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Endocrinology Vol. 142, No. 6 2409-2418
Copyright © 2001 by The Endocrine Society

ARTICLES

Cloning and Characterization of a Novel Gene That Is Regulated by Estrogen and Is Associated with Mammary Gland Carcinogenesis1

Daniela Marcantonio, Lorraine E. Chalifour, Moulay A. Alaoui-Jamali, Lesley Alpert and Hung The Huynh

Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Departments of Medicine (D.M., L.E.C., M.A.A.-J., H.T.H.), Pharmacology and Therapeutics (M.A.A.-J.), and Pathology and Oncology (L.A.), McGill University, Montréal, Québec, Canada H3T 1E2

Address all correspondence and requests for reprints to: Lorraine E. Chalifour, Ph.D., Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Department of Medicine, McGill University, 3755 Chemin Côte Ste Catherine, Montréal, Québec, Canada H3T 1E2. E-mail: lorraine.chalifour{at}mcgill.ca Or Hung The Huynh at his current


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens play a role in mammary gland function and are implicated in mammary carcinogenesis. We report the cloning of a novel gene [steroid-sensitive gene 1 (SSG1)] that is regulated by E2 in the rat uterus and mammary gland. The full-length SSG1 complementary DNA has an open reading frame of 1158 nucleotides encoding a putative protein of 385 amino acids. A SSG1-specific antibody recognizes a 40-kDa protein localized to myoepithelial cells of normal mammary tissue and to endothelial cells of 7,12-dimethylbenz(a)antracene-induced mammary tumors. Treatment of rats with E2 at 1.2 or 2.4 µg/kg·day for 21 days increases SSG1 protein levels in mammary tissue by 16-fold compared with controls. Removal of E2 after a 14-day treatment decreases SSG1 protein levels 6-fold and 3-fold at 120 and 144 h, respectively. Treatment of rats with the estrogen antagonists tamoxifen or ICI 182,780 did not affect SSG1 protein levels compared with controls. SSG1 protein levels in 7,12-dimethylbenz(a)antracene-induced rat mammary tumors were 23-fold greater than SSG1 levels in resting mammary tissue, and 8-fold higher than protein levels expressed in lactating mammary glands. We propose that SSG1 plays a role in estrogen functions, and its overexpression is correlated with mammary carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
17ß-ESTRADIOL (E2) is a steroid hormone that is primarily synthesized and secreted by the ovaries (1). E2 exerts various effects on many tissues in the female reproductive and nonreproductive systems. In the mammary gland, E2 is required for cellular differentiation, development, and physiological function. E2 also influences uterine physiology, regulates bone density and lipid and cholesterol homeostasis, and exerts cardio-protective effects (2, 3, 4). The biological activity of E2 is regulated by its interaction with specific high affinity nuclear estrogen receptors (ER), ER{alpha} and ERß (2). The E2-ER complexes bind to estrogen response element (ERE) DNA sequences to induce transcriptional control of estrogen responsive genes (5). E2-ER acts directly on mammary tissue to regulate cell proliferation by inducing early response genes including c-fos, c-myc, ras, cell cycle associated genes including cyclin D1, and the expression of insulin-like growth factor II, epidermal growth factor, transforming growth factor {alpha}, as well as their receptors (6, 7, 8, 9). It has been shown that E2 stimulates the nuclear localization of cyclin D1 and cdk4 in breast cancer cells (7). E2 is also associated with the activation of cyclin E- and cyclin {alpha}-cdk2-dependent kinases and hyperphosphorylation of pRb and p107 (7). Ultimately, E2 increases the rate of cell proliferation by recruiting noncycling cells into the cell cycle and by shortening the overall cell cycle time by reducing the length of the G1 phase (6). In addition to the classical mechanism of ER activation through E2-ER complexes, ER can activate transcription in a ligand-independent manner (10).

Antiestrogens antagonize the biological effects of E2 by competitively inhibiting the binding of E2 to the ER. Type 1 antiestrogens, such as tamoxifen or its metabolites, have mixed estrogenic/antiestrogenic activity. They form imperfect complexes upon binding to the ER and are unable to induce transcription of E2 target genes in certain cellular contexts (11). Tamoxifen is an E2 antagonist in the mammary gland and is currently used in the prevention and treatment of breast cancer (12). Type 2 antiestrogens, which include ICI 182,780, have no estrogen-like properties in vivo or in vitro (11). Type 2 compounds antagonize E2 at two levels. First, type 2 antiestrogen-ER complexes bind to EREs but do not induce gene transcription. Secondly, these compounds disable the ER by binding to the newly synthesized receptor in the cytoplasm and prevent its transport to the nucleus. The paralyzed receptor complex is later degraded (13).

E2 is believed to be a major player in the development and progression of breast cancer. E2 removal by ovariectomy has a protective effect against breast cancer (14). Epidemiological studies have shown that the incidence of breast cancer increases with age yet drops off sharply at the age of menopause (15). In rodents, prolonged treatment with high levels of E2 can lead to the development of mammary tumors and E2 is mitogenic to ER-positive breast cancer cells. Antiestrogens are effective agents in suppressing the E2 stimulated proliferation and metastatic activity of ER positive breast cancers (16, 17, 18). Recently, tamoxifen has shown encouraging results in clinical trials as a preventative agent for breast cancer, which further supports a role for E2 in breast cancer development and progression (19).

Several E2-regulated genes are overexpressed in breast cancer cells and may play a role in breast cancer development and progression. These include transforming growth factor {alpha}, cathepsin D, pS2, heat shock protein 27, and vascular endothelial growth factor (20, 21, 22, 23, 24). In 7,12-dimethylbenz(a)antracene (DMBA)-induced hormone-dependent rat mammary tumors, E2 is required for tumor formation and plays a role in endothelial cell proliferation and in angiogenesis (25, 26, 27). To identify novel E2 responsive genes, we used differential display to compare uterine tissues from ovariectomized and ovariectomized and E2-treated rats. We identified steroid-sensitive gene 1 (SSG1) as a novel gene that is regulated by E2 in vivo and is associated with mammary carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal studies
All animal studies were performed in accordance with the regulations of the Canadian Council of Animal Care and the Animal Care Committee of the Lady Davis Institute for Medical Research. All rats were purchased from Charles River Laboratories, Inc. (St-Constant, Québec, Canada). To prepare samples for differential display, ovariectomized, mature, Sprague Dawley female rats were either dorsally implanted with empty SILASTIC brand silicon tubing (Dow Corning Corp., Midland, MI) or were dorsally implanted with SILASTIC brand silicon tubing containing E2 (Sigma, St. Louis, MO) to release 4.8 µg/kg·day, 2 weeks following ovariectomy. Animals were killed 2 weeks after hormone implantation and the uterus of each animal was removed. To demonstrate the regulation of SSG1 by E2 in the rat uterus, mature Sprague Dawley female rats were either left intact (three animals per group) or received SILASTIC brand silicon tubing implants packed with E2 in the dorsal area to release 3.6 µg/kg·day for 21 days. Uterine tissue was collected, snap frozen in liquid nitrogen, and stored at -80 C until needed.

To study E2 regulation in rat mammary gland, mature, intact female Sprague Dawley rats (six animals per group) were dorsally implanted with SILASTIC brand silicon tubing packed with E2 releasing 1.2, 2.4, or 3.6 µg/kg·day, and control animals received empty tubing. Animals were killed 21 days after hormone implantation.

To study the effect of E2 withdrawal on SSG1 expression, mature, intact Sprague Dawley female rats (three animals per group) were dorsally implanted with SILASTIC brand silicon tubing to deliver 2.4 mg/kg·day E2. After 14 days of treatment, the tubing was surgically removed and rats were killed at 0, 72, 120, and 144 h following withdrawal of E2.

To study the effect of antiestrogens on SSG1 expression, mature, intact Sprague Dawley female rats received dorsally implanted SILASTIC brand silicon tubing packed with tamoxifen (Sigma) to deliver 100 or 200 mg/kg·day for 21 days (four animals per group). Control animals received empty tubing. Rats treated with preformulated ICI 182,780 (Astra-Zeneca Pharmaceuticals, Mississauga, Canada) received weekly sc injections of either 1.0, 1.5, or 2.0 mg/kg for 3 weeks (six animals per group). Control rats received castor oil.

To induce mammary tumors, 20 mg DMBA (Sigma) in peanut oil was administered by gavage to 55-day-old, virgin, intact Sprague Dawley female rats. Palpable tumors (>0.5 cm) were observed by day 90 following DMBA administration (four to seven animals per group). Tumors were collected from rats on day 105 and immediately frozen in liquid nitrogen. Normal mammary tissue was obtained from vehicle-treated rats.

Differential display
Total RNA was extracted from the uteri of ovariectomized rats and from ovariectomized rats treated with E2 using the RNAzol B method (Tel-Test, Friendswood, TX) as directed by the manufacturer. Differential display was performed essentially as described in Refs. 28 and 29 . The primer sequences used to amplify the differential display products were 5'-T11MG-3' and 5'-AGCCAGCGAA-3'. Differentially expressed complementary DNA (cDNA) sequences of interest were eluted from the gel, reamplified, and used as a probe in Northern blotting to confirm E2 regulation. DNA fragments of interest were cloned into a T-tailed EcoRV linearized pBluescript vector and both strands were sequenced. The cDNA sequences were compared with GenBank databases. One sequence, which led to the identification of SSG1, was a novel cDNA fragment.

cDNA isolation
Poly-A-enriched RNA was isolated from rat uterus and cloned into the pcDNA3 vector (Invitrogen, Carlsbad, CA) at the NotI site to create a rat uterus cDNA library. The novel differential display fragment radiolabeled with [{alpha}-32P]dCTP, was used to screen the cDNA library according to standard protocols. The clone containing the largest insert of 3.719 kb was sequenced on both strands. The SSG1 cDNA sequence was analyzed using GeneRunner (Hastings Software, Inc., Hudson, NY) for open reading frames.

In vitro transcription-translation
In vitro transcription-translation was performed using the TNT Promega kit (Promega Corp., Madison, WI) as directed by the manufacturer using the expression construct, SSG1 cDNA cloned into the pcDNA 3.1 HisC vector. [35S]Methionine was incorporated into nascent protein. Two microliters of the total reaction was resolved by 10% SDS-PAGE and subjected to autoradiography.

Northern blot analysis
Forty micrograms of total RNA were electrophoresed through a 1% formaldehyde-agarose gel and RNA was transferred by downward alkaline capillary blotting onto Zeta Probe GT nylon membranes (Bio-Rad Laboratories, Inc., Montréal, Québec, Canada). Northern blotting using a [{alpha}-32P]dCTP-labeled SSG1-specific probe was performed as described in (30). SSG1 expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The optical density of the hybridization signals was quantified using a densitometer (HPScanJet, Image Quant Software, Hewlett Packard, Vancouver, WA).

Generation of an SSG1-specific antibody
Synthetic peptides corresponding to predicted amino acids 3–29 of the rat SSG1 gene (TVRGPSVSEQLYPLPRKEQQREKPQA) were synthesized and coupled to preactivated keyhole limpet hemocyanin (Sheldon Biotechnology, Montréal, Quebéc, Canada). Rabbit polyclonal antibodies were produced according to standard protocols. Affinity purified serum following the sixth boost was used in these studies.

Protein extraction and Western blot analysis
About 100 mg of tissue was homogenized in buffer containing 1% Triton X-100, 300 mM NaCl, 20 mM Tris (pH 7.4), 2 mM EDTA, 0.5 mM sodium orthovanadate, and 0.5% Nonidet P-40. Samples were centrifuged at 14,000 x g at 4 C for 15 min and the supernatant containing the protein was collected. Seventy-five milligrams of protein was resolved by 10% SDS-PAGE, electrotransferred onto nitrocellulose (Bio-Rad Laboratories, Inc.), and used for Western blotting by standard means using an affinity purified anti-SSG1 rabbit polyclonal antibody (1:1000) and a goat-antirabbit IgG coupled to horseradish peroxidase (1:5000). Immune complexes were detected using the enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Baie d’Urfé, Canada). Blots were stripped then immunoblotted with anti-{alpha}-tubulin as above to normalize for protein loading.

Immunofluorescence
Deparaffinized tissue sections were blocked with 0.2% gelatin, 2% BSA, and 2% normal goat serum in PBS for 1.5 h. Tissue was incubated with anti-SSG1 (1:50) in blocking solution overnight at 4 C, washed with PBS containing 0.1% BSA, and incubated with antirabbit antibody conjugated to Texas Red (1:1000). Tissue was washed with PBS containing 0.1% BSA and then with PBS. Samples were mounted and photographs were taken at a magnification of x400 using an Olympus Corp. BH2 microscope equipped with epifluorescence (Olympus Corp., Lake Success, WA).

Statistics
Values are expressed as the mean ± SEM. Statistical analyses of significance were performed by ANOVA using GraphPad Software, Inc. (San Diego, CA) Prism 2.0. P less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of SSG1
To isolate uterine genes regulated by E2, we used the differential display technique and compared RNA samples collected from uteri of ovariectomized and ovariectomized + E2-treated rats. We selected a novel differential display fragment, as determined by DNA sequence analysis, that is highly expressed in the uteri of ovariectomized rats but down-regulated in the uteri of ovariectomized + E2-treated rats. We used this cDNA fragment to screen a rat uterus cDNA library to isolate the full-length cDNA sequence of SSG1, which is shown in Fig. 1Go. Analysis of the nucleotide sequence of the cDNA using the GenBank database showed no corresponding homologous gene. However, the sequence did have high homology (>85%) to expressed sequence tags (ESTs) isolated from mouse (Accession No. BE284696), rat (accession no. AA891240), rabbit (Accession No. C83580), and human (Accession No. AI751036) tissues. A search for motifs within the SSG1 nucleotide sequence detected multiple direct repeat ERE half sites (RGGTCA) located at nucleotide positions -1061 to -1066, -745 to -750, 321–326, and 911–916, and are double underlined in Fig. 1Go. An SSG1-specific probe was used to detect the SSG1 transcript on a blot containing the RNA prepared from uteri of control-treated and E2-treated rats. We detected a messenger RNA of 3.8 kb that was down-regulated by E2 in rat uterine tissue, which is in agreement with the results obtained from the differential display, but not in the rat lung following E2 treatment (Fig. 2A).



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  		Figure 1. Rat SSG1 nucleotide and predicted amino acid sequence. A 3719-bp cDNA sequence was isolated. The longest open reading frame of SSG1 yields a predicted amino acid sequence of 385 residues. The ATG start site was designated as position 1. SSG1 protein sequence contains a lysine-rich domain located between amino acids 85 and 187. Lysine residues in this region are highlighted. The positions of the putative ERE half sites are double underlined. The positions of putative classical NLS are indicated in bold and bipartite NLS are underlined. The complete nucleotide sequence of the rat SSG1 cDNA has been deposited into GenBank as Accession No. AF223677.

 
The longest open reading frame beginning with an ATG and terminating with an in frame stop codon encodes a putative protein of 385 amino acids (Fig. 1Go). This open reading frame predicts a 41.8-kDa protein. In vitro transcription-translation studies of an expression construct containing the coding sequence of SSG1 resulted in the translation of a protein product migrating to about 40 kDa (Fig. 2BGo, lane 3), consistent with initiation of translation at the predicted start codon. The SSG1 amino acid sequence contains a high content of lysine residues. Overall, the amino acid sequence of SSG1 consists of 13.2% lysine residues (51 lysines in a total of 385 residues). Importantly, 66.6% of the total lysine residues are located between amino acids 85 and 187 and are highlighted. These account for 33% of all residues within this stretch of amino acids (34 lysines in a total of 102 amino acids).



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  		Figure 2. SSG1 mRNA is down-regulated by E2 in the rat uterus and translates a 40-kDa protein. A, Northern blotting was performed on 30 µg of total RNA extracted from the uterus and lung of intact (i) or E2-treated (E2) rats. Blots were hybridized using radioactive SSG1 and GAPDH-specific cDNA probes as indicated. B, In vitro transcription-translation was performed using [35S]methionine as described in Materials and Methods. Control reactions were carried out using a luciferase construct as a positive control (lane 1) and the empty pcDNAHisC vector as a negative control (lane 2). When the SSG1 cDNA-pcDNAHisC construct was used in the reaction, a protein migrating to 40 kDa was translated (lane 3, arrow). Molecular mass standards are indicated on the left in kilodaltons.

 
A search of GenBank protein databases showed that a portion of the SSG1 protein was similar to the rat drs protein (31). Residues 207–354 of the SSG1 protein were 28% identical and 43% similar to the rat drs protein sequence, which contains sushi repeats. However, the SSG1 protein sequence does not contain the conserved cysteine residues found in the sushi repeat motif family members. Therefore, we conclude that SSG1 is not functionally related to the rat drs protein.

Examination of the lysine-rich stretch of amino acids identified 5 classical nuclear localization signals (NLS) located at amino acid 92 (PKKK), (PKKKEKI), 143 (PEKEKKK), 147 (KKKK), 181 (KKKK), shown in bold in Fig. 1Go. Five potential bipartite NLS located at amino acid positions 18 (RKEQQREKPQATRRPNK), 135 (KKHEKPEKPEKEKKKKG), 167 (KKAEKKSKQEKEKTKKK), 171 (KKSKQEKEKTKKKKAGK), and 206 (RKSVADLLGSFEGKRRL) were also identified and are underlined in Fig. 1Go.

SSG1 mRNA and protein expression in rat tissues
The tissue specificity of SSG1 gene expression was investigated by Northern blot analysis of total RNA isolated from various tissues from intact rats to an SSG1-specific radiolabeled cDNA probe (Fig. 3AGo). SSG1 mRNA, about 3.8 kb in length, was detected in ovary, uterus, mammary gland, liver, lung, spleen, kidney, heart, bladder, intestine, skeletal muscle, and brain, with varying levels of expression. To characterize the SSG1 protein, a rabbit polyclonal antiserum was raised against a synthetic peptide corresponding to predicted amino acids 3–29 of the rat SSG1 protein. Western blotting experiments performed on protein extracted from mature intact female Sprague Dawley rats using the SSG1 antibody recognized a protein that migrated at around 40 kDa. This is in agreement with the predicted size of the SSG1 protein as well as the in vitro transcription-translation studies using the pcDNA3-HisC-SSG1 cDNA construct shown in Fig. 2BGo. The SSG1 protein was expressed in uterus, liver, lung, spleen, kidney heart, bladder, skeletal muscle, and brain. Protein was very low or undetectable in mammary gland and intestine despite the presence of mRNA expression (Fig. 3BGo). To demonstrate the specificity of the SSG1 antibody, the same blot was probed with preimmune serum. The protein migrating at 40 kDa is not detected (Fig. 3CGo). The specificity of the antibody was also confirmed by its ability to recognize the SSG1 protein in transfected cells (our unpublished observations).



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  		Figure 3. SSG1 mRNA and protein expression in various rat tissues. A, Northern blots of total RNA (30 µg) isolated from various rat tissues, as indicated, were hybridized to a SSG1-specific radioactive cDNA probe as described in Materials and Methods. An mRNA migrating to 3.8 kb was detected in all tissues. Ethidium bromide staining of the agarose gel is shown. B, Western blotting was performed on 75 µg total protein extracted from various rat tissues, as indicated. Immunoblotting with an anti-SSG1 antibody identified a protein migrating to 40 kDa. C, Immunoblotting using the preimmune serum was performed on the same blot shown in B. The SSG-1-specific signal migrating at 40 kDa was not observed indicating specificity of the SSG1 antibody.

 
SSG1 mRNA is unchanged and SSG1 protein is increased by E2 treatment in the rat mammary gland
Several experiments were performed to characterize the responsiveness of SSG1 to E2 treatment. Intact rats were treated with 0, 1.2, 2.4, or 3.6 µg/kg·day of E2 for 21 days and total RNA was extracted from the mammary glands. Northern blotting indicated that there was no significant regulation of SSG1 mRNA expression in E2-treated samples of mammary gland vs. the control group (Fig. 4AGo), despite mRNA down-regulation by E2 in the rat uterus (Fig. 2AGo). Corresponding protein samples were extracted from mammary gland tissues and immunoblotting was performed using an affinity purified anti-SSG1 antibody. Despite high levels of SSG1 mRNA expression, SSG1 protein was very low or undetectable in control untreated mammary tissue. In contrast, SSG1 protein expression was significantly increased by 16-fold in E2-treated rats at doses of 1.2 and 2.4 mg/kg·day of E2 and 12-fold at 3.6 µg/kg·day of E2 compared with control tissue (Fig. 4Go, B and C).



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  		Figure 4. SSG1 protein expression is increased in E2-treated rat mammary glands and is decreased following withdrawal of E2. A, Northern blotting was performed on 40 µg total RNA extracted from rat mammary glands treated with 0 (lanes 1, 2, 3), 1.2 (lanes 4 and 5), 2.4 (lanes 6, 7, and 8), and 3.6 (lanes 9, 10, and 11) µg/kg·day of E2 for 21 days. Blots were hybridized using radioactive SSG1 and GAPDH-specific cDNA probes. B, Total protein (75 mg), extracted from rat mammary glands treated with 0 (lanes 1 and 2), 1.2 (lanes 3 and 4), 2.4 (lanes 5 and 6), and 3.6 (lanes 7 and 8) µg/kg·day for 21 days, was separated on a 10% SDS-PAGE gel. Immunoblotting was performed using anti-SSG1 and anti {alpha}-tubulin antibodies. Densitometry of x-ray films from SSG1 and {alpha}-tubulin hybridizations was performed. The expression of SSG-1 was normalized to {alpha}-tubulin expression and is plotted in C. Values are expressed in arbitrary units with the means ± SEM shown. Statistical significance, (P < 0.01), of four to six independent observations is indicated by an asterisk. D, Protein was extracted from untreated rat mammary gland (-, lanes 1 and 2) and from tissue treated with 2.4 µg/kg·day for 14 days (+, lanes 3 and 4). Protein was extracted from tissue treated with 2.4 µg/kg·day for 14 days followed by removal of exogenous E2 for 72 (lanes 5 and 6), 120 (lanes 7 and 8), and 144 (lanes 9 and 10) h. Western blotting was performed on 75 mg total protein separated on a 10% SDS-PAGE gel using an affinity purified anti-SSG1 antibody. Loading was normalized to {alpha}-tubulin expression. E, Densitometry was performed and the SSG1 to {alpha}-tubulin ratio is plotted in arbitrary units with the means ± SEM shown. Statistical significance, (P < 0.01), of three independent observations is indicated by an asterisk.

 
SSG1 protein expression is decreased in response to E2 withdrawal in the mammary gland
To demonstrate the dependence of SSG1 protein expression on E2, Western blotting was performed on protein extracted from rat mammary tissue following the surgical removal of E2 implants after 14 days of treatment with 2.4 µg/kg·day E2. Removal of E2 resulted in a time-dependent down-regulation of SSG1 protein expression (Fig. 4DGo). At 120 and 144 h following E2 removal, SSG1 protein levels were significantly decreased by 6- and 3-fold, respectively, when compared with the E2-treated control group (Fig. 4EGo).

SSG1 protein expression is not altered following antiestrogen treatment in the rat mammary gland
We determined the effects of tamoxifen and ICI 182,780 on SSG1 mRNA and protein expression in the mammary gland. SSG1 mRNA levels in the mammary glands of rats treated for 21 days with 100 or 200 mg/kg·day of tamoxifen were not significantly different from mRNA levels expressed in control untreated rats (P > 0.05) (Fig. 5AGo). SSG1 protein levels remained low following tamoxifen treatment and were similar to protein levels expressed in control tissue (Fig. 5BGo). Treatment with 1.0, 1.5, or 2.0 mg/kg·week of ICI 182,780 for 3 weeks resulted in a 2.5-fold up-regulation of mammary gland SSG1 mRNA at all doses compared with vehicle-treated rats (Fig. 5Go, C and D) but SSG1 protein levels remained low and were not significantly different from SSG1 protein levels expressed in control untreated mammary glands (P > 0.05) (Fig. 5EGo). Therefore, the antiestrogens tamoxifen and ICI 182,780 do not significantly stimulate SSG1 protein expression in the mammary gland.



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  		Figure 5. SSG1 mRNA and protein expression in the rat mammary gland following tamoxifen and ICI 182,780 treatment. A, Northern blotting was performed on 30 µg total RNA extracted from rat mammary glands treated with 0 (lanes 1–3), 100 (lanes 4–6), or 200 (lanes 7–9) µg/kg·day of tamoxifen for 21 days. Blots were hybridized to radioactive SSG1 and GAPDH-specific cDNA probes. B, Total protein (75 µg) extracted from rat mammary glands treated with 0 (lanes 1 and 2), 100 (lanes 5 and 6), and 200 (lanes 7 and 8) µg/kg·day tamoxifen or 2.4 µg/kg·day of E2 (lanes 3 and 4) for 21 days were separated on a 10% SDS-PAGE gel. Immunoblotting was performed using anti-SSG1 or anti {alpha}-tubulin antibodies. C, Northern blotting was performed on 30 mg total RNA extracted from rat mammary glands treated with 0 (lanes 1 and 2), 1.0 (lanes 3 and 4), 1.5 (lanes 5 and 6), and 2.0 (lanes 7 and 8) µg/kg·week ICI 182,780. Blots were hybridized to radioactive SSG1 and GAPDH-specific cDNA probes. D, Densitometry was performed and the SSG1 to GAPDH ratio is plotted. Values are expressed in arbitrary units with the means ± SEM shown. Statistical significance (P < 0.05) of six independent observations is indicated by an asterisk. E, Total protein (75 mg) extracted from rat mammary gland treated with 0 (lanes 1, 2, and 3), 1.0 (lanes 4–6), 1.5 (lanes 7–9), 2.0 (lanes 10, 11, and 12) µg/kg·week ICI 182,780 or 2.4 µg/kg·day E2 (lanes 13 and 14) for 3 weeks, was separated on a 10% SDS-PAGE gel. Immunoblotting was performed using anti-SSG1 or anti {alpha}-tubulin antibodies.

 
SSG1 protein levels are consistently elevated in DMBA induced rat mammary tumors
We measured the levels of SSG1 protein expression in 11 DMBA-induced mammary tumors collected from female Sprague Dawley rats (Fig. 6Go). Immunoblotting analysis showed that all DMBA tumors, without exception, expressed elevated levels of the SSG1 protein when compared with protein expression in the resting normal mammary gland (Fig. 6AGo, lanes 1–5). DMBA tumors express about 23-fold more SSG1 protein compared with levels in the resting mammary gland (Fig. 6BGo). SSG1 protein levels in the lactating mammary gland were compared with SSG1 protein levels in DMBA tumors induced in pregnant rats (Fig. 6AGo, lanes 6–13). DMBA tumors express approximately 8-fold more SSG1 protein than the lactating mammary gland (Fig. 6BGo).



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  		Figure 6. Western blot analysis of SSG1 protein expression in DMBA induced rat mammary carcinomas. A, Total protein was extracted from mammary tissue of untreated virgin rats (lane 1), from individual DMBA-induced mammary tumors induced in virgin rats (lanes 2–5), from lactating mammary glands (lane 6), and from individual DMBA induced mammary tumors in pregnant rats (lanes 7–13). Total protein (75 µg) was separated on a 10% polyacrylamide gel and immunoblotting was performed using anti-SSG1 or anti {alpha}-tubulin antibodies. B, Densitometry was performed and the SSG1 to {alpha}-tubulin ratio is plotted in arbitrary units. Results are expressed as the mean ± SEM. Statistical significance (P < 0.01) of four to seven independent experiments is indicated by an asterisk. SSG1 protein expression in DMBA tumors induced in virgin rats is indicated by empty shading and SSG1 protein expression in DMBA tumors induced in pregnant rats is indicated by black shading. SSG1 expression in control resting mammary glands (C), lactating mammary glands (L), or in DMBA tumor tissue is indicated.

 
Immunofluorescent localization of the SSG1 protein in normal rat mammary tissue, E2-treated mammary tissue and in DMBA-induced rat mammary tumors
To determine cell type distribution of SSG1 protein within mammary tissue, immunofluorescent analysis was performed using an anti-SSG1-specific antibody. Specific immunofluorescent labeling was localized to the myoepithelial cells of the normal rat mammary ducts and to E2-treated tissue (Fig. 7Go, A and C, respectively). SSG1 appears to localize to the cytoplasmic compartment of the cell in the control tissue. In the absence of primary antibody, no specific immunofluorescent labeling was detected (Fig. 7Go, B and D). In contrast to normal rat mammary tissue and E2-treated mammary tissue, significant levels of immunofluorescent labeling were localized to the endothelial cells of the vasculature of DMBA-induced rat mammary tumors (Fig. 7EGo). When immunofluorescent analysis was performed in the absence of the primary SSG1 antibody, no SSG1 labeling was observed in tumor tissue (Fig. 7FGo).



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  		Figure 7. Immunofluorescent localization of the SSG1 protein in normal rat mammary tissue, in E2-treated mammary tissue and in rat DMBA-induced rat mammary tumor tissue. Immunofluorescent analysis of SSG1 expression was performed on normal rat mammary tissue in the presence (A) of primary anti-SSG1 antibody and in the absence (B) of primary antibody. Immunofluorescent localization of SSG1 is shown on E2-treated mammary tissue in the presence (C) or absence (D) of primary anti-SSG1 antibody. Immunofluorescent analysis of SSG1 expression was performed on DMBA-induced rat mammary tumor tissue in the presence (E) or in the absence (F) of primary antibody. A and C, The arrow indicates myoepithelial cells. E, The arrow indicates endothelial cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we report on the isolation of a novel gene of 3.719 kb whose expression is down-regulated by E2 in the rat uterus. This gene, designated SSG1, contains a 1.158-kb open reading frame and predicts a protein of 41.8 kDa. A rabbit polyclonal antibody raised against a peptide contained in this predicted sequence recognizes a protein migrating to about 40 kDa. This is consistent with the open reading frame analysis and with in vitro transcription-translation experiments, which transcribed a protein migrating to about 40 kDa.

GenBank database searches did not reveal any significant homology of SSG1 to existing sequences and, therefore, SSG1 could not be classified into any known superfamily of proteins. SSG1 is likely to be ubiquitous as comparison of the SSG1 cDNA sequence against an EST database indicates that it shares high nucleotide identity with ESTs isolated from mouse (Accession No. BE284696), rat (Accession No. AA891240), rabbit (Accession No. C83580), and human (Accession No. AI751036) and the SSG1-specific antibody recognizes a protein of the predicted size in rat, mouse, and human (our unpublished observations) cell lines. A lysine-rich region in the SSG1 protein, between residues 85 and 187, encodes 5 putative classical and 5 putative bipartite NLS motifs, suggesting that SSG1 may be a nuclear protein. Despite these NLS motifs, SSG1 localized to the cytoplasmic compartment of myoepithelial cells in the rat mammary gland. These data suggest either that these NLS sequences are nonfunctional or that SSG1 is being retained in the cytosol. Specific protein-protein interactions may be required for nuclear entry of SSG1. At this time, the signal(s) permitting nuclear transport of SSG1 and reversing its cytoplasmic localization, if any, is not known.

SSG1 protein expression is tightly regulated by E2 in the normal rat mammary gland in vivo. Following E2 treatment, the SSG1 protein was significantly increased by 16-fold vs. control untreated mammary glands, whereas mRNA levels were not significantly altered. A similar pattern of discordant regulation of mRNA and protein was observed in the NMDAR1 gene in the rat hypocampus following E2 treatment (32). Increased NMDAR1 protein levels are thought to result from posttranslational modifications, which may include an increased rate of protein translation and/or alterations in the rate of protein degradation (32). E2 may regulate SSG1 protein expression in a similar manner. Although SSG1 protein expression displays a reproducible pattern of E2 responsiveness in the rat mammary gland, accumulation of the SSG1 protein is not a generalized response to E2. In rat uterus, SSG1 mRNA was down-regulated in a dose-dependent manner by E2 treatment yet protein expression remained unchanged (our unpublished observations). In lung, neither SSG1 mRNA nor protein expression were altered following E2 treatment indicating that a particular cellular context is required for E2-mediated SSG1 protein accumulation.

The epithelial compartment of ducts and lobules of the mammary gland is embedded in a mesenchymal compartment of fatty stroma, permeated by blood vessels and nerves (33, 34). Mammary ducts consist of luminal epithelial cells and myoepithelial cells (35). We show that E2 treatment results in a 16-fold increase in SSG1 protein expression and that SSG1 is located to the myoepithelial cells of the ducts of normal mammary tissue. Although myoepithelial cells completely lack expression of ER{alpha}, they express ERß and recently Jin et al. (37) showed that estradiol diproprionate at a dose of 500 µg/kg·week for 1 week induced myoepithelial cell proliferation (36, 37). Therefore, E2 may directly act on myoepithelial cells via ERß and induce SSG1 protein expression. Antiestrogens counteract E2-ER mediated effects in the mammary gland and tamoxifen has antiproliferative effects on myoepithelial cells (36). In our study, tamoxifen did not significantly affect SSG1 mRNA or protein levels when compared with expression in control animals. Further, protein levels remained low following treatment with the pure antiestrogen ICI 182,780. Because potentially important relationships might exist between myoepithelial cells and epithelial cells in both normal and precancerous disease states, the discovery of novel genes regulated by steroid hormones in myoepithelial cells are of importance.

Despite the wide agreement regarding the involvement of estrogen in the etiology of breast cancer, the molecular mechanisms involved in the evolution of normal breast tissue to benign and malignant breast tumors in vivo are not well understood. Many genes involved in mammary carcinogenesis are overexpressed in response to E2 and are considered essential to tumor development and progression. We investigated the expression of SSG1 protein in DMBA-induced rat mammary tumors, which are strongly hormone-dependent for both induction and growth and are well vascularized tumors (26, 38). All DMBA tumors overexpressed the SSG1 protein when compared with resting or lactating mammary glands. Further, we identified SSG1 as expressed in the ER-positive endothelial cells of the vasculature of DMBA-induced tumors but not in the endothelial cells of normal rat mammary tissue. Growth and formation of capillary blood vessels is essential for solid tumor growth (39, 40). Fukuda et al. (41) observed capillary endothelial cell growth in DMBA-induced tumors that was estrogen dependent, and that treatment of rats exposed to DMBA with E2 after ovariectomy inhibits tumor necrosis and maintains high rates of endothelial cell proliferation. The increase in SSG1 observed in DMBA-induced tumors vs. normal mammary tissue may be the result of the increased number of endothelial cells expressing SSG1. This may be secondary to increased vascularization of DMBA mammary tumors. At this time, the function of SSG1 in normal mammary and cancer tissue is unknown and the potential involvement of SSG1 in mammary tumorgenesis remains to be demonstrated.


   Acknowledgments
 
We thank Drs. B. Vose and A. Wakeling (Zeneca Pharmaceuticals) for ICI 182,780. We also thank Dr. Sylvie Mader for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the U.S. Army Grant DAMD 17-97-1-7084 (to H.T.H.), MRC Grant MOP-37986 (to L.E.C.), and Canadian Breast Cancer Initiative Grant CBCRI-008491 (to M.A.A.-J.). Back

Received August 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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