Equilibrative nucleoside transporter 1 expression in primary human hepatocytes is highly variable and determines uptake of ribavirin

Abstract

Aims

Ribavirin is a nucleoside analogue and remains a necessary component of both interferon-based and directly acting anti-viral regimens for the treatment of hepatitis C virus infection. The achievable concentration of ribavirin within hepatocytes is likely to be an important determinant of therapeutic outcome. In vitro expression levels of equilibrative nucleoside transporter 1 (ENT1) has been shown to be a predictor of treatment response in patients receiving nucleoside-based chemotherapeutic agents. We therefore investigated whether a similar relationship existed between ENT1 expression and ribavirin uptake in freshly isolated primary hepatocytes.

Methods

Primary hepatocytes were cultured on collagen-coated plates and exposed to ribavirin. Parallel samples were taken for high-performance liquid chromatography to assess ribavirin uptake and for quantitative polymerase chain reaction to evaluate ENT1 expression. Similar assays were performed on the human hepatoma cell line (Huh7). ENT1 gene sequence was analysed by cloning of polymerase chain reaction amplified complementary DNA followed by direct sequencing.

Results

There was a strong direct correlation between expression of ENT1 in primary hepatocytes and ribavirin uptake at 24 hr. Huh7 cells expressed ENT1 at similar levels to the majority of primary hepatocytes, but did not take up ribavirin. Sequencing revealed that ENT1 in Huh7 cells is wild type.

Conclusions

In this study, we clearly demonstrate that ribavirin uptake in primary human hepatocytes is variable and correlates with ENT1 expression. This variation in ENT1 expression may account for differences in response rate in patients receiving ribavirin-based anti-hepatitis C virus therapy.

Introduction

Ribavirin (RV) is an essential component of interferon-based regimens for the treatment of hepatitis C virus (HCV) infection.^1–4 These regimens are low cost and remain in use in many parts of the world. The recent introduction of directly acting anti-viral agents (DAA) has transformed outcomes for HCV-infected individuals,^5–9 but optimum results continue to require the use of RV in many patient groups, particularly in those with cirrhosis. ¹⁰ RV is associated with potentially severe side effects, including haemolytic anaemia caused by the accumulation of drug in the red blood cell compartment. ¹¹ Of the proposed mechanisms for RV action, most require the drug to be transported into cells. ¹² In these circumstances, it is legitimate to interrogate factors which regulate RV entry into hepatocytes, particularly those which may provide a mechanistic basis for variations in side effect profile or the outcome of therapy.

RV is a nucleoside analogue and transport into human hepatocytes is mainly mediated through equilibrative nucleoside transporter 1 (ENT1). ¹³ Primary hepatocytes contain high levels of ENT1 mRNA. ¹⁴ A study using three cryopreserved hepatocyte lines suggested that ENT1 is a major transporter of RV uptake in hepatocytes. ¹⁵ Interestingly, there is now good evidence that low ENT1 expression levels predict a poor response to Gemcitabine (a nucleoside analogue) in patients with pancreatic ductal carcinoma, ¹⁶ thus suggesting that ENT1 expression in human pancreas may vary and that this variation may determine the outcome of treatment. Against this background, we elected to test the hypothesis that ENT1 expression in human hepatocytes is variable and regulates the uptake of RV.

Materials and methods

Isolation of primary human hepatocytes and cell cultures

Primary human hepatocytes were isolated using a modified two step collagenase perfusion method. ¹⁷ Details of the method and donors are provided in Supplementary information. All liver tissue was obtained from donors undergoing liver resection with full ethical approval and informed patient consent. This work was done in collaboration with the FRAME group (Biomedical Sciences, University of Nottingham). Cell number and viability were assessed by trypan blue (TB) exclusion and extractions with viability of less than 85% were discarded. Cell cultures were resuspended in plating medium and seeded in six well plates on either collagen-coated surfaces or untreated tissue culture plastic. All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Human hepatoma cell line Huh7 was maintained in culture medium made up of Dulbecco’s modified Eagle’s medium (GibcoBRL, UK) supplemented with 10% foetal calf serum (Sigma UK), 2 mM L-glutamine (Gibco, UK) and antibiotic/antimycotic solution (Hyclone Thermoscientific). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and passaged when ∼80–90% confluent.

RV solution was prepared by dissolving 10 mg of powder (Sigma, UK) in 10 ml of water to obtain a concentration of 1 mg/ml and added to the cell culture medium at a final concentration of 12 µM/well for 24 h. The concentration of RV used corresponds to that found in patients receiving therapy. ¹⁸

High-performance liquid chromatography (HPLC)

Culture medium was aspirated and cells detached using trypsin–ethylene diaminetetraacetic acid (EDTA) solution (Lonza). The resulting cell suspension was pelleted by centrifugation at 250 g for 5 min and lysed by adding 2% Triton X-100 (Sigma) in phosphate-buffered saline and vortexing the tubes for 5 min until no debris was visible. After centrifugation at 1000 r/min for 5 min, supernatant was transferred to fresh tubes and stored at −20°C. Two hundred microlitres of stored cell suspension were treated with 300 µl of 30 µM Tris-HCL buffer (pH 7), 25 µl of 1 M sodium acetate (Sigma) pH 4.0 and 2.5 µl of sweet potato acid phosphatase type IV (500 U, Sigma) and incubated at 37°C for 1 h. The reaction was stopped by adding 2.5 µl of KOH (10 M) and supernatant harvested for column extraction. Phenylboronic acid cartridges (PBA Bond Elute, Varian) were used to remove impurities from HPLC samples. Cartridges were washed with 1 ml of methanol containing 0.5% v/v H₃PO₄ (pH = 2) and 2 ml of ammonium phosphate buffer ((NH₄)H₂PO₄ (250 mM, pH 8.5)). Samples treated with ammonium phosphate buffer ((NH₄)H₂PO₄ (250 mM, pH 8.5)) and 2.5 µl (1 mg/ml) of internal standard (3-methylcytidine methosulphate) applied to the column and washed with ammonium phosphate buffer and methanol. Finally, RV and the internal standard were eluted into glass tubes with 2 ml of methanol containing 2.5% formic acid. Effluents were dried under nitrogen gas and reconstituted in 200 µl of water and analysed in 20 µl aliquots. Quantification of RV was based on equations derived from the calibration curve (see Supplementary information) and expressed as RV concentration (µg/ml), which was then divided by total protein content (mg/ml) in each sample to normalise for input cell number. Details of the HPLC system used are given in Supplementary information.

Total RNA extraction

RNA was extracted by RNeasy mini kit (Qiagen) following manufacturers instruction. RNA was eluted in 30–50 µl of RNase free water and quantified by a NanoDrop ND-1000 UV-Vis Spectrophotometer (LabTech International Ltd, Ringmer, UK). Eluted RNA was stored at −80°C until used.

Parallel samples were taken for HPLC, RNA and assessment of cell viability after 4, 8 and 24 h for liver samples 1–4. All analyses were conducted at 24 h for liver samples 5 and 6.

Polymerase chain reactions (PCRs)

Complementary DNA (cDNA) was synthesised by reverse transcription of RNA. Briefly, 1 µg of RNA was prepared in 20 µl of water and 1 µl of random hexamers (pD(N)₆) and incubated at 70°C for 10 min as initial denaturation step. Samples were placed on ice and master mix was prepared by adding 1 µl (200 units) of M-MLV Virus Reverse Transcriptase enzyme (Invitrogen, UK), 50 mM of dithiothreitol (Invitrogen, UK) and 1.5 µl of dNTP mix. In RT negative (RT−) samples, water was added to replace enzyme. Master Mix was added to each sample up to a final volume of 50 µl and incubated at 37°C for 1 h followed by 10 min incubation at 95°C.

Primers for quantitative real time PCR were designed by the primer 3 web 0.040 programme and targeted at exon–exon junctions (primer sequences are given in Supplementary information). PCR amplification was performed using a SYBR green II (reporter dye) based assay (Stratagene, UK). Reaction mixture consisted of 12.5 µl of 1X SYBR Green Master Mix (Stratagene), 1 µl of each forward and reverse primer (final concentration of 250 nM), 0.38 µl of ROX dye and 5 µl of DNA template (10 ng/5 µl). Cycling conditions were 10 min denaturation at 95°C followed by 40 cycles of: 30 s denaturation at 95°C; 30 s annealing at a temperature according to the primer used; 30 s extension at 72°C and a final melt for 60 s. The reaction was conducted using thermal cycler (MX3005P Stratagene) and data analysed by Mxpro-QPCR software version 3.20. A standard curve was generated using serial dilution of neat cDNA. No template control (NTC) and no RT (RT−) controls were included in each reaction.

Direct sequencing of ENT1 coding sequence

cDNA was extracted from Huh7 cells as described. Gene specific primers were designed and blast analysis was performed to confirm specificity. Primer sequences were: forward primer: 5′-ATGACAACCAGTCACC-3′; reverse primer: 5′-TCACACAATTGCCCGGAACAGG-3′. One-tenth of the cDNA reaction mixture was amplified using Phusion (pfu) high-fidelity DNA polymerase (Finzyme, NEB, UK) to produce blunt ended PCR product according to manufacturer’s instructions. The PCR reaction contained primers at a final concentration of 500 nM and was performed in thermal cycler (Perkin Elmer GeneAmp PCR system 2400) using the following conditions: Initial denaturation at 98°C for 30 s followed by: 40 cycles of denaturation 98°C for 10 s; annealing 60 ± 10°C (gradient) for 15 s and extension 72°C for 90 s and final extension at 72°C. At an optimal annealing temperature of 62.1°C, a single product of the correct size (1.3 kb) was amplified. The amplimer was purified by column filtration using QIAquick PCR Purification Kit, following manufacturer’s instructions. DNA was visualised by agarose gel electrophoresis and quantified by NanoDrop ND-1000 UV-Vis Spectrophotometer (LabTech International Ltd, Ringmer, UK). PCR products were used for cloning by TOPO TA cloning kit (Invitrogen) and analysed by direct sequencing.

Statistical methods

Non-parametric Spearman’s rank correlation test (r_s) was used to correlate RV uptake with total ENT1 expression. All the graphs and data analysis were done in GraphPad Prism version 4. Bar graphs shows means ± standard deviation from three repeat experiments.

Results

Ten liver resections were used. Primary human hepatocytes from six of these resections met the criteria for yield and cell viability and were plated in six well plates at a density of 1.68 million/well. Cells grown on collagen-coated plates maintained the typical cuboidal shape, prominent nuclei and defined cell boundaries characteristic of healthy hepatocytes throughout the culture period and formed three dimensional spheroids (Figure 1(a)). In contrast, hepatocytes grown on tissue culture plastic lost their cuboidal shape and did not form spheroids (Figure 1(b)).

Figure 1.

Primary human hepatocytes on day 3 of in vitro culture. Human hepatocytes cultured on collagen coated plates (a) or tissue culture plastic (b). Black arrows show hepatocytes attached to culture plate maintaining typical cuboidal liver cell morphology and white arrows indicate areas of spheroid formation. In contrast, cells maintained on tissue culture plastic have lost their cuboidal pattern and do not form spheroids.

Time course for RV uptake and ENT1 expression

Human hepatocytes from human livers 1–4 were cultured on six-well collagen-coated plates and treated with RV at a final concentration of 12 µM. Samples were harvested at 4, 8 and 24 h. Livers 1, 2 and 4 showed a progressive increase in drug uptake over a period of 24 h whereas liver 3 showed stable uptake (Figure 2). Twenty-four hours was selected as the time point for further comparison of ENT1 expression and RV uptake. Total ENT1 expression at 24 h was quantified by RT-QPCR and compared with RV uptake by all six human livers at the same time point. Each experiment was repeated a minimum of three times. There was a threefold variation in the mean level of ENT1 expression in six human livers which was associated with a clearly observable difference in RV uptake (Figure 3(a) and (b)). As RV uptake levels do not follow a normal distribution, the non-parametric Spearman correlation test abbreviated by r_s was done to assess the significance of our observations. Despite an apparent outlier activity in liver 5, the correlation between ENT1 expression and RV uptake was highly significant (correlation test value of r equal to 0.94 and a p value of < 0.01). In order to confirm that correlation is specific to ENT1, RV uptake levels were compared to ENT2 transporter expression but no significant correlation was found (r_s = −0.54 and p value = 0.297) (Figure 3(c)).

Figure 2.

Time course for ribavirin uptake and ENT1 expression. Human hepatocytes from liver 1–4 were cultured on collagen coated plates and exposed to ribavirin diluted in culture medium. After 4, 8 and 24 h, cells were harvested and analysed for ribavirin uptake (a) and ENT1 expression (b). Bars indicate the mean and standard deviation is represented by error bars.

Figure 3.

Correlation of ribavirin uptake and ENT1 expression. Human hepatocytes from livers 1–6 and Huh7 cells were exposed to ribavirin and drug uptake and ENT1 expression levels quantified at 24 h. Ribavirin concentration plotted on left y-axis indicates µg of drug over mg of total proteins in the sample; total ENT1 plotted on right y-axis shows quantity in ng and is normalised to the house keeping gene HPRT (a). Bar graphs indicate the mean of three repeat experiments and error bars denote standard deviation. (b) Scatter plot of ribavirin uptake (y axis) versus ENT1 expression (x axis). The relationship between ENT2 quantity in nanograms normalised to HPRT and ribavirin concentration is shown in (c).

The human hepatoma cell line Huh7 was used as a control. We observed that Huh7 cells did not take up RV despite expressing levels of ENT1 higher than all but one of the primary hepatocyte cultures (Figure 3).

Analysis of Huh7 ENT1 (SLC29A1) gene sequence

The lack of RV uptake by the Huh7 cell line despite high levels of ENT1 expression suggests either that the receptor itself is non-functional, or that other requirements for RV uptake are not present in this cell line. In order to screen for mutation, Huh7 ENT1 coding sequence was amplified from ENT1 cDNA and analysed by cloning into pCR® 2.1-TOPO® TA cloning vector/kit (Invitrogen), followed by direct sequencing. As shown in Figure 4(a), there was a single PCR product of the right size (1.3 kb) without any non-specific bands. Amplimers were purified by column purification (Figure 4(b)) and cloned using TOPO TA cloning kit (Invitrogen). The results of plasmid analysis by direct sequencing were compared to wild type SLC29A1 sequence (Genebank). ENT1 gene expressed by Huh7 cell line was found to be identical to wild type. The nucleotide sequence is given in Supplementary information and a representative chromatogram is shown in Figure 4(c).

Figure 4.

Purification and sequencing of ENT1 gene product. ENT1 coding sequence was amplified using specific primers and analysed by agarose gel electrophoresis. (a) Gradient PCR to select optimal annealing temperature (lanes 1–10 and temperature range 60 ± 10°C). (b) Temperature with highest amplification was used for PCR amplification and resulting products were purified for cloning (lane 11 & 12) followed by sequencing (c). M: DNA marker; NTC: non-template control.

Effect of culture conditions on RV uptake and ENT1 expression

In order to determine whether the observed variability of ENT1 expression may be caused by differences in the degree of three-dimensional spheroid formation and therefore the proportion of hepatocytes maintained in the polarised state, we compared ENT1 expression by hepatocytes from livers 5 and 6 in: (i) cultures on collagen-coated surfaces, which promote the formation of three-dimensional spheroids which are known to promote hepatocyte polarisation and (ii) hepatocytes cultured on untreated plastic surfaces, which leads to rapid loss of hepatocyte morphology and polarisation. In our system, we found that culturing cells on tissue culture plastic or collagen-coated plates did not affect either levels of receptor expression or RV uptake (Figure 5).

Figure 5.

The effect of culture conditions on ENT1 gene expression. Hepatocytes from human livers 5 and 6 were cultured either on tissue culture plastic or on collagen-coated plates. Ribavirin quantity and ENT1 expression measured after 24 h were compared from cells grown on two culture surfaces (T/C: tissue culture surface; C/G: collagen-coated surface). Bar graphs indicate an average of three repeat experiments and error bars denote standard deviation.

Discussion

There is good evidence that the anti-viral effects of RV are dose related and that response to treatment can be improved by increasing RV delivery to hepatocytes. ¹⁹ An unavoidable consequence of increasing RV dose, however, is an increase in drug toxicity which often requires dose reduction or temporary cessation of therapy. In these circumstances, a greater understanding of the factors which determine RV uptake into hepatocytes would permit individualisation of antiviral regimens. We here demonstrate an up to threefold variation in ENT1 gene expression between different sets of primary human hepatocytes in culture and show that this variation is associated with significant differences in RV uptake when cells are exposed to drug levels which correspond to those found in patients receiving therapy. ¹⁸ No such relationship was observed between expression of ENT2 and RV uptake. These findings suggest that constitutive differences in host ENT1 receptor expression may be a major determinant of the outcome of antiviral therapy in HCV infection.

ENTs are facilitative transporters, in which substrate concentration is the driving force, and are divided into two major classes defined by their sensitivity to nitrobenzylthioinosine (NBMPR). ²⁰ ENT1 is inhibited by nanomolar concentration of NBMPR, whereas ENT2 requires exposure to micromolar concentration of NBMPR to be inhibited. Additional family members ENT3 and ENT4 are less well characterised. ²¹ Human ENT1 is a 456-residue glycoprotein comprising 11 transmembrane domains (TMDs), and has been identified as the major RV transporter in a variety of cell types.^22–24 A number of splice variants have been reported for the ENT1 gene. The study by Fukuchi et al. explored the ENT1 promoter region and showed that four different promoters give rise to at least 12 different ENT1 isoforms. ¹⁵ They also suggested that expression levels of d1–d4 isoforms were higher in hepatocytes having higher drug uptake. However, the primers used in the Fukuchi study picked up ENT1 variants other than isoforms linked to higher drug uptake. In view of the complexity of splice variants of ENT1, we elected to explore the relationship between ENT1 and RV uptake using a primer pair which captures all variants.

The differences in ENT1 expression and RV concentration between cultures of primary hepatocytes found in our study did not exceed threefold. Two lines of argument, however, suggest that differences of this magnitude may be highly significant. Firstly, the efficacy of RV in the treatment of HCV is tightly dose dependent, and a two- to threefold change of dose in clinical practice would be expected to have a major impact on both outcomes and toxicity.^1–4 Secondly, there is consistent evidence from a number of studies that variation in human ENT1 expression in cancer cells correlates with sensitivity of the tumour to nucleoside analogues and predicts the duration of disease free survival.^16,25 While interpretation of these studies is limited by the lack of standardised scoring for ENT1 expression, those which quantified ENT1 expression, such as that by Fujita et al., ²⁶ found that differences in expression comparable to those found in our study were predictive of differences in outcome.

Although there are well-defined limitations to cell growth and the maintenance of functions in vitro, primary human cells remain the best model for the study of key hepatocyte-specific functions. In particular, the maintenance of metabolism and transporter systems facilitate drug-based studies in the short term. ²⁷ Further, as humans are the only natural host for hepatitis C, primary human cells provide the most relevant tool to study response to anti-virals and host virus interaction. ²⁷ Another advantage of human cells is that expression of sinusoidal transport proteins remains relatively constant and mimics closely that found in vivo when compared to rat hepatocytes. ²⁸ In this study, we successfully isolated and cultured human hepatocytes, validating a method described previously. ¹⁷ Good cell viability was achieved and cultured cells maintained a typical phenotype throughout the study. Time course experiments demonstrated that hepatocytes continued to take up RV, which itself is evidence that transport systems are intact in this in vitro culture model. Our finding that ENT1 expression varies significantly in primary hepatocytes obtained from different donors and that there is a highly significant correlation between RV uptake and the level of ENT1 may therefore provide a basis for modelling therapeutic responses in vivo.

Within the liver, hepatocytes are maintained in a polarised state, which is lost in most culture models and may be a factor determining transporter expression and function. In contrast to growth on tissue culture plastic, hepatocytes cultured on collagen-coated surfaces form spheroids (Figure 1) which contribute to maintenance of the polarised state. ²⁹ To investigate whether differences in ENT1 expression and RV uptake could simply be a consequence of differences in the proportion of hepatocytes within spheroids, we compared ENT1 expression and RV uptake in hepatocytes from the same donor cultured on either tissue culture plastic or on collagen-coated plates. No differences were observed in either transporter expression or drug uptake by primary hepatocytes in these different models.

Finally, an interesting observation in this study was that the Huh7 cell line used in our study, despite having ENT1 expression levels which were at least as high as the majority of the livers tested, failed to take up any RV. Huh7 cells were selected as the positive control for our study as they have previously been shown to express ENT1³⁰ and have been widely used to demonstrate the anti-viral activity of RV on HCV replication in in vitro models of infection. While we did not set out to investigate ENT1 expression in Huh7 cells, there appears to be a discordance in our results which requires further interrogation. Mutations in the ENT1 gene can lead to defects in transporter function resulting in poor uptake of nucleosides and their analogues. It has, for example, been shown that ENT1 TMDs 3–6 are implicated in nucleoside binding and transport. ³⁰ A study by Zimmerman et al. suggested that mutation in Glycine 24 in TMD 1 of human ENT, a highly conserved amino acid among different ENT isoforms including ENT1 and ENT2, abolished transporter function without affecting gene expression and its plasma membrane localisation. ²⁵ We therefore first interrogated the coding sequence of the Huh7 ENT1 gene, as determined by direct sequencing of amplification products of ENT1 cDNA, for evidence of mutations or splicing events which may explain defects in RV uptake. The ENT1 cDNA sequence in our Huh7 cell lines was homogeneous and corresponded exactly to wild type SLC29A1 sequence (Genebank), thus excluding mutation and suggesting that regulation by splicing is unlikely. A recent study has interrogated the membrane localisation of ENT1 in Huh7 cells and has demonstrated that ENT1 can be trafficked to internal membranes, in a clathrin-dependent manner, with loss of RV uptake. ³¹ Further work has shown that hepatocyte-derived cell lines recurrently exposed to RV develop a RV-resistant phenotype in the continued presence of high levels of ENT1 expression. ³² Therefore there is no necessary linkage between ENT1 expression and RV uptake in hepatocyte-derived cell lines. We consider our RV assays to be extremely robust (Supplementary Table S2 and Figures S1 and S2) and think it likely that ENT1 expression and RV uptake has become unlinked in our particular cell line.

Overall, our observations suggest that intrinsic differences in ENT1 expression in primary hepatocytes may determine the uptake of RV and have important consequences for the outcomes of RV containing antiviral regimens for hepatitis C infection.

Authors contribution

KB, BJT, MI and WI planned the study and supervised the study team. KB, LC and LD carried out the experimental work. KB, AAM and BJT analysed the data. KB, AAM and BJT wrote the manuscript.