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Tumor Biology |
Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy [R. O., F. T., P. C., C. R., S. M.], and Istitute for Biomedical Technologies-CNR, 20090 Segrate, Milan, Italy [M. C., I. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SEL1L resides on chromosome 14q24.3–31 near the insulin-dependent diabetes mellitus I locus (IDDM11) but unlinked to a locus for this disease (10, 11, 12, 13) or to a locus for autoimmune thyroid diseases residing in the same chromosomal interval (14) . SEL1L is primarily expressed in embryonic and adult pancreas (15) , and is down-regulated or undetectable in several primary adenocarcinomas of the pancreas (1) and in gastric cancers (16) . Comparative sequence analysis of SEL1L in different species has revealed remarkable conservation, suggesting an important or even an essential role for this gene (17) .
We observed previously a differential pattern of SEL1L expression in mammary carcinoma cell lines, and in breast cancer and normal tissues (16) suggesting a role for SEL1L in breast tumor development. In the present study we analyzed a large series of human primary mammary carcinomas using a MAb3 raised against a recombinant SEL1L protein and examined the relationship between SEL1L expression levels and patient survival. We also examined the effect of inducibly up-regulated SEL1L expression on anchorage-dependent growth and the colony formation ability of human breast carcinoma MCF7 cells, which express SEL1L at low levels (16) . Our results suggest a role for the SEL1L protein in breast tumor growth and aggressiveness.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemistry
Immunoperoxidase assay was carried out by a sensitive peroxidase-streptavidin method on formalin-fixed, paraffin-embedded sections of breast carcinomas. Briefly, 1–2 µm consecutive sections were cut, deparaffinized, rehydrated, and pretreated using the heat-induced epitope retrieval method (19)
. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in methanol for 30 min. After several washes in PBS and treatment with normal goat serum (1:50) for 30 min at room temperature, sections were incubated overnight at 4°C with 3 µg/ml MSel1 mouse MAb that specifically recognizes the SEL1L protein,4
followed by biotinylated antimouse IgG and streptavidin-conjugated horseradish peroxidase (Dako). Peroxidase activity was detected using 3,3'-diaminobenzidine as substrate. Negative controls were incubated without MAb MSel1. Images were obtained using a Nikon Eclipse 600 equipped with a digital camera. A x60 oil immersion objective was used.
Reproducibility of the immunohistochemistry analysis was assessed in the preliminary set up of the immunoperoxidase assays with MAb MSel1, which included selection of MAb concentration, scoring system with intra- and interobservers evaluation, and reproducibility on serial slides of the same cases.
Statistical Analysis
Association of SEL1L expression in tumor tissue with breast cancer patient survival was evaluated using the 
2 test. The actuarial probability of mortality was estimated by Kaplan-Meier analysis, and differences were assessed using the log-rank test. The Cox proportional hazards model was used to identify the clinicopathological variables independently associated with mortality (20)
. All of the statistical tests were two-tailed (significance at P < 0.05) and performed using SAS software packages (SAS Institute Inc., Cary, NC).
Cell Culture
Human breast adenocarcinoma MCF-7 cells were grown in RPMI (Microbiological Associates, Walkersville, MD) supplemented with 10% FCS (Hyclone), and penicillin and streptomycin (100 IU/ml) in a humidified chamber (95% air and 5% CO2) at 37°C.
SEL1L-pDEX.1 Construct and Transfection
Full-length SEL1L cDNA (2.4 kb) was obtained by PCR amplification of reverse-transcribed pancreatic mRNA with primers designed to contain a 5' KpnI restriction site and cloned into the PCR 2.1 vector (Invitrogen). Plasmid DNA was digested with KpnI and EcoRI, gel-purified (JetSorb; Invitrogen), and recloned into the polylinker of vector pDEX/1. Transcription of SEL1L was driven by the ß-globin promoter contained within the plasmid. The pDEX/1 vector also contained six glucorticoid-sensitive repeats followed by a TATA box and a G418 resistance gene serving as a selectable marker.5
Cells were stably transfected with the SEL1L-pDEX/1 or control pDEX/1 constructs by electroporation (Bio-Rad gene pulsar) at 960 µF and 250 V for three pulses. Exponentially growing cells (107) in RPMI plus 10% FCS were incubated with 10 µg of XmnI-linearized SEL1L-pDEX/1 or control pDEX/1 for 20 min at 4°C. G418 (400 µg/ml) was added at 24 h after transfection. Individual neomycin-resistant clones were isolated and expanded for additional analysis.
DNA Isolation, Southern Blot, and PCR Analysis
High molecular weight DNA was extracted from cells using standard procedures (21)
. DNA (8 µg) was digested with BamHI, fractionated on a 0.7% agarose gel, transferred to Hybond N+ filters (Amersham), and hybridized with the [32P]dCTP-labeled SEL1L cDNA probe. The presence of the ß-globin promoter in the transfected clones was confirmed by PCR amplification using a set of primers designed for the promoter and the 3' end of SEL1L (see below). For PCR, 20 pmol of vector-specific sense pDEX/1 primer and SEL1L-specific antisense (IBD8) primer were added to 300 ng of genomic DNA; amplification was carried out for 30 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 3 min in a Perkin-Elmer Corp. thermal cycler.
RNA Isolation and RT-PCR Analysis
Total RNA was extracted from cells using the guanidine thiocynate method (1)
. Total RNA was treated with 1 unit/mg of RNase-free DNase I (Clontech) at 37°C for 15 min.
cDNA Synthesis.
Total RNA (1 µg) was used in a 20-µl reaction containing 5 µM of MgCl2, 2 µl 10 x reaction buffer [500 mM Tris-HCl (pH 8.8), 80 nM MgCl2, 300 mM KCl, 10 mM dithiothreithol], 1 µM dNTPs, 50 units of RNase inhibitor (RNAsin), 0.8 µg oligo-p(dT)15 primer, 1.6 µg p(dN)6 random primers, and 20 units of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim). The reaction mixture was incubated for 10 min at 25°C and for 60 min at 42°C. The enzyme was denatured at 99°C for 5 min and chilled on ice.
PCR Conditions.
The 30-µl reaction volume contained 2 µl of cDNA, 3 µl of 10x buffer [100 mM Tris-HCl (pH 8.8), 15 mM MgCl2, 500 mM KCl, 1% Triton X-100], 0.3 µl of each 25 µM dNTPs, 0.2 µl of vector-specific sense pDEX/1 primers and SEL1L antisense IBD5 primer (100 µM), and 1 unit of AmpliTaq DNA polymerase (DYNAZYme). The mixture was heated to 94°C for 3 min followed by 30 cycles of amplification using the following conditions: denaturing 94°C for 1 min; annealing at 60°C for 1 min; and extension at 72°C for 1 min. Amplifications included an initial denaturation at 94°C for 5 min and final extension for 10 min at 72°C. Reaction products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. The quality of the neo-synthesized cDNA was verified using HPRT primers.
Primers Used
PDEX/1: 5'-AAGGCAGGATGATGACCAGG-3'
IBD8: 5'-GCTGGATCCAGTGCCTATTACTGTGG-3'
IBD5: 5'-TCTGCTTCCTGCATCTGCCGTCTC-3'
HPRT: 5'-AATTATGGACAGGACTGAACGTC-3'
5'-CGTGGGGTCCTTTTCACCAGCAAG-3'
In Vitro Induction of SEL1L Transcription
MCF-7 clones containing the SEL1L-pDEX/1 construct in a stable form were treated with 100 nM of DEX for 1–7 days after which the cells were harvested, and the RNA isolated and analyzed by RT-PCR.
Immunofluorescence Analysis
Trypsin-detached MCF-SEL1L or MCF-pDEX/1 cells were incubated with 3% paraformaldehyde for 10 min at 0°C and permeabilized by a 30-min treatment at 0°C with 0.1% saponin and 0.1% BSA in PBS. Cells were washed and incubated for 30 min at 0°C with 5–10 µg of MAb MSel1 in PBS containing 0.1% BSA and 0.1% saponin. After several washes, cells were additionally incubated for 30 min at 0°C with FITC-goat antimouse antibody (Kierkegaard and Perry Labs.) and assessed for fluorescence using a FACScalibur (Becton Dickinson) and CellQuest software including the Kolmogorov-Smirnov statistics. To analyze the pattern of integrin expression, trypsin-detached cells were incubated with mouse MAbs MAR6 (anti-
6), MAR4 (anti-ß1), or MLuc5 (anti-67LR) produced in our laboratory, or MAB1964 (anti-Hu integrin ß4) obtained by Chemicon and processed as described above.
BT-474 cells were seeded on glass coverslips, fixed with acetone, treated with 0.1% saponin and 0.1% BSA in PBS for 20 min, and incubated with 10 µg of MAb MSel1 followed by FITC-goat antimouse antibody. Cells were then analyzed using a Radiance 2000 (Bio-Rad) confocal microscope using a FITC filter set.
Western Blotting
Cells were lysed using a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 0.5% NP40, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml phenylmethylsulfonyl fluoride. Protein concentration was determined by Comassie Plus Protein Assay (Pierce, Rockford, IL). Total cell lysates were resolved on 10% SDS-polyacrylamide gel, and Western blotting was performed using standard procedures. MAb MSel1 was used at 10 µg/ml.
Anchorage-dependent Proliferation Assays
SRB.
Cells treated with DEX for 1 week were trypsin-detached and seeded in 96-well plates (4 x 103 cells/well; 5 replicates) in 200 µl culture medium supplemented with 300 nM DEX and grown for 1, 3, 4, and 7 days. After fixation with 10% trichloroacetic acid at 4°C for 1 h and five washes with distilled water, 0.4% SRB (Sigma Chemical Co.) in 1% acetic acid was added and incubation continued for 30 min at room temperature. After several washes with 1% acetic acid, SRB bound to cellular proteins was dissolved in 10 mM Tris-HCl, pH 10.5. Absorbance at 490 nm, proportional to the number of cells attached to the culture plate, was measured by spectrophotometry.
Colony Assay
Cells treated with DEX for 1 week or control cells were trypsin-detached, seeded in six-well plates at 1000 cells/ml, and cultured for 1 week. Clones were fixed with methanol and stained with 10% Giemsa.
Anchorage-independent Clonogenicity Assays
DEX-treated or control cells were seeded in six-well or 24-well plates in semisolid medium containing 0.3% Bacto-Agar (Difco) supplemented with 30% FCS and 300 µg/ml of G418 over a 0.6% agarose layer. Medium containing 30% FCS was added weekly. Colonies were scored after 1–2 week incubation at 37°C in 5% CO2 in air. For cloning in Matrigel, cells were suspended in Matrigel diluted 1:2 in culture medium and seeded in 24-well plates at a concentration of 22,000 cells/well.
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RESULTS |
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. BT-474 human breast carcinoma cells expressing high level of SEL1L revealed intense cytoplasmic immunoreactivity with MSel1 in intracellular vesicles (Fig. 1, a and b)
. SEL1L protein was detected with a strong staining in 37% of invasive breast carcinomas analyzed (Fig. 1, c and d)
and with a weak staining in 35% (Fig. 1, e and f)
. The remaining 28% scored negative (Fig. 1g)
. Staining was always cytoplasmic, with a punctuate pattern suggestive of cytoplasmic vesicles. Normal breast epithelial tissues adjacent to the tumors were always positive. Two MSel1-positive normal mammary ducts are shown in Fig. 1
, panels d and e, and MSel1-positive normal breast acini are shown in Fig. 1
, panel h. MSel1 also reacted strongly with plasma cells (Fig. 1, g and h)
. The "in situ" carcinomas were also positive, often staining more intensely stronger than their invasive counterparts (Fig. 1f)
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. The association with the histological subtype was not applicable, because 86% of cases were of ductal or mixed histotype. Interestingly, 4 of 5 pure lobular cases were SEL1L-negative.
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were not. Among the prognostic variables that were statistically significant in univariate analysis, only nodal status maintained its prognostic significance in multivariate analysis using proportional hazards model by Cox (Table 2)
, whereas SEL1L expression was not statistically significant.
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Southern Blot and Genomic PCR Analysis of MCF7-SEL1L and MCF-pDEX/1 Clones.
High molecular weight DNA was digested with BamHI, transferred to nitrocellulose filters, and hybridized with radiolabeled SEL1L cDNA insert ("Materials and Methods"). BamHI was used because it cuts the entire cDNA ligated to the promoter, releasing a 3.25-Kb fragment. Fig. 3
shows a Southern blot of three representative clones and from a mock-transfected clone. Clones A (Fig. 3
, Lane 2), B (Fig. 3
, Lane 3), and C (Fig. 3
, Lane 4) but not the control (Fig. 3
, Lane 1) showed the expected DNA fragment. The constitutive SEL1L genomic fragment was present, as expected, in all of the DNAs. The 8-kb fragment present in clone C (Fig. 3
, Lane 4) may indicate construct chromosomal rearrangements. To determine the status of the promoter-cDNA junction within the genome of the selected clones, PCR amplification was carried out using a primer set designed on the 3'-end of the cDNA (antisense IBD8) and on the pDEX/1 promoter (sense pDEX/1). As shown in Fig. 3
, clones A, B, and C (Fig. 3
, Lanes 1, 2, and 3, respectively) retained the entire SEL1L-cDNA construct in-frame with the promoter (2.8 kb). DNA from a mock-transfected clone was used as a negative control (Fig. 3
, Lane 4).
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, Lanes 1 and 2) and a mock-transfected clone (Fig. 4
, Lanes 3 and 4) showed no SEL1L signal before or after DEX induction, whereas clone A (Fig. 4
, Lanes 5 and 6), B (Fig. 4
, Lanes 7 and 8), and C (Fig. 4
, Lanes 9 and 10) clearly showed a marked increase of the 760-bp transcript only after induction. The presence of a faint band in these uninduced samples suggests promoter leakage resulting in a low DEX-independent expression of the SEL1L-transfected gene. As control, amplification of retrotranscribed RNAs was performed with HPRT-specific primers. A 200-bp band was present in all of the RNAs, with slight variation in intensity.
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, Lanes 1–6). Appreciable SEL1L levels were seen beginning at day 5 (Fig. 4
, Lane 5) and were sustained by day 7 (Fig. 4
, Lane 6). Amplification of total RNA was used as control for DNA contamination (Fig. 4
, Lanes 8–13). Fig. 4
, Lanes 7 and 14 reflect loading of negative controls, whereas Fig. 4
, Lane 15 shows the positive control.
Flow Cytometry Analysis of SEL1L Protein Levels in MCF-7-SEL1L Clones.
Induction of exogenous SEL1L protein in the SEL1L clones was measured by indirect immunofluorescence using MAb MSel1. Fig. 5
shows SEL1L protein levels expressed by clones A and B before and after DEX addition. Despite basal fluorescence attributable to endogenous SEL1L, a significant mean increase in fluorescence was observed in both clones after DEX induction, as determined using Kolmogorov-Smirnov statistical analysis. Mock-transfected controls (only one clone is shown) showed no significant increase. Fig. 5
panel shows a Western blot containing lysates from uninduced and DEX-induced clone A confirming the increase of SEL1L levels after DEX treatment.
Effect of SEL1L Expression Levels on Anchorage-dependent Growth of MCF7-SEL1L Clones
MCF7-SEL1L clones A, B, and C, and a mock-transfected control clone were grown in the absence or presence of DEX for 1 week to induce the transcription of exogenous SEL1L and to increase SEL1L cellular levels. Cells were then grown in adherence for 1, 3, 4, and 7 days, fixed, and assessed for protein content as described in "Materials and Methods." DEX-induced SEL1L expression leads to 50%, 58%, and 60% inhibition of growth rate at 7 days in clones A, B, and C, respectively (Fig. 6)
. DEX did not significantly affect growth of the two MCF7-pDEX/1 mock-transfected clones (shown in Fig. 6
for one clone). Comparable results were obtained using MCF7-SEL1L clones pretreated with DEX for 2 weeks. Chronic DEX treatment caused growth arrest and cell detachment of MCF7-SEL1 clones after 3–4 weeks treatment, with no affect on the growth of control MCF7-pDEX/1 cells. Terminal deoxynucleotidyl transferase (Tdt) -mediated nick end labeling analysis of adherent cells treated with DEX for 1 or 2 weeks revealed no increase in the number of apoptotic cells (
5%) in MCF7-SEL1 clones as compared with uninduced cells or mock-transfected DEX-treated cells. The ability of MCF7-SEL1L clones to form adherent colonies was also impaired. In a representative experiment, clone A seeded at 1000 cell/ml formed 24 colonies when DEX-induced but formed 173 colonies when uninduced. A mock-transfected clone formed 172 and 176 colonies with and without induction, respectively. In a series of three colony assay experiments, the number of colonies from DEX-treated clone A was significantly lower than from the untreated clone A or from the DEX-treated or untreated mock-transfected clone (P < 0.0001; Student’s t test).
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. Even wild-type MCF7 cells, grown with or without DEX and subjected to soft agar assay, showed no significant difference in cloning efficiency (Fig. 7
, top). Similar results were obtained when the induction time was increased from 3 to 7 days and when the number of cells seeded was increased from 5 x 104 to 1 x 105. The percentage of colonies formed in DEX-induced in regard to the uninduced cells ranged from 3% to 30% (mean = 12%) for MCF7-SEL1L cells and from 83% to 105% (mean = 92%) for control cells.
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shows a representative experiment with clone A indicating that the same number of colonies were obtained in Matrigel independent of SEL1 induction, whereas the same DEX-induced cells were highly impaired in regard to colony formation in soft agar. The control clone grew in Matrigel as well as in soft agar. Immunofluorescence analysis of expression of laminin-binding integrins (6 and ß1) and the 67LR laminin receptor revealed no differences between clone A, induced or noninduced, and a mock-transfected clone (Table 3)
. The only appreciable difference (+56%) was the level of SEL1L protein, detected with MAb MSel1, in induced MCF7-SEL1L-A cells, as expected. Similar results were obtained with clones B and C.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemical analysis of mammary breast normal and tumor tissues, and confocal microscopy analysis of breast cancer cell lines clearly indicated the cytoplasmic localization of SEL1L in vacuoles consistent with the compartmentalization of transfected SEL1L/green fluorescent protein recombinant gene expression in cytoplasmic vesicles (3) . This localization is also in line with a possible role for SEL1L in protein trafficking.
To investigate the involvement of SEL1L in breast carcinoma, we transfected the MCF-7 human breast carcinoma cells, found previously to constitutively express low levels of SEL1L, with a construct containing the SEL1L entire open reading frame under an inducible promoter. Increased SEL1L expression strongly reduced anchorage-dependent growth and almost completely abolished the capacity of these cells to grow in soft agar. These data suggest that loss of SEL1L expression in breast cancer leads to enhanced tumor cell proliferation and aggressiveness. The effect induced by the programmed expression of SEL1L appears to be irreversible, because only a few days of exposure to DEX are sufficient to trigger a permanent decrease in MCF7 cell growth.
Analysis of more than 30 tumor cell lines of different origin by RT-PCR, Northern blot, or fluorescence-activated cell sorter (16) 6 has consistently revealed some SEL1L expression. Indeed, we identified breast carcinoma cell lines with weak expression (MCF7) and with high expression (BT-474) but as yet have found no cell lines corresponding to the 28% of SEL1L-negative tumor samples. Taken in to account that SEL1L may be considered an "essential" gene (17) and that SEL1L may be expressed in in vivo tumor cells at levels not detectable by immunohistochemistry, we suggest that cells require SEL1L protein expression. After SEL1L cDNA transfection and DEX-induction, the SEL1L levels in MCF7-SEL1L cells are still lower than in BT474 cells, as evaluated by cytofluorimetric analysis. Therefore, we exclude that in our model the increased SEL1L levels may lead to an excess of SEL1L protein with conseguent toxic effect. In fact, DEX-induced MCF7 cells may survive for weeks, although they showed a marked decrease in cell growth ability. We believe that the increased level of SEL1L in DEX-induced MCF7-SEL1L cells is a re-expression of the SEL1L molecule at physiological level that causes a decrease in tumor aggressiveness.
The extracellular matrix profoundly influences the growth and differentiation of breast epithelial cells both in culture and in vivo (23)
, and regulates apoptosis and cell cycle progression through an integrin-dependent signaling (24)
. Interestingly, colony-forming ability of MCF7-SEL1L clones was restored when cells were grown in anchorage-independent conditions in the presence of Matrigel. It is possible that SEL1L influences the interaction of the cells with the extracellular matrix, for example, by modulating membrane adhesion-receptors. The increased level of SEL1L in the stained cells adjacent to the basal membrane of the in situ carcinoma (Fig. 1f)
is consistent with this possibility. The finding that SEL1L expression did not modulate the expression of two different laminin receptor expression does not rule out this hypothesis considering the large repertoire of molecules involved in cell/matrix interaction. Alternatively, SEL1L expression may inhibit endogenous synthesis of extracellular matrix by the tumor cells. Indeed, if laminin is not produced by the SEL1L-expressing cells, impairment of anchorage-independent growth and also of adherence would be expected, whereas a substrate rich in laminin such as Matrigel could still favor the growth of SEL1L-expressing cells. This is in keeping with a possible role of SEL1L in a secretory pathway as also suggested by the localization of SEL1L to intracellular vesicles. Gene expression profiling by microarray analysis and two-dimensional gel electrophoresis followed by mass spectroscopy of our biological system may shed light on this issue.
In conclusion, our data suggest that SEL1L is a cytoplasmic protein involved in cell growth control of breast cancer cells, and its down-modulation in breast carcinomas is associated with tumor aggressiveness. The mechanism involved is still unknown. Recent studies suggest that activin A induces SEL1L expression during morphogenesis of the salivary gland (25) and leads to a dramatic decrease in growth of T47D human breast cancer cells (26) . On the basis of these findings and our data, we speculate that SEL1L may be regulated by the transforming growth factor-ß family of growth factors and receptors, and may exert its function by influencing cell-matrix interactions and/or cytoplasmic regulation of membrane receptors that control cell growth and differentiation such as lin12/Notch/TAN.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Associazione Italiana per la Ricerca sul Cancro. M. C. was supported by a fellowship from Fondazione Italiana Ricerca sul Cancro. 
2 To whom requests for reprints should be addressed, at Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-23-90-25-71; Fax: 39-23-90-30-73; E-mail: menard{at}istitutotumori.mi.it
3 The abbreviations used are: MAb, monoclonal antibody; DEX, dexamethasone; RT-PCR, reverse transcription-PCR; SRB, sulforhodamine B; HPRT, hypoxanthine phosphoribosyltransferase.
4 R. Orlandi, Production of a monoclonal antibody directed against the recombinant SEL1L protein, manuscript in preparation.
5 G. Bunone, personal communication.
Received 7/18/01. Accepted 11/ 6/01.
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, high SEL1L expressors, 
, low SEL1L expressors, 
SEL1L-negative cases.
) and trypsinized, counted, seeded in 96-well plates, and grown for 1, 3, 4, and 7 days. Proliferation was assessed spectrophotometrically based on absorbance at 490 nm of SRB bound to cellular protein. Data are given as mean of quintuplicate determinations; bars, ± SD.
