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Experimental Biology and Medicine 228:1078-1082 (2003)
© 2003 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Pendrin Transporter Carries Out Iodide Uptake into MCF-7 Human Mammary Cancer Cells

James A. Rillema1 and Melissa A. Hill

Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201–1928


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that iodide is actively taken up into mammary alveolar epithelial cells and secreted into milk. In the present studies we demonstrate that 125I also accumulates in MCF-7 cells against a concentration gradient; distribution ratios of greater than 30 were achieved. Iodide uptake into MCF-7 cells is transient, with peak accumulations occurring in about 5 min. The iodide is rapidly metabolized, probably to iodine, and it then exits the cells. The iodide transporter identified in MCF-7 cells is pendrin. DIDS, a nonspecific inhibitor of anion exchange, inhibits iodide uptake. Iodide uptake is impaired at reduced temperature, but is not dependent on sodium. Inhibitors of the sodium-iodide symporter (NIS) as well as ouabain did not affect the extent of iodide uptake. The pendrin transporter but not NIS was identified via western blotting techniques. Pendrin appears to be the primary iodide transporter in the MCF-7 cell line stocks that were employed for these studies.

Key Words: mammary • MCF-7 cells • iodide transport • pendrin


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The levels of iodide in the milk of a variety of species are 10- to 30-fold higher than those present in the maternal plasma (1–5). The iodide in milk is of critical importance for the growth and development of the neonate, particularly because iodide is essential for the synthesis of the thyroid hormones. We as well as several other investigators have identified and characterized a sodium-iodide symporter (NIS) that actively transports iodide from the maternal plasma into the alveolar epithelial cells of the mammary gland (6–12). This transporter has also been reported to be expressed in MCF-7 breast cancer cells; retinoic acid stimulated the expression of NIS (13). More recently, Shennan (14), as well as our laboratory (15), have identified a DIDS-sensitive anion exchange transporter in the mammary gland; this transporter accepts iodide as a substrate and was identified via western blotting techniques as pendrin in thyroid cells (16–18). The present studies were designed to assess the possible contribution of the pendrin iodide transporter in human MCF-7 mammary cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials used in these studies were from the following sources: penicillin and streptomycin from Eli Lilly (Indianapolis, IN); 3HOH from New England Nuclear Inc. (Boston, MA); 125I (423 mCi/mMol) from Amersham Life Science Inc. (Arlington Heights, IL); all other culture supplies and chemicals were from Sigma-Aldrich, Inc. (St. Louis, MO). Stocks of the MCF-7 human mammary epithelial cell line were obtained from the Michigan Cancer Foundation (now Karmanos Cancer Institute, Detroit, MI) and maintained in continuous culture; passages 30 to 195 were employed in these studies. Pendrin antibody initially was a gift from Dr. Eric A. Green, NIH, Bethesda, MD; most experiments were carried out with rabbit antipeptide antibodies, raised against human pendrin sequence 630-643 (PTKEIEIQUDWNSE; GenBank AF 167412) as specified by Royaux et al. (17); the antibody was prepared by Zymed Laboratories, Inc., South San Francisco, CA. The human NIS antibody was provided by Dr. Sissy M. Jhiang of the Ohio State University.

The MCF-7 cells were cultured (19) in Eagle’s minimal essential medium (EMEM) supplemented with 2x nonessential amino acids, L-glutamine (292 mg/l), 10% newborn bovine serum (inactivated), penicillin (100 U/ml), streptomycin (100 µg/ml), and insulin (10-6 M). Cultures in log growth were harvested with 0.25% trypsin in phosphate-buffered saline (pH = 7.4). The cells were seeded at a density of 0.5-1 x 106/60 mm plate in supplemented Eagle’s minimal essential medium (Earle’s salts) and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air. The medium was aspirated and replaced on day 2. On day 4, the medium was removed and the cells washed with 5 ml/plate Hanks’ balanced salts solution; at this time the cells were at about 50% confluence. The cells were then cultured for specified times 0°C or 37°C with 5 ml HBSS containing 1 µCi/ml 125I and drugs where specified. The cells were then rinsed with 5 ml HBSS and homogenized in 3 ml 5% trichloroacetic acid (TCA). After centrifugation for 10 min at 2,000 g, the radioactivity in 1 ml aliquots of the supernatants was determined by liquid scintillation. 125I in the TCA-pellet fraction was determined after solubilization of the pellets in 2 ml 1N NaOH; only trace amounts of 125I were found in the pellet fraction indicating a limited incorporation into cellular protein. In each experiment, intracellular water was quantitated by equilibrating 3 plates of cells at 37°C for 1 hr with 3HOH (1 µCi/ml) contained in 5 ml HBSS; in preliminary studies the 3HOH was found to equilibrate within 10 min of incubation. After the incubation with 3HOH, the cells were rinsed with HBSS and then homogenized in 3 ml 5% TCA. Afterwards centrifugation radioactive [3H] in the TCA supernatant was determined and water content of the cells calculated. Using these data, the results of the iodide uptake studies are expressed as a distribution ratio, which represents the ratio of the intracellular specific activity divided by the extracellular specific activity of the radioactive iodide.

For western blotting studies with the pendrin antibody, plates of MCF-7 cells were homogenized in 1 ml lysis buffer in a ground glass homogenizer; the lysis buffer contained 2% NP40, 10 mM Tris, 50 mM NaCl, 30 mM sodium pyrophosphate, 2.5 mM EDTA, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, at pH 7.6. After 30 min on a rocking platform, lysates were centrifuged (100,000 g) for 30 min at 4°C. After protein determination by the method of Bradford, the resulting supernatants, containing greater than 95% of extractable protein, were separated by SDS-PAGE (8%–20% linear gradient) under reducing conditions and transferred to polyvinylidine fluoride (PVDF) membranes (Schleicher and Schuell). Membranes were probed with 1:2,500 human anti-pendrin for 2 hrs, followed by treatment with anti-rabbit IgG HRP conjugate (Amersham NA934; 25 ml at 1:3,000 dilution for 1.5 hrs). Detection was accomplished by incubation with enhanced chemiluminescence reagents (Amersham) and exposure to photographic film. Statistical comparisons were made with Student’s t test for comparing two means, or with an analysis of variance followed by Scheffe’s test for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go shows the time course for 125I accumulation in MCF-7 cells. Peak uptakes were observed after culture periods between 2.5 and 15 mins; thereafter the 125I progressively exited the cells. Peak distribution ratios of greater than 30 were achieved during the early minutes of uptake determinations, indicating a concentrating mechanism for iodide accumulation. Radioactivity in the culture media remained constant during the time course studies (data not included). Although significant amounts of 125I were not incorporated into cellular proteins (in the TCA-precipitable fraction), 125I incorporation into serum proteins occurred when 125I uptake was determined with the 125I in serum-containing medium rather than in HBSS; the conversion of radioactive iodide to iodine, which then effluxes from the cells is thus suggested (data not included). Figure 2Go shows the effect of iodide concentration on 125I accumulation after a 10-min uptake of 125I in MCF-7 cells. The distribution ratio decreases progressively as the iodide concentration increases, but distribution ratios of greater than 25 were still achieved with iodide at a concentration of 25 mM. The 125I concentrating mechanism in MCF-7 cells can thus handle a very high concentration of iodide. The temperature dependence of the iodide uptake mechanism in the MCF-7 cells is presented in Figure 3Go. When uptake of 125I was determined with the cells at 0°C, it took 30 to 60 mins to achieve a maximum cellular accumulation, and an efflux from the cells was only observed after 60 mins (data not presented).



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  		Figure 1. Time-course for 125I uptake in MCF-7 cells. Cells were cultured for the times indicated with 1 µCi/ml 125I. Intracellular 125I was then determined. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 


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  		Figure 2. Effect of iodide concentration on 125I uptake in MCF-7 cells. Cells were cultured for 10 mins with 125I (1 µCi/ml) with iodide at the concentrations indicated. Intracellular 125I was then determined. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 


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  		Figure 3. Effect of temperature on 125I uptake in MCF-7 cells. Cells were cultured for 0 to 60 min at 0°C or 37°C with 1 µCi/ml 125I. Intracellular 125I was then determined. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 
The experiment shown in Figure 4Go was carried out to assess the reason for the efflux of 125I from MCF-7 cells employing culture times of greater than 15 minutes at 37°C. Control uptakes at 10 and 60 mins are shown in the first two bars respectively. The uptake in the cells of bar 3 was assessed after a 1-hr incubation with 125I followed by a 10-min incubation with fresh 125I; these data show that the cells exposed to 125I for 1 hr are as capable of accumulating 125I during a 10-min incubation as are the cells in bar one. The fourth bar contains fresh MCF-7 cells that were cultured for 10 min with the "spent" media of the cells in bar 2 that were cultured with 125I for 1 hr. The fact that the fresh cells in bar 4 did not accumulate 125I from the "spent" media of bar 2 indicates that the 125I in this media has been chemically altered, probably to iodine via an oxidation-reduction reaction.



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  		Figure 4. Metabolism of 125I in MCF-7 cells. 125I uptake was assessed for 10 mins (bar 1) or 1 hr (bar 2) as in Figure 1Go. In bar 3, the cells were incubated for 1 hr with 125I as in Figure 1Go, and then an additional 10 mins with fresh media containing 1 µCi/ml 125I. In bar 4, fresh MCF-7 cells were incubated for 10 mins with the "spent" media from the cells in bar 2. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 
To determine the mechanism by which 125I is taken up into MCF-7 cells the following experiments were carried out. Figure 5Go shows an experiment in which iodide uptake was assessed in the absence of sodium. Clearly, after a 10 or 60 min incubation with 125I, the absence of sodium had no effect on the distribution ratio, indicating that the 125I uptake is not sodium dependent. The data in Figure 6Go show that 3 inhibitors of the NIS, as well as an inhibitor of sodium-potassium ATPase (ouabain), had no effect on 125I accumulation during a 10-min uptake period. Even in experiments where we precultured tissues for 1 hr with these drugs prior to the 10-min 125I uptake determination in the presence of these drugs, inhibitory effects were not observed (data not included). In the MCF-7 cells that we employed for these studies, the NIS transporter appears to be of little importance.



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  		Figure 5. Effect of sodium on 125I accumulation in MCF-7 cells. Cells were cultured for 10 mins or 60 mins with 1 µCi/ml 125I contained in EMEM, KRB, or sodium-free KRB. Intracellular 125I was then determined. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 


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  		Figure 6. Effect of NIS inhibitors and ouabain on 125I uptake in MCF-7 cells. Cells were incubated for 10 mins with 1 µCi/ml 125I plus the inhibitors specified in the figure. Intracellular 125I was then determined. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 
In western blotting studies, the only iodide transporter that we could identify was the pendrin iodide transporter with a molecular weight of about 90 kDa (Fig. 7Go). Using an NIS antibody that we used in earlier studies to identify the NIS transporter in mouse mammary tissues (8), we could not identify the NIS transporter in extracts of the MCF-7 cells. In further studies we determined the dose-response effect of an anion exchange inhibitor, DIDS, on 125I accumulation in MCF-7 cells; DIDS has earlier been shown to inhibit the pendrin iodide transporter. Figure 8Go shows that DIDS inhibited iodide accumulation in a concentration-dependent fashion.



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  		Figure 7. Western blot of pendrin in proteins from MCF-7 cells. Proteins from MCF-7 cells were subjected to western blot analysis as described in the Materials and Methods section. Lanes 1 and 2 (15 µg protein); lanes 3 and 4 (45 µg protein). The band appeared in the gels indicating a MW of about 91 kDa.

 


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  		Figure 8. Dose-response effect of DIDS on 125I uptake in MCF-7 cells. Cells were cultured for 10 mins with 1 µCi/ml 125I plus DIDS at the concentrations indicated in the figure. Intracellular 125I was then determined. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 
Figure 9Go shows the data from a time course experiment in which RNA and protein synthesis were inhibited by actinomycin D and cycloheximide respectively. The turnover of the transporter mechanism, probably the pendrin protein, appears to be quite slow since even after 8 hrs in the presence of cycloheximide there is only about a 25% reduction in 125I accumulation.



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  		Figure 9. Effect of actinomycin D and cycloheximide on 125I uptake in MCF-7 cells. Cells were cultured for the times indicated with 2 µg/ml actinomycin D or 5 µg/ml cycloheximide. Intracellular 125I wsa then determined. Numbers represent the mean ± SE of distribution ratios from 4 plates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies clearly show that the pendrin iodide transporter is expressed in human MCF-7 mammary cancer cells. The pendrin transporter was identified via western blotting analysis as a 90 kDa protein. In addition, DIDS, which is known to inhibit the pendrin transporter as well as other anion exchange transporters (14–18), inhibited iodide uptake in the MCF-7 cells. Iodide uptake was temperature dependent, but sodium independent; these are additional characteristics of the pendrin transporter. Since the MCF-7 cell line was originally developed at the Michigan Cancer Foundation, which is affiliated with our institution, more than 5 stocks of the MCF-7 cells were employed in these studies; each of these took up iodide in a similar fashion. In further studies, we were unable to identify the NIS transporter in proteins from the MCF-7 cells. Via western blotting techniques employing a human NIS antibody that we had used in earlier studies (8), the NIS protein was not identified. Nor did several NIS transporter inhibitors impair iodide uptake. Further, the NIS transporter is sodium dependent, but iodide uptake was not impaired in the absence of sodium. In view of the results of Kogai et al. (13) in which the NIS transporter, but not the pendrin transporter, was clearly identified and characterized in MCF-7 cells, it is quite apparent that there may be variants of the MCF-7 cell line that express different iodide transporters or the specific culturing conditions employed may allow the expression of the genes for the NIS or pendrin transporters. Kogai et al. further reported a retinoic acid stimulation of iodide uptake and NIS expression in their studies.

125I uptake in the MCF-7 cells is transient with a peak accumulation occurring between 2.5 and 15 mins. The transient nature of the 125I accumulation is likely due to the conversion of the iodide to iodine, as cells in culture are known to have high levels of peroxidase activity. The efflux of 125I-iodine would then explain the efflux of radioactivity from the cells during extended culture times. The total radioactivity in the culture media was not altered in the time course studies, but when the "spent" media was added to fresh cells, 125I uptake was impaired. All the data therefore seem to indicate that iodide is rapidly converted to iodine in the MCF-7 cells. The fact that iodide-uptake distribution ratios of greater than 50 were observed in the MCF-7 cells is suggestive of an active uptake mechanism via the pendrin transporter. However, in view of the rapid metabolism of 125I, after being taken up into the MCF-7 cells, it is not possible to conclude that iodide is transported into the cells against a concentration gradient.


   Footnotes
 
This work was supported by funds from the Children’s Hospital of Michigan and NIH Grant RR08167.

1 To whom requests for reprints should be addressed at Department of Physiology, Wayne State University School of Medicine, Room 4251, Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201–1928. E-mail: jrillema{at}med.wayne.edu Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brown-Grant K. Extrathyroidal iodide concentrating mechanisms. Physiol Rev 41:189–213, 1961.[Free Full Text]
  2. Brown-Grant K. Iodide concentrating mechanisms of the mammary gland. J Physiol 135:644–654, 1957.
  3. Honour AJ, Myant NB, Rowlands EN. Secretion of radioiodine in digestive juices and milk in man. Clin Sci 11:447–462, 1952.
  4. Van Middlesworth L. Iodide metabolism of mammary gland. J Clin Endocrinol 16:989, 1956.
  5. Wright WE, Christain JE, Andrews FN. Mammary elimination of radioiodine. J Dairy Sci 38:131–136, 1955.[Abstract/Free Full Text]
  6. Rillema JA, Rowady DL. Characteristics of the prolactin stimulation of iodide uptake into mouse mammary gland explants. Proc Soc Exp Biol Med 215:366–369, 1997.[Medline]
  7. Rillema JA, Yu TX. Prolactin stimulation of iodide uptake into mouse mammary gland explants. Am J Physiol 271:E879–E882, 1996.
  8. Rillema JA, Yu TX, Jhiang SM. Effect of prolactin on sodium iodide symporter expression in mouse mammary gland. Am J Physiol 279:E769–E772, 2000.
  9. Dai G, Levy O, Carrasco N. Cloning and characterization of the thyroid iodide transporter. Nature 379:458–460, 1996.[Medline]
  10. Spitzweg C, Joba W, Eisenmenger W, Heufelder AE. Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complimentary deoxyribonucleic acids from salivary gland, mammary gland and gastric mucosa. J Clin Endocrinol Metab 83:1746–1751, 1998.[Abstract/Free Full Text]
  11. Cho J-Y, Leveille R, Kao R, Rousset B, Parlow AF, Burak WE Jr, Mazzaferri EL, Jhiang SM. Hormonal regulation of radioiodide uptake activity and Na+-I- symporter expression in mammary glands. Endocrinol 85:2936–2943, 2000.
  12. Tazebay UH, Wapnir IL, Levy O, Dohan O, Zuckier LS, Zhoa QH, Deng HF, Ameta PS, Fineburg S, Pestell RG, Carrasco N. The mammary gland iodide transporter is expressed during lactation and in breast cancer. Nat Med 6:871–878, 2000.[Medline]
  13. Kogai T, Schultz JJ, Johnson LS, Huang M, Brent GA. Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line. Proc Natl Acad Sci 97:8519–8524, 2000.[Abstract/Free Full Text]
  14. Shennan DB. Iodide transport in lactating rat mammary tissue via a pathway independent from the Na+-I- cotransporter: evidence for sulfate/iodide exchange. Biochem Biophys Res Commun 280:1359–1363, 2001.[Medline]
  15. Rillema JA, Hill MA. Prolactin regulation of the pendrin iodide transporter in the mammary gland. Am J Physiol 284:E25–E28, 2003.
  16. Scott DA, Wang R, Kreman TM, Sheffield VC, Karnisk LP. The Pendred syndrome encodes a chloride-iodide transport protein. Nat Genet 21:440–443, 1999.[Medline]
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  18. Fugazzola L, Cerutti N, Mannavola D, Vannucchi G, Beck-Peccoz P. The role of pendrin in iodide regulation. Exp Clin Endocrinol Diabetes 109:18–22, 2001.[Medline]
  19. Linebaugh BE, Rillema JA. Actions of insulin on MCF-7 cells that are synchronized with hydroxyurea. Mol Cell Endocrinol 52:227–232, 1987.[Medline]
Received for publication April 15, 2003. Accepted for publication June 16, 2003.




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