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Original Studies |
University College Dublin, St. Vincent’s University Hospital, Dublin 4, Ireland; and University of Sheffield, Sheffield S57AV, United Kingdom
Address all correspondence and requests for reprints to: Dr. P. P. A. Smyth, Endocrine Laboratory, Department of Medicine and Therapeutics, Woodview, University College Dublin, Dublin 4, Ireland. E-mail: PPA.SMYTH{at}UCD.IE
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A role for iodine in the prevention of breast dysplasia and hyperplasia has previously been described (10). More recently, it has been demonstrated that elemental iodine (I2) rather that iodide (I-) when administered together with the carcinogen dimethylbenzanthracene resulted in a significant reduction in the incidence and size of multiple mammary tumors that developed after carcinogenesis (11, 12). The latter group (12) reported that a higher tumor iodine content together with a significantly reduced tumor size were evident in rats treated with medroxyprogesterone acetate and I2 than in those treated with medroxyprogesterone acetate alone, suggesting that the active uptake of iodine had a suppressive effect on tumor growth. The objective of this study was to investigate NIS expression and the iodine content of human breast tissues of both benign and malignant pathologies. In addition, a modification of a method developed using a CHO-K1 cell line stably transfected with the human NIS (hNIS) (9) was applied to study the ability of serum obtained from patients with both breast cancer and benign breast disease to inhibit iodide uptake.
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Subjects and Methods |
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NIS expression was investigated in two fibroadenoma and seven breast carcinoma tissues. Tissue specimens were also obtained from patients undergoing subtotal thyroidectomy (n = 2) as well as specimens of skin, vein, and prostate, which served as positive and negative controls, respectively. Total RNA was extracted from frozen thyroid and breast cancer tissues using the Ultraspec II total RNA isolation kit (Biocytex Laboratories, Inc., Houston, TX) and was diluted to 1 µg/µL in ribonuclease-free water, following the determination of RNA concentration at 260 nm. RNA was reverse transcribed using oligo(deoxythymidine) (15-mer) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). One microgram of total RNA and 1 µg oligo(deoxythymidine) primers (15 mer) in a 5-µL reaction volume were heated to 65 C for 10 min, cooled on ice, and subsequently incubated for 1 h at 37 C with 15 µL of a pool containing 4 µL 5-fold concentrated RT buffer [250 mmol/L Tris-HCl (pH 8.3), 375 mmol/L KCl, and 15 mmol/L MgCl2], 10 mmol/L dithiothreitol, 1 µL RNAsin (40 U/µL), 1 µL deoxy-NTP (10 mmol/L), and 1 µL Moloney murine leukemia virus reverse transcriptase (200 U/µL). The quality of the cDNA was investigated by performing PCR amplification, under the conditions described below, with a pair of primers from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequence (sense, 5'-TCCATGACAACTTTGGCATCGTGG-3'; antisense, 5'-GTTGCTGTTGAAGTCACAGGAGAC-3') predicted to amplify a 380-bp fragment upstream from the polyadenylated tract of the cDNA. The primer pairs selected (sense, 5'-CTTCTGAACTCGGTCCTCAC-3'; antisense, 5'-CCATGCCTGAGAGTAACTACG-3') for the amplification of a 454-bp fragment of the human NIS were those previously described (5). Five microliters of cDNA from RT was used as template for PCR. The PCR reaction was carried out in 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 0.1% Triton X-100, 1.25 mmol/L MgCl2, 1.25 mmol/L deoxy-NTP, 0.25 µg/µL primers, and 0.05 U/µL Taq polymerase. The reaction profile consisted of 1 cycle at 94 C for 3 min, followed by 35 cycles at 94 C for 30 s, 56 C for 30 s, and 72 C for 2 min. After amplification, 20 µL of the reaction mix were separated on a 2% agarose gel, and the resulting bands were visualized under UV light after ethidium bromide staining (0.1 µg/µL). Molecular weights were compared to a 1-kb ladder. In all PCR experiments, a negative control, in which water replaced the cDNA template, was included.
Tissue iodine determination
Surgical specimens were collected on ice and homogenized in 0.05 mol/L KH2PO4, containing 10 M KOH. Homogenates were stored at -20 C until assay. Total tissue iodine concentration was determined in 23 benign tumors (fibroadenomata) and 19 breast carcinoma, including tumor tissue and specimens taken from a site remote from the tumor but within the tumor-bearing breast. Surgical specimens of thyroid tissue (n = 2) served as positive controls. The technique used was a modification (13) of the alkaline incineration method of Foss et al. (14). Homogenates were incinerated in a muffel furnace at 600 C for 180 min. The ash was dissolved in ddH2O and quantified spectrophotometrically at 490 nm using the Sandell-Kolthoff reaction. To account for differences in tissue cellularity, results were expressed as nanograms of I per mg protein.
Iodide uptake inhibition assay
A total of 273 sera were tested for iodide uptake inhibitory
activity, of whom 105 had histologically confirmed evidence of breast
carcinoma and 49 had benign fibroadenomata. Both breast study groups
were asymptomatic for thyroid disease. Sera were also obtained from 33
age-matched healthy female controls and 86 Graves’ disease patients.
Sera were tested for iodide uptake inhibitory activity, using a
modification of the method of Ajjan et al. (9). This assay
used a CHO-K1 cell line stably transfected with the hNIS (designated
CHO-NIS 9). Cells were first incubated with test serum. After an
initial 1-h incubation, an additional 500 µL Ham’s F-12 medium
containing 3–4 kilobecquerels 125I were added,
and the cells were incubated for another 30 min. After washing to
remove exogenous 125I, the cells were solubilized
using 1.0 mL 1 mol/L NaOH, and radioactivity was counted using a
-counter for 1 min to determine the degree of
125I incorporated. Results were expressed as a
percentage of inhibition of iodide uptake. The upper limit of the
reference range (30% inhibition of 125I uptake)
was determined on the basis of the mean value for 33 control sera
± 3 SD. To investigate whether
125I uptake inhibition was IgG mediated, sera
from patients with breast disease (n = 4), Graves disease (n
= 6), and normal controls (n = 4) were run through a protein
G-Sepharose column with 10 mmol/L phosphate buffer at pH 7.0. These
patients were selected on the basis of positive or negative serum
125I uptake inhibitory activity. Bound IgG was
eluted with 100 mmol/L glycine-HCl at pH 2.8 and extensively dialyzed
against PBS before use in the assay.
Serum TPO antibody (TPO.Ab) determination
Sera from 93 patients with breast cancer and 42 patients with benign breast disease in which 125I uptake inhibition had been studied were also assessed for serum TPO.Ab titers. Serum TPO.Ab were measured using a highly sensitive direct RIA (RSR, Cardiff, UK). TPO.Ab titers were recorded as units per mL Medical Research Council Standard 65/93. The upper limit of the reference range was 0.3 U/mL (15).
Statistical analysis
Data were analyzed using Mann-Whitney U and
2 tests.
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Results |
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Figure 1
shows the results of NIS
RT-PCR analysis after separation on a 2% agarose gel. The upper gel
shows GAPDH mRNA amplified from the 14 tissues. The lower gel shows
that an amplified fragment of 454 bp sized against a 1-kb ladder
corresponding to NIS was observed in 6 of 7 breast cancer specimens
(lanes 2–8). Similar bands were observed in 2 fibroadenomata (lanes 9
and 10) as well as in an extract prepared from thyroid tissue (lane
14), which served as a positive control. In contrast, no bands
corresponding to the NIS were seen in vein, prostate, and skin tissue
samples (lanes 11–13) or from a single breast carcinoma tissue sample
(lane 5).
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Results of tissue iodine measurements in breast and thyroid tissue
homogenates are shown in scattergraph form in Fig. 2. There was a wide distribution of
individual values, but levels in 23 fibroadenomata (median, 69 ng I/mg
protein; range, 21–173) were significantly greater than those in 19
breast carcinoma (median, 22 ng I/mg protein; range, 10–78) or remote
tissue specimens (median, 15 ng I/mg protein; range, 12–81;
P < 0.01 in each case). Tissue iodine concentrations
in breast carcinoma were also significantly lower than those in the
remote breast group (P < 0.05). All breast tissue
iodine concentrations were an order of magnitude lower than those in
two thyroid specimens, which had iodine concentrations of 704 and 850
ng I/mg protein, respectively.
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The distribution of individual values as well as the percent
prevalence of 125I uptake inhibition (>30%) in
patient and control sera are shown in scattergraph form (Fig. 3). Thirty-three normal sera were tested
for iodide uptake inhibitory activity (median, 17.4%; range,
1.2–38.0%). Of the 105 breast carcinoma sera tested, 20 (19.0%)
exhibited significant inhibitory activity (median, 19.0%; range,
4.7–53.5%). In sera from patients with benign breast disease, 8 of 49
(16.3%) showed more than 30% inhibition of iodide uptake (median,
20.0%; range, 5.1–46.3%). These prevalences were not significantly
different from each other or from those observed in sera obtained from
Graves’ patients of whom 27 of 86 (31.4%) showed positive inhibitory
activity (median, 22.7%; range, 1.7–55.1%). Differences in the
frequency of iodide uptake inhibition in the breast carcinoma and
Graves’ disease patient groups were significantly greater than those
in controls, of whom only 1 of 33 (3.0%) was positive
(P < 0.05). In contrast, differences in the frequency
of 125I uptake inhibition between sera from
patients with benign breast disease and healthy controls failed to
reach statistical significance.
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Figure 4 shows results of
125I uptake inhibition produced by purified IgG
(1 mg/mL) prepared in sera from patients with breast disease, Graves’
disease, and normal controls. IgGs from three patients with breast
disease previously shown to have significant serum
125I uptake inhibitory activity (36.4–45.7%)
uptake also inhibited iodide uptake by 7.3–18.2%. IgGs prepared from
six patients with Graves’ disease showed inhibition of
125I uptake ranging from 8.0–37.5%. In
contrast, purified IgG prepared from a single breast disease patient
and four controls who were negative for 125I
uptake inhibition did not show significant IgG-mediated
125I uptake inhibition (range, -2.6% to
2.1%).
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Table 1 shows a significant
association between TPO.Ab positivity and 125I
uptake inhibition in sera from 93 breast cancer patients and 42 with
benign breast disease. Of the breast cancer patients, 31 were TPO.Ab
positive, and 62 were antibody negative. Significant inhibition of
125I uptake was observed in 11 of 31 sera
positive for TPO.Ab compared to 6 of 62 TPO.Ab-negative sera
(P < 0.01). No significant association was observed
between 125I uptake inhibition and TPO.Ab
positivity in sera from patients with benign breast disease; 2 of 11
sera positive for TPO.Ab showed significant inhibition of
125I uptake compared to 5 of 31 TPO.Ab-negative
sera.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Studies were focused on the hNIS, the extrathyroidal expression of which has recently been demonstrated (6, 7) using RT-PCR and Southern hybridization in tissues including normal human mammary glands. In the present study expression of the NIS was found to be a feature of both fibroadenomata and breast carcinoma tissues, which suggests that variations in tissue iodine content according to breast pathology may reflect differences in the functional capacity of the hNIS to take up iodide.
The concept of a diminished iodide uptake by breast cancer tissue is supported by the presence of significant iodide uptake inhibitory activity in serum from breast carcinoma patients (19.6%) compared to that in a healthy control population (3.0%). Such inhibitory activity was present in 31.4% of 86 Graves’ patients, a prevalence identical to that previously reported using a similar assay methodology (9), but differing from an earlier report demonstrating that approximately 15% and 84% of Hashimoto’s thyroiditis and Graves’ disease sera, respectively, bound to recombinant rat NIS in Western blotting experiments (20). However, the significance of the presence of NIS antibodies in patients with Graves’ disease remains a matter of conjecture. It has been suggested that such antibodies may partially counter the effect of TSAb in Graves’ disease and thus ameliorate the severity of hyperthyroidism (9). Although no such explanation is pertinent in breast cancer, a possible explanation for diminished I- uptake by CHO-NIS 9 cells would be excessive I- content of patient sera. Although iodine excretion was not measured in the individual patients studied, a previous report from this laboratory (21) found no difference in urinary iodine excretion between patients with breast disease and age-matched controls. There is no reason to suspect that patients in the current study cohort had any greater exposure to iodine. Another potential contributor to observed anomalies in 125I uptake is NIS structure. The CHO-NIS 9 cell line expresses a truncated symporter (amino acids 1–612) and therefore may not be the best tool to detect blocking antibodies. However, in preliminary experiments we have performed transient transfection of COS-7 cells with full-length symporter and found complete correspondence between inhibition assays with full-length and truncated symporter in 49 Graves’ disease sera. In particular, no additional sera were positive with the full-length symporter, indicating that CHO-NIS 9 cells are satisfactory for measurement of inhibitory antibodies.
The finding that 125I uptake inhibition, when present in sera from patients with breast disease, resided in the IgG fraction is consistent with the view that such inhibition was immunologically mediated. This is supported by studies using purified IgGs from patients with Graves’ disease, which suggested that NIS inhibitory activity was antibody related. The possibility of an autoimmune-mediated mechanism being responsible for inhibition of iodide uptake in the thyroid has been previously reported (8, 20, 22). This is further supported by the demonstration in the present study of a significant association between TPO.Ab and 125I uptake inhibition positivity in the breast cancer patients tested. Previous studies from our own (23) and other (24, 25) groups have demonstrated an increased prevalence of circulating thyroid antibodies in breast cancer, thus emphasizing a possible link with thyroid autoimmunity. Whether this is a consequence of the disease or part of its pathogenesis is unclear. As tissue iodine content, NIS mRNA, and 125I uptake inhibition analysis were not carried out on the same individual patients, any hypothesis based on such disparate observations must be speculative. However, we postulate that the low iodine content observed in some breast cancers may arise from the presence in patients’ serum of NIS blocking 125I uptake inhibitory activity, perhaps of immunogenic origin. This postulate is more difficult to sustain in the case of benign breast disease, in which higher tissue iodine content compared to either breast cancer or remote tissue was observed despite the finding of iodide uptake blocking activity in 16.5% of the subjects tested. This may reflect the heterogeneity of fibroadenomata (and, hence, NIS expression) or the likelihood that iodide uptake blockade is only one of many mechanisms influencing iodide uptake by the breast. Although, as previously stated, a beneficial role for elemental I2 in experimental tumorigenesis has been demonstrated in the rat (12, 19), the involvement of iodine in the natural history of breast carcinoma remains to be explored.
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Acknowledgments |
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Footnotes |
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Received April 27, 1999.
Revised July 27, 1999.
Revised October 7, 1999.
Accepted November 19, 1999.
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References |
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