-independent and has a Kt value of 620 lM for OCT1 and 210 lM for OCT2. However, OCT3 does not recognize choline as a substrate [16,18]. Therefore, we examined whether choline uptake into HT-29 cells is mediated by OCTs. Real-time PCR data showed that mRNA for OCT1 and OCT2 was only slightly expressed in HT-29 cells. The choline transport system in HT-29 cells resembles OCTs with regard to some pharmacological and functional properties, such as sensitivity of the organic cation compound, Na+-independence and pH-sensitivity. In the present study, however, TEA, a prototypical OCT substrate, interacted with a very low affinity (Ki = 1813 lM). TEA is very often used as a high-affinity reference compound for OCTs, and has Ki values of 46.6 and 52.2 lM for OCT1 and OCT2, respectively [31]. These biochemical and pharmacological data show that choline uptake in HT-29 cells does not occur via OCTs. Next, we searched for a new transporter that could be part of a Na+-independent choline transport system. Recently, a novel family of choline transporters, called choline transporter-like protein 1 (CTL1), has been cloned from Torpedo marmorata, and several homologous genes have also been found in mammals (CTL1-5) [19,21]. Rat CTL1 was also cloned as a homologous rat gene of the CTL protein family and exhibited saturable, HC-3-inhibitable (Ki value > 10 lM) and weak Na+-dependent uptake of choline [20,21,32]. Unlike CTL1, the function of other members of the CTL family (CTL2-5) is unknown. However, there have been several reports on the tissue distribution of the CTL family [19,21]. Human CTL1 protein is expressed in a variety of tissues, including brain, heart, small intestine, kidney, liver, lung, skeletal muscle, pancreas, spleen, ovary, and testis [33]. Rat CTL1a mRNA is expressed in the ileum and colon, whereas rat CTL1b mRNA is expressed exclusively in the brain. CTL2 is predominantly expressed in the tongue, muscle, kidney, heart and lung, as well as, to a lesser extent, the intestine, testis and stomach. CTL3 is expressed in the colon, kidney and ileum, while CTL4 is found at high levels in the intestine, kidney and stomach. CTL5 gene was identified by database screening in the human and macaque genome databases [21], but its tissue distribution is unclear. We examined whether the CTL family medi-
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Fig. 5. Real-time RT-PCR analysis of the expression of CHT1, CTLs, OCTs and NHEs mRNA in HT-29 cells. The percentage of relative mRNA expression is expressed as the ratio of target mRNA to GAPDH mRNA. Experiments were performed in duplicate. BLQ, below the limit of quantification.
ates choline uptake in HT-29 cells. In the present study, CTL1, CTL2 and CTL4 mRNA were mainly expressed in HT-29. Furthermore, kinetics data indicated that the Km value of [3H]choline uptake in HT-29 cells was very close to the Km value for CTL1. The Eadie–Hofstee plot shows a single straight line, suggesting that choline uptake is mediated by a single transport system. Together, these results suggest that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We sought to confirm the expression of CTL1 protein in HT-29 cells by Western blot analysis and immunocytochemistry. Western blotting indicated that a major band of 80 kDa was present in HT-29 cells. The estimated size of human CTL1 from the deduced amino acid sequences was 73.3 kDa for deglycosylated CTL1 (Swiss-Prot),
which is very similar to the size of the HT-29 cells described here. Furthermore, we achieved the first direct visualization of CTL1 proteins expressed in plasma membrane of HT-29 cells by immunocytochemical staining using anti-CTL1 affinity-purified polyclonal antibody. Thus, CTL1 protein was obviously expressed in HT-29 cells. CTL1 is strongly expressed in oligodendrocytes, myelin-synthesizing cells, and cancer cells such as breast cancer and lung adenocarcinoma which require high levels of choline to synthesize sphingomyelin, phosphocholine and phosphatidylcholine [11,34,35]. In light of our present work, further studies are needed to examine the expression status of CTL1 in both cancer and noncancerous colon tissues. Interestingly, a very similar pH profile (pH-dependent uptake) was observed for choline transport in rat astrocytes, which is mediated by CTL1 [20]. [3H]Choline uptake in HT-29 cells was decreased by acidification of the extracellular medium and by intracellular alkalinization. In addition, collapse of the plasmamembrane H+ electrochemical gradient by a protonophore inhibited choline uptake, indicating that the transport of choline is H+dependent. These data suggest that choline may be transported by a choline/H+ antiport system. Moreover, removal of Na+ from the uptake buffer strongly enhanced [3H]choline uptake in HT-29 cells. The absence of Na+ in uptake buffer stimulates reverse-mode NHEs, which results in an increase in the concentration of intracellular H+, and this H+ gradient in turn accelerates the choline transport mediated by choline/H+ antiporter. We investigated the functional correlation of choline transporter and NHEs in HT-29 cells. We found that HT-29 cells highly expressed NHE1 mRNA, and the increase in choline uptake under the Na+-free condition was inhibited by NHE1 inhibitor (DMA or EIPA). These data indicate that H+-coupled choline transport mediated by CTL1 might be modulated by NHE1 activity. In normal cells, NHE1 is quiescent at the steady-state resting intracellular pH and is activated only upon cytosolic acidification. In transformed and cancer cells, NHE1 is hyperactive even at resting pH and the resulting change in cellular alkalinity has been shown to be directly related in most cases to the permanent and uncontrolled proliferation that is characteristic of neoplastic cells [36]. Recently, compelling evidence has indicated that the activity of NHE1 is also a critical factor in the activation of proliferation, motility and invasion of cancer cells derived from various tissues [37]. Thus, the choline transporter CTL1 functions in co-operation with NHE1; the two transporters functioning together to mediate choline uptake and cellular alkalinity may be correlated with cell proliferation, motility and invasion of cancer cells. Finally, we explored the possible correlation between choline uptake and cell proliferation, i.e., the effect of choline transporter inhibitors on cell proliferation. Choline uptake inhibitors such as THA and HC-3 can inhibit HT-29 cell proliferation, suggesting that cell proliferation may require an increased supply of choline. It is possible that cell growth can be restrained by obstructing the function of CTL1. Recently, it was reported that targeting CTL1 might be an effective way to block choline transport and thus inhibit cell proliferation in human lung adenocarcinoma [35]. Previous studies have shown that choline deficiency triggers apoptosis via a p53independent pathway [38]. The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content [39]. Aberrant choline phospholipid metabolism of cancer cells and its strong correlation with malignant progression has been observed in several studies. Human breast cancer cells exhibit consistently elevated phosphatidylcholine levels [40]. In addition, choline kinase has been proposed to play a relevant role in the onset of human cancer, since it is overexpressed in breast, lung, colon and prostate tumors [2,3]. These data indicate that diverse molecular alterations arrive at common end-points in choline phospholipid metabolism in cancer cells. Thus, intracellular choline uptake through CTL1 is the
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Fig. 6. Detection of CTL1 protein in HT-29 cells by Western blot analysis. (A) Total protein extracts from HT-29 cells were separated on 7.5% SDS–PAGE and transferred to PVDF membrane. The membrane was incubated with affinity-purified anti-CTL1 rabbit polyclonal antibody and then incubated with an EnVision alkaline phosphatase polymer. The alkaline phosphatase reaction was performed with BCIP/NBT substrate. M.W. color prestained molecular marker. Immunocytochemical detection of CTL1 protein in HT-29 cells. (B) HT-29 cells were incubated with 4 lg/ml anti-CTL1 rabbit polyclonal antibody and then incubated with 10 lg/mL Alexa Fluor 568 goat anti-rabbit IgG as described in the Material and methods. The panel (a) shows phase-contrast micrograph. Presence of CTL1 is shown in red (b), staining of nuclei with DAPI in blue (c), and merged images are shown in panel (d). Scale bars: 20 lm.
Fig. 7. Effect of the choline uptake inhibition by HC-3 and THA on cell proliferation. The effect of transporter inhibitors HC-3 and THA on cell proliferation was examined by luminescence ATP detection assays. Cells were plated at 25,000 cells per well in 24-well plates. Various concentrations of HC-3 and THA were added immediately after cell plating and every day for 4 days. Cell numbers were measured using an ATPLiteTM. Each column represents the mean + S.D. of four determinations.
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rate-limiting step in choline phospholipid metabolism, and a prerequisite for cancer cell proliferation. Acknowledgment This work was supported by Tokyo Medical University, Cancer Research Foundation. References [1] J. Klein, R. Gonzalez, A. Köppen, K. Löffelholz, Neurochem. Int. 22 (1993) 293– 300. [2] A. Ramírez, A. de Molina, R. Rodríguez-González, L. Gutiérrez, J. MartínezPiñeiro, F. Sánchez, R. Bonilla, L. Rosell, L. Lacal, Biochem. Biophys. Res. Commun. 296 (2002) 580–583. [3] A. Ramírez de Molina, D. Gallego-Ortega, J. Sarmentero-Estrada, D. Lagares, T. Gómez Del Pulgar, E. Bandrés, J. García-Foncillas, J.C. Lacal, Int. J. Biochem. Cell. Biol. 40 (2008) 1753–1763. [4] R. Katz-Brull, H. Degani, Anticancer Res. 16 (1996) 1375–1380. [5] R. Katz-Brull, D. Seger, D. Rivenson-Segal, E. Rushkin, H. Degani, Cancer Res. 62 (2002) 1966–1970. [6] T. Hara, A. Bansal, T.R. DeGrado, Mol. Imaging 5 (2006) 498–509. [7] H. Cai, P. Erhardt, J. Troppmair, M.T. Diaz-Meco, G. Sithanandam, U.R. Rapp, J. Moscat, G.M. Cooper, Mol. Cell. Biol. 13 (1993) 7645–7651. [8] K. Glunde, E. Ackerstaff, N. Mori, M.A. Jacobs, Z.M. Bhujwalla, Mol. Pharm. 3 (2006) 496–506. [9] T. Hara, J. Nucl. Med. 42 (2001) 1815–1817. [10] S.A. Kwee, H. Wei, I. Sesterhenn, D. Yun, M.N. Coel, J. Nucl. Med. 47 (2006) 262– 269. [11] G. Eliyahu, T. Kreizman, H. Degani, Int. J. Cancer 120 (2007) 1721–1730. [12] K. Glunde, Z.M. Bhujwalla, Lancet Oncol. 8 (2007) 855–857. [13] S. Apparsundaram, S.M. Ferguson, A.L. George Jr., R.D. Blakely, Biochem. Biophys. Res. Commun. 276 (2000) 862–867. [14] T. Okuda, T. Haga, Y. Kanai, H. Endou, T. Ishihara, I. Katsura, Nat. Neurosci. 3 (2000) 120–125. [15] H. Koepsell, B.M. Schmitt, V. Gorboulev, Rev. Physiol. Biochem. Pharmacol. 150 (2003) 36–90. [16] D. Gründemann, B. Schechinger, G.A. Rappold, E. Schömig, Nat. Neurosci. 1 (1998) 349–351. [17] D. Gründemann, G. Liebich, N. Kiefer, S. Köster, E. Schömig, Mol. Pharmacol. 56 (1999) 1–10.
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Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells Hironobu Kouji a,c, Masato Inazu a,b,*, Tomoko Yamada a, Hirohisa Tajima a, Tatsuya Aoki c, Teruhiko Matsumiya a,b a
Department of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan Department of Preventive Medicine (SONOKO), Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan c Third Department of Surgery, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan b
a r t i c l e
i n f o
Article history: Received 6 November 2008 and in revised form 12 December 2008 Available online 25 December 2008 Keywords: Choline Transporter Na+/H+ exchanger Colon carcinoma Cell proliferation
a b s t r a c t We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. 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 component of choline uptake under Na+-free conditions was inhibited by a Na+/H+ exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H+ electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H+ gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention. Ó 2008 Elsevier Inc. All rights reserved.
Introduction Choline is an organic cation that plays a critical role in the structure and function of biological membranes in all cells as an essential component of membrane phospholipid, phosphatidylcholine and sphingomyelin. Extensive choline uptake and phosphatidylcholine biosynthesis is necessary for the synthesis of new membranes. Choline also acts as a precursor for synthesis of the neurotransmitter acetylcholine (ACh)1 [1]. Recently, choline kinase (CK) has been proposed to play a role in the onset of human cancer,
* Corresponding author. Address: Department of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. Fax: +81 3 3352 0316. E-mail address: [email protected] (M. Inazu). 1 Abbreviations used: ACh, acetylcholine; CK, choline kinase; NHE1, Na+/H+ exchanger 1; HC-3, hemicholinium-3; CTL1, choline transporter-like protein 1; PCho, phosphocholine; OCTs, organic cation transporters; TBA, tetrabutylammonium chloride; THA, tetrahexylammonium chloride; PAH, p-aminohippuric acid; DMA, 5-(N,Ndimethyl)-amiloride; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; CCCP, carbonyl cyanide-m-chlorophenylhydrazone; FCCP, carbonyl cyanide-p-trifluromethoxyphenylhydrazone; NMDG, N-methyl-D-glucamine. 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.12.008
since it is overexpressed in breast, lung, colon, and prostate tumors [2,3]. CK catalyzes the phosphorylation of choline by ATP in the presence of Mg2+, to give phosphocholine (PCho). Growth factors, chemical carcinogens, and/or ras oncogene transfection have been shown to cause the induction of CK activity, leading to an accumulation of PCho. Uptake studies have shown that enhanced choline transport may play a role in the elevation of PCho levels in cancer cells [4– 6]. The elevation of PCho and total choline is one of the most widely established characteristics of cancer cells. PCho is both a precursor and a breakdown product of phosphatidylcholine, the most abundant phospholipid in biological membranes. Biosynthesis and hydrolysis of phosphatidylcholine are essential processes for mitogenic signal transduction events in cells [7]. Previous studies have demonstrated abnormal choline phospholipid metabolism in cancer cells by positron emission tomography and magnetic resonance spectroscopy [8–10]. The aberrant choline metabolism in cancer cells is strongly correlated with the progression of malignancy [11,12]. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in choline phospholipid metabolism, and a prerequisite for cancer cell proliferation. However, the
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uptake system for choline and the functional expression of choline transporters in human colon cancer are poorly understood. To date, the choline transport system has been categorized into three transporter families according to their affinity for choline. A high-affinity choline transporter (CHT1) has recently been cloned and characterized, and is thought to be unique to cholinergic neurons [13,14]. CHT1 is a Cl–- and Na+-dependent co-transporter that is highly sensitive to the choline analogue HC-3, and is thought to be part of the rate-limiting step in ACh synthesis. As an organic cation, choline is known to be a substrate for carriers of organic cation transporters (OCTs). To date, three different OCTs (OCT1-3) have been cloned and their function, which involves a Na+-independent uptake mechanism, has been characterized [15]. These transporters recognize a multitude of endogenous and exogenous organic cations as substrates and exhibit considerable overlap in substrate specificity. Choline also interacts with these transporters with varying degrees of affinity. OCT1 and OCT2 accept choline as a substrate with comparatively low affinity. However, OCT3 does not recognize choline as a substrate [16–18]. Recently, a choline transporter called choline transporter-like protein (human CTL1-5), has been shown to be present in various human tissues [19]. CTL1 has been cloned from Torpedo marmorata, and was first cloned as a suppressor of a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation [19]. Functional characterization studies with CTL1 have shown that CTL1 is a Na+-independent, intermediate-affinity transporter of choline that can be completely inhibited by a high concentration of HC-3 [20–22]. However, the functions of other transporters in this family, CTL2-5, are completely unknown. In this study, we examined the functional characterization of choline uptake and sought to identify the transporters that mediate choline uptake in the human colon cancer cell line HT-29. We also examined the correlation between choline uptake and cell proliferation.
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medicals Inc. Aurora, OH) and 20 mg/L kanamycin (Gibco BRL, Carlsbad, CA) in non-coated flasks and 24-well plates (BD Biosciences, Bedford, MA). Cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C, and the medium was changed every 3 days. [3H]Choline uptake into HT-29 cells Culture medium was removed from the 24-well culture plates by aspiration and the cells were washed twice with uptake buffer consisting of 125 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl2, 1.2 mM KH2PO4, 5.6 mM glucose, 1.2 mM MgSO4 and 25 mM Hepes adjusted to pH 7.4 with Tris. Uptake was then started by the addition of 250 ll of uptake buffer containing [3H]choline. After incubation, cells were washed twice with ice-cold uptake buffer and then dissolved in 0.1 M NaOH and 0.1% Triton X-100, and aliquots were taken for liquid scintillation counting and protein assay. The radioactivity was measured by a liquid scintillation counter (TriCarbÒ 2100TR, Packard Instrument Company, Meriden, CT, USA). The concentration of Na+ in the uptake buffer was modified by replacing NaCl with an equimolar concentration of N-methyl-Dglucamine chloride. Uptake buffers of varying pH (pH 6.0, 6.5, 7.0, 7.5 and 8.0) were prepared by mixing 25 mM MES (pH 6.0) and 25 mM Tris (pH 8.0). Both buffers contained 125 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl2, 1.2 mM KH2PO4, 5.6 mM glucose and 1.2 mM MgSO4. In experiments dealing with saturation kinetics, the concentration of [3H]choline was kept constant at 10 nM and unlabeled choline was added to give the desired choline concentrations. The nonspecific component of uptake was determined from the radioactivity associated with the cells when incubation was performed with [3H]choline in the presence of 30 mM unlabeled choline. Total uptake at each concentration of choline was adjusted for this nonspecific uptake to calculate the saturable component. Data were analyzed by nonlinear regression and confirmed by linear regression. Protein concentrations were determined using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA).
Materials and methods Materials [Methyl-3H]choline chloride (specific activity: 3182 GBq/mmol) was obtained from PerkinElmer Life Sciences, Inc. (Boston, MA). Hemicholinium-3 (HC-3), choline chloride, acetylcholine, TEA, tetrabutylammonium chloride (TBA), tetrahexylammonium chloride (THA), clonidine, quinine, quinidine, desipramine, diphenhydramine, p-aminohippuric acid (PAH), 5-(N,N-dimethyl)-amiloride (DMA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), carbonyl cyanide-m-chlorophenylhydrazone (CCCP), carbonyl cyanide-p-trifluromethoxyphenylhydrazone (FCCP), NH4Cl, Triton X-100 and Nmethyl-D-glucamine (NMDG) were obtained from Sigma–Aldrich Inc. (St. Louis, MO). QIA shredder and RNeasy Mini Kit were obtained from QIAGEN Inc. (Valencia, CA). TaqManÒ RNA-to-CTTM 1-Step Kit and TaqManÒ Gene Expression Assays were obtained from Applied Biosystems (Foster City, CA). Milk Diluent/Blocking Solution and Wash Solution were purchased from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD). VECTASHIELD mounting medium with DAPI was purchased from Vector Laboratories, Inc. (Burlingame, CA). Alexa Fluor 568 goat anti-rabbit IgG was purchased from Molecular Probes Inc. (Eugene, OR). The human colon carcinoma cell line HT-29 was purchased from the American Type Culture Collection (Manassas, VA). All other reagents were of analytical grade. Cell culture HT-29 cells were grown in MoCoy’s 5A medium (Gibco BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum (ICN Bio-
RNA extraction and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay HT-29 cells were washed with sterile Dulbecco’s phosphatebuffered saline (D-PBS) and total RNA was extracted from the cells using QIA shredder and RNeasy Mini Kit (QIAGEN, Valencia, CA). The pairs of primers and the TaqMan probes for the target mRNAs (CHT1, OCT1-3, CTL1-5, NHE1-5 and housekeeping gene GAPDH) were designed based on the human mRNA sequence using TaqManÒ Gene Expression Assays (Applied Biosystems, Assay ID: CHT1, Hs00222367_m1 OCT1, Hs00427554_m1; OCT2, Hs01010723_m1; OCT3, Hs01009568_m1; CTL1, Hs00939357_m1; CTL2, Hs01105938_m1; CTL3, Hs01118290_m1; CTL4, Hs01046784_ml; CTL5, Hs01120485_ml; NHE1, Hs00384604_m1; NHE2, Hs00268166_m1; NHE3, Hs00903842_m1; NHE4, Hs00962495_m1; NHE5, Hs00234441_m1; GAPDH, Hs99999905_ml). For the one-step real-time RT-PCR, 5 lL of total RNA (10 ng) was added to 15 lL of a master mixture by using the TaqManÒ RNA-to-CTTM 1-Step Kit. The final master mixture was contained TaqMan RT Enzyme Mixture, TaqManÒ RT-PCR Mixture and TaqManÒ Gene Expression Assay. The cycling conditions used were 48 °C for 15 min to synthesize cDNA, followed by 1 cycle at 95 °C for 10 min to inactivate the RT and activate the Taq polymerase, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min for amplification. Analysis of the real-time RT-PCR data was performed with the Applied Biosystems StepOne PlusTM Real-Time PCR System. The relative mRNA expression levels of the target genes in HT29 cells were calculated using the comparative cycle time (Ct)
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method. The threshold Ct is the fractional PCR cycle number at which the fluorescent signal reaches the detection threshold. The target PCR Ct value is normalized to the GAPDH PCR Ct value by subtracting the GAPDH Ct value from the target PCR Ct value, which gives the 4Ct value. From this 4Ct value, the relative mRNA expression level to GAPDH for each target PCR can be calculated using the following equation: relative mRNA expression = 2(Ct target–Ct GAPDH) 100%. Antibody production The rabbit polyclonal anti-CTL1 antibody was prepared by immunizing a rabbit with synthetic peptide corresponding to amino acids 261–274 (LYAKQRRSPKETVI), which was coupled to KLH by MBS cross-linking to free sulfhydryl groups. For antibody production, primary immunization with synthetic peptide-KLH conjugate (400 lg) emulsified in complete Freund’s adjuvant was followed by 14, 28 and 42 d of booster injections (200 lg) in incomplete Freund’s adjuvant. Thereafter, monthly boosts were given at 200 lg in incomplete Freund’s adjuvant. Blood was collected, and serum was isolated 2 weeks after boosts and stored at 80 °C. Antisera were tested for approximate antibody titer by ELISA against the immunizing synthetic peptide. Antibody was affinity-purified on a Sepharose-4B column, which was coupled with synthetic peptide. Approximately 2 mg of synthetic peptide was coupled to 3.5 mL of CNBr-activated Sepharose-4B. Antibodies were purified by applying 10 mL of antisera to the column. The column was washed with PBS until the optical density at 280 nm was below 0.05. The antibodies were then eluted with 0.1 M glycine– HCl buffer (pH 2.5). The eluate was neutralized and dialyzed against PBS. The antibody was aliquoted and stored at 80 °C. Western blotting analysis HT-29 cells were washed three times with D-PBS, harvested in D-PBS, and recovered by centrifugation. Cell pellets were extracted on ice in a RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris–HCl) containing a protease inhibitor cocktail and centrifuged (14,000g) for 15 min at 4 C. The supernatant was incubated for 30 min at room temperature in a 1: 1 ratio (v:v) of gel-loading buffer (20 mM Tris–HCl, 2 mM EDTA, 10% ß-mercaptoethanol, 2% SDS, 20% glycerol and 0.2% bromophenol blue, pH 6.8). The samples were then stored at 80 °C until electrophoresis. The proteins in the samples were resolved by one-dimensional gel electrophoresis on 7.5% SDS gel with molecular weight standards. Gels, Whatman filter paper, and PVDF membranes were soaked in electroblotting buffer (25 mM Tris–HCl, 193 mM glycine, 20% methanol) for 15 min prior to transfer. Proteins that had been separated on 7.5% SDS–PAGE were transferred to PVDF membranes by electroblotting in 25 mM Tris–HCl, 192 mM glycine, and 20% methanol at 15 V for 60 min. Following protein transfer, the membrane was blocked with casein solution overnight at 4 °C and incubated with 2 lg/mL rabbit anti-CTL1 polyclonal antibody in new casein solution overnight at 4 °C. The membrane was then washed three times in wash solution. After being washed with wash buffer, membrane was incubated with an EnVision systems-alkaline phosphatase (Dako, Glostrup, Denmark). The alkaline phosphatase reaction was performed with BCIP/NBT substrate. Molecular weight standards were used to determine the molecular weight of immunoreactive species. Immunocytochemistry HT-29 cells grown on glass bottom culture dishes (MatTek Corporation, Ashland, MA) were washed twice with D-PBS and fixed with 100% methanol for 10 min at room temperature. Cells were
permeabilized with D-PBS containing 0.2% Triton X-100 for 5 min and then washed three times with D-PBS. Fixed cells were incubated with non-fat dry milk blocking solution overnight at 4 °C. After being washed with wash solution, cells were incubated with 4 lg/mL rabbit anti-CTL1 polyclonal antibody in new blocking solution overnight at 4 °C. After being washed with wash solution, the cells were incubated with 10 lg/mL Alexa Fluor 568 goat antirabbit IgG for 1 h at room temperature in an area protected against light. After excess antibody was washed out three times with wash solution, the specimens were mounted using VECTASHIELD mounting medium with DAPI. Fluorescence images were collected by a BZ-8000 fluorescence microscope (KEYENCE, Osaka, Japan). Cell proliferation assay Cells were plated at 25,000 cells per well in 24-well plates. Choline uptake inhibitors (THA and HC-3) were added immediately after cell plating, and the final medium volume of each well was 1.0 mL. Inhibitors were added every day for 4 days, and cell numbers were measured using an ATPLiteTM (PerkinElmer Life and Analytical Sciences, Boston, MA). A luminescence ATP detection assay was carried out as described in the manufacturer’s instructions. Luminescence was measured on a GloMaxTM 20/20n Luminometer (Promega Corporation, Madison, WI).
Results Time-course and kinetics of [3H]choline uptake in HT-29 cells We first examined the time-course of [3H]choline uptake at a concentration of 10 nM in the presence and absence of Na+ in HT-29 cells for 60 min (Fig. 1a). [3H]Choline uptake in HT-29 cells increased in a time-dependent manner; it was linear with time at up to 30 min (r2 = 0.9921, p = 0.004 in the presence of Na+, r2 = 0.9959, p = 0.002 in the absence of Na+). Based on these findings, subsequent experiments were performed using an uptake period of 10 min. When NaCl in the uptake buffer was replaced by NMDG, the uptake of [3H]choline under Na+-free conditions was strongly enhanced as compared with control uptake under normal conditions. The characteristics of the kinetics of [3H]choline uptake by HT29 cells were determined (Fig. 1b). HT-29 cells were incubated for 10 min with [3H]choline at a concentration of from 0.78 to 50 lM. Kinetic analysis of [3H]choline uptake data (specific uptake), as computed by a non-linear regression analysis, yielded a Michaelis–Menten constant (Km) of 12.1 ± 1.2 lM and a maximal velocity (Vmax) of 387 ± 15 pmol/mg protein/min. The Eadie–Hofstee plot (Fig. 1b, inset) shows a single straight line (r2 = 0.9857, p < 0.0001), suggesting that [3H]choline uptake by HT-29 cells is mediated by a single transport system. Effects of various compounds on [3H]choline uptake in HT-29 cells An inhibition study was performed to determine the substrateselectivity of the choline transport system in HT-29 cells (Fig. 2). The Ki values for the inhibition of [3H]choline uptake were calculated from the corresponding inhibition curves and are shown in Table 1. [3H]Choline uptake was strongly inhibited by THA, unlabeled choline, HC-3, desipramine, quinidine and quinine, in a concentration-dependent manner. Moderate inhibition was seen with diphenhydramine and acetylcholine and only weak inhibition was seen with TEA, corticosterone and TBA. In addition, the Ki values for the inhibition of [3H]choline uptake by MPP+, carnitine and betaine were over 1 mM, and PAH did not cause any significant inhibition at up to 10 mM.
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Fig. 1. Time-course of 10 nM [3H]choline uptake in the presence (d) and absence (s) of Na+ in HT-29 cells for 60 min. (a) Na+-free buffer was modified by replacing NaCl with an equimolar concentration of N-methyl-D-glucamine chloride. Each point represents the mean ± S.D. of three determinations. The time-course of [3H]choline uptake was fitted to the experimental data by a non-linear regression analysis. The kinetic characteristics of [3H]choline uptake in HT-29 cells. (b) HT-29 cells were incubated for 10 min with [3H]choline at concentrations from 0.78 to 50 lM. Each point represents the mean ± S.D. of four determinations. Specific [3H]choline uptake is saturable with a Km of 12.1 ± 1.2 lM and a Vmax of 387 ± 15 pmol/mg protein/min. Inset: Eadie–Hofstee plots of [3H]choline uptake. The correlation is highly significant (r2 = 0.9920, p < 0.0001).
Fig. 2. Effects of various organic cation compounds and PAH on [3H]choline uptake in HT-29 cells. Cells were pre-incubated with test compounds for 20 min and the uptake of 10 nM [3H]choline was measured for 10 min. Results are given as a percentage of control uptake measured in the presence of vehicle. Each point represents the mean ± S.D. of four determinations.
Effects of extra- and intracellular pH on [3H]choline uptake in HT-29 cells The effect of extracellular pH on the uptake of [3H]choline in HT-29 cells was examined by varying the pH of the pre-incubation and incubation media between pH 6.0 and 8.0 (Fig. 3a). [3H]Choline uptake significantly decreased when the extracellular pH was changed from a physiological value of 7.5–6.0. We further investigated the effect of intracellular pH on [3H]choline uptake in HT-29 cells. It is well known that exposure to NH4Cl in Na+-free conditions cause rapid cytosolic alkalinization [23]. Cells were pre-incubated in Na+-free uptake buffer containing various concentrations of NH4Cl for 5 min, and [3H]choline uptake was measured for 10 min. The increase in [3H]choline uptake under the Na+-free condition was inhibited by the extracellular application of NH4Cl in a concentration-dependent manner (Fig. 3b). These results indicate
that choline uptake in HT-29 cells was decreased by acidification of the extracellular medium and by intracellular alkalinization. In addition, we also examined the effects of protonophores, CCCP and FCCP, which known to dissipate transmembrane H+ gradients, on [3H]choline uptake in HT-29 cells. Upon collapse of the plasmamembrane H+ electrochemical gradient with 50 lM CCCP and 50 lM FCCP, the uptake of [3H]choline was significantly reduced to 52.3 ± 5.7% and 56.1 ± 4.9%, respectively (Fig. 3c). Effects of NHE1 inhibitors on [3H]choline uptake in HT-29 cells We demonstrated that choline transport activity was enhanced by the removal of Na+ ion from uptake buffer and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization, suggesting that the activity of NHE1 might affect choline transport activity. Therefore, we investigated
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Table 1 Ki values of various organic cation compounds and PAH on [3H]choline uptake in HT29 cells. Compound
Ki value (lM)
Tetrahexylammonium chloride (THA) Choline Hemicholinium-3 (HC-3) Desipramine Quinidine Diphenhydramine Quinine Acetylcholine Tetraethylammonium chloride (TEA) Corticosterone Tetrabutylammonium chloride (TBA) 1-Methyl-4-phenylpyridinium (MPP+) Carnitine Betaine PAH (p-aminohippric acid)
6.6 7.9 15.4 50.5 64.5 149.1 216 533 1813 3350 5618 >1000 >1000 >1000 >10000
the effects of NHE1 inhibitors, such as DMA and EIPA, on [3H]choline uptake into HT-29 cells (Fig. 4). The increase in [3H]choline uptake under Na+-free conditions was inhibited by 10 lM DMA and EIPA. However, DMA and EIPA did not cause any significant inhibition of [3H]choline uptake in the normal condition. Expression of mRNA for CHT1, CTLs, OCTs and NHEs in HT-29 cells The expression of mRNA for CHT1, CTL1-5, OCT1-3 and NHE15 in HT-29 cells was investigated by Real-time RT-PCR analysis.
The amplified PCR products of transporters were analyzed to determine the expression profiles of mRNA for choline transporters, such as CHT1 and CTLs, in HT-29 cells. CTL1, CTL2 and CTL4 mRNAs are mainly expressed in HT-29. In addition, CHT1, CTL3 and CTL5 mRNAs are expressed at low levels (Fig. 5a). As an organic cation, choline is a substrate for the OCT family. Therefore, we investigated the expression of mRNAs for OCTs. OCT1-3 mRNAs were expressed, albeit OCT1 and OCT2 mRNAs were expressed at low levels (Fig. 5b). We demonstrated that the increase in choline uptake under the Na+-free condition was inhibited by NHE1 inhibitors. Therefore, we determined the expression profile of NHEs in HT-29 cells. As shown in Fig. 5c, NHE1 mRNA is mainly expressed, and NHE2, NHE3 and NHE5 mRNAs are expressed at low levels. Under similar conditions, NHE4 was not detectable in HT-29 cells. Detection of CTL1 protein expression in HT-29 cells by Western blotting analysis and immunocytochemistry Extracts of HT-29 cells were immunoblotted with anti-CTL1 polyclonal antibody. Western blotting analysis indicated that anti-CTL1 polyclonal antibody recognized a band of 80 kDa (Fig. 6A). In addition, CTL1 immunoreactivity in HT-29 cells was detected by immunocytochemical staining using anti-CTL1 antibody (Fig. 6B), which was consistent with the results of RT-PCR and Western blotting. Immunocytochemical staining using anti-CTL1 antibody in HT-29 cells revealed that the plasma membrane showed a considerable level of immunoreactivity.
Fig. 3. Effect of pH on [3H]choline uptake in HT-29 cells. (a) Effect of extracellular pH on [3H]choline uptake. The uptake of 10 nM [3H]choline was measured for 10 min under different pH conditions. Each column represents the mean ± S.D. of four determinations; **p < 0.01 compared with pH 7.5. (b) Effect of intracellular pH on [3H]choline uptake. Cells were pre-incubated in Na+-free uptake buffer containing various concentrations of NH4Cl for 5 min, and then [3H]choline uptake was measured for 10 min. Each column represents the mean ± S.D. of three determinations. ***p < 0.001 compared with the [3H]choline uptake in the normal condition, #p < 0.05, ##p < 0.01 and ###p < 0.001 compared with 0 mM NH4Cl in the Na+-free condition. (c) Effects of protonophore on [3H]choline uptake in HT-29 cells. The plasma-membrane H+ electrochemical gradient was collapsed by 50 lM CCCP and 50 lM FCCP for 20 min and [3H]choline uptake was then measured for 10 min. Each column represents the mean ± S.D. of three determinations. ***p < 0.001 compared with the vehicle control.
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Fig. 4. Effects of Na+/H+ exchange (NHE) inhibitor, DMA (a) and EIPA (b) on [3H]choline uptake in HT-29 cells. HT-29 cells were pre-incubated with test compounds and vehicle for 20 min in the presence and absence of Na+, and [3H]choline uptake was then measured for 10 min. Results are given as a percentage of vehicle control uptake measured under the normal condition. Each column represents the mean ± S.D. of three determinations. *p < 0.05 and **p < 0.01 compared with choline uptake under the normal condition. N.S.; not significant.
Effects of choline uptake inhibition by THA and HC-3 on cell proliferation To explore the possible correlation between choline uptake and cell growth, the effect of transporter inhibitors on cell proliferation was examined by luminescence ATP detection assays. As shown in Fig. 7, potent choline uptake inhibitors such as THA and HC-3 both inhibited HT-29 cell proliferation in a concentration-dependent and time-dependent manner, with relative inhibitory potencies as follows: THA > HC-3. The inhibitory potencies of these inhibitors on cell growth were consistent with the inhibitory potencies on choline uptake inhibition. Discussion Colorectal carcinoma is the most common cancer in both men and women worldwide. Despite the use of aggressive surgical resection and chemotherapy, nearly 50% of patients with colorectal carcinoma develop recurrent disease. Thus, novel approaches to the medical treatment of human colon cancer are required. Intracellular choline accumulation through choline transporters appears to be a prerequisite for cancer cell proliferation based on CK activity [6,24,25]. In this sense, the identification and characterization of choline transporters in cancer may offer a new target for the design of anti-tumor strategies. Therefore, it is important to identify choline transporters in cancer cells. In this study, we first found that the human colon carcinoma cell line HT-29 takes up [3H]choline by a saturable process that is mediated by a single transport system. The Km value was 12.1 lM, which is similar to the blood concentration of choline (10–30 lM). The kinetic parameters are very close to those of the choline transport system in endothelial cells of the blood–brain barrier [26], keratinocytes [27] and astrocytes [20], and these reports have shown conclusively that choline is taken up into these cells by a specific transporter that is not OCT1-3 or CHT1. In astrocytes, the inhibition of CTL1 mRNA expression using siRNA inhibited Na+-independent choline uptake, suggesting that CTL1 is responsible for Na+-independent choline uptake in these cells. The Km value of choline uptake in CTL1-expressing cells was approximately 30 lM [21], which is very close to the Km value in this study, in contrast to those for CHT1 (Km = 0.5 2 lM) and the OCT family of transporters (Km = 100 450 lM). The kinetics data indicate that choline transport in HT-29 cells does not occur via CHT1 or the OCT family. Unfortunately, the functions of CTL25 are unclear. CHT1 is a choline transporter that has been reported to be found at cholinergic neuronal membranes; it is Na+-dependent and can be blocked by HC-3 at a very low concentration (Ki value = 50– 100 nM). Recently, CHT1 has also been identified in non-neuronal cholinergic cells, i.e., keratinocytes [28], and in ciliated cells of
the tracheal epithelium [29]. CHT1 is a novel component of the intrinsic non-neuronal cholinergic system. We found that CHT1 mRNA is expressed in HT-29 cells at low levels. However, the Ki value of HC-3 in HT-29 cells (Ki value = 15.4 lM) is much higher than the values in the literature (Ki value = 50–100 nM), and [3H]choline uptake could also be observed under Na+-free conditions. These findings strongly suggest that CHT1 plays no role in the uptake of choline by HT-29 cells. As an organic cation, choline is a substrate for the OCT family. These transporters, known as OCT1, OCT2 and OCT3, exhibit a broad and overlapping substrate-specificity toward organic cations [16,18,30]. OCT1 and OCT2 accept choline as a substrate with comparatively low affinity. The transport process is Na+-independent and has a Kt value of 620 lM for OCT1 and 210 lM for OCT2. However, OCT3 does not recognize choline as a substrate [16,18]. Therefore, we examined whether choline uptake into HT-29 cells is mediated by OCTs. Real-time PCR data showed that mRNA for OCT1 and OCT2 was only slightly expressed in HT-29 cells. The choline transport system in HT-29 cells resembles OCTs with regard to some pharmacological and functional properties, such as sensitivity of the organic cation compound, Na+-independence and pH-sensitivity. In the present study, however, TEA, a prototypical OCT substrate, interacted with a very low affinity (Ki = 1813 lM). TEA is very often used as a high-affinity reference compound for OCTs, and has Ki values of 46.6 and 52.2 lM for OCT1 and OCT2, respectively [31]. These biochemical and pharmacological data show that choline uptake in HT-29 cells does not occur via OCTs. Next, we searched for a new transporter that could be part of a Na+-independent choline transport system. Recently, a novel family of choline transporters, called choline transporter-like protein 1 (CTL1), has been cloned from Torpedo marmorata, and several homologous genes have also been found in mammals (CTL1-5) [19,21]. Rat CTL1 was also cloned as a homologous rat gene of the CTL protein family and exhibited saturable, HC-3-inhibitable (Ki value > 10 lM) and weak Na+-dependent uptake of choline [20,21,32]. Unlike CTL1, the function of other members of the CTL family (CTL2-5) is unknown. However, there have been several reports on the tissue distribution of the CTL family [19,21]. Human CTL1 protein is expressed in a variety of tissues, including brain, heart, small intestine, kidney, liver, lung, skeletal muscle, pancreas, spleen, ovary, and testis [33]. Rat CTL1a mRNA is expressed in the ileum and colon, whereas rat CTL1b mRNA is expressed exclusively in the brain. CTL2 is predominantly expressed in the tongue, muscle, kidney, heart and lung, as well as, to a lesser extent, the intestine, testis and stomach. CTL3 is expressed in the colon, kidney and ileum, while CTL4 is found at high levels in the intestine, kidney and stomach. CTL5 gene was identified by database screening in the human and macaque genome databases [21], but its tissue distribution is unclear. We examined whether the CTL family medi-
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Fig. 5. Real-time RT-PCR analysis of the expression of CHT1, CTLs, OCTs and NHEs mRNA in HT-29 cells. The percentage of relative mRNA expression is expressed as the ratio of target mRNA to GAPDH mRNA. Experiments were performed in duplicate. BLQ, below the limit of quantification.
ates choline uptake in HT-29 cells. In the present study, CTL1, CTL2 and CTL4 mRNA were mainly expressed in HT-29. Furthermore, kinetics data indicated that the Km value of [3H]choline uptake in HT-29 cells was very close to the Km value for CTL1. The Eadie–Hofstee plot shows a single straight line, suggesting that choline uptake is mediated by a single transport system. Together, these results suggest that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We sought to confirm the expression of CTL1 protein in HT-29 cells by Western blot analysis and immunocytochemistry. Western blotting indicated that a major band of 80 kDa was present in HT-29 cells. The estimated size of human CTL1 from the deduced amino acid sequences was 73.3 kDa for deglycosylated CTL1 (Swiss-Prot),
which is very similar to the size of the HT-29 cells described here. Furthermore, we achieved the first direct visualization of CTL1 proteins expressed in plasma membrane of HT-29 cells by immunocytochemical staining using anti-CTL1 affinity-purified polyclonal antibody. Thus, CTL1 protein was obviously expressed in HT-29 cells. CTL1 is strongly expressed in oligodendrocytes, myelin-synthesizing cells, and cancer cells such as breast cancer and lung adenocarcinoma which require high levels of choline to synthesize sphingomyelin, phosphocholine and phosphatidylcholine [11,34,35]. In light of our present work, further studies are needed to examine the expression status of CTL1 in both cancer and noncancerous colon tissues. Interestingly, a very similar pH profile (pH-dependent uptake) was observed for choline transport in rat astrocytes, which is mediated by CTL1 [20]. [3H]Choline uptake in HT-29 cells was decreased by acidification of the extracellular medium and by intracellular alkalinization. In addition, collapse of the plasmamembrane H+ electrochemical gradient by a protonophore inhibited choline uptake, indicating that the transport of choline is H+dependent. These data suggest that choline may be transported by a choline/H+ antiport system. Moreover, removal of Na+ from the uptake buffer strongly enhanced [3H]choline uptake in HT-29 cells. The absence of Na+ in uptake buffer stimulates reverse-mode NHEs, which results in an increase in the concentration of intracellular H+, and this H+ gradient in turn accelerates the choline transport mediated by choline/H+ antiporter. We investigated the functional correlation of choline transporter and NHEs in HT-29 cells. We found that HT-29 cells highly expressed NHE1 mRNA, and the increase in choline uptake under the Na+-free condition was inhibited by NHE1 inhibitor (DMA or EIPA). These data indicate that H+-coupled choline transport mediated by CTL1 might be modulated by NHE1 activity. In normal cells, NHE1 is quiescent at the steady-state resting intracellular pH and is activated only upon cytosolic acidification. In transformed and cancer cells, NHE1 is hyperactive even at resting pH and the resulting change in cellular alkalinity has been shown to be directly related in most cases to the permanent and uncontrolled proliferation that is characteristic of neoplastic cells [36]. Recently, compelling evidence has indicated that the activity of NHE1 is also a critical factor in the activation of proliferation, motility and invasion of cancer cells derived from various tissues [37]. Thus, the choline transporter CTL1 functions in co-operation with NHE1; the two transporters functioning together to mediate choline uptake and cellular alkalinity may be correlated with cell proliferation, motility and invasion of cancer cells. Finally, we explored the possible correlation between choline uptake and cell proliferation, i.e., the effect of choline transporter inhibitors on cell proliferation. Choline uptake inhibitors such as THA and HC-3 can inhibit HT-29 cell proliferation, suggesting that cell proliferation may require an increased supply of choline. It is possible that cell growth can be restrained by obstructing the function of CTL1. Recently, it was reported that targeting CTL1 might be an effective way to block choline transport and thus inhibit cell proliferation in human lung adenocarcinoma [35]. Previous studies have shown that choline deficiency triggers apoptosis via a p53independent pathway [38]. The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content [39]. Aberrant choline phospholipid metabolism of cancer cells and its strong correlation with malignant progression has been observed in several studies. Human breast cancer cells exhibit consistently elevated phosphatidylcholine levels [40]. In addition, choline kinase has been proposed to play a relevant role in the onset of human cancer, since it is overexpressed in breast, lung, colon and prostate tumors [2,3]. These data indicate that diverse molecular alterations arrive at common end-points in choline phospholipid metabolism in cancer cells. Thus, intracellular choline uptake through CTL1 is the
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Fig. 6. Detection of CTL1 protein in HT-29 cells by Western blot analysis. (A) Total protein extracts from HT-29 cells were separated on 7.5% SDS–PAGE and transferred to PVDF membrane. The membrane was incubated with affinity-purified anti-CTL1 rabbit polyclonal antibody and then incubated with an EnVision alkaline phosphatase polymer. The alkaline phosphatase reaction was performed with BCIP/NBT substrate. M.W. color prestained molecular marker. Immunocytochemical detection of CTL1 protein in HT-29 cells. (B) HT-29 cells were incubated with 4 lg/ml anti-CTL1 rabbit polyclonal antibody and then incubated with 10 lg/mL Alexa Fluor 568 goat anti-rabbit IgG as described in the Material and methods. The panel (a) shows phase-contrast micrograph. Presence of CTL1 is shown in red (b), staining of nuclei with DAPI in blue (c), and merged images are shown in panel (d). Scale bars: 20 lm.
Fig. 7. Effect of the choline uptake inhibition by HC-3 and THA on cell proliferation. The effect of transporter inhibitors HC-3 and THA on cell proliferation was examined by luminescence ATP detection assays. Cells were plated at 25,000 cells per well in 24-well plates. Various concentrations of HC-3 and THA were added immediately after cell plating and every day for 4 days. Cell numbers were measured using an ATPLiteTM. Each column represents the mean + S.D. of four determinations.
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