We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsα expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells(poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsα, but not HP1Hsβ or HP1Hsγ, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsα staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsα localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsα expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsαprotein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsα in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsα in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsα normally silences genes involved in breast cancer invasion and metastasis.

Heterochromatin has been shown to have a silencing effect on euchromatic gene expression (1). In Drosophila,a repression of euchromatic gene expression was observed when chromosomal rearrangements juxtaposed euchromatic genes near constitutive heterochromatin (2). This silencing effect has been shown to be mediated in part by heterochromatin-associated proteins, of which the best characterized is HP14(3). HP1 is a nonhistone chromosomal protein that localizes primarily to centric, telomeric, and specific euchromatic sites on polytene chromosomes in Drosophila(4).

Direct evidence that HP1 is involved in gene silencing stems from experiments in which the levels of HP1 were modulated. Mutations in the gene encoding HP1 are homozygous lethal in Drosophila(5). HP1 heterozygotes show a loss of silencing of transgenes at centric locations, which correlates with a more“open” (euchromatic-like) chromatin structure (6). In contrast, HP1 overexpression leads to an increase in gene silencing(5, 7). In mice, increased silencing of a centric transgene was observed upon overexpression of a mouse HP1-like protein,M1 (8). Therefore, gene expression appears to be sensitive to HP1 dosage (7); however, the exact mechanism by which HP1 exerts its suppressive effect is not known.

Three HP1-like proteins have also been identified in humans. HP1Hsα and HP1Hsβlocalize to pericentric heterochromatin and minor sites within euchromatic regions (9, 10, 11). In contrast,HP1Hsγ localizes primarily to euchromatin(10). Drosophila and mammalian HP1 interact with many different types of proteins (5), and recent evidence demonstrates that mammalian HP1 is part of a transcriptional repression complex (8, 12, 13, 14). Taken together, these results suggest that the function of mammalian HP1-like gene products is similar to that of Drosophila HP1 and may play a key role in the regulation of genes essential for proper development.

We demonstrate here that HP1Hsα mRNA and protein expression are down-regulated in breast cancer cells with an invasive/metastatic phenotype. HP1Hsα protein expression was decreased in seven of nine metastatic tumor tissues from breast cancer patients, and stable transfection of the HP1Hsα gene into MDA-MB-231 cells decreased their in vitro invasive potential. These data suggest that a reduction in HP1Hsα expression is directly associated with tumor cell invasion and metastasis.

Cells and Culture Conditions.

MCF-7 cells were kindly supplied by Dr. F. Miller (Michigan Cancer Foundation, Detroit, MI) and MDA-MB-231, T-47D, and Hs-578T cell lines were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were maintained in complete medium: RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 10 mmHEPES (Life Technologies, Inc.), and 10 mml-glutamine (Life Technologies, Inc.). Transfected cell lines were maintained in complete medium containing 200 μg/ml G418(MediaTech, Inc., Herndon, VA).

RNA Isolation and Northern Hybridization Analysis.

Total RNA was isolated from breast cancer cell lines using Trizol RNA isolation reagent (Life Technologies, Inc.) according to the manufacturer’s specifications. Northern blot analysis of HP1Hsα mRNA expression was performed on breast cancer cell lines of different invasive and metastatic potential, as described previously (15). Each experiment was repeated twice with similar results.

Semiquantitative RT-PCR Analysis.

Reverse transcription of total RNA from breast cancer cell lines was performed using the Advantage PCR kit according to the manufacturer’s instructions (Clontech, Palo Alto, CA). PCR amplifications were performed with HP1Hsβ- and HP1Hsγ-specific primers(HP1Hsβ forward primer,5′-AAAACAAGAAGAAAGTAGAGGAGGTGC-3′; HP1Hsβ reverse primer,5′-CAGATGTGACAGGGGCTGG-3′; HP1Hsγforward primer, 5′-GCGTTTCTTAAACTCTCAGAAAGC-3′; HP1Hsγ reverse primer,5′-TTATTATTTGTTCAAAGTAACCAGTGCT-3′). Annealing temperature and the number of amplification cycles were optimized at 62°C and 28 cycles,respectively, using cDNA from MCF-7 and MDA-MB-231 cells. PCR amplification reactions were performed in a Robocycler gradient 96 thermocycler (Stratagene, La Jolla, CA) under the following conditions:1 cycle at 94°C for 1 min; 28 cycles at 94°C for 1 min, 62°C for 2.5 min, 72°C for 1 min; and 1 cycle at 72°C for 5 min. GAPDH primers (Clontech) were used as controls for PCR amplification. These experiments were repeated twice, and similar results were obtained.

PCR fragments were ligated into the pCR2.1-TOPO sequencing plasmid according to the manufacturer’s instructions (Invitrogen, Carlsbad,CA). Plasmid DNA was isolated and subjected to DNA sequencing analysis using the Thermo Sequenase cycle sequencing kit (Amersham Life Science,Cleveland, OH) according to the manufacturer’s instructions. Two plasmids were sequenced from each primer set and showed identical sequence.

Western Blot Analysis.

Breast cancer cells were lysed (30 min, 4°C) in 100 mmTris (pH 7.5), 150 mm NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 2 mm phenylmethylsulfonyl fluoride, and 1 mm Pefabloc (Boehringer Mannheim, Indianapolis, IN). Cell lysates were briefly sonicated and centrifuged, and the total protein concentration was determined using the bicinchoninic acid protein assay(Pierce, Rockford, IL). Twenty five μg of protein for each sample were electrophoresed on a 12.5% polyacrylamide gel under denaturing conditions, and proteins were transferred onto Immobilon-P membranes(Millipore, Beford, MA).

Membranes were blocked with Tris-buffered saline containing 5% BSA(Sigma, St. Louis, MO), and HP1Hsα was detected using a mouse polyclonal HP1Hsα-specific antibody (16) and a horseradish peroxidase-conjugated goat antimouse secondary antibody (Amersham Pharmacia, Piscataway, NJ). To control for the amount of protein loaded per lane, an actin monoclonal antibody (ICN Biomedicals, Aurora, OH) was used. HP1Hsα and actin proteins were visualized using the enhanced chemiluminescence detection system according to the manufacturer’s instructions (Amersham Pharmacia). Each experiment was repeated at least twice with similar results.

HP1Hsα Immunofluorescence.

Breast cancer cell lines (5 × 104cells/well) were plated onto 12-mm round glass coverslips and grown to approximately 50% confluence. Cells were fixed in ice-cold methanol for 5 min. HP1Hsα was detected using a mouse polyclonal HP1Hsα-specific antibody(16) and a rhodamine-conjugated goat antimouse IgG secondary antibody (ICN Pharmaceuticals). Coverslips were mounted onto glass slides for analysis by confocal microscopy using Laser Scanning Microscope 510 (Carl Zeiss, Inc., Thornwood, NY) with optical sectioning ranging from 13–27 slices covering a 6–25-μm cell thickness. These experiments were repeated twice with similar results.

HP1Hsα Expression Vector Construction and Transfection.

The HP1Hsα complete coding sequence was obtained by PCR from MCF-7 cells using a HP1Hsα-specific forward primer containing an EcoRI restriction site(5′-GCTCAAGCGAATTCATGGGAAAGAAAACCAAGCGGAC-3′) and a HP1Hsα-specific reverse primer containing BamHI and HindIII restriction sites(5′-GAGTCCGGAAGCTTTGGATCCCGGCTCTTTGCTGTTTCTTTCTCTTTG-3′). The HP1Hsα PCR product was ligated into the pCR2.1-TOPO TA cloning vector (Invitrogen) and then ligated into the EcoRI/BamHI restriction sites of the pEGFP-C2 mammalian expression vector (Clontech), creating a GFP-HP1Hsα fusion protein. The GFP-HP1Hsα construct was transfected into MDA-MB-231 cells using LipofectAMINE reagent according to the manufacturer’s instructions (Life Technologies, Inc.). GFP-HP1Hsα-expressing cells were selected in complete medium containing 400 μg/ml G418.

In Vitro Invasion Analysis.

Analysis of in vitro invasion of breast cancer cell lines was performed using the MICS assay as described previously(17). The percentage of invasion was calculated as the total number of invading cells/total number of cells seeded × 100. Each parameter was assayed in triplicate, and each experiment was repeated twice with similar results.

HP1Hsα Immunohistochemistry.

Frozen and formalin-fixed, paraffin-embedded sections of human benign mammary tissue, primary breast cancer, and metastatic tissues were obtained from Dr. Ruth Lininger (University of North Carolina,Chapel Hill, NC). The Vectastain Elite ABC kit (Vector Laboratories,Burlingame, CA) was used to stain frozen tissue sections according to the manufacturer’s specifications, except that frozen tissue sections were quenched with 0.3%H2O2 in ice-cold methanol for 5 min at room temperature. Tissue sections were mounted with Aquamount (Lerner Laboratories, Pittsburgh, PA) for staining analysis.

Formalin-fixed, paraffin-embedded sections of primary and metastatic breast cancer tissues were deparaffinized, rehydrated in distilled water, and incubated in 20 mm Tris-HCl buffer (pH 1.5) for 18 h at 60°C. Tissues were washed in distilled water and then blocked sequentially with avidin, biotin, and serum-free protein block for 10 min each (LSAB+Alkphos Kit; DAKO, Carpinteria, CA). Tissues were incubated sequentially with anti-HP1Hsαantibody (16), biotinylated linking antibody, and enzyme-conjugated streptavidin. Sections were stained with Vector red chromagen (Vector Laboratories) and counterstained with 0.2% light green stain. Tissues were evaluated for the percentage of cells showing HP1Hsα immunoreactivity and the intensity of immunoreactivity, which was graded on a scale from 1+ to 3+, with 3+representing the highest intensity.

HP1 Expression in Breast Cancer Cell Lines.

HP1Hsα was identified previously among 19 differentially expressed genes whose expression was down-regulated in an invasive/metastatic breast cancer cell line (MDA-MB-231) compared with a poorly invasive/nonmetastatic breast cancer cell line (MCF-7;Ref. 15). Since these initial studies, we noted that the MDA-MB-231 cell line had become heterogeneous with respect to cellular morphology and contained a distinct subpopulation of large,epithelial-like cells. The density of this subpopulation of large cells increased with increasing passage of the cell line in culture. Furthermore, these large epithelial-like cells were positive for HP1Hsα expression, and HP1Hsα mRNA levels in bulk MDA-MB-231 cell cultures increased over time (data not shown). It is hypothesized that these large, epithelial-like cells are revertants to a noninvasive/nonmetastatic phenotype.5Therefore, the MDA-MB-231 cell line was enriched for invasive cells by two rounds of selection through a collagen IV/laminin/gelatin matrix to establish the MDA-MB-231–2 cell line (Fig. 1,A). The MDA-MB-231–2 cell line did not contain any large epithelial-like cells, and HP1HsαmRNA expression was reduced 34% in the MDA-MB-231-2 cell line compared with the MCF-7 and T-47D cell lines (Fig. 1,B). In addition,HP1Hsα protein levels were barely detectable in MDA-MB-231-2 and Hs578T cells by Western blot analysis (Fig. 1 C). These results demonstrate a down-regulation of HP1Hsα expression at the mRNA and protein levels in breast cancer cells with an invasive/metastatic phenotype.

To determine whether the down-regulation was specific for HP1Hsα or whether it included other family members, expression of HP1Hsβ and HP1Hsγ mRNA in breast cancer cell lines was also investigated using RT-PCR analysis. As shown in Fig. 1 D, virtually no difference in expression of either HP1Hsβ or HP1Hsγ mRNA was observed in the breast cancer cell lines tested, using GAPDH mRNA levels to normalize for RNA loading. These results suggest that only the down-regulation of HP1Hsα is associated with the breast cancer invasive/metastatic phenotype.

Localization of HP1Hsα in Breast Cancer Cell Lines.

Breast cancer cell lines were stained with a mouse polyclonal antibody to human HP1Hsα and analyzed by confocal microscopy to determine HP1Hsα expression and cellular localization patterns. A heavy punctate staining, which was localized to the nucleus and nuclear membrane, was observed in MCF-7 cells, T-47D cells, and in the poorly invasive subpopulation(consisting of large epithelial-like cells) within the MDA-MB-231 cell line before enrichment for more invasive subpopulations (Fig. 2). In contrast, a low-level diffuse nuclear staining pattern with little or no localization in the nuclear membrane was observed in Hs578T,MDA-MB-231 (in the invasive smaller cells before enrichment for more invasive subpopulations), and the MDA-MB-231-2 cells (enriched for highly invasive potential; Fig. 2). These results corroborate the reduction in HP1Hsα observed in whole cell lysates and demonstrate changes in HP1Hsαnuclear localization patterns in breast cancer cells with an invasive/metastatic phenotype.

Effect of GFP-HP1Hsα Transfection on in Vitro Invasion.

A mammalian expression vector containing the HP1Hsα gene downstream to a GFP gene was constructed (GFP-HP1Hsα)and stably transfected into MDA-MB-231 cells. GFP-HP1Hsα-transfected MDA-MB-231 cells showed GFP fluorescence that localized only to the nucleus,compared with control GFP-transfected MDA-MB-231 cells that showed GFP fluorescence predominantly located in the cytoplasm (data not shown). The ability of control GFP- and GFP-HP1Hsα-transfected MDA-MB-231 cells to degrade and invade a laminin/collagen IV/gelatin-coated polycarbonate membrane was investigated. As is shown in Fig. 3, a 43% and 49% reduction in invasion was observed in two GFP-HP1Hsα-transfected MDA-MB-231 clones, compared with control GFP-transfected MDA-MB-231 cells. In addition, analysis of the invasive cells (underside of the polycarbonate membrane) revealed that approximately 95% were nonfluorescent (data not shown). These results provide direct evidence that down-regulation of HP1Hsαexpression contributes to breast cancer cell invasion.

HP1Hsα in Human Tissues.

To determine that the decrease in HP1Hsαobserved in the invasive/metastatic breast cancer cell lines was not limited to cultured cells, HP1Hsα protein expression was analyzed in clinically relevant tumor tissues. Frozen or formalin-fixed, paraffin-embedded normal mammary tissue and tissues from ductal and lobular carcinoma in situ, invasive ductal carcinoma, and metastatic lesions from distant sites (lymph node, lung,skin, and omentum) were analyzed for both the percentage of tumor cells positive for HP1Hsα and the intensity of staining. All of the tumor tissues from nonmetastatic lesions contained>80% HP1Hsα immunoreactive cells with a staining intensity of up to 3+, as shown in Table 1. In contrast, seven of nine metastatic tissues from distant sites contained up to 95% less HP1Hsα-positive cells with a staining intensity of 1–2+ (Table 1). Fig. 4 demonstrates HP1Hsα nuclear staining of normal epithelial cells and, to a lesser extent, stromal and myoepithelial cells in a normal lactating mammary tissue (Fig. 4,A) and tumor cells in an infiltrating ductal carcinoma (Fig. 4,C). In contrast, little or no HP1Hsα staining was observed in the nuclei of tumor cells in lung metastases from two breast cancer patients (Fig. 4, E and G). These results demonstrate the low level of HP1Hsαprotein expression in tumor cells from metastatic lesions.

Many studies have investigated biochemical, molecular, and morphological parameters for their value as prognostic markers to predict the metastatic potential of breast cancers. Included in this list are chromosomal anomalies (18) and nuclear morphology(19, 20). Changes in nuclear shape and size and heterochromatin content are associated with more aggressive breast tumors, including patients with recurrent disease. In one study, a combination of computer-assisted image analysis of heterochromatin content and nuclear texture and shape measurements was capable of correctly predicting prognosis in 92% of breast cancer patients(20). These results suggest that patterns of chromatin condensation change during metastatic progression.

One of the proteins known to be involved in chromatin packaging and gene regulation is HP1, which we identified as being down-regulated in the invasive/metastatic human breast cancer cell line MDA-MB-231(15). In this study, we observed a down-regulation in HP1Hsα mRNA and protein expression only in highly invasive/metastatic breast cancer cell lines. This down-regulation was limited to HP1Hsα expression because HP1Hsβ and HP1Hsγ mRNA expression was similar in all cell lines tested, regardless of invasive/metastatic phenotype,suggesting that these genes are transcriptionally regulated differently from HP1Hsα. In addition, we observed that endogenous HP1Hsαexpression was greatly increased in MDA-MB-231 cells that have an epithelial-like morphology (presumably reverted to a noninvasive phenotype) and that these HP1Hsα-positive epithelial-like cells are poorly invasive in vitro. Furthermore, MDA-MB-231 cells transfected with GFP-HP1Hsα have a decreased in vitro invasive potential compared with GFP-transfected MDA-MB-231 controls. Taken together, these results support our hypothesis that HP1Hsα expression is capable of modulating the invasive/metastatic phenotype in breast cancer cells.

Consistent with the data generated from cultured breast cancer cell lines, clinical pathology findings indicated a reduction in HP1Hsα in tumor cells from seven of nine distant metastatic lesions from breast cancer patients compared with normal mammary tissues and primary breast cancer tumors. There are several possible explanations for why a reduction in HP1Hsα was not observed in tumor cells from one lung and a lymph node metastatic lesion. The substantial localization of HP1Hsα in the lung tissue of one patient may be indicative of a tumor lesion that has arrested or reverted to a nonmetastatic phenotype. This hypothesis might also apply to certain metastatic lesions in lymph nodes, which may not represent true metastases. Through localized trauma, cancer cells (whether in situ or invasive) could be transferred by draining lymph to a regional lymph node, where they could be trapped and possibly grow. This could explain the uncommon observation of in situcarcinoma of the breast associated with lymph node metastasis with no identifiable invasive tumor (21). Additionally, it could explain why not all patients with lymph node metastases develop distant metastases and die of their disease. It is also conceivable that not all mechanisms of metastatic progression in breast cancer cells are mediated through pathways requiring loss or down-regulation of HP1Hsα expression.

This is the first report that demonstrates an association between a metastatic phenotype and a reduction in HP1Hsα. We theorize that HP1Hsα mediates silencing of genes involved in the EMT in differentiated normal cells and breast cancer cells maintaining a nonmetastatic phenotype. Cells that have undergone EMT have been shown to have a highly invasive/metastatic phenotype (22, 23), concomitant with a down-regulation in HP1Hsα mRNA and protein. In our model of breast cancer metastatic progression, release of HP1Hsα-mediated silencing would allow an increase in expression of EMT-related genes and induce the metastatic phenotype. Whether down-regulation of HP1Hsα is the cause of metastasis-related gene expression or rather a consequence of other genetic alterations remains to be determined. However, a clear association between a reduction in HP1Hsα and a breast cancer metastatic phenotype has been shown and may be useful as a molecular marker in predicting long-term prognosis in breast cancer patients.

Fig. 1.

Down-regulation of HP1Hsα expression in invasive/metastatic breast cancer cell lines. A, the invasive phenotype of breast cancer cell lines was determined by seeding cells into the upper wells of a MICS chamber. Invading cells were harvested from the lower wells after 24 h and counted. The percentage of invasion was calculated as the total number of invading cells/total number of cells seeded × 100. B, total RNA was isolated from breast cancer cell lines and probed with a 32P-labeled full-length HP1Hsα PCR product andβ-actin to control for equal loading. Numbersrepresent the percentage of HP1Hsα mRNA expression relative to that in MCF-7, which was set at 100%. C, HP1Hsα detected by Western analysis using whole cell protein lysates and a HP1Hsα polyclonal antibody. A monoclonal actin antibody was used to normalize for protein loading. D, total RNA from breast cancer cells was subjected to RT-PCR analysis using HP1Hsβ- and HP1Hsγ-specific primers. GAPDH-specific primers were used to normalize for the amount of RNA. PCR products were electrophoresed on a 1% agarose gel containing 3μg/ml ethidium bromide.

Fig. 1.

Down-regulation of HP1Hsα expression in invasive/metastatic breast cancer cell lines. A, the invasive phenotype of breast cancer cell lines was determined by seeding cells into the upper wells of a MICS chamber. Invading cells were harvested from the lower wells after 24 h and counted. The percentage of invasion was calculated as the total number of invading cells/total number of cells seeded × 100. B, total RNA was isolated from breast cancer cell lines and probed with a 32P-labeled full-length HP1Hsα PCR product andβ-actin to control for equal loading. Numbersrepresent the percentage of HP1Hsα mRNA expression relative to that in MCF-7, which was set at 100%. C, HP1Hsα detected by Western analysis using whole cell protein lysates and a HP1Hsα polyclonal antibody. A monoclonal actin antibody was used to normalize for protein loading. D, total RNA from breast cancer cells was subjected to RT-PCR analysis using HP1Hsβ- and HP1Hsγ-specific primers. GAPDH-specific primers were used to normalize for the amount of RNA. PCR products were electrophoresed on a 1% agarose gel containing 3μg/ml ethidium bromide.

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Fig. 2.

Cellular localization of HP1Hsα in breast cancer cell lines. Poorly invasive/nonmetastatic (MCF-7 and T-47D) and invasive/metastatic (Hs578T, MB-231, and MB-231-2) breast cancer cell lines (5 × 104 cells/well) were plated onto 12-mm round glass coverslips, fixed in ice-cold methanol, and stained for HP1Hsα expression using a mouse polyclonal anti-HP1Hsα antibody and a rhodamine-conjugated secondary antibody (left panels) or secondary antibody alone(right panels). Cells were analyzed by confocal microscopy using a Laser Scanning Microscope 510 with optical sectioning ranging from 13–27 slices covering a 6–25-μm cell thickness.

Fig. 2.

Cellular localization of HP1Hsα in breast cancer cell lines. Poorly invasive/nonmetastatic (MCF-7 and T-47D) and invasive/metastatic (Hs578T, MB-231, and MB-231-2) breast cancer cell lines (5 × 104 cells/well) were plated onto 12-mm round glass coverslips, fixed in ice-cold methanol, and stained for HP1Hsα expression using a mouse polyclonal anti-HP1Hsα antibody and a rhodamine-conjugated secondary antibody (left panels) or secondary antibody alone(right panels). Cells were analyzed by confocal microscopy using a Laser Scanning Microscope 510 with optical sectioning ranging from 13–27 slices covering a 6–25-μm cell thickness.

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Fig. 3.

Effect of GFP-HP1Hsα transfection on MDA-MB-231 in vitro invasion. MDA-MB-231 cells were transfected, using LipofectAMINE reagent, with a mammalian expression vector containing GFP alone or with a GFP-HP1Hsα fusion. After selection for GFP expression, two clones were established,MB-231-GFP-HP1-C1 and MB-231-GFP-HP1-C2. In vitroinvasive potential was analyzed by seeding cells into the upper wells of a MICS chamber. Invading cells were harvested from the lower wells after 24 h and counted. The percentage of invasion was calculated as the total number of invading cells/total number of cells seeded × 100.

Fig. 3.

Effect of GFP-HP1Hsα transfection on MDA-MB-231 in vitro invasion. MDA-MB-231 cells were transfected, using LipofectAMINE reagent, with a mammalian expression vector containing GFP alone or with a GFP-HP1Hsα fusion. After selection for GFP expression, two clones were established,MB-231-GFP-HP1-C1 and MB-231-GFP-HP1-C2. In vitroinvasive potential was analyzed by seeding cells into the upper wells of a MICS chamber. Invading cells were harvested from the lower wells after 24 h and counted. The percentage of invasion was calculated as the total number of invading cells/total number of cells seeded × 100.

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Fig. 4.

HP1Hsα expression in human normal mammary,primary, and metastatic breast cancer tissues. Frozen normal lactating mammary tissue (A and B), formalin-fixed paraffin-embedded sections of invasive ductal carcinoma(C and D), and two lung metastatic lesions (E−H) were stained for HP1Hsα expression using a mouse polyclonal HP1Hsα antibody and Vector red chromagen(A). Formalin-fixed paraffin-embedded sections were also counterstained with 0.2% light green stain (C, E, and G). In addition, a serial tissue section was stained with H&E (B, D, F, and H). Tissues were evaluated for the percentage of HP1Hsα immunoreactive tumor cells, and intensity of nuclear immunoreactivity graded on a scale of 1+ to 3+.

Fig. 4.

HP1Hsα expression in human normal mammary,primary, and metastatic breast cancer tissues. Frozen normal lactating mammary tissue (A and B), formalin-fixed paraffin-embedded sections of invasive ductal carcinoma(C and D), and two lung metastatic lesions (E−H) were stained for HP1Hsα expression using a mouse polyclonal HP1Hsα antibody and Vector red chromagen(A). Formalin-fixed paraffin-embedded sections were also counterstained with 0.2% light green stain (C, E, and G). In addition, a serial tissue section was stained with H&E (B, D, F, and H). Tissues were evaluated for the percentage of HP1Hsα immunoreactive tumor cells, and intensity of nuclear immunoreactivity graded on a scale of 1+ to 3+.

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH/National Cancer Institute Grant CA59702 (to M. J. C. H.), the University of Iowa Carver Collaborative Award (to D. A. K. and L. L. W.), and The Wellcome Trust (A. M. A. and W. C. E.).

4

The abbreviations used are: HP1,heterochromatin-associated protein 1; GFP, green fluorescent protein;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-PCR; EMT, epithelial to mesenchymal transformation; MICS,Membrane Invasion Culture System.

5

Dr. Janet Price, personal communication.

Table 1

Immunohistochemical evaluation of HP1Hsα in primary and metastatic breast carcinoma

Lesiona% cells stainingbIntensityc
Ductal carcinoma in situ >80 1–3+ 
Lobular carcinoma in situ >90 2–3+ 
Invasive ductal carcinoma, grade 2 >80 1–3+ 
Invasive ductal carcinoma, grade 2–3 >95 2–3+ 
Invasive ductal carcinoma, grade 3 80 2–3+ 
Invasive lobular carcinoma, grade 1 >90 1–3+ 
Invasive lobular carcinoma, grade 2 >90 1–3+ 
Invasive lobular carcinoma, grade 3 >90 2–3+ 
Metastasis to lymph node >90 2–3+ 
Metastasis to skin (scalp) 40 1–2+ 
Metastasis to lung 5–10 1+ 
Metastasis to lung <5 1+ 
Metastasis to omentum 30 1–2+ 
Metastasis to soft tissue (hip) 20 1–2+ 
Metastasis to lung ≪5 1+ 
Metastasis to lung 60 1–2+ 
Metastasis to lung 80 2–3+ 
Lesiona% cells stainingbIntensityc
Ductal carcinoma in situ >80 1–3+ 
Lobular carcinoma in situ >90 2–3+ 
Invasive ductal carcinoma, grade 2 >80 1–3+ 
Invasive ductal carcinoma, grade 2–3 >95 2–3+ 
Invasive ductal carcinoma, grade 3 80 2–3+ 
Invasive lobular carcinoma, grade 1 >90 1–3+ 
Invasive lobular carcinoma, grade 2 >90 1–3+ 
Invasive lobular carcinoma, grade 3 >90 2–3+ 
Metastasis to lymph node >90 2–3+ 
Metastasis to skin (scalp) 40 1–2+ 
Metastasis to lung 5–10 1+ 
Metastasis to lung <5 1+ 
Metastasis to omentum 30 1–2+ 
Metastasis to soft tissue (hip) 20 1–2+ 
Metastasis to lung ≪5 1+ 
Metastasis to lung 60 1–2+ 
Metastasis to lung 80 2–3+ 
a

Frozen and formalin-fixed paraffin-embedded tissue sections were fixed and stained as described in “Materials and Methods.”

b

The percentage of tumor cells showing HP1Hsα immunoreactivity was visualized and estimated in values of 5% increments.

c

Intensity of HP1Hsαimmunoreactivity was visually graded on a scale from 1+ to 3+, with 3+representing the highest intensity.

We thank Dr. Richard Seftor for scientific expertise in confocal microscopy, Dr. Michael Cohen for generously supplying normal lactating mammary frozen tissue sections, and Dr. David Schultz for helpful scientific discussions.

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