* Department of Pathology School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202; Department of Surgery, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58202
1 To whom correspondence should be addressed at Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, 501 N. Columbia Road, Grand Forks, ND 582029037. Fax: 7017773108. E-mail: msens{at}medicine.nodak.edu.
Received January 19, 2005; accepted March 17, 2005
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ABSTRACT |
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Key Words: metallothionein; breast cancer; cadmium; MCF-10A; ductal epithelium; myoepithelium; microdissection.
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INTRODUCTION |
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The metallothioneins (MT) are a family of low molecular weight (67 kDa), cysteine-rich proteins that are believed to play an important role in the homeostasis of essential metals such as Zn+2 and Cu+2. The MT-1 and MT-2 family members have been extensively studied and are widely recognized for their ability to attenuate Cd+2-induced effects through binding and sequestering of the metal within the cell (Andrews, 2000; Cherian et al., 1994
; Hamer, 1986
; Kägi, 1993
). The MT-1 and MT-2 isoforms exhibit a ubiquitous pattern of tissue expression upon metal challenge and are highly inducible by a large number of stimuli (Andrews, 2000
; Hamer, 1986
; Kägi, 1993
; Kägi and Hunziker, 1989
). The expression of the MT-1 and MT-2 proteins has been routinely visualized immunohistochemically using antibodies raised against the E9 epitope, which is conserved in both the MT-1 and MT-2 isoform proteins (Jasani and Schmid, 1997
). Studies employing this antibody have shown that only the outer myoepithelial cells of the bilayered ductales/acini are immunoreactive for the MT-1/2 protein, with very strong staining localized to both the nucleus and the cytoplasm (Bier et al., 1994
; Fresno et al., 1993
; Jin et al., 2001
). Only very rarely was an immunoreactive cell profile found in the epithelial cells lining the large ducts. In contrast, a significant number of ductal breast cancers exhibit MT-1/2 immunoreactivity within the malignant ductal epithelium (Bier et al., 1994
; Douglas-Jones et al., 1995
; Fresno et al., 1993
; Goulding et al., 1995
; Haerslev et al., 1994
; Ioachim et al., 1999
; Oyama et al., 1996
; Schmid et al., 1993
).
These observations, in conjunction with the findings that Cd+2 acts as an estrogen mimic, define a need to further examine MT-1/2 expression in the normal and malignant breast epithelial cell. To date, the expression of the MT-1 and MT-2 isoformspecific mRNAs have not been determined in the normal human breast epithelial cell. Likewise, no cell culture model of the normal human breast epithelial cell has been shown to retain the in vivo expression patterns of MT-1 and MT-2 mRNA and protein. The goals of the present study were to determine the expression of the MT-1 and MT-2 isoformspecific mRNAs in the ductal epithelium of the human breast; to determine if the MCF-10A cell line recapitulates the pattern of MT-1 and MT-2 expression found in the human breast duct epithelial cell; and to determine the effect on MT-1 and MT-2 expression when the MCF-10A cell line is exposed to Cd+2.
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MATERIALS AND METHODS |
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Immunohistochemical localization of MT-1/2.
The specimens of normal human breast obtained from the pathology archives were routinely fixed in neutral buffered formalin for 1618 h. The tissue was transferred to 70% ethanol and dehydrated in 100% ethanol. The dehydrated tissue was cleared in xylene, infiltrated, and embedded in paraffin. Serial sections were cut at 35 µm for use in immunohistochemical protocols. Prior to immunostaining, sections were pretreated in a microwave at 700W in 10 mM citrate buffer (pH 6.0) for 5 min. Sections were allowed to cool for 5 min at room temperature, microwaved again for 5 min, and slowly immersed into distilled water. Metallothionein was localized using a monoclonal mouse anti-horse MT-1, MT-2 antibody (DAKO-MT, E9, in 0.05M Tris/HCl, 15 mM NaN3, pH 7.5, 1% BSA, DAKO Corporation, Carpinteria, CA) as the primary antibody. The primary anti-MT antibody was localized using the avidin-biotin-peroxidase complex (ABC) procedure (Vectastain Kit, Vector Laboratories, Burlingame, CA) with diaminobenzidine used for visualization (Stable DAB, Research Genetics, Huntsville, AL). Slides were rinsed in distilled water, dehydrated in graded ethanols, cleared in xylene, and coverslipped. The positive control was the demonstration of MT staining localized to the proximal tubule in human archival kidney specimens. The negative control consisted of replacement of primary antibody from the immunohistochemical ABC sequence with non-immune serum.
RNA isolation and RT-PCR from fresh tissues and cell lines.
Breast tissue was ground to a powder under liquid nitrogen. The MCF-10A cell line was grown in plastic flasks and plates. Total RNA was isolated from the cultured cells and powdered tissue according to the protocol supplied with TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH) as described previously (Garrett et al., 2000). The concentration and purity of the RNA samples were determined using spectrophotometer scan in the ultraviolet (UV) region and ethidium bromide (EtBr) visualization of intact 18S and 28S RNA bands after agarose gel electrophoresis. Total RNA (0.5 µg) was reverse transcribed (RT) with MuLV reverse transcriptase (50 U) in 10x polymerase chain reaction (PCR) buffer (500 mM KCl and 100 mM Tris-HCl, pH 8.3), 5 mM MgCl2, 20 U RNase inhibitor, 1 mM each of the dNTPs, and 2.5 µM random hexanucleotide primers. The samples underwent RT for 20 min at 42°C, followed by a 5-min denaturation step at 99°C in a DNA thermocycler (Gene Amp 9700, Applied Biosystems, Foster City, CA). The RT product was used for PCR amplification with the AmpliTaq DNA polymerase enzyme (2.5 U) and the specific upstream and downstream primers at a concentration of 0.1 µM each. The primers developed for analysis of each of the active MT genes have been previously described (Garrett et al., 1998a
, 1998b
). The thermocycler was programmed to cycle at 95°C for a 2-min initial step, at 95°C for 30 s, and at 68°C for 30 s. Controls for each PCR included a no-template control where, water was added instead of the RNA, and a no-RT control, where water was added instead of the enzyme. Samples were removed at 25, 30, 35, and 40 PCR cycles to ensure that the reaction remained in the linear region. The final PCR products were electrophoresed on 2% agarose gels containing EtBr along with DNA markers. The intensity (integrated optical density, IOD) of the PCR product bands was determined on a Dell workstation configured with Kontron KS 400 image-analysis software.
Microdissection of tissue from paraffin blocks, RNA isolation, and real time RT-PCR.
The procedures for laser capture microdissection of tissue from paraffin blocks and micro RNA isolation has been described previously (Garrett et al., 2000). Briefly, 5-µm-thick sections were cut from formalin-fixed, paraffin-embedded tissue blocks with a microtome and mounted on plain glass slides. After microdissection, total RNA was extracted from samples with the micro RNA isolation kit (Stratagene, Catalog No: 200344). The expression of the MT-1X, MT-1E, MT-2A, and ß-actin mRNAs were determined using real-time PCR. Briefly, one-half of the total RNA obtained from the microdissection was reverse transcribed in a 20-µl reaction by the iScript cDNA synthesis kit (Bio-Rad Laboratories Hercules, CA). The reaction was incubated for 5 min at 25°C, followed by 30 min at 42°C and 5 min at 85°C. Real-time PCR was performed with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) utilizing the iQ SYBR Green Supermix kit (Bio-Rad Laboratories). Amplification was monitored by SYBR Green fluorescence and compared to that of a standard curve of the PCR product cloned into pcDNA3.1/hygro (+) and linearized with Fsp I. The PCR product was verified by sequencing. The reaction conditions consisted of 2 µl of cDNA and 0.2 µM primers in a final volume of 20 µl of supermix, and the cycling parameters consisted of annealing and extension at 65°C for 45 s and denaturation at 95°C for 30 s. The primers for MT-1X, MT-1E, and ß-actin were as described above, and those for the MT-2A gene product were upper primer GCGTGCAACCTGTCCCGACTC and lower primer TGGGATCCATGGCGTGCT; yielding a product size of 47 base pairs.
Cell culture.
The MCF-10A cell line was obtained from the American Type Culture Collection and grown in a 1:1 mixture of Ham's F-12 medium and Dulbecco's minimum essential medium (DMEM) supplemented with 5% (v/v) fetal calf serum, 10 µg/ml insulin, 0.5 µg/ml hydrocortisone, 20 ng/ml epidermal growth factor, and 0.1 µg/ml cholera toxin. The cells were fed fresh growth medium every 3 days, and at confluence (normally 612 days post-subculture), the cells were subcultured at a 1:10 ratio using trypsinethylenediamine tetraacetic acid (EDTA; 0.25%, 1 mM). For use in experimental protocols, cells were subcultured at a 1:10 ratio, allowed to reach confluence (12 days after subculture), and then used in the described experiments. Preliminary experiments were performed to determine the approximate concentrations of CdCl2 that would result in MCF-10A cell toxicity over a 16-day period of exposure. From this preliminary determination, three concentrations of CdCl2 (7, 15, and 30 µM) were chosen for a short-term exposure of 48 h, and four concentrations (3, 7, 15, and 30 µM) were used for an extended exposure of 16 days. These concentrations were chosen such that over a 16-day time course, one concentration would result in no cell death and another would result in appreciable cell death early in the time course. The viability of confluent cell monolayers was determined by the automated counting of 4', 6-diamidino-2-phenylindole (DAPI)stained nuclei as described previously by this laboratory (Garrett et al., 1998a). Triplicate cultures were analyzed for each time point and concentration.
MT protein determination.
The immunoblot protocol used for the determination of the co-expressed levels of the MT-1 and MT-2 protein in cell lysates has been described previously by this laboratory (Garrett et al., 1998a, 2000
). For cultured cells, the cells were rinsed twice with phosphate buffered saline (PBS) and harvested in 10 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol. For tissue, the samples were ground to a powder under liquid nitrogen, harvested in 10 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol, and homogenized. The cells were lysed by two cycles of freezing and thawing under liquid nitrogen and a 37°C water bath. The lysates were centrifuged in a microfuge and the supernatants were stored at 70°C. The MT-1 and MT-2 proteins were detected by immunoblotting using a mouse anti-horse antibody (DAKO-MT, E9) as the primary antibody. This antibody detects both the MT-1 and MT-2 isoforms, and, in this report, the product detected is referred to as "MT-1/2." Using image analysis software (KS 400, Kontron), MT-1/2 protein was quantified by comparing the optical density of the sample dots to that of the standard MT curve. Rabbit liver Cd/Zn metallothionein-1 was applied to each blot to generate standard curves. This assay has detection limits in the range of 0.10.5 ng MT-1/2 per microgram of total protein.
Statistical analysis.
All experiments were performed in triplicate except as noted. Statistical analyses were performed with Systat software, using separate variance t-tests and analysis of variance (ANOVA) with Tukey post hoc testing. Unless otherwise stated, the level of significance was 0.05.
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RESULTS |
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DISCUSSION |
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The MT-1/2 protein was also analyzed in normal breast tissue and in a variety of benign, pre-invasive, and malignant breast lesions (Bier et al., 1994). Of significance was the finding that in 12/24 ductal in situ carcinomas and in 9/20 invasive ductal carcinomas there was MT-1/2 overexpression, suggesting that the in situ components within invasive ductal carcinomas generally reflected the MT-1/2 status of their invasive counterpart. It was concluded that breast carcinoma cases with MT-1/2 protein overexpression arise from lesions that also demonstrate MT-1/2 overexpression and that MT-1/2 overexpression is a genuine feature of the tumor cells and not simply related to endogenous or exogenous factors known to induce MT-1/2 synthesis. This is in agreement with studies by Douglas-Jones and coworkers (1995)
, which analyzed MT-1/2 expression in duct carcinoma in situ and found MT overexpression to arise from early lesions that also overexpress MT-1/2.
Despite these compelling observations, there is no information regarding the expression of the mRNAs for the eight active MT-1 and MT-2 genes in normal breast duct epithelium. Without such information, no conclusions can be drawn from examinations of MT-1 and MT-2 isoformspecific mRNA expression in human ductal breast cancers. The first goal of this study was to determine the expression of the MT-1 and MT-2 isoformspecific mRNAs in the ductal epithelium of the normal breast. As part of this initial goal, the previous immunohistochemical reports that demonstrated intense staining for the MT-1/2 protein in the myoepithelium of the normal breast and a corresponding lack of immunoreactivity in adjacent ductal epithelial cells were confirmed in this laboratory (Bier et al., 1994; Fresno et al., 1993
; Jin et al., 2001
). Laser capture microdissection was used to separate the ductal and myoepithelial cells into separate components, with subsequent isolation of total RNA and RT-PCR to determine expression of MT-1 and MT-2 isoform-specific mRNAs in each fraction. Rather than the expected finding that there would be limited expression of MT isoformspecific mRNAs in the ductal fraction, in agreement with the absence of immunoreactive MT-1/2 protein, the results of this analysis demonstrated that both the identity of the MT isoformspecific genes expressed (MT-2A and MT-1X), and their relative levels of expression were similar between the myoepithelial and ductal components. These findings showed that the ductal and myoepithelial components of the human breast express similar amounts of MT-2A and MT-1X mRNAs, but that they have distinctly different expression of the MT-1/2 protein. The finding of similar MT mRNA expression, but vastly different MT protein expression, in two adjacent cell types provides initial evidence for a component of post-transcriptional regulation of MT-1 and MT-2 protein expression in the human breast.
The finding that MT-1/2 immunonegative normal ductal epithelial cells express MT-2A and MT-1X mRNA in amounts similar to the highly MT-1/2 immunoreactive myoepithelial cells has several impacts on the observation that MT-1/2 protein expression is increased in a subset of ductal breast cancers. The first impact is that no assumption can be made that the increased expression of the MT-1/2 protein in breast cancer is due solely to an induction of transcription of the MT-1 and MT-2 mRNAs, but rather could be due to post-transcriptional regulation of MT-1/2 protein accumulation. Historically, evidence indicates that the MT-1 and MT-2 genes are primarily regulated at the level of transcription (Samson and Gedamu, 1998). However, there is recent evidence in rodents after treatment with Cu+2 and Cd+2 that shows that MT-1/2 protein levels can be regulated at the post-transcriptional level (Vasconcelos et al., 1996
, 2002
). To determine if post-transcriptional control of MT-1/2 protein expression could occur in ductal epithelium, the MCF-10A cell line was analyzed for its basal and Cd+2-induced expression of the MT-1 and MT-2 isoformspecific mRNAs and protein. The spontaneously immortalized MCF-10A cell line is widely employed as a model of the normal human breast epithelial cell (Soule et al., 1990
). First, the basal expression pattern of the MT-1/2 protein and the MT-1 and 2 isoformspecific mRNAs was determined to establish if the cell line would have similar expression patterns to that found for in situ ductal epithelium. The basal expression of MT-1/2 protein was modest, being approximately 3.3 ng of MT-1/2 protein per microgram of total MCF-10A protein. The MCF-10A cells expressed mRNA for the MT-1X and MT-2A genes, in agreement with the gene expression found for in situ ductal epithelium. Furthermore, the MT-1X and MT-2A mRNAs were found to be very abundant transcripts and in large excess to what would be expected for the accumulation of the low amount of MT-1/2 protein. The only discrepancy between the MCF-10A cells and the in situ epithelium was in the level of expression of the MT-1E gene. The expression of the MT-1E was noted at 30 cycles of PCR in the MCF-10A cells and at the limits of detection in the in situ ductal epithelium. Overall, theses findings demonstrated a similarity to ductal epithelium in the expression of the MT-1/2 proteins and isoform-specific mRNAs.
The MCF-10A cells were exposed to lethal and sublethal levels of Cd+2 to determine if evidence could be found for post-transcriptional regulation of MT-1/2 protein accumulation. It was demonstrated that Cd+2 elicited only a marginal induction of MT-1E, MT-1X, or MT-2A mRNAs in the MCF-10A cells at either lethal or sublethal levels of exposure and under both acute and extended periods of exposure. In marked contrast, it was demonstrated that Cd+2 exposure under identical conditions resulted in a large increase in MT-1/2 protein expression, reaching levels of 6% of total cell protein under conditions of extended exposure to Cd+2. The likely mechanism underlying these findings is the classic observation that apoMT (metal-free MT) is rapidly degraded by the cell and that metal binding stabilizes the MT protein against such degradation (Hamer, 1986; Kägi, 1993
; Kägi and Hunziker, 1989
). These findings are strong evidence that post-transcriptional regulation could be one mechanism determining MT-1/2 protein levels in malignant human breast duct epithelial cells.
One can only speculate how the present findings might influence the role of cadmium in the development of breast cancer. Cadmium is a pollutant produced almost exclusively by the Industrial Revolution. Because of this relatively recent time frame of cadmium accumulation, it is certain that the pattern of MT expression found in the human breast duct has no evolutionary correlation to the presence of cadmium as an environmental pollutant. Rather, it is more likely that the pattern of MT expression in the breast duct reflects a past nutritional need to rapidly sequester zinc, perhaps under certain conditions of zinc deficiency during lactation. A limited, but rapid need to sequester zinc is suggested because this would be most consistent with the pattern of MT expression found in the breast duct. The high basal expression of MT mRNA, coupled with a post-transcriptional stabilization of the protein by zinc, would provide the cell a means to rapidly accumulate zinc without the need to induce new transcription. To have a link to cadmium and the development of breast cancer, one would have to speculate that at some stage of the transformation process, the duct cell acquires a zinc- and cadmium-accumulating phenotype as noted through stabilization of the MT protein. Evidence for this hypothesis comes from whole-body autoradiography of lactating mice injected with 109CdCl2 which documents that breast tissue is a major organ of cadmium accumulation and that the duct epithelial cells are the accumulating cell type (Grawe and Okarsson, 2000). Fractionation of cytosolic proteins in this study also showed that most of the accumulated metal is bound to MT. Brako and co-workers have also shown an increase in cadmium accumulation in lactating breast and that MT levels increase in the mammary gland during lactation in cadmium-treated animals (Brako et al., 2003
). Thus, either there is a lactating-specific induction of MT or there is a post-transcriptional regulation of MT by cadmium similar to that shown in the present study. An interaction of cadmium, MT and breast cancer is also implicated by the recent observation that some smokers have increased expression of MT protein in invasive breast cancers, and it is well-established that cigarette smoking produces a high level of exposure to cadmium (Gallicchio et al., 2004
). The inherent ability of ductal epithelium to accumulate cadmium and MT under lactating conditions, coupled with the chronic low-level activation of the estrogen receptor by this metal, could have a potential role in cadmium-induced breast carcinogenesis.
There have been only limited examinations of the expression of the mRNAs supporting MT-1/2 protein overexpression in breast cancer, and none have examined a relationship to disease outcome. The examination of MT-2A mRNA expression in fresh surgical specimens of ductal breast cancer found that increased expression correlated with higher histological grade but not with patient age or lymph node status (Jin et al., 2002). The level of expression of MT-2A mRNA was not compared to that found in normal breast duct epithelium. The expression of the MT-1E isoform was also examined in a similar series of breast cancers and was found to be highly expressed in estrogen-receptornegative breast cancers compared to estrogen-receptorpositive breast cancers (Jin et al., 2000
). This observation is important because, in conjunction with the current findings, it would indicate that these breast cancers had gained the expression of the MT-1E gene compared to the normal ductal epithelial cell. Such an increase in expression would have to be initiated at the level of transcription. The relationship between the expression of the MT-1E gene and estrogen receptor status was first noted in breast cancer cell lines (Friedline et al., 1998
). In these studies, the estrogen-receptorpositive cell lines MCF-7 and T-47D were shown to express only MT-2A and MT-1X mRNA, whereas the estrogen-receptornegative cell lines Hs578T and MDA-MB-231 were shown to express MT-1E in addition to the MT-2A and MT-1X genes. Together, these findings suggest that elucidation of the expression pattern of the underlying MT-1 and MT-2 genes in breast cancers could increase the prognostic significance beyond that of just knowing the expression of the MT-1/2 protein.
The present study suggests that the mechanism underlying the finding of increased MT-1/2 protein expression in ductal breast cancer and its relationship to disease prognosis may be very complex, involving the post-transcriptional regulation of MT-1/2 protein expression from normally expressed MT-1X and MT-2A mRNAs and new, induced transcription from at least one additional MT-1 isoform-specific gene.
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NOTES |
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ACKNOWLEDGMENTS |
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