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METHODS IN CELL PHYSIOLOGY
1Laboratory of Medical Genetics, Department of Internal Medicine, Cardiology and Hepatology, University of Bologna, Bologna; and 2Laboratory of Molecular Genetics, Istituto Giannina Gaslini, Genova, Italy
Submitted 26 May 2006 ; accepted in final form 15 September 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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thyroid; fluorescence microscopy; FRTL-5 cells
Alterations in NIS expression and function are a feature of thyroid diseases. The intrinsic ability of the thyroid to accumulate I– has long been exploited clinically in the diagnosis and treatment of thyroid cancer with radioiodide. However, a significant proportion of patients with aggressive thyroid cancer fail to respond to radioiodide therapy, and no alternative treatment exists. The causes are unclear but may involve defects in NIS expression, maturation, and plasma membrane targeting (6, 19, 28). Autoimmune thyroid disease is also associated with changes in NIS expression (3) and, in some cases, with the presence in serum of NIS autoantibodies (9). Furthermore, NIS mutations in the "iodide transport defect" are a rare cause of congenital hypothyroidism (23).
The role of NIS extends beyond the thyroid gland. NIS is normally expressed in extrathyroidal tissues including the lactating breast, salivary glands, gastric mucosa, placenta, and kidney (reviewed in Ref. 7). The detection of NIS in breast and other extrathyroidal tumors, as well as the induction of NIS expression through gene transfer, raises the possibility of imaging and treating a wide range of cancers with radioiodide (27, 32). In addition, NIS has been used as a noninvasive in vivo nuclear imaging reporter of gene expression, graft success, and stem cell migration (1, 2, 16).
The role of NIS in thyroid physiology and pathophysiology and growing importance of NIS in cancer treatment and nuclear imaging highlights the need for cellular studies to define the mechanisms that regulate NIS expression and function, both in thyroidal and nonthyroidal cells. Critical to such studies is a cell-based assay by which to measure NIS activity. I– accumulation in thyrocytes is commonly measured with radiotracers (125I–) and electrophysiological techniques, and both approaches have been used to characterize the kinetics of I– transport by NIS (4, 10, 18, 33). The advent of optical probes has facilitated the measurement of other ions. A quinolinium-based fluorophore LZQ is sensitive to I– (but not Cl–) and permits the measurement of I–/Cl– exchange in cell lines expressing the cystic fibrosis transmembrane conductance regulator (CFTR) (14). The disadvantage of small-molecule probes is their requirement for cell loading and their poor retention within cells, problems that can been overcome by the use of genetically encodable fluorescent proteins that can be transcriptionally expressed in cells. Yellow fluorescent protein (YFP) is sensitive to halides (31), and two variants with enhanced halide sensitivity, YFP-H148Q and YFP-H148Q/I152L, have been used to measure cellular I–/Cl– exchange via CFTR (11, 12, 15). The high affinity of YFP-H148Q/I152L for I– and 30-fold selectivity for I– over Cl– (KI of 1.9 mM and KCl of 85 mM at pH 7.4) (11) makes it an interesting candidate as a potential sensor of NIS-mediated I– accumulation in the presence of physiological intracellular Cl–.
The aim of the present study was to assess the utility of YFP-H148Q/I152L as a biosensor of cellular I– uptake via NIS. Rat thyroid FRTL-5 cells were chosen as the model in which to test this novel assay, since these cells express NIS endogenously and have been extensively used to characterize the kinetics of I– uptake with radiotracers and voltage clamp (18, 30, 33, 37). In fact, the coding sequence of the NIS gene was first deduced from cDNA derived from FRTL-5 cells (4). Specific criteria were selected by which fluorescence changes induced by I– in these cells could be ascribed to NIS, including 1) sensitivity to micromolar extracellular I–, 2) inhibition by perchlorate, 3) extracellular Na+ dependence, and 4) regulation by TSH. The assay was further validated in COS-7 cells expressing human NIS (hNIS) to confirm the specificity of I–-induced fluorescence changes.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Fischer rat thyroid FRTL-5 cells (donated by F. Curcio and S. Ambesi-Impiombato, University of Udine, Italy) were cultured in Coon's modified F12 medium supplemented with 5% donor calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and a six-hormone mix (6H) containing 1 µg/ml insulin, 3.6 ng/ml hydrocortisone, 5 µg/ml apotransferrin, 10 ng/ml glycyl-L-histidyl-L-lysine acetate, 10 ng/ml somatostatin, and 1 mU/ml TSH. FRTL-5 cells were passaged with "CTC" solution consisting of 20 U/ml collagenase, 0.75 mg/ml trypsin, and 2% dialyzed chicken serum in Ca2+- and Mg2+-free Hanks' balanced salt solution. COS-7 cells (donated by S. Ferroni, University of Bologna) were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, and were passaged with 0.05% trypsin-EDTA solution.
Transfection
The pcDNA3.1 vector containing the YFP-H148Q/I152L coding sequence (pcDNA3.1-YFP-H148Q/I152L) was donated by A. Verkman (University of California), and the pcDNA3 vector containing the full-length hNIS coding sequence was donated by S. Jhiang (Ohio State University). The hNIS sequence was subcloned into a pcDNA3.1/Zeo vector at KpnI/XbaI sites and designated as pcDNA3.1/Zeo-hNIS. FRTL-5 cells grown in 24-well plates were transfected with 0.8 µg/well of pcDNA3.1-YFP-H148Q/I152L using Lipofectamine 2000 (Invitrogen) at a ratio of 4 µl liposome to 1 µg DNA. Clonal populations of FRTL-5 cells stably expressing the fluorescent protein were generated by selection of antibiotic-resistant cells with 500 µg/ml G418 for 2 wk and limited dilution of surviving cells. COS-7 cells were cotransfected with pcDNA3.1-YFP-H148Q/I152L and either pcDNA3.1/Zeo-hNIS or the empty vector pcDNA3.1/Zeo. For transient transfection, COS-7 cells were grown in six-well plates containing round glass coverslips and double transfections carried out with 0.5 mg of each plasmid (1 mg/well total) and Fugene (Stratagene) at a ratio of 3 µl Fugene to 1 µg DNA. Fluorescence measurements were performed 48–72 h after transient transfection.
Cellular YFP-H148Q/I152L Fluorescence Measurement
Fluorescence intensity was monitored continuously with a Zeiss Axiovert 200 inverted microscope equipped with an XBO xenon 75-W short arc lamp, Lambda 10C filter changer (Sutter Instrument), and XF104–2 YFP filter set for excitation at 500 ± 12.5 nm and emission at 545 ± 17.5 nm (Omega Optical). Cellular YFP-H148Q/I152L fluorescence was observed with a x40 oil-immersion objective, and images were typically acquired for 100 ms every 10 s. Images were acquired with a Coolsnap HQ CCD camera (Roper Scientific) and processed with Metafluor software (Universal Imaging). Cells were grown on 25-mm diameter round glass coverslips and mounted in a thermostatically controlled imaging chamber (Warner Instruments) maintained at 36.5–37°C. Cells were continuously perfused at 4–5 ml min–1 with phosphate-buffered saline (PBS) composed of 137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, and 10 mM glucose (pH 7.4). Cells were exposed to I– concentrations ranging from 1 µM to 100 mM by replacing specified amounts of NaCl with equimolar NaI and maintaining the total concentration of NaCl plus NaI at 137 mM. To examine the effect of extracellular Na+ on fluorescence changes induced by I–, experiments were performed in a HEPES-buffered solution (HBS) composed of 145 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). Low-Na+ HBS containing 10 mM Na+ was prepared by replacing NaCl with equimolar choline chloride. The effect of pH on cellular YFP-H148Q/I152L fluorescence in FRTL-5 cells was determined by exposing cells to a high-K+ solution (10 mM HEPES, 130 mM KCl, 0.7 mM CaCl2) at pH 6.5–7.5 in the presence of 7 µM nigericin and 5 µM valinomycin.
Analysis of Cellular YFP-H148Q/I152L Fluorescence
Typically, measurements of fluorescence intensity were performed on groups of 20–100 FRTL-5 cells and 5–15 COS-7 cells, and represent the average intensity of all fluorescent cells within the selected region of interest. Cell-to-cell variability was determined by selecting 50 individual cells within a representative group of cells. Background fluorescence, measured in an area of the coverslip without cells, was subtracted from cellular fluorescence and was typically <10% of total fluorescence (mean background fluorescence 8.7 ± 0.5%, n = 45). Autofluorescence of cells not transfected with YFP-H148Q/I152L was negligible compared with the fluorescence of the coverslip itself. Photobleaching resulted in a one-phase exponential decrease in cellular YFP-H148Q/I152L fluorescence, occurring with a mean rate constant of 0.0033 ± 0.0003 min–1 (n = 45) with exposure times of 100 ms every 10 s. To correct for photobleaching, fluorescence intensity in the absence of iodide (F0) was fitted to a one-phase exponential decay function to obtain a theoretical curve of F0 at each time point. Fluorescence intensity during I– exposure (F) was then normalized to F0 to obtain a measure of relative fluorescence (RF) at each time point (i.e., RF = F/F0). Changes in fluorescence induced by I– are expressed in terms of the maximal rate of fluorescence decrease (
RF/t) and the steady-state decrease measured at 5 min of I– exposure (RF5min). Both are negative values, reflecting a decrease in fluorescence, but are reported by their absolute values throughout. RF/t was estimated as the initial or maximal slope of the best-fit curve when the I–induced decrease in RF was fitted by nonlinear regression to one-phase or, more commonly, a two-phase exponential decay function.
The concentration dependence of I–-induced changes in fluorescence was analyzed by nonlinear regression fitting to a biphasic Hill equation
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RF/t; Ymin and Ymax are the minimal and maximal responses, respectively; KH and KL are the affinity constants (defined as the extracellular I– concentrations producing a half-maximal response) for the high- and low-affinity components of the response; nH and nL are the Hill coefficients for high- and low-affinity components; and 
H is the fraction of the response mediated by the high-affinity component. Curve fitting and other data analysis was performed with Prism 4 (GraphPad Software). I– Sensitivity and pH Sensitivity of YFP-H148Q/I152L in Solution
The I– sensitivity and pH sensitivity of thyrocyte-derived YFP-H148Q/I152L was determined in solution. YFP-H148Q/I152L-expressing FRTL-5 cells were cultured in 150-cm2 flasks (10–20 x 106 cells/flask), trypsinized, and centrifuged at 1,500 rpm. Pelleted cells were lysed by resuspension in 100 µl of 10 mM HEPES and two freeze-thaw cycles. The lysate was centrifuged at 12,000 rpm, and the supernatant was collected. Droplets of a volume of 12 µl of the YFP-H148Q/I152L-containing supernatant were deposited on a coverslip, and fluorescence images acquired with a x10 objective (to allow >90% of the droplet to be imaged). The pH sensitivity of YFP-H148Q/I152L fluorescence was determined by adjusting the pH of the supernatant to various pH values with NaOH (final Na+ < 3 mM). Fluorescence intensity at each pH value was normalized to that at pH 7, and the pKa (pH at half-maximal fluorescence) was derived by nonlinear regression fitting to a sigmoidal curve. I– sensitivity was determined by adding 0–100 mM NaI or reciprocal amounts of sodium gluconate to the supernatant, maintaining the total concentration of NaI plus sodium gluconate at 100 mM. Fluorescence intensity in the presence of I– was normalized to fluorescence in the absence of I– and fitted to the one-site binding equation Y = 1 – a[I–]/(KI + [I–]), where Y is the relative fluorescence in the presence of I–, a is the maximal decrease in relative fluorescence produced by a high I– concentration, and KI is the I– concentration producing a half-maximal fluorescence decrease.
Intracellular pH Measurement
Intracellular pH (pHi) was measured with BCECF in FRTL-5 cells not expressing YFP-H148Q/I152L. Cells were loaded with 10 µM BCECF-AM for 30–60 min in culture medium, maintained for a further 20 min in culture medium to allow hydrolysis of BCECF-AM, and mounted as above in the imaging chamber. Fluorescence was monitored by fluorescence microscopy with the same filter set as YFP-H148Q/I152L (excitation 500 ± 12.5 nm, emission 545 ± 17.5 nm). Because of the higher intensity and photobleaching-induced decay of BCECF fluorescence, cells were visualized with a x20 objective, rather than the x40 objective used for YFP-H148Q/I152L experiments. Images were acquired for 200 ms every 10 s. The effect of I– on BCECF fluorescence was determined by replacing 1 mM NaCl in PBS with 1 mM NaI. The effect of pH was determined by exposing cells to a high-K+ solution (10 mM HEPES, 130 mM KCl, 0.7 mM CaCl2) at pH 6.5–7.5 in the presence of 7 µM nigericin and 5 µM valinomycin. Fluorescence changes were expressed relative to resting fluorescence, which in the absence of specific manipulations, was stable for at least 30 min.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FRTL-5 cells stably expressing YFP-H148Q/I152L were exposed to 100 µM extracellular I– (Figs. 1 and 2). I– produced a rapid decrease in cellular fluorescence that began within 10 s and reached a steady-state level in 5–10 min. The fluorescence decrease was reversible, with complete recovery of resting fluorescence occurring within 10 min of removing extracellular I–. Fluorescence changes induced by I– were reproducible within the same experiment, with three consecutive applications of I– producing similar responses (data not shown). Addition of I– to YFP-H148Q/I152L in solution (derived from a lysate of FRTL-5 cells) suppressed fluorescence with a time constant <1 s, suggesting that the rate of fluorescence decrease in intact cells is not limited by the rate of I– binding to YFP-H148Q/I152L (data not shown).
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RF/t) and the steady-state decrease measured at 5 min of I– exposure (RF5min). Neither parameter correlated with the resting fluorescence of cells (Fig. 2E), suggesting that cell-to-cell variability in I–-induced responses is independent of differences in YFP-H148Q/I152L expression between cells. In contrast, there was a significant correlation between RF/t and RF5min (P < 0.001, R2 = 0.59) (Fig. 2F), suggesting that cell-to-cell differences in the rate of I– influx result in parallel differences in intracellular I– concentration. Criteria for the Identification of NIS Activity in FRTL-5 Cells
I– concentration dependence. I– consistently decreased YFP-H148Q/I152L fluorescence in FRTL-5 cells at concentrations ranging from 3 µM to 100 mM, and at 1 µM I– produced a response in 4/7 experiments (Fig. 3A). The maximal rate of fluorescence decrease exhibited a biphasic I– concentration dependence, with high- and low-affinity constants of 35 µM and >1 mM, respectively (Fig. 3B).
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I–-induced changes in fluorescence of COS-7 cells were examined 48–72 h following cotransfection with YFP-H148Q/I152L cDNA and either hNIS cDNA or empty vector. I– (100 µM) decreased YFP-H148Q/I152L fluorescence in cells transfected with hNIS cDNA but had no effect on cells lacking hNIS (Fig. 7, A and B). This response was prevented by pretreatment of cells with 100 µM perchlorate, confirming that it is specifically mediated by NIS (Fig. 7C). I– (10 mM) also decreased YFP-H148Q/I152L fluorescence in cells lacking hNIS, suggesting that the higher concentration of I– activates influx into COS-7 cells via additional lower affinity pathways.
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YFPs are pH-sensitive, with acidification suppressing fluorescence emission. The role of pH in mediating or modulating I–-induced changes in YFP-H148Q/I152L fluorescence was therefore investigated. First, effect of extracellular I– on pHi of FRTL-5 cells was assessed to exclude the possibility that I– decreases pHi and thereby reduces YFP-H148Q/I152L fluorescence independently of I– influx. pHi was monitored with BCECF in FRTL-5 cells not expressing YFP-H148Q/I152L. I– (1 mM) had no effect on BCECF fluorescence (Fig. 8A). In contrast, altering pHi to values of 7.5 and 6.5 with a high-K+/nigericin/valinomycin buffer, respectively, increased and decreased BCECF fluorescence, confirming its pH sensitivity. Second, the effect of pHi alterations on YFP-H148Q/I152L fluorescence in FRTL-5 cells was examined by exposing cells to the same high-K+/nigericin/valinomycin buffer, and this resulted in qualitatively similar changes in fluorescence as with BCECF. Thus, raising pHi to 7.5 increased YFP-H148Q/I152L fluorescence, and decreasing pHi to 6.5 reduced fluorescence (Fig. 8B). Next, the pH sensitivity of thyrocyte-derived YFP-H148Q/I152L fluorescence was measured in solution and yielded a pKa of 7.02 (Fig. 8C). Considering a typical pHi of 7.0, a physiological pH change of ±0.2 units would result in a ±15% change in resting YFP-H148Q/I152L fluorescence. Finally, the effect of pH on the I– sensitivity of thyrocyte-derived YFP-H148Q/I152L fluorescence was measured in solution over a physiological pH range of 6.8–7.2. The affinity of YFP-H148Q/I152L for I– (KI) was 1.4, 2.0, and 2.4 mM at pH values of 6.8, 7, and 7.2, respectively (Fig. 8D), suggesting a small decrease in I– sensitivity with increasing pH. Considering a typical pHi of 7.0, a physiological pH change of ±0.2 units would cause, at most, a 15% change in the magnitude of the fluorescence decrease induced by millimolar intracellular I–, with acidification increasing the response to I–, and alkalinization decreasing it.
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DISCUSSION |
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Validation of New Method
I–-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells fit specific criteria for NIS-mediated I–-transport including 1) sensitivity to micromolar extracellular I–, 2) inhibition by perchlorate, 3) extracellular Na+ dependence, and 4) regulation by TSH.
Extracellular I– decreased YFP-H148Q/I152L fluorescence in FRTL-5 cells with affinity constants of 35 µM and >1 mM. The Km of NIS for I– is 
30 µM based upon 125I– uptake and I–-induced currents in FRTL-5 cells and in NIS-transfected nonthyroidal cells (4, 10, 18, 33). In this study, I– exposure was carried out isotonically by replacing specified amounts of NaCl with equimolar NaI while maintaining the total concentration of NaCl plus NaI constant. For solutions containing less than 1 mM I–, substitution of NaCl with NaI produces a negligible change in Cl– concentration, and fluorescence changes can be considered to be solely due to I– accumulation. However, solutions containing more than 1 mM I– have a reduced Cl– concentration, and fluorescence changes reflect the combined effects of I– influx and Cl– efflux (I–/Cl– exchange). The high-affinity component of I–-induced fluorescence changes activated by micromolar I– is consistent with I– influx via NIS, whereas the low-affinity component activated by millimolar I– may reflect I–/Cl– exchange via additional channels or transporters. The lowest concentration of I– to consistently suppress YFP-H148Q/I152L fluorescence in FRTL-5 cells was 3 µM, with 1 µM causing <5% decrease in fluorescence in 4/7 experiments. 125I– uptake in vitro can be detected with 0.1–1 µM I– (8, 17, 23), suggesting that radiotracer uptake is more sensitive to I– concentration than the fluorescence assay.
Inhibition of NIS with perchlorate or Na+ removal prevented the fluorescence decrease induced by low concentrations (<1 mM) of I– in FRTL-5 cells. This is consistent with inhibition of 125I– uptake and I–-induced currents in FRTL-5 cells and NIS-transfected nonthyroidal cells by these two manipulations (4, 10, 18, 33, 36, 37). Perchlorate also causes the loss of accumulated 125I– in FRTL-5 cells (33) and in this study reversed the fluorescence changes induced by I–. In the absence of I– organification, perchlorate results in the discharge of I– from the thyroid in vivo, and this represents the basis for the perchlorate discharge test used to detect I– organification defects in patients (13).
TSH is the main regulator of thyroid function and is necessary to maintain the I–-concentrating capability of FRTL-5 cells. TSH regulates I– uptake through transcriptional and posttranscriptional mechanisms, including regulation of NIS mRNA expression and protein synthesis and subsequent targeting to and retention at the plasma membrane (17, 24). TSH withdrawal for 1 wk results in the loss of 125I– accumulation in FRTL-5 cells (33) and, in this study, the loss of the fluorescence response to 100 µM I–, a concentration that is selective for NIS-mediated transport.
The specificity of NIS-dependent changes in cellular YFP-H148Q/I152L fluorescence was further confirmed in COS-7 cells expressing recombinant hNIS. Cells cotransfected transiently with hNIS and YFP-H148Q/I152L cDNAs exhibited a perchlorate-sensitive decrease in fluorescence in response to 100 µM I–, whereas cells expressing only the fluorochrome did not. These results are in agreement with the demonstration of perchlorate-inhibitable 125I uptake in COS-7 cells expressing hNIS (26). Fluorescence responses in NIS-expressing COS-7 cells were smaller than in FRTL-5 cells, suggesting lower NIS activity.
A potential concern with the use of YFP-H148Q/I152L as an I– biosensor is its pH sensitivity, with acidification causing a reduction in fluorescence emission and an increase in I– sensitivity. Thyrocyte-derived YFP-H148Q/I152L in solution exhibited an apparent pKa of 7.02, similar to the pKa of 6.92 of purified bacterial protein (11). The I– sensitivity of thyrocyte-derived YFP-H148Q/I152L was also pH dependent, with a shift in affinity from 1.4 mM at pH 6.8 to 2.4 mM at pH 7.2. Suppression of YFP fluorescence by halides is due to protonation of the chromophore (31) and involves ground-state binding to a unique site close to the chromophore, where it alters the electrostatic environment producing a change in apparent pKa (15). It is therefore possible that drug treatments and other manipulations may alter the response of YFP-H148Q/I152L to I– by changing pHi and hence the apparent affinity for the halide. We estimated that a pH change of 0.2 units causes, at most, a 15% change in YFP-H148Q/I152L fluorescence intensity and in I–-induced fluorescence responses, suggesting that physiological variations in pHi have a small impact upon fluorescence responses of intact cells. Nevertheless, independent measurements of pHi are necessary to rule out pH as a factor in modulating the response of YFP-H148Q/I152L to I– when cells are exposed to drugs or other treatments. I– itself had no effect on pHi in FRTL-5 cells measured with BCECF, confirming that suppression of cellular YFP-H148Q/I152L fluorescence by extracellular I– is not due to intracellular acidification.
Advantages and Applications of New Method
Live cell imaging with YFP-H148Q/I152L enables rapid changes in intracellular I– concentration to be monitored. In FRTL-5 cells, exposure to extracellular I– causes a decrease in YFP-H148Q/I152L fluorescence within 10 s. Radiotracer uptake studies typically measure I– uptake over time periods ranging from a few minutes to over an hour (4, 18, 30, 33) and are inaccurate at shorter time intervals because of the need to remove extracellular radioactivity. The fluorescence assay is therefore particularly useful in detecting early time points of I– uptake that are difficult to measure with radiotracers.
Assays based upon cell imaging techniques allow measurements on single cells and permit functional differences between cells in a population to be assessed. In a clonal population of FRTL-5 cells, there was a 3.5-fold variation in the rate of I– uptake via NIS (measured as RF/t) and a 2.5-fold variation in intracellular I– concentration (measured as RF5min) at constant extracellular I– concentration. Variation was unrelated to resting fluorescence suggesting that YFP-H148Q/I152L expression itself does not influence transport kinetics. Although YFP-H148Q/I152L fluorescence and I–-induced changes in fluorescence are both influenced by pH, it is unlikely that physiological variations in pHi can account for the large differences among cells in resting fluorescence, RF/t and RF5min.
The ability to monitor I– accumulation in individual cells may be useful to characterize I– accumulation in primary cultures of human thyrocytes where the number of cells available for study is limited. Primary cultures represent a heterogeneous population of cellular phenotypes, and transient expression of YFP-H148Q/I152L may allow the characterization of NIS activity in a population of cells with a varied ability to accumulate I–. NIS expression in the thyroid gland in vivo is heterogeneous, with 20–30% of follicular cells in normal tissue exhibiting NIS immunoreactivity (3). The major obstacle to the application of the fluorescence assay to cells in primary culture is the difficulty of transfecting these cells. This may be overcome with virus-mediated gene delivery, with a 95% transduction efficiency reported for adenoviral transfer of the LacZ gene into normal human thyroid cells (21). Adenoviral delivery also permits thyroid-targeted gene expression in primary human thyrocytes using the thyroglobulin promoter and a Cre/loxP system. (20). Given that cell imaging allows I– concentration to be monitored only in YFP-H148Q/I152L-expressing cells, a high transduction efficiency is needed to ensure that fluorescent cells are representative of the entire population. Transient virus-mediated expression of YFP-H148Q/I152L in primary cultures derived from thyroid carcinomas may also benefit the characterization of the changes in NIS activity occurring in thyroid cancer.
NIS gene transfer is being increasingly considered as a means of rendering tumors sensitive to radioiodide therapy (27) and as a nuclear imaging reporter of gene expression, graft success, and stem cell migration (1, 2, 16). Cell imaging with YFP-H148Q/I152L may be a useful in vitro assay by which to assess the success of NIS gene transfer by providing an estimate of the proportion of cells with NIS activity. This is not possible with radiotracers, which measure mean I– uptake in a sample containing thousands of cells. Furthermore, the fluorescence assay may be useful in testing the ability of tissue-specific promoters to selectively induce NIS expression in specific cell types. Targeted NIS expression is necessary for cancer gene therapy to limit radioiodide ablation to malignant cells.
YFP-H148Q/I152L may be useful not only to study NIS-mediated I– uptake but also to characterize pathways of I– efflux. Steady-state fluorescence in the presence of extracellular I– reflects the balance of I– influx and efflux, and preventing further influx with perchlorate or by removing extracellular I– results in the complete recovery of resting fluorescence due to I– efflux. Several pathways may mediate I– efflux including pendrin (25), a TSH-stimulated Ca2+-dependent pathway (34), and a TSH/cAMP-activated Cl–/I– channel (38). In addition, thyrocytes express Cl– transporters such as CFTR (5) and ClC-5 (29) which may directly or indirectly affect I– efflux. Real-time detection of intracellular I– concentration permits the rates of I– influx and efflux to be measured within a single experiment (not possible with radiotracer studies) and facilitates the characterization of I– efflux pathways in cultured cells. The role of specific channels or transporters may be studied with pharmacological inhibitors or by modifying gene expression with molecular techniques such as RNA interference.
One of the major applications of fluorescence assays is their adaptability to a high-throughput platform for drug screening. Indeed, screening of small molecule libraries with a YFP-H148Q/I152L-based cellular assay has identified novel correctors of defective F508-CFTR cellular processing and channel gating that may be useful in the treatment of cystic fibrosis caused by the F508 mutation (22, 35). Compounds that enhance I– accumulation in the thyroid, by increasing the expression, plasma membrane targeting, and activity of NIS, may be useful in restoring the effectiveness of radioiodide therapy in patients with unresponsive thyroid cancer. NIS in extrathyroidal tumors is predominantly intracellular (32), and agents that promote surface localization may be useful in rendering these tumors sensitive to radioiodide imaging and ablation. Furthermore, compounds that inhibit I– efflux may be useful in promoting radioiodide retention by metastatic thyroid tumor cells and by nonthyroidal cells expressing NIS endogenously or following gene transfer. In these cells, the absence of a follicular structure and I– organification results in rapid loss of cellular radioactivity, which limits the effectiveness of radioiodide therapy. Identification of compounds that alter I–-induced fluorescence responses in a cell-based YFP-H148Q/I152L assay must be followed by secondary assays to eliminate false positives. Other assays include measurement of pHi to rule out pH-mediated changes in the I– sensitivity of YFP-H148Q/I152L fluorescence and measurement of YFP-H148Q/I152L fluorescence in solution to rule out a direct interaction of compounds with YFP-H148Q/I152L. Given these precautions, high-throughput screening with YFP-H148Q/I152L may be useful in identifying therapeutic compounds that increase cellular uptake and retention of radioiodide for the diagnosis and treatment of thyroidal and extra-thyroidal cancer.
In conclusion, YFP-H148Q/I152L is a promising biosensor of I– accumulation in cells expressing NIS endogenously or following gene transfer and represents a new tool with which to study NIS function and identify novel therapeutic compounds for the treatment of defective I– transport.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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