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Cell Growth & Differentiation Vol. 10, 131-140, February 1999
© 1999 American Association for Cancer Research

Met-HGF/SF Mediates Growth Arrest and Differentiation in T47D Breast Cancer Cells1

Dvora Ronen, Rom T. Altstock, Michal Firon, Leonid Mittelman, Tama Sobe, James H. Resau, George F. Vande Woude and Ilan Tsarfaty2

Department of Human Microbiology [D.R., R.T.A., M.F., T.S., I.T.] and Interdepartmental Core Facility [L.M.], Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 69978, and ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201 [J.H.R., G.F.V.W.]


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Hepatocyte growth factor/scatter factor (HGF/SF) is a pluripotent growth factor that exerts mitogenic, motogenic, and morphogenic effects. To elucidate the cellular mechanisms underlying the pluripotent function of this growth factor, T47D human breast cancer cells were transfected with human hgf/sf. The hgf/sf-positive clones exhibited different levels of biologically functional HGF/SF expression and up-regulation of endogenous Met (HGF/SF receptor) expression. In addition, a constitutive phosphorylation of the receptor on tyrosine residues was detected, establishing a Met-HGF/SF autocrine loop. The autocrine activation of Met caused marked inhibition in cell growth accompanied by cell accumulation at G0/G1. These cells underwent terminal cell differentiation as determined by morphological changes, synthesis of milk proteins such as ß-casein and {alpha}-lactalbumin, and production of lipid vesicles. Our results demonstrate that Met-HGF/SF, an oncogenic signal transduction pathway, is capable of inducing growth arrest and differentiation in certain breast cancer cells and, thus, may have potential as therapeutic and/or prognostic tools in breast cancer treatment.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Most breast cancers evolve over a long latent period, proceeding through a stepwise progression from preneoplastic disease into fully invasive cancer capable of lymphatic spread and hematogenous metastasis (1) . These processes require cellular growth (mitogenicity), migration (motogenesis), and differentiation. However, the precise cellular mechanisms and the regulatory molecules that mediate these developmental stages remain mostly undetermined.

Several growth factors and their receptors play important roles in mammary differentiation and have been implicated in mammary carcinogenesis. Alteration in their structure, quantity, or subcellular localization may result in uncontrolled cell growth and/or modification of cell differentiation, which can lead to tumor formation (2) . Recently, attention has focused on the role of the tyrosine kinase growth factor receptor Met and its ligand HGF/SF3 in mammary development and carcinogenesis (3, 4, 5, 6) . HGF/SF is a paracrine factor, produced primarily by mesenchymal cells, that induces mitogenic, motogenic, and morphogenic behavior, as well as antiproliferative effects on a variety of epithelial cells. The diverse biological effects of HGF/SF are mediated by Met, which is preferentially expressed on epithelial cells (7) . HGF/SF is a potent mitogen for rat and human hepatocytes (8, 9, 10, 11) , human mammary epithelial cells (12, 13, 14) , keratinocytes (14 , 15) , human melanocytes (13 , 15 , 16) , and melanoma cells (14 , 15) . It also stimulates invasiveness of certain epithelial tumor cell lines (17 , 18) . Null mutations of HGF/SF and Met are embryonic lethal (19 , 20) . Although HGF/SF is characterized as a growth factor, it induces antiproliferative effects on rat and human hepatocellular carcinoma cells (21 , 22) and has a moderate cytotoxic effect on MCF7 human breast carcinoma cells. This antiproliferative effect has not been fully characterized, and little is known about the molecular mechanism(s) underlying it.

Met-HGF/SF plays a major role in the development of tubular/lumenal structures. HGF/SF induces MDCK cells cultured in three-dimensional collagen gels to form long branching structures reminiscent of kidney tubules (23) . We have shown that in vitro treatment of human colon and breast epithelial carcinoma cell lines with HGF/SF results in the formation of lumenal structures (3) . HGF/SF induces tubular branching of mammary cell lines in collagen gels (24 , 25) .

Met and HGF/SF have been implicated in breast cancer (3, 4, 5, 6 , 26 , 27) . We and others have shown that Met is expressed and activated in normal breast ducts and reduced in certain breast tumors (3 , 5 , 6 , 25) . In primary human breast cancer, loss of heterozygosity at the met locus highly correlates with risk of relapse, metastatic disease, and reduced overall survival (26) . Nagy et al. (27) have shown increased Met expression in tumor tissue compared with benign breast diseased tissues, but no correlation with prognosis, whereas Jin et al. (28) reported overexpression of Met in breast cancer and coexpression of Met and HGF/SF in breast cancer tissue. Other studies show that a high expression level of HGF/SF is a strong and independent predictor of recurrence and reduced survival in human breast cancer (4 , 29) . These results show that additional studies are required to determine the exact role of Met-HGF/SF in breast cancer.

Constitutive activation of growth factor receptors through an autocrine mechanism frequently occurs in human cancers and is thought to play an important role in breast carcinogenesis (30) . We previously demonstrated that when human Met and its ligand are coexpressed in a variety of mouse and human cells of mesenchymal origin, the cells become highly tumorigenic in nude mice through an autocrine mechanism (31, 32, 33) . Despite the epithelial specificity of lumen formation, the mesenchymal tumor explants of these cells acquire lumen-like morphology in vitro and in vivo. Furthermore, histopathological examination of paraffin-embedded tumor sections shows, in addition to lumenal structures, carcinoma-like focal areas (34) . Previous studies describe Met-HGF/SF autocrine loops inducing in vitro invasiveness in murine mammary carcinoma cells (35 , 36) .

In the present study, we show that generation of an autocrine Met-HGF/SF loop in the T47D breast cancer cell line suppresses growth and induces a differentiated phenotype, as opposed to increased tumorigenicity and metastasis. In addition, this constitutive activation of Met receptor leads to accumulation of the cells in G0/G1. Thus, Met-HGF/SF signal transduction can play opposing roles in breast cancer by activating an intrinsic cellular program that elicits either proliferation or differentiation.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Expression of HGF/SF in T47D Breast Cancer Cells.
To investigate the cellular consequence of autocrine Met activation, T47D human breast cancer cells, which express low levels of endogenous Met, were cotransfected with human hgf/sf cDNA and pSV2neo plasmids. Thirty hgf/sf-transfected, G418-resistant clones were collected from two separate transfections and screened for HGF/SF expression using immunofluorescence analyzed by CLSM and WB analysis. Six clones showed positive staining for HGF/SF (Fig. 1A)Citation . These hgf/sf-transfected T47D clones (designated B2, B3, B4, B5, B6, and B7) expressed heterogeneous levels of HGF/SF, with clone B5 displaying the highest level of HGF/SF (Fig. 1A)Citation . Most of the cells in clones B5, B6, and B7 were homogeneous for high HGF/SF expression. Clone B2 expressed varying levels of HGF/SF, with a minority of the cells exhibiting very high levels, whereas the majority expressed low to moderate HGF/SF levels (Fig. 1A, B2)Citation . Clones B3 and B4 expressed relatively low homogeneous levels of HGF/SF (Fig. 1A, B3 and B4)Citation . The control clones, pSV2neo-transfected and G418-resistant, designated D9 and D12, were negative for HGF/SF staining (Fig. 1A, D9 and D12)Citation . Similar negative staining was observed in the parental T47D cells (data not shown). These results were corroborated by WB of heparin-sepharose-precipitated supernatants collected from each clone. Detection of 34 kDa ß subunit of HGF/SF (37) by WB analysis, shows that clones B2, B5, B6, and B7 (Fig. 1BCitation , Lanes 3, 6, 7, and 8, respectively) are higher expressers of HGF/SF, whereas clones B3 and B4 (Fig. 1BCitation , Lanes 4 and 5, respectively) exhibit lower levels of HGF/SF expression. HMH (NIH/3T3 cells transfected with human met and human hgf/sf) supernatant served as positive control (Fig. 1BCitation , Lane 1). Quantitative analysis of HGF/SF levels, determined by CLSM and image analysis software, confirmed the observation that B5 expresses the highest levels of HGF/SF (37% PPA). B6 and B7 also expressed high levels of HGF/SF (28% PPA and 22% PPA, respectively). B2 exhibits moderate levels of HGF/SF (18% PPA), and B3 and B4 express low levels of HGF/SF (3.2% and 4% PPA, respectively; Fig. 1ACitation ). Moreover, the HGF/SF secreted by the positive clones was biologically active, as determined by its ability to induce scattering of MDCK cells. A demonstration of scatter activity in supernatants collected from B5-transfected cells is shown in Fig. 1C b.Citation Scatter activity of HGF/SF secreted in conditioned medium collected from different HGF/SF-transfected clones is shown in Table 1Citation . These results indicate that the biological activity of HGF/SF secreted from the various clones correlates with the levels detected within the cells.



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Fig. 1. HGF/SF expression in hgf/sf-transfected T47D cells. A, immunofluorescence staining of HGF/SF. Cells from HGF/SF-transfected clones (B2–B7) and control transfected clones (D9 and D12) were grown in 8-well Lab Tek chamber slides, fixed, reacted with rabbit anti-HGF/SF antibody, and labeled using FITC-conjugated antirabbit antibody. Images were obtained by CLSM (magnification, x110). Relative HGF/SF levels were calculated using PPA for each clone, as described in "Materials and Methods," and displayed in bar graph (n = three independent experiments). B, WB analysis of secreted HGF/SF. Supernatants collected from HGF/SF transfectants and control transfected clones were concentrated by HS, subjected to 12% SDS-PAGE, and analyzed by WB using rabbit anti-HGF/SF antibodies: supernatant from HMH cells (Lane 1); supernatant from D12 (Lane 2); supernatants from B2–B7 cells (Lanes 3–8, respectively); and supernatants from T47D parental cells (Lane 9). C, scatter assay using MDCK cells. Supernatants collected from cultures of D12 cells (a) and B5 cells (b) were diluted 1:32 and added to MDCK cell cultures. After an overnight incubation, MDCK cells were fixed, stained, and observed by inverted light microscope (magnification, x50).

 

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Table 1 Scatter activity in conditioned medium collected from HGF/SF-transfected clones

 
Met Up-Regulation and Activation in hgf/sf-transfected T47D Clones.
To investigate the effect of constitutive HGF/SF expression on endogenous Met levels, we examined the HGF/SF-producing clones for Met expression using immunofluorescence staining, CLSM, and image analysis. Control clones D9 and D12 showed a very low basal level of endogenous Met expression. The high HGF/SF producer clones (B2, B5, B6, and B7), exhibited increased Met expression levels by 6- (P < 0.0005), 8- (P < 0.001), 7.5- (P < 0.0005), and 7- (P < 0.0001)-fold, respectively, compared with control clones. A moderate increase in Met expression was also observed in the low HGF/SF producer clones B3 and B4, by 2- (P < 0.05) and 6- (P < 0.0001)-fold, respectively (Fig. 2A)Citation . WB analysis confirmed the immunofluorescence-detected Met up-regulation in the HGF/SF-transfected clones. Total cell extracts obtained by pooling several plates of each HGF/SF-transfected clone, control D12 cells and T47D parental cells were IP using 19S anti-Met antibody and immunoblotted with C28 antihuman Met antibody. Results show a faint signal corresponding to p140h-Met in D12 and T47D cell extracts, indicating a very low basal level of endogenous Met (Fig. 2BCitation , Lanes 2 and 9, respectively). All cell extracts obtained from HGF/SF-transfected clones (B2–B7) show significantly elevated signal for p140h-Met mature protein, (Fig. 2BCitation , Lanes 3–8, respectively) compared with control extracts (Fig. 2BCitation , Lanes 2 and 9). Having established HGF/SF secretion and up-regulation of its receptor, Met, we proceeded to confirm the presence of an autocrine loop by determining Met phosphorylation in HGF/SF-transfected clones. Results of IP with anti-Met antibody and WB using antipTyr antibody show tyrosine phosphorylation on p140h-Met in HGF/SF-transfected cell extracts (Fig. 2CCitation , Lanes 3–8) compared with control cell extracts (Fig. 2CCitation , Lanes 2 and 9). In addition, B4, B5, B6, and B7 (Fig. 2CCitation , Lanes 5–8) extracts show an increase in p110 and p90 phosphorylation, possibly indicating association and phosphorylation of Met substrates (under further investigation). These results indicate that the ectopic expression of HGF/SF in T47D cells establishes an active Met-HGF/SF autocrine loop.



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Fig. 2. Met expression in hgf/sf-transfected T47D cells. A, immunofluorescence staining of Met. HGF/SF-transfected clones (B2–B7) and control transfected clones (D9 and D12) were grown in 8-well Lab Tek chamber slides, fixed, reacted with anti-Met C28 antibody, and labeled using FITC-conjugated antirabbit antibody. Images were obtained by CLSM (magnification, x110). Relative Met levels were calculated using PPA for each clone, as described in "Materials and Methods," and displayed in bar graph (n = three independent experiments). B, WB analysis of Met. HMH cell extract (0.03 mg; Lane 1) and 1 mg of cell extract of each of the clones D12 (Lane 2), B2 (Lane 3), B3 (Lane 4), B4 (Lane 5), B5 (Lane 6), B6 (Lane 7), B7 (Lane 8), and T47D cells (Lane 9) were IP with anti-Met 19S antibody. Precipitated samples were subjected to 7.5% SDS-PAGE and WB analysis using anti-Met C28 antibody. C, WB analysis of Met phosphorylation. HMH cell extract (0.03 mg; Lane 1) and 1 mg of cell extract of each of the clones D12 (Lane 2), B2 (Lane 3), B3 (Lane 4), B4 (Lane 5), B5 (Lane 6), B6 (Lane 7), B7 (Lane 8) and T47D cells (Lane 9) were IP with anti-Met 19S antibody. Precipitated samples were subjected to 7.5% SDS-PAGE and WB analysis using anti-pTyr antibody. D, RNA fluorescence in-situ hybridization of Met. Cells from clones B5 (a and b) and D12 (c and d) were plated in 8-well Lab Tek chamber slides, fixed, and probed with antisense (a and c) or sense (b and d) met oligo probes. Images were analyzed by CLSM (magnification, x130).

 
To further characterize the increase in Met expression observed in HGF/SF-transfected clones, we examined the levels of Met mRNA in one representative clone—B5. Met transcripts in B5 and D12 cells were detected by in situ hybridization with antisense met-specific biotin-labeled oligonucleotides, stained with streptavidine conjugated to FITC and analyzed by CLSM. Sense met oligonucleotides were used as controls. B5 cells expressed higher levels of met mRNA (20-fold as calculated by PPA) compared with control D12 cells (Fig. 2DCitation , a and c, respectively). The control sense met oligo probe had only a low background signal in both B5 and D12 cells, confirming the specificity of the hybridization (Fig. 2DCitation , b and d, respectively).

Growth Arrest of hgf/sf-transfected T47D Cells.
To determine the consequence of Met-HGF/SF autocrine loop on T47D cell growth, we analyzed HGF/SF-transfected T47D clones growth rate. Cells (5 x 104) of each clone were seeded in 24-well plates and monitored for cell viability on days 2, 4, 7, and 11. Subsequently, the generation time for each clone was calculated (38) . The number of D9 and D12 control cells increased with time and continued to grow even after 11 days in culture ((Fig. 3A, a)Citation , with a generation time of 40 h in both clones (Fig. 3A, b)Citation . HGF/SF-transfected clones showed a marked inhibition in growth rate (Fig. 3A, a and b)Citation . Generation time of B5 cells was dramatically prolonged (220 h), by 5.5-fold compared with that found in D9 and D12 cells. Clones B2 and B6 also exhibited a prolonged generation time of 84 h and 72 h, respectively. Clones B3, B4, and B7 had generation times of 53 h, 48 h, and 54 h, respectively. The growth inhibition observed in the various transfected clones correlated with HGF/SF expression levels.



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Fig. 3. Growth arrest of hgf/sf-transfected T47D cells. A, growth rate of T47D transfectants: a, growth curve. Cells from clones B2 (—{square}—), B3 (—{triangleup}—), B4 (X), B5 (—{circ}—), B6 (—{blacktriangleup}—), B7 (+), D9 (—•—), and D12 (—{diamondsuit}—) were seeded in 24-well plates at 5 x 104/ml. On days 2, 4, 7, and 11 after seeding, cells were harvested, stained with trypan blue, and counted. b, generation time. Bar graph depicting the generation time for each of the T47D-transfected clones calculated, as described in "Materials and Methods." B, BrdUrd incorporation in hgf/sf-transfected T47D cells. Cells from clones D12 (a) and B5 (b) were analyzed for BrdUrd incorporation. After incubation with BrdUrd, cells were fixed, reacted, with mouse anti-BrdUrd antibody, and labeled with FITC-conjugated antimouse antibody. Images were obtained by CLSM (magnification, x170). C, cell cycle analysis of T47D transfectants. Cells from clones D12 (a) and B5 (b) were fixed, stained with propidium iodide, and analyzed by flow cytometry. Cells in stages G0/G1 of the cell cycle are depicted by the first peak (2N). The number of cells at G2/M is illustrated by the second peak (4N). The low dark gray area between the two peaks accounts for the cells in stage S phase.

 
The prolonged generation time detected in B5 cells led us to examine the effect of HGF/SF on DNA synthesis. Analysis of DNA synthesis using BrdUrd incorporation showed that although 10–15% of the cells in control clone D12 incorporated BrdUrd, only 1–2% of the cells in clone B5 incorporated the drug (Fig. 3B, a and bCitation respectively). These results suggest that the inhibition in cell growth, induced by Met-HGF/SF interaction, was a result of the inhibition in cellular DNA synthesis.

To further understand the inhibition in cell growth we compared cell cycle distribution of B5 and D12 cells using propidium iodide staining and FACS analysis. Fifty-nine percent of D12 control cells had 2N DNA content indicative of G0/G1, 23% of the cells were in S phase, and 18% had 4N DNA content indicative of G2/M (Fig. 3C, a)Citation . Cell cycle analysis of B5 cells showed that 74% of the cells had a 2N DNA content, indicating that this cell population was predominantly in the G0/G1 stage of the cell cycle. Thirteen percent were at S phase, and 13% were at G2/M phase of the cell cycle (Fig. 3C, b)Citation . These results further demonstrate the growth inhibition of hgf/sf-transfected cells, with most of the cells retarded at G0/G1 phase and their ability to progress to S phase and G2/M phase inhibited.

Met-HGF Autocrine Loop Induces Differentiation of T47D Cells.
The observation that HGF/SF caused growth arrest in the transfected clones at the G0/G1 phase of the cell cycle raised the question whether the cell growth arrest led to apoptosis or terminal differentiation. Examination for cellular apoptosis by FACS (Fig. 3C, b)Citation and by 4',6-diamidino-2-phenylindole (DAPI) staining (data not shown) revealed no apoptosis in the hgf/sf-transfected clones. We subsequently examined the transfected cells for progression toward terminal differentiation. Because differentiation and maturation of luminal breast epithelial cells are characterized by synthesis of milk proteins such as ß-casein (39, 40, 41) and {alpha}-lactalbumin (41, 42, 43) , we examined the expression of these proteins in T47D transfectants using immunofluorescence staining and CLSM analysis. ß-casein was highly and homogeneously expressed in the cytoplasm of B2, B5, B6, and B7 cells compared with the very low expression levels in D9 and D12 control cells (Fig. 4A)Citation . Quantification of the relative levels of ß-casein expression showed 64% PPA (P < 0.001), 50% PPA (P < 0.0001), 42% PPA (P < 0.002), and 48% PPA (P < 0.001) in clones B2, B5, B6, and B7, respectively, (Fig. 4A)Citation . The low HGF/SF-producing clone B3 showed 26% PPA (P < 0.05) and B4 showed 8% PPA (P < 0.07) (not significant), compared with 0.7% PPA in D9 and D12 (Fig. 4A)Citation . Examination of expression of another cell differentiation marker, {alpha}-lactalbumin, revealed a high induction of this protein in clones B2 (52% PPA, P < 0.002), B5 (76% PPA, P < 0.0001), B6 (57% PPA, P < 0.001), and B7 (54% PPA, P < 0.001), compared with 6% PPA in D9 and D12 control cells (Fig. 4B)Citation . A slight increase in {alpha}-lactalbumin was observed in B3 and B4 (not statistically significant), compared with control clones (Fig. 4B)Citation .



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Fig. 4. Differentiation of hgf/sf-transfected T47D cells. A, immunofluorescence staining for ß-casein. Cells from HGF/SF-transfected clones and control transfected clones were grown in 8-well Lab Tek chamber slides, fixed, reacted with antihuman ß-casein antibody, and labeled using Texas-Red-conjugated antimouse antibody. Images were obtained by CLSM (magnification, x200). Relative ß-casein levels were calculated using PPA for each clone, as described in "Materials and Methods," and displayed in bar graph (n = three independent experiments). B, immunofluorescence staining for {alpha}-lactalbumin. Cells from HGF/SF-transfected clones (B2–B7) and control (D9 and D12) transfected clones were grown in 8-well Lab Tek chamber slides, fixed, reacted with antihuman {alpha}-lactalbumin antibody, and labeled using Texas-Red-conjugated antirabbit antibody. Images were obtained by CLSM (magnification, x200). Relative {alpha}-lactalbumin levels were calculated using PPA for each clone, as described in "Materials and Methods," and displayed in bar graph (n = three independent experiments). C, WB analysis of differentiation markers of HGF/SF-transfected T47D cells. Extract (10 µg) of lactating human breast tissue (Lane 1) and 60 µg extract of each of the clones D12 (Lane 2), B2 (Lane 3), B3 (Lane 4), B4 (Lane 5), B5 (Lane 6), B6 (Lane 7), B7 (Lane 8), and T47D cells (Lane 9) were subjected to a 15% SDS-PAGE and analyzed by WB using antibodies for ß-casein (a), {alpha}-lactalbumin (b), and actin (c). D, Oil Red-O staining of hgf/sf-transfected T47D cells. B5 and D12 cells were cultured in 4-well plates. After fixation, cells were stained for lipid droplets by Oil Red-O and viewed by inverted light microscope (magnification, x260).

 
The induction of these cell differentiation markers in the HGF/SF-transfected clones was confirmed by WB analysis. Using anti-ß-casein antiserum, a 26 kDa band corresponding to ß-casein was detected in HGF/SF producer clones (Fig. 4C, aCitation , Lanes 3–8). These bands correlate to results exhibited by lactating human breast tissue extract (Fig. 4C, aCitation , Lane 1). No detectable ß-casein induction was observed in extracts obtained from D12 or T47D cells (Fig. 4C, aCitation , Lanes 2 and 9). WB analysis using anti-{alpha}-lactalbumin verified the significant increase in {alpha}-lactalbumin (16 kDa) in the HGF/SF-transfected cells (Fig. 4C, bCitation , Lanes 3–8), compared with the D12 control or T47D parental cells (Fig. 4C, bCitation , Lanes 2 and 9). The observed results correlate with the band exhibited by lactating human breast tissue extract. Actin expression in the different cell extracts served as control for equal protein levels (Fig. 4C, cCitation , Lanes 2–9).

Lipid synthesis was analyzed using "Oil Red-O" staining and microscopic examination of at least 100 cells. Oil Red-O-positive staining was found in 70% of B5 cells, whereas only 5% of D12 cells exhibited Oil Red-O-stained droplets (Fig. 4D)Citation . These results indicate that the Met-HGF/SF autocrine loop in T47D cells induces cell differentiation that correlates with HGF/SF expression level.

Changes in Cellular Morphology in hgf/sf-transfected T47D Cells.
Prolonged maintenance of clone B5, through continuous passages, revealed remarkable morphological changes in cell shape compared with D12 cells. D12 control cells grew in packed colonies with morphology of immature cells, characterized by compact nuclei enclosed by a fine layer of cytoplasm (Fig. 5, C and D)Citation . In contrast, B5 cells grew sparsely, with a typical scattered morphology and no cell colony formation. The cells exhibited mature cell morphology characterized by lacy nuclei surrounded by sizeable cytoplasms (Fig. 5, A and B)Citation . Measurement of the nucleus:cytoplasm ratio showed a value of 0.4 and 0.1 for D12 and B5 clones, respectively. This decrease in nucleus:cytoplasm ratio is also consistent with cell differentiation toward maturation.



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Fig. 5. Morphological changes in hgf/sf-transfected T47D cells. B5 (A and B) and D12 (C and D) cells grown in tissue culture plates were fixed, Giemsa-stained, and viewed by inverted light microscope. Images were obtained using Olympus microphotographic system (Olympus) at magnification x50 (A and C) and magnification x280 (B and D).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The present study focuses on understanding the effects of Met activation by its ligand HGF/SF, via an autocrine loop, on epithelial breast cancer cells. We generated an in vitro autocrine model system by stable expression of HGF/SF in T47D breast cancer cells, which express low endogenous levels of Met.

The stable hgf/sf-transfected cells expressed and secreted the mature form of HGF/SF. This HGF/SF was biologically active, as determined by scatter assay on MDCK cells. All T47D hgf/sf-transfected clones expressing HGF/SF exhibited significant endogenous Met up-regulation. The magnitude of increased Met expression correlated directly with the level of HGF/SF expressed. This up-regulation of Met was associated with an increase in met mRNA, accompanied by an elevated level of p140h-Met mature protein. A similar induction of Met by HGF/SF was described in human lung adenocarcinoma cells. HGF/SF treatment of these cells induced an up-regulation of met mRNA, followed by an increase in protein expression (44) . This effect, determined to be time- and dose-dependent, occurred due to the existence of transcriptional responsive elements located 297 bp upstream to the met transcriptional start site (44 , 45) . Ligand-induced up-regulation of a receptor was also described for the insulin receptor in MCF7 cells, followed by growth arrest, where insulin induced an increase in receptor mRNA levels via a posttranscriptional mechanism that increased mRNA stability (46) . Previously, it was shown that HGF/SF treatment down-regulated Met expression in other systems where Met is associated with cell proliferation (47) . The decrease in Met levels was caused by specific degradation of the receptor by the cellular ubiquitin-proteosome system (47) . It is, thus, conceivable that in the T47D system Met up-regulation could result from suppression of cellular ubiquitin system. It is also possible that the relatively low endogenous levels of Met, compared with the higher levels in previously described systems, are significant to the outcome of HGF/SF activation.

In addition to Met up-regulation we found a constitutive phosphorylation of Met tyrosine residues, indicating a functionally active Met-HGF/SF autocrine loop capable of eliciting signaling events. We show here that this autocrine loop induces growth arrest and cell differentiation. T47D cells can be induced either to proliferate or differentiate, depending on the stimulator. Prolactin and androgens induce alteration in cell morphology and cause lipid droplet accumulation in T47D cells via their own receptors (48 , 49) . A similar differentiation process can be achieved in T47D cells by treatment with retinoic acid without affecting the level of retinoic acid receptor (50) . In contrast, stimulation of the same cells with NDF/Heregulin results in activation of its receptor and cell proliferation (51) .

Opposing phenotypes induced by Met-HGF/SF signal transduction were also observed in different cell systems. An endogenous autocrine Met-HGF/SF loop in S1 murine mammary tumor cells led to enhancement of cell growth and invasive phenotype of the cells (35) . Other studies showed antiproliferative effect of HGF/SF on several tumor cell lines, including carcinomas and sarcomas (21 , 22 , 52) . Met-HGF/SF autocrine loop generated in T47D cells induced growth arrest with accumulation of cells at the G0/G1 stage of the cell cycle and acquisition of differentiated phenotype. The hgf/sf-transfected T47D cells underwent an apparent morphological and functional terminal differentiation process. The cells showed morphological changes exhibiting a more mature phenotype accompanied by the synthesis of differentiation markers such as ß-casein, {alpha}-lactalbumin, and oil droplets. Previous studies showed that in mammary gland organ culture, HGF/SF promoted branching of ductal trees, but inhibited the production of secretory proteins and ß-casein (6 , 53) . In the T47D autocrine system, HGF/SF does not induce extensive branching, but does induce growth arrest and milk protein production. The different responses could be attributed to the cellular heterogeneity of the mammary organ culture and the transformed phenotype of the T47D cells. The organ culture comprises a variety of cells that secrete a wide range of substances (e.g., hormones—prolactin) that alter HGF/SF activity, whereas T47D HGF/SF autocrine system comprises cloned cells affected primarily by the HGF/SF signal transduction. Induction of differentiation markers such as ß-casein and {alpha}-lactalbumin was also described in HC11 mammary epithelial cells after treatment with lactogenic hormones (39 , 40 , 54) . Similarly to the T47D HGF/SF system, an endogenous Met-HGF/SF autocrine loop was shown to promote the differentiation of human monocytes to macrophages (55) . In contrast, constitutive activation of Met in C2 mouse myoblasts provided a negative stimulus, which inhibited myogenesis (56) . These findings indicate that the autocrine nature of Met-HGF/SF drives cells to opposing phenotypes, depending on the cell origin.

Activation of Met-HGF/SF signal transduction in breast tumor cells can induce either cell proliferation and invasiveness or, as observed here, growth arrest and cell differentiation. These opposing effects of HGF/SF on tumor cells indicate that its activity depends strongly on the initial differentiative state of the cell, which might be determined by the availability of specific Met substrates. It was shown that Met-induced activation of the signal transducers and activators of transcription (STAT) pathway leads to tubulogenesis (57) , whereas activation of Ras and Src signaling is involved in mitogenicity and motogenicity (58, 59, 60) . Thus, it can be suggested that in poorly differentiated cells where low levels of substrates for "differentiative" signal transduction exist, Met-HGF/SF might signal for cell proliferation. In more differentiated cells, such as T47D, where intrinsic factors for cell differentiation are present, Met-HGF/SF interaction induces growth arrest and cell differentiation.

Understanding the cellular mechanisms and intrinsic cellular status of cell differentiation underlying these opposing effects induced by Met-HGF/SF signaling may shed light on the molecular processes leading to breast cancer progression. This, in turn, may contribute to the development of therapeutic modalities that induce cell differentiation of breast cancer tumors.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines, cDNA Plasmids and Antibodies.
T47D mammary carcinoma cell line (61) was grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (Life Technologies, Inc.). MDCK epithelial cells (type 2) were provided by Dr. K. E. Mostov (University of California, San Francisco, CA). MDCK cells were grown in DMEM (Life Technologies, Inc.) supplemented with 5% heat-inactivated FCS (Life Technologies, Inc.). HMH cells, NIH/3T3 cells transfected with human met and human hgf/sf, were maintained as described previously (31) . Human hgf/sf plasmid was constructed by inserting the 2.3-kb BamHI-KpnI fragment of the human HGF/SF sequence (62) into the BamHI-KpnI site of pMEX expression vector (31) . The following antibodies were used: rabbit polyclonal C28 antihuman Met peptide antibody (Ref. 63 ; Santa Cruz Biotechnology, Santa Cruz, CA), 19S antihuman Met mAb (64) , SP260 antimouse Met antibody (Ref. 65 ; Santa Cruz Biotechnology), rabbit polyclonal antirecombinant human HGF/SF antibody (66) , mouse monoclonal anti-pTyr 4G10 mAb (UBI, Lake Placid, NY), rabbit antihuman {alpha}-lactalbumin (Accurate Chemical and Scientific Corporation, Westbury, NY), mouse monoclonal antihuman ß-casein (Accurate Chemical and Scientific Corporation), and mouse monoclonal antiactin (Boehringer Mannheim, Mannheim, Germany).

DNA Transfection.
The Lipofectin reagent kit (Life Technologies, Inc.) was used for DNA transfection, as suggested by the manufacturer. In short, T47D cells at 70% confluency were either cotransfected with 4 µg of human hgf/sf and pSV2neo plasmids (1:1) or with pSV2neo (31) . The plasmids were suspended in buffer solutions and added to the T47D cells. After 18 h of incubation at 37°C, the transfecting mixture was replaced with DMEM medium containing 10% FCS. After 72 h of incubation at 37°C, the transfected cells were divided 1:5, grown in DMEM medium containing 10% FCS and 1 mg/ml G418 (Life Technologies, Inc.), and 30 resistant clones were isolated.

Immunofluorescence Staining and Confocal Analysis.
Lab-Tek chamber slides (8-well; Nunc, Roskilde, Denmark) were seeded with 104 cells/well in appropriate medium. Cells were fixed in absolute cold methanol for 10 min and consequently permeabilized with cold acetone for 10 min. After blocking (5% BSA, 10% normal donkey serum in PBS) for 10 min at room temperature, the cells were incubated with primary antibody for 1 h at room temperature. After three washes in PBS, cells were stained with either donkey antirabbit or antimouse antibody conjugated to either FITC or Texas Red (Jackson Immuno Research Laboratories, West Grove, PA) diluted 1:50 for 1 h at room temperature. Slides were washed three times in PBS and mounted with coverslips using GelMount (Biomeda, Foster City, CA). Immunostained cells and sections were analyzed using a 410 Zeiss (Oberkochen, Germany) CLSM with the following configuration: 25 mW Krypton/Argon (488, 568 nm) and HeNe (633 nm) laser lines. Images were printed using Codonics 1600 dye sublimation color printer (Codonics, Middleburg Heights, OH). When comparing fluorescence intensities, identical CLSM parameters (e.g., scanning line, laser light, contrast, and brightness) were used. To compare the relative levels of protein expression, we used the PPA image analysis procedure previously described (3) . In short, PPA was calculated as a ratio between the positive stained area and the total cellular area. The positive stained area was determined by measuring the fluorescent intensity of the image, which is above the positive cutoff intensity. Positive cutoff intensities were determined based on the fluorescence intensities histogram for each antibody staining. Total cellular area was determined by measuring the fluorescent intensity above the surrounding background of the image and depicts the cellular auto-fluorescence. The PPA calculation allows for the quantitative comparison of protein expression by cell populations or in tissue sections. The PPA data shown represents the calculated average of at least three different CLSM fields. Statistical significance was calculated using the Student’s t test in Microsoft Excel (Microsoft, Redmond, WA).

In Situ Hybridization.
In situ hybridization was performed as previously described (67) . Briefly, cells were spotted on 2% 3 aminopropyltriethoxy-silane (AES) (Sigma Chemical Co., St. Louis, MO)-coated slides, fixed in 4% paraformaldehyde for 20 min, and treated with 0.3% Triton X100 solution for 15 min. Permeabilization was performed with 1 µg/ml proteinase K (Amresco, Solon, OH) in TE buffer for 10 min at room temperature. Cells were then washed in 2 x SSC for 1 min and a graded series of ethanol washes (70%, 80%, 90%, and 100%) for 1 min in each. Prehybridization was performed by incubating slides at 37°C in OmniSlide (Hybaid, Teddington, England) for 2 h in hybridization buffer (50% formamide, 1 x Denhart, 4 x SSC, Salmon sperm ssDNA, 25 µg/ml yeast tRNA, and 10% dextransulfate). Slides were hybridized using the same buffer either with two biotinylated sense met oligoprobes (mouse met positions 1918–1939, 2298–2320, 500 ng/ml each) or with two biotinylated antisense met oligoprobes of the same positions at 37°C for 16 h. After hybridization, the slides were washed four times in 2 x SSC and 50% formamide at 40°C, twice in 2 x SSC and 50% formamide, and once in 1 x SSC for 1 h at 22°C. After rinsing in ethanol, the slides were incubated with streptavidin fluorescein (Amersham Corp., Arlington Heights, IL) for 1 h at room temperature, washed in PBS, mounted, and analyzed by CLSM.

WB Analysis of HGF/SF.
Supernatants were collected from confluent cells, filtered, and incubated with 0.2 mg/ml HS (Pharmacia-Bio-tec, Uppsala Sweden) for 2 h at room temperature. After incubation, the HS was washed three times with PBS. Pellets were suspended in sample buffer, resolved on 12% SDS-PAGE, and analyzed by WB analysis using rabbit anti-HGF/SF antibody diluted 1:350. Visualization was achieved using horseradish peroxidase-conjugated antimouse IgG antibody (1:5000; Amersham Corp.), enhanced chemiluminescence reaction, and exposure to X-ray film (Fuji, Tokyo, Japan).

Immunoprecipitation and WB Analysis of Met and Met Tyrosine Phosphorylation.
Near confluent cells in 90-mm diameter dishes were washed twice with cold PBS and lysed in 0.2 ml of lysis buffer [20 mM Tris-HCL (pH 7.8), 100 mM NaCl, 50 mM NaF, 1% NP40, 0.1% SDS, 2 mM EDTA, and 10% glycerol] with protease inhibitor mixture (Boehringer Mannheim) and 1 mM sodium orthovanadate. Cell lysates pooled from several plates, were clarified by centrifugation, and 1 mg of cell lysate protein was IP with 19S anti-Met mAb. The immunoprecipitates were evaluated by WB analysis using either C28 peptide anti-Met antibody (1:300) or 4G10 anti-pTyr (1:1500). Visualization was achieved using horseradish peroxidase-conjugated antimouse IgG antibody (1:5000; Amersham Corp.), enhanced chemiluminescence reaction, and exposure to X-ray film (Fuji).

Scatter Assay.
Scatter assay was carried out as previously described (68) . MDCK cells (5000 cells/well) were grown in DMEM supplemented with 5% FCS in 96-well plates (Nunc). Supernatants collected from hgf/sf-transfected T47D cells and T47Dneo cells were added to the wells, and plates were incubated overnight. Cells were washed with PBS and fixed in methanol, air dried, stained with Giemsa in water (1:4), and microscopically examined for scattering (spread and dispersion of epithelial colonies). The highest dilution at which scatter effect was observed was defined as 0.5 HGF/SF scatter unit/ml. HGF/SF concentration in T47Dneo and hgf/sf-transfected T47D cell supernatants was calculated accordingly.

BrdUrd Labeling.
BrdUrd labeling was carried out as previously described (69) . hgf/sf-transfected T47D cells and T47Dneo cells were grown in 8-well Lab-Tek slides (Nunc) for 20 h and labeled with BrdUrd for 3 h using the Amersham cell proliferation kit (Amersham Corp.). The cells were washed and fixed in 5% acetic acid and 90% ethanol for 30 min at room temperature and then washed extensively with PBS. Fixed cells were incubated for 1 h at room temperature with anti-BrdUrd antibody diluted 1:100 and nuclease (as specified by manufacturer). Cells were then washed in PBS and incubated at room temperature with FITC-labeled donkey antimouse antibody. After extensive washing, cells were mounted on slides with Gel Mount and examined by CLSM.

FACS Analysis.
Cells (1 x 106) were fixed in 70% ethanol for 30 min at 4°C. Cells were washed twice with PBS, resuspended in PBS, and incubated with 10 mg of DNase-free RNase (Boehringer Mannheim) for 30 min at 4°C. Propidium iodide (50 µg/ml; Sigma Chemical Co.) was added, and the samples were scanned on a FACSort counter (Becton Dickinson, Mountainview, CA) using a 610 bandpass filter in peak versus area signal to exclude aggregates. Histograms were analyzed using MPLUS Phoenix software.

Lipid Visualization.
A modified Oil Red O staining (70) was used to visualize neutral lipids in cells. Briefly, after the medium was removed, cells were fixed in 1% CaCl2, 10% formamide. After washing in water, cells were stained in Oil Red Solution (0.3% Oil Red in 60% isopropanol) for 15 min at room temperature. Slides were then rinsed with deionized water and counterstained with hematoxylin blue. Red staining of neutral lipids was examined by transmitted light microscope (Olympus, Tokyo, Japan).


    Footnotes
 
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 Partially supported by United States-Israel Binational Science Foundation Grant 93-00072 and by the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities. Back

2 To whom requests for reprints should be addressed, at Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv/Tel Aviv 69978, Box 39040, Israel. Phone: 972-3-640-7015; Fax: 972-3-640-9160; E-mail: ilants{at}post.tau.ac.il Back

3 The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; MDCK, Madin-Darby canine kidney; WB, Western blot; CLSM, confocal laser scan microscope; PPA, percentage of positive area; FACS, fluorescence-activated cell sorter; IP, immunoprecipitated; mAb, monoclonal antibody; pTyr, phosphotyrosine; HS, heparin sepharose. Back

Received for publication 6/ 1/98. Revision received 11/30/98. Accepted for publication 1/ 5/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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Chronic exposure to fulvestrant promotes overexpression of the c-Met receptor in breast cancer cells: implications for tumour-stroma interactions
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S. Sellappan, R. Grijalva, X. Zhou, W. Yang, M. B. Eli, G. B. Mills, and D. Yu
Lineage Infidelity of MDA-MB-435 Cells: Expression of Melanocyte Proteins in a Breast Cancer Cell Line
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J Biol ChemHome page
Y. Andegeko, L. Moyal, L. Mittelman, I. Tsarfaty, Y. Shiloh, and G. Rotman
Nuclear Retention of ATM at Sites of DNA Double Strand Breaks
J. Biol. Chem., October 12, 2001; 276(41): 38224 - 38230.
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H. Kataoka, R. Hamasuna, H. Itoh, N. Kitamura, and M. Koono
Activation of Hepatocyte Growth Factor/Scatter Factor in Colorectal Carcinoma
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J Biol ChemHome page
Y. Andegeko, L. Moyal, L. Mitelman, I. Tsarfaty, Y. Shiloh, and G. Rotman
Nuclear retention of ATM at sites of DNA double strand breaks
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