Identification of genes preferentially expressed in mammary epithelial cells of Copenhagen rat using subtractive hybridization and microarrays

Chengshi Quan1,3 and Shi-Jiang Lu1,2,4

1 Department of Pediatrics, 2 Cancer Center, College of Medicine, University of Illinois at Chicago, 840 South Wood Street, Chicago, IL 60612, USA and 3 Department of Pathology, Jilin University, Changchun, Jilin, China


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rats, like humans, vary considerably in susceptibility for mammary cancer development among different strains. The Copenhagen (Cop) rat is extremely resistant to mammary cancer development induced by a variety of carcinogens. Multiple genetic loci have been linked to the resistant phenotype, but the genes have yet to be cloned and the mechanisms underlying the resistance still remain unknown. Transplantation experiments, however, have demonstrated that these genes act only in the epithelial cells of mammary parenchyma; they do not act systemically. In the present study, we analyzed genes differentially expressed in mammary epithelial cells obtained from pubescent female Cop and susceptible Buffalo (Buf) rats, using PCR-based suppressive subtractive hybridization and cDNA microarray approaches. Our results showed a high degree of similarity in the expression profiles of about 4000 genes between Cop and Buf rats, with a few exceptions. We found that the interleukin-2 receptor {alpha} (IL-2R{alpha}) chain gene and claudin-6 gene were preferentially expressed in mammary epithelial cells purified from Cop rats. We further demonstrated that IL-2R{alpha} message was undetectable in two rat mammary cancer cell lines and in two human breast cancer cell lines. The level of claudin-6 mRNA was undetectable in two rat mammary cancer cell lines and was lower in two human breast cancer cell lines and one breast cancer sample than that in normal breast tissues. These results suggest that IL-2R{alpha} and claudin-6 may function as tumor suppressors, particularly for breast cancer. However, this possibility needs further investigation.

Abbreviations: Buf, Buffalo rat; Cop, Copenhagen rat; IL-2R{alpha}, interleukin-2 receptor {alpha}; mcs, mammary cancer suppressor genes; SSH, suppressive subtractive hybridization; TJs, tight junctions


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is the most common cancer in women worldwide, affecting 10% of all women in the US. Familial and genetic factors play an important role in determining the susceptibility of individuals to the development of this disease. Studies of families with hereditary breast cancer have resulted in the identification of abnormalities in several genes such as Brca-1, Brca-2 and p53 that confer susceptibility to the disease. Resistance to breast cancer, on the other hand, is difficult to study in humans and, as a result, little is known about the contributing genetic factors. Chemical induction of mammary tumors in rats has provided an excellent model to study resistance (1). Rats, like humans, vary considerably in susceptibility for mammary cancer development among different strains. For example, Buffalo (Buf), Wistar-Furth, inbred and outbred Sprague–Dawley rats are highly susceptible, developing multiple mammary carcinomas even after a single exposure to a number of carcinogens. Fischer 344, August and ACI rats are less susceptible. Copenhagen (Cop) and Wistar-Kyoto rats are extremely resistant to the induction of macroscopically detectable mammary carcinomas by a diverse class of carcinogens (1,2). Genetic studies have linked multiple loci to the resistant phenotype in Cop and Wistar-Kyoto rats (3,4), but the genes have not been cloned and the mechanism of resistance remains largely unknown. Transplantation experiments have demonstrated that the products of putative mammary cancer suppressor (mcs) genes inherited in the Cop rat are localized within the epithelial cells of mammary parenchyma; they do not act systemically (5,6). These results are in agreement with carcinogenic studies that a relatively high incidence of mammary sarcomas was reported in Cop rats by direct carcinogen exposure, and Cop rats did develop tumors at other sites following carcinogen exposures (7,8). The mcs genes are also more penetrant in adult than in neonatal rats (8).

Several studies have demonstrated that the putative mcs genes do not prevent initiation, such as Ha-ras oncogene activation and intraductal proliferation, a pre-neoplastic lesion, in Cop rats, but rather block the expansion of initiated cells and pre-neoplastic lesions to progress into macroscopically visible tumors (9,10). These observations suggest that the mcs genes are expressed in target cells of Cop rats early during the process of carcinogenesis, but may be absent or mutated in susceptible rats. Therefore, comparison of gene expression profiles in target cells from susceptible and resistant rat mammary glands may help to identify and clone the putative mcs genes.

Rat mammary gland is a complex organ composed of, at least, stromal fibroblasts, endothelial cells, adipocytes, epithelial and myoepithelial cells (11). The majority of rat mammary carcinomas express phenotypic markers consistent with an origin of luminal epithelial cells (12). In order to identify differentially expressed genes in mammary epithelial cells of susceptible Buf and resistant Cop rats, we first purified mammary epithelial cells from 50-day-old virgin female Cop and Buf rats by collagenase digestion and immunomagnetic selection. Differentially expressed genes in the two strains were then identified and isolated by PCR-based suppressive subtractive hybridization and cDNA microarray approaches. Our results showed a high degree of similarity in the expression of about 4000 genes between Buf and Cop rats. However, we found that the interleukin-2 receptor {alpha} (IL-2R{alpha}) chain gene and claudin-6 gene were preferentially expressed in the mammary epithelial cells purified from Cop rats. We further demonstrated that IL-2R{alpha} message was undetectable in two rat mammary cancer cell lines and two human breast cancer cell lines, and that claudin-6 expression was not detected in two rat mammary cancer cell lines and was low in two human breast cancer cell lines.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mammary epithelial cell purification and RNA isolation
Mammary epithelial cells were purified from 50-day-old inbred virgin female Buf and Cop rats (Harlan Sprague-Dawley, Indianapolis, IN) using a modified method of Kim and Clifton (13) as described previously (14). A total of 6 x 106 mammary epithelial cells from Buf rat mammary glands (from five rats) and 3.5 x 106 mammary epithelial cells from Cop rat mammary tissues (from five rats) were obtained. Cytoplasmic RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) following the procedure recommended by the supplier and stored at -70°C before use. cDNA pools were constructed from RNA isolated from Cop and Buf mammary epithelial cells using the SMART cDNA synthesis kit (Clontech, Palo Alto, CA) and stored at -70°C. Similarly, cytoplasmic RNA was isolated from rat mammary cancer cell lines RBA and NMU (ATCC, Rockville, MD), and human breast cancer cell lines MCF-7 and BT474, and cDNA pools were constructed as described above. Normal human mammary tissue (pooled from two Caucasians, ages 26 and 27) and breast tumor (an infiltrating ductal carcinoma with lymph node metastasis from a 49-year-old Caucasian) RNAs were obtained from Clontech.

Suppressive subtractive hybridization
Suppressive subtractive hybridization (SSH) was performed between Cop and Buf using the PCR-Select Subtraction kit (Clontech) following the supplier's instructions. In the forward subtraction, Cop was used as tester and Buf as driver (Cop-Buf); whereas in the reverse subtraction, Buf was used as tester and Cop as driver (Buf-Cop). In brief, 2 µg of PCR-amplified cDNA were purified by column chromatography to remove primers, nucleotides and other contaminants, and digested by restriction enzyme RsaI. For forward subtraction (Cop-Buf), RsaI digested Cop cDNA was ligated to adaptors 1 and 2R, respectively; for reverse subtraction (Buf-Cop), RsaI digested Buf cDNA was ligated to the adaptors. Then two rounds of subtraction and suppression PCR amplification were performed to enrich for genes uniquely expressed in Cop or Buf mammary epithelial cells according to the manufacturer's instructions. The subtracted cDNA libraries of forward (Cop-Buf) and reverse (Buf-Cop) subtractions were constructed by cloning subtracted cDNAs into pGEM-T Easy vector (Promega, Madison, WI) with blue and white color selection.

Differential screening and clone identification
A total of 1920 forward and 96 reverse subtracted individual recombinant clones were picked up and plasmids were prepared. Plasmid DNA was denatured and applied onto four identical Zeta-Probe nylon membranes by a dot blot apparatus (Bio-Rad, Hercules, CA) under conditions recommended by the supplier. Samples of PCR amplified cDNA pools from Cop-Buf (forward) and Buf-Cop (reverse) subtractions, and from unsubtracted Cop and Buf RNA were digested by RsaI enzyme to cleave the common primers used in PCR amplification and purified by QIAquick PCR purification kit (Qiagen). The quantities of each cDNA sample were estimated by separating on a 1% agarose gel and visualizing under UV light. Aliquots of the purified cDNA pools were labeled with [{alpha}-32P]dCTP (ICN, Aurora, OH) using random-primed method and purified by Sephadex-G50 column chromatography. The four identical DNA array filters were separately hybridized with one of the following probes: forward (Cop-Buf) subtracted cDNAs; reverse (Buf-Cop) subtracted cDNAs; unsubtracted Cop cDNAs; and unsubtracted Buf cDNAs under stringent conditions. The filters were washed, exposed to Kodak XAR film with an intensifying screen at -70°C for 2, 8 and 16 h.

Hybridization of cDNA probes to Atlas cDNA arrays
cDNA(2 µg) from mammary epithelial cells of Buf and Cop rats were digested with RsaI and purified with Qiagen PCR purification kit. Restriction digestion with RsaI released the primers used for cDNA synthesis, and therefore eliminated hybridization background. The cDNA pools were labeled with [{alpha}-33P]dATP (ICN) and purified with the kit supplied by Clontech. Equal amounts of labeled probes for Cop and Buf rats were hybridized to two rat cDNA arrays (Atlas rat 1.2 array and Atlas rat 1.2 array II, each with four identical filters; Clontech) according to the manufacturer's instructions. After completing the washing procedure, the arrays were exposed with a PhosphorImaging screen (Molecular Dynamics, Sunnyvale, CA) for 2–3 days. The signal intensity was detected using a Storm PhosphorImaging system (Molecular Dynamics) at 100-µ resolution, analyzed using AtlasImage 1.5 software (Clontech) following the manufacturer's guidelines. The background signals were normalized using global normalization with the sum method and the expression of a cDNA spot between the Buf and Cop probes that were hybridized to the same set of filters was compared, and the threshold was set at 2. The data were exported as Excel files for further analysis. The whole procedure was repeated using the other two filters in the same set of the array.

Cloning of rat Claudin-6 gene by 5'-RACE
Primers corresponding to the sequence of the 430 bp cDNA clone obtained from SSH analyses were used to initiate the 5'-RACE procedure according to the manufacturer's instructions (BRL, Rockville, MD). The protocol was repeated four times to obtain 1.5 kb sequence, with each cycle obtaining 500–600 bp. The 1.5 kb sequence contained a candidate translation start codon and a downstream in-frame stop codon, encoding a peptide of 219 amino acids.

RT–PCR
As a result of the limited number of purified mammary epithelial cells, confirmation of expression for genes (claudin-6, IL-2R{alpha} and two novel clones we designated as MC12 and MC32) obtained from SSH and array analyses was performed by RT–PCR analyses. Primers were designed based on the sequences obtained from clones of SSH analyses or cDNA clones that were included on the Atlas arrays. The quantities of DNA in each cDNA pool were adjusted accordingly as described previously (15). In brief, 5 µl of each of the cDNA pools were diluted 100-fold with TE buffer, and various quantities of the diluted cDNA pools were used for PCR amplification, using primers for {alpha}-tubulin gene, in a total volume of 50 µl. Ten microliters of the PCR products were separated on 1.5% agarose gels and stained with ethidium bromide. The signal intensity of the corresponding bands was estimated visually under a UV transilluminator. The DNA template quantities in the cDNA pools were equalized by dilution with TE buffer based on the signal intensities of the {alpha}-tubulin bands.

PCR primers for rat claudin-6 gene were: sense 5'-CTA TCG CTC CGT TAC TGG CAT-3', antisense 5'-ACA GCT GGG ACC GTA TGT GG-3', with a PCR product of 345 bp. Human claudin-6 primers were: sense 5'-TTC ATC GGC AAC AGC ATC GT-3', antisense, 5'-GGT TAT AGA AGT CCC GGA TGA-3', with a PCR product of 345 bp. Rat IL-2R{alpha} gene primers were: sense 5'-GGT TCA CCT GGC AAC ATA GAT-3', antisense 5'-CGA AAA GTG ACC ACA CCA TC-3', with a PCR product of 367 bp. Human IL-2R{alpha} gene primers were: sense 5'-CCT TCC AGG TCA CTG CAG GGA-3', antisense 5'-CAG CCG GCC ACT GCT ACC TG-3', with a PCR product of 357 bp. Rat somatostatin receptor 3 primers were: sense 5'-CCA GGC AAG CTA CTG CTC AC-3', antisense 5'-TTG GAC TGC TTA CTT CCT GAC C-3', with PCR product of 290 bp. Rat MC12 primers were: sense 5'-ACG ACC CTG TCT CAT CAA CG-3', antisense 5'-AGA AAA CCA CGC GTT ATC TGC-3', with a PCR product of 300 bp. Rat MC32 primers were: sense 5'-ACG CAG GAT TCT TGG CTA GA-3', antisense 5'-GGG TCT CAC GTA GTC CAG GT-3', with a PCR product of 296 bp. Primers for both human and rat {alpha}-tubulin gene were: sense 5'-CAC CCG TCT TCA GGG CTT CTT GGT TT-3' and antisense 5'-CAT TTC ACC ATC TGG TTG GCT GGC TC-3', which produced a 527-bp PCR product.

PCR reaction was performed in a volume of 50 µl containing 20 mM Tris–HCl (pH 8.0), 50 mM KCl, 0.2 mM dNTP and 0.25 µg of each primer, 2.5 U of Taq DNA polymerase with optimal concentrations of MgCl2 for each specific gene. Optimal cycles for each gene of PCR amplification were carried out, with 1 min for denaturation at 94°C, 1.5 min for annealing at variable temperatures for each specific gene, and 2 min for polymerization at 72°C. Ten microliters of PCR products were separated on 1.5% agarose gel and visualized by ethidium bromide staining.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolation of differentially expressed genes by SSH
To clone known and novel genes differentially expressed in mammary epithelial cells of Cop and Buf rats, two rounds of forward and backward subtractions and suppression PCR amplifications were carried out, and the subtracted cDNA libraries were hybridized with four cDNA pool probes. Clones that hybridized to Cop-Buf and Cop cDNAs, but not to Buf-Cop and Buf probes corresponded to genes that were likely over-expressed in the Cop rats. Clones that hybridized only to Cop-Buf cDNAs were strong candidates of genes preferentially expressed in Cop rats with low-abundance transcripts that were enriched during the subtraction process, whereas clones that hybridized to Buf-Cop and Buf cDNAs, but not to Cop-Buf and Cop cDNA probes, corresponded to genes that were likely to be over-expressed in Buf rats, and clones hybridized only to Buf-Cop probes were strong candidates of genes preferentially expressed in Buf rats with low-abundance messages. Using the above criteria, 1920 colonies from the Cop-Buf subtraction library and 96 clones from the Buf-Cop library were screened using cDNA probes prepared from Cop-Buf subtraction, Buf-Cop subtraction, unsubtracted Cop and Buf cDNA pools to identify clones with differential levels of expression. Ninety-seven clones from the Cop-Buf library were found to hybridize preferentially with cDNA generated from mammary epithelial cells of Cop rats. Sequence analyses of the inserts revealed that 12 of them showed a high degree of sequence homology with the 3' untranslated region of mouse claudin-6 gene, and the other 85 clones showed no homology with known genes. Homology search for these 85 clones were also carried out against an EST database, which demonstrated high homology of all clones with rat or mouse EST sequences, suggesting they were derived from mRNA, rather than from genomic DNA contamination. Twelve clones from the Buf-Cop library were found to hybridize strongly with probes of Buf rats, but no high degree of homology with known genes was observed. Again all 12 clones were found to share a high degree of homology with mouse and rat EST clones.

To clone rat claudin-6 gene, the 5'-RACE procedure was performed. After four rounds, a 1.5 kb full-length cDNA sequence was obtained for rat claudin-6 gene and a 219 amino acid protein was predicted by GCG program. Protein homology comparison of the predicted protein revealed 95% identity to mouse claudin-6 and 87% identity to human claudin-6 (Figure 1).



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Fig. 1. Comparison of rat claudin-6 protein with mouse and human claudin-6 proteins. Below the three sequences, the amino acid positions identical in the three molecules are indicated by stars, whereas identities between any two molecules are indicated by dots. Gap was introduced to maximize the alignment.

 
To verify the expression status of these clones, primers were designed based on obtained sequence information, and semi-quantitative RT–PCR analysis was performed on all the clones and the claudin-6 gene. The analyses revealed that most of the clones showed no or negligible difference in their expression levels between mammary epithelial cells derived from Cop and Buf rats, with only few exceptions. Notably, clones MC12 and MC32 were found to be expressed preferentially in the Cop rat mammary epithelial cells, and the claudin-6 gene showed a 3- to 5-fold increase in its expression in cells derived from Cop rats as compared with cells derived from Buf rats (Figure 2). No clone was found to be over-expressed in mammary epithelial cells derived from Buf rats. This may be due to the fact that only a small number of clones (96 versus 1920 clones) from the Buf-Cop subtraction library were screened.



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Fig. 2. Analyses of gene expression in mammary epithelial cells derived from pubescent female Buf and Cop rats. Cytoplasmic RNA from mammary epithelial cells was used to construct cDNA pools, and the expression of genes was examined by semi-quantitative PCR. The number at the top of each lane indicates the amount (µl) of cDNA used in the 50 µl PCR reaction. M: 1 kb plus DNA marker (BRL).

 
Identification of differentially expressed genes by cDNA arrays
In order to identify known genes that were preferentially expressed in mammary epithelial cells of Cop or Buf rat, probes of cDNA pools from Cop and Buf rats were used to hybridize with Atlas rat cDNA 1.2 array and rat 1.2 array II. These two cDNA arrays were spotted with more than 2350 cDNA probes, including regulators of cell growth and cell cycle; apoptosis genes, oncogenes and tumor suppressor genes; DNA damage, repair and recombination genes; growth factors, cytokines and their receptors; cell adhesion molecules and cell–cell interaction proteins; angiogenic factors and other genes. To minimize the possibility of false positive, the process was repeated using four identical filters of the same set cDNA array, and only reproducible results from two independent experiments were collected and analyzed. Our results demonstrated a remarkable high degree of similarity in the expression of 2350 genes between Cop and Buf rats, and only less than 20 genes showed a 2-fold difference (Table I). No tumor suppressor genes, oncogenes or genes whose alterations have been shown to be associated with malignant development showed expression difference, except for the IL-2 receptor {alpha} chain gene. Therefore, the expression of the IL-2R{alpha} gene was subjected to verification by semi-quantitative RT–PCR analysis. The results showed that the IL-2R{alpha} chain gene was expressed at a substantial level in mammary epithelial cells from Cop rats, whereas negligible message of this gene was observed in cells from Buf rats, in accordance with the results obtained from array analyses (Figure 2). The expression of somatostatin receptor 3 was also subjected to RT–PCR analysis and no difference in its expression was found between the Buf and Cop rats (data not shown). It has been reported that the measurement of the level of a cDNA using filter hybridization is related to many factors, including the amount of DNA placed on the filter (which cannot be accurately controlled) and the efficiency of hybridization (16), which may contribute to the discrepant results of somatostatin receptor 3 gene obtained by RT–PCR and cDNA array analyses.


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Table I. Gene expression analyses by cDNA arrays

 
Expression of claudin-6, IL-2R{alpha}, MC12 and MC32 in mammary tumors
To examine whether the expression levels of the claudin-6 and IL-2R{alpha} genes as well as clones MC12 and MC32 were also down-regulated in mammary cancers, cytoplasmic RNA was isolated from rat NMU and RBA cancer cell lines, which were derived from mammary tumors induced in susceptible rats by different carcinogens. Similarly, RNA from human normal breast tissues, one breast cancer, and MCF-7 and BT474 breast cancer cell lines was also obtained. cDNA pools were constructed and the expression of claudin-6 and IL-2R{alpha} was examined by semi-quantitative RT–PCR. As shown in Figure 3, no apparent expression of claudin-6 and IL-2R{alpha} was observed in NMU and RBA cells, even with maximum allowable amounts of starting materials and 40 cycles of PCR amplification. Similarly, a very low or negligible level of clone MC32 and a decreased level of clone MC12 were observed in the two rat mammary cancer cell lines. The expression status of claudin-6 and IL-2R{alpha} in human breast cancers was found to be variable as well. As shown in Figure 4, no detectable level of IL-2R{alpha} message was observed in human MCF-7 and BT474 breast cancer cells, whereas a similar level of IL-2R{alpha} expression was demonstrated between normal breast tissues and breast cancer. The caludin-6 message was detected in RNAs isolated from all origins. However, the expression level was lower in breast cancer sample and MCF-7 and BT474 breast cancer cells than that in normal breast tissue (Figure 4). The expression of MC12 and MC32 was not examined in human samples due to the lack of sequence information.



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Fig. 3. Analyses of gene expression in NMU and RBA rat mammary cancer cell lines. Cytoplasmic RNA was isolated from Cop rat mammary epithelial cells and from NMU and RBA rat mammary cancer cell lines. cDNA pools were constructed and the expression of genes was examined by semi-quantitative PCR. The number at the top of each lane indicates the amount (µl) of cDNA used in the 50 µl PCR reaction. M: 1 kb plus DNA marker (BRL).

 


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Fig. 4. Analyses of gene expression in human normal mammary glands, breast cancer sample, MCF-7 and BT474 breast cancer cell lines. Total RNA of normal mammary tissues (N.M. gland) and breast cancer sample (B. cancer) was obtained from Clontech. Cytoplasmic RNA was isolated from MCF-7 and BT474 breast cancer cell lines. cDNA pools were constructed and the expression of genes was examined by semi-quantitative PCR. The number at the top of each lane indicates the amount (µl) of cDNA used in the 50 µl PCR reaction. M: 1 kb plus DNA marker (BRL).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are a number of strategies available to isolate genes differentially expressed in biological samples; mRNA differential display, SSH, serial analysis of gene expression and DNA microarrays are among the most common ones. In the present study, we utilized SSH and cDNA microarrays to identify and clone genes that are differentially expressed in mammary epithelial cells derived from susceptible Buf and resistant Cop rats. Our results showed a remarkably high degree of similarity in gene expression profiles between these two strains, with only a few exceptions. These observations are in accordance with their very similar biological properties except for their susceptibility to mammary carcinogenesis (2,6). By analyzing about 2000 SSH clones and more than 2350 known genes, we only found two novel and two known genes exhibiting different expression patterns in Cop and Buf rats. BLAST search using partial sequences obtained for clones MC12 and MC32 revealed no homology with any known genes. However, a high degree of homology with a number of EST clones was noted, suggesting that both clones might be from mRNA transcripts, rather than from genomic contamination. Molecular cloning of full-length cDNA for these two clones should provide more information on their biochemical properties, especially on their potential role in mammary carcinogenesis.

One known gene that was repeatedly identified by SSH analyses (12 out of 97) was the claudin-6 gene, a member of the recently described claudin gene family with at least 20 members (17). Claudins are integral constituents of tight junctions (TJs) and play a central role in TJ formation by forming homodimers and heterodimers (1820). TJs are intercellular junctions that serve a major role in cell-to-cell adhesion for endothelial and epithelial cells (21). TJs also function as major barriers that allow selective passage of molecules and ions, and separate distinct protein and lipid components on the apical and basolateral plasma membranes to maintain cell polarity (21). In the mammary gland, the epithelial TJs are dynamic and regulated by a number of factors. TJs of the mammary gland in pregnant animals are leaky, but are impermeable during lactation. Both intramammary pressure and hormones such as prolactin, progesterone and glucocorticoids are involved in the regulation of mammary TJs (22). It has been known for years that TJ abnormalities, such as decrease in number or aberrant organization, are associated with cancer development (2325). Disruption of TJ functions is believed to allow growth regulatory proteins to cross barriers to bind their receptors, leading to neoplastic transformation (26). One component of TJs, ZO-1, is down-regulated in breast cancer cell lines, and loss or reduction of its expression is correlated with poor tumor differentiation (23). In Drosophila, mutations resulting in the absence of the dlg protein, the mammalian TJ equivalent, lead to neoplastic growth of epithelial cells (27). Moreover, introduction of Raf-1 oncogene into cancer cells resulted in down-regulation of claudin-1 expression and loss of TJ function (28). Loss or significant reduction of claudin-1 expression has been found in several established breast cancer cell lines and primary breast tumors (29,30). Incubation of murine embryonic carcinoma cells with retinoic acid resulted in the formation of TJ structures and the expression of ZO-1, claudin-6 and claudin-7 (31).

In the current study, we demonstrated a substantial level of claudin-6 expression in rat mammary epithelial cells. More importantly, a 3- to 4-fold higher expression level was observed in cells isolated from resistant Cop rats when compared with cells derived from susceptible Buf rats. In addition, claudin-6 expression level in NMU and RBA rat mammary cancer cells was below the detection limit of RT–PCR assays. The claudin-6 mRNA levels were also lower in two human breast cancer cell lines and one breast cancer sample than those in normal breast tissues. These findings suggest that claudin-6 may function as a tumor suppressor, particularly for breast cancer. However, this possibility needs further investigation.

Another interesting finding from cDNA microarray analyses is that cytokine receptor IL-2R{alpha} is preferentially expressed in mammary epithelial cells from the resistant Cop rats. The IL-2R is multimeric, consisting of two common subunits shared by other types of cytokine receptors, IL-2Rß (CD122) and {gamma}c (CD132), and an IL-2R-specific subunit IL-2R{alpha} (CD25) (32). Co-expression of all three ({alpha}, ß and {gamma}c) subunits results in the formation of high-affinity IL-2R (32). Although IL-2R{alpha} by itself has very low affinity to IL-2, mice with a disrupted IL-2R{alpha} gene show an abnormal phenotype resembling that observed in mice lacking IL-2 itself (33,34). Nemoto et al. (35) have also demonstrated that {alpha} chain is absolutely required for fully functioning IL-2R in mice. These observations suggest that IL-2R{alpha} is critically important for IL-2 response.

Functional IL-2R has been shown to be present in cells of non-hematopoietic origins and in various types of tumor cells (31,3640). Treatment with IL-2 inhibits the growth of IL-2R positive tumor cells both in vitro and in vivo (41,42). Porta et al. (43) and Yasumura et al. (44) have demonstrated that IL-2 inhibited tumor cell growth by altering the cell cycle kinetics and inducing apoptosis. Furthermore, the cell line with the most sensitive response to the cytotoxic activity of IL-2 was found to express the IL-2R{alpha} gene (43), and introduction of the IL-2R{alpha} gene into carcinoma cells increased their sensitivity to the growth-inhibitory activity of IL-2 in vitro (45,46). These results indicate that IL-2R{alpha} plays a critical role in transducing the inhibitory signal of IL-2 in tumor cells. In line with these observations, our data show that mammary epithelial cells derived from susceptible Buf rats express very low to negligible level of IL-2R{alpha}, whereas substantial IL-2R{alpha} expression is observed in cell preparations from resistant Cop rats. We have also demonstrated no apparent expression of the IL-2R{alpha} gene in two mammary carcinoma cell lines derived from susceptible rats and in two human breast cancer cell lines. It is thus reasonable to speculate that preferential expression of the IL-2R{alpha} chain in mammary epithelial cells in Cop rat may facilitate the formation of a fully functional and high-affinity IL-2R to prohibit neoplastically initiated cells from expansion via the action of IL-2 under the physiological conditions.

In summary, we have identified two novel and two known genes that are preferentially expressed in mammary epithelial cells derived from resistant Cop rats. The messages of these genes are low or undetectable in rat and human mammary cancer cells. Whether these identified genes possess tumor suppressing potential, especially for breast cancer, is under further investigation.


    Notes
 
4 To whom correspondence should be addressed Email: sjlu{at}uic.edu Back


    Acknowledgments
 
The authors thank Dr Hanlin L.Wang (Washington University School of Medicine, St Louis, MO) for critically reading and commenting on the manuscript, and Dr Rajeshwari Mehta (University of Illinois at Chicago, Chicago, IL) for providing the human breast cancer cell lines MCF-7 and BT474 used in this study. This research was supported by grant DAMD 17-00-1-0678 from the US Army Medical Research and Material Command.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 24, 2003; revised June 24, 2003; accepted July 23, 2003.