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Endocrinology Vol. 142, No. 2 767-777
Copyright © 2001 by The Endocrine Society

ARTICLES

The Effects of Autocrine Human Growth Hormone (hGH) on Human Mammary Carcinoma Cell Behavior Are Mediated via the hGH Receptor1

Karmal K. Kaulsay2, Tao Zhu2, William F. Bennett, Kok-Onn Lee and Peter E. Lobie

Department of Medicine, National University of Singapore (K.K.K., K.O.L.), Singapore 119074, Republic of Singapore; and Institute of Molecular and Cell Biology (T.Z., P.E.L.), Singapore 117609, Republic of Singapore; and Sensus Drug Development Corporation (W.F.B.), Austin, Texas 78701

Address all correspondence and requests for reprints to: Peter E. Lobie, M.D., Ph.D., Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore. E-mail: mcbpel{at}imcb.nus.edu.sg


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human GH (hGH) antagonist B2036 combines a single amino acid substitution impairing receptor binding site 2 (G120K) with eight additional amino acid substitutions that improve binding site 1 affinity. B2036 does not bind, activate, or antagonize the human PRL receptor and therefore is suitable to determine cellular effects mediated specifically through the hGH receptor. We have used this hGH receptor specific antagonist in MCF-7 cells stably transfected with either the hGH gene (MCF-hGH) or a translation deficient hGH gene (MCF-MUT) to determine whether the effects of autocrine hGH on mammary carcinoma cell behavior are mediated via the hGH receptor. Enhanced JAK2 tyrosine phosphorylation observed in MCF-hGH cells compared with MCF-MUT cells is abrogated by B2036 as is the autocrine hGH stimulated increase in total cell number and DNA synthesis. Interestingly, autocrine hGH functions as a potent inhibitor of apoptosis induced by serum withdrawal compared with exogenously added hGH, and the protection against apoptosis afforded by autocrine hGH is abrogated by B2036. B2036 also inhibited autocrine hGH stimulated transcriptional activation mediated by either STAT5, CHOP (p38 MAP kinase specific) or Elk-1 (p44/42 MAP kinase specific). Finally, B2036 inhibited the autocrine hGH-dependent enhancement of the rate of mammary carcinoma cell spreading on a collagen matrix. Thus, the effects of autocrine hGH on human mammary carcinoma cell behavior are mediated via the hGH receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GH GENE gene is expressed in the normal and tumorous mammary gland of the cat and dog (1). In human mammary gland, hGH messenger RNA (mRNA) identical to pituitary hGH is also expressed by nontumorous tissue and by benign and malignant tumoral tissue, immunoreactive hGH being restricted to epithelial cells (2). Furthermore, several human mammary carcinoma cell lines have been demonstrated to express hGH mRNA when cultured in the presence of serum (E. Van Garderen, personal communication). The pituitary and mammary gland GH gene transcripts originate from the same transcription start site but are regulated differentially because mammary gland GH gene transcription does not require Pit-1 (3). GH receptor (GHR) mRNA and protein have also been detected in the mammary gland epithelia of murine and rabbit (4, 5, 6), bovine (7), and human species (8, 9). Both endocrine GH and autocrine produced GH therefore possess the capacity to exert a direct effect on the development and differentiation of mammary epithelia in vitro (10) and in vivo (11). We have recently generated a model system to study the role of autocrine produced hGH in mammary carcinoma by stable transfection of either the hGH gene or a translation-deficient hGH gene into human mammary carcinoma (MCF-7) cells (12). The autocrine hGH producing cells display a marked IGF-1-independent increase in cell number in both serum-free and serum-containing conditions as well as a specific increase in STAT5-mediated transcription (12). Also, autocrine hGH production results in enhancement of the rate of mammary carcinoma cell spreading on a collagen substrate (13). Thus, autocrine production of hGH by mammary carcinoma cells may direct mammary carcinoma cell behavior to impact on the final clinical prognosis.

GH signal transduction is thought to be initiated by ligand induced receptor dimerization (14, 15). This has permitted the generation and development of GH antagonists in several species by introduction of a single point mutation within the second binding site of the hormone, which consequently prevents receptor dimerization (16, 17, 18). In hGH, this is achieved by substitution of glycine 120 with an arginine or lysine residue (G120R or G120K respectively) (19, 20). hGH has also been reported to bind to both the hGH receptor and the hPRL receptor (21). Unlike hPRL, however, hGH requires Zn2+ to bind to the hPRL receptor via site 1 (22), although it has recently been reported that hGH may activate the hPRL receptor in the absence of Zn2+ (23). The structural basis for the interaction between hGH and the hPRL receptor has been determined by x-ray crystallography (24). Thus, the previously described hGH receptor antagonist hGH-G120R could inhibit hGH responses mediated through the hPRL receptor (25). Recently, a human GH (hGH) antagonist (B2036) has been engineered, which combines the single amino acid substitution impairing receptor binding site 2 (G120K) with eight additional amino acid substitutions that improve binding site 1 affinity (26). The eight amino acid substitutions within binding site 1 provide binding specificity directed toward the human GH receptor (26). It has been demonstrated that B2036 does not bind, activate, or antagonize PRL receptors of either rat or human origin (27).

We have therefore used the hGH antagonist B2036 to determine which effects of autocrine (and exogenously added) hGH on mammary carcinoma cell behavior are mediated via the hGH receptor. We demonstrate that under the conditions used in this study, B2036 is able to antagonize the proliferative, antiapoptotic, transcriptional, and morphological effects of autocrine hGH in mammary carcinoma cells. The potential clinical utility of hGH antagonists in treatment of mammary carcinoma should therefore be considered.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat tail type 1 collagen and DOTAP transfection reagent (N-[1-(2,3-dioleoylloxy)propyl]-N,N,N-trimethylammonium methylsulfate) were obtained from Roche Molecular Biochemicals (GmbH, Mannheim, Germany). The Cell Titer 96 cell proliferation kit was obtained from Promega Corp. (Madison, WI). Ca2+- and Mg2+-free PBS solution (HBSS) used for cell dissociation was obtained from Life Technologies, Inc. (Grand Island, NY). All other tissue culture materials were obtained from HyClone Laboratories, Inc. (Logan, UT). The in situ death detection kit (TUNEL) and JAK2 polyclonal antiserum (directed against amino acids 758–776 of murine JAK2) used for western immunoblotting and immunoprecipitation were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antiphosphotyrosine mAb PY20 used for Western immunoblot was obtained from Transduction Laboratories, Inc. (Lexington, KY). Peroxidase-conjugated antirabbit and antimouse IgGs were obtained from Pierce Chemical Co. (Rockford, IL). ECL detection reagents were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). The fusion trans-activator plasmids (pFA-CHOP and pFA2-Elk-1) consisting of the DNA binding domain of Gal4 (residue 1–147) and the transactivation domain of CHOP or Elk-1, respectively, were purchased from Stratagene (La Jolla, CA). pFC2-dbd plasmid is the negative control for the pFA plasmid to ensure the observed effects are not due to the Gal4 DNA binding domain, and was also obtained from Stratagene. Protein G cell suspension, Hoescht dye 33528 and 5'-bromo-2'-deoxyuridine was obtained from Sigma (St. Louis, MO). The BrdU staining kit was obtained from Zymed Laboratories, Inc. (South San Francisco, CA).

The MCF-7 cell line was obtained from the ATCC and stably transfected with an expression plasmid containing the wild-type hGH gene (pMT-hGH) under the control of the metallothionein 1a promoter (28) [designated MCF-hGH]. For control purposes, the ATG start site in pMT-hGH was disabled via a mutation to TTG generated by standard techniques (pMT-MUT), and MCF-7 cells stably transfected with this plasmid were designated MCF-MUT. MCF-MUT cells therefore transcribe the hGH gene but do not translate the mRNA into protein. A detailed description of the characterization of these cell lines has been previously published (12). Neither MCF-7 nor MCF-MUT cells produce detectable amounts of hGH protein when cultured under serum-free conditions, whereas MCF-hGH cells secrete approximately 100 pM hGH into 2 ml of media over a 24-h period under the culture conditions described here. MCF-7 and MCF-MUT cells behave identically to each other in terms of proliferation, transcriptional activation (12) and cell spreading (13).

Cell culture
MCF-hGH and MCF-MUT cells (12) were cultured at 37 C in 5% CO2 in RPMI supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine.

Immunoprecipitation of proteins from cell extracts
MCF-hGH and MCF-MUT cells were grown to confluence in 10% serum-supplemented medium and incubated for 12 h in serum-free medium. Cells were lysed at 4 C in 1 ml lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.2 mM sodium orthovanadate, 0.5% Nonidet P-40, 0.1% phenylmethylsulfonyl fluoride) for 30 min with regular vortices. Cell lysates were then centrifuged at 14,000 x g for 15 min, the resulting supernatants were collected, and protein concentration determined by the Lowry method using BSA as a standard. Eight hundred micrograms total protein was used for each immunoprecipitation. Immunoprecipitation was performed routinely by incubating cell lysates with 4 µg/ml of anti-JAK2 antibody for 2 h at 4 C. Immunocomplexes were collected by incubation with 50 ml protein G cell suspension for 1 h at 4 C and subsequent centrifugation of lysates at 14,000 x g for 5 min. Immunoprecipitates were washed three times in ice-cold lysis buffer. The pellets were resuspended in 2x SDS-sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 2% ß-mercaptoethanol, and bromophenol blue), boiled for 10 min, and centrifuged at 14,000 x g for 5 min. The supernatants were collected and subjected to 7% SDS-PAGE. Proteins were transferred to nitrocellulose membranes using a standard semidry electroblotting apparatus in Laemlli buffer containing 10% methanol.

Western blot analysis
Nitrocellulose membranes were blocked with 5% insulin-free BSA in PBS with 0.1% Tween 20 (PBST) for 2 h at 22 C. Blots were then immunolabeled for 1 h at 22 C with either mouse phosphotyrosine antiserum (1:1000) or rabbit JAK2 antiserum (1:800). After 6 washes for 10 min each in PBST, membranes were incubated in either goat antimouse (1:1000) or goat antirabbit IgG (1:10,000) HRP-conjugated second antibodies, respectively, for 1 h at 22 C. Membranes were further washed 6 times for 10 min each in PBST before immunolabeling was detected by ECL according to the manufacturer’s instructions.

Cell proliferation assays
Total cell number was estimated by use of the Cell Titer 96 kit as previously described (12). Briefly, MCF-MUT and MCF-hGH cell lines were maintained in 10% FBS-supplemented RPMI before being serum deprived for 12 h. All cell lines were resuspended in SFM and plated to a final concentration of 1 x 104 cells/well in a total volume of 100 µl/well, according to the indicated serum conditions and time periods. At the end of the respective time periods, 20 µl/well of assay reagent was added to the plates to measure total cell number. Briefly, Cell Titer 96 assay solution is composed of a tetrazolium salt, MTS, and an electron coupling reagent, PMS. The conversion of MTS into its aqueous soluble formazan product is accomplished by dehydrogenase enzymes found only in metabolically active cells. The quantity of formazan product (as measured by absorbance at 490 nm) is directly proportional to the number of living cells in culture. Plates were then incubated at 37 C for 1–2 h in a humidified 5% CO2 atmosphere before being directly assayed at an absorbance of 490 nm using an ELISA plate reader. Background absorbance was corrected for by subtracting the average 490 nm absorbance from triplicate wells containing RPMI only from all other absorbance values.

Mitogenesis was directly assayed by either measuring incorporation of [3H]-thymidine (29) or by incorporation of 5'-bromo-2'-deoxyuridine (BrdU) during DNA synthesis (30). For measurement of [3H]-thymidine incorporation, subconfluent MCF-MUT and MCF-hGH cells in 24-well plates were grown in serum-free RPMI at 37 C for 16 h. Cells were then incubated for 24 h in RPMI ± B2036 to a final concentration of 600 nM. Each cell line was plated in triplicate for each treatment. [3H]-thymidine (1 mCi per well; 1 Ci = 37 GBq, Amersham Pharmacia Biotech) was added, and the cells were incubated at 37 C for a further 8 h. Cells were rinsed twice with ice-cold PBS and incubated with 1 ml ice cold 5% trichloroacetic acid for 30 min at 4 C and 0.5 ml 0.5 N NaOH/0.5% SDS was added subsequently at room temperature. Solubilized samples were subjected to liquid scintillation counting in a scintillation counter. For incorporation of 5'-bromo-2'-deoxyuridine (BrdU), subconfluent MCF-MUT and MCF-hGH cells were washed twice with PBS and seeded to glass coverslips in either serum-free RPMI medium or serum-free medium supplemented with 100 nM hGH or serum-free medium supplemented with 10% FBS for 24 h. Both cell types were pulse-labeled with 20 µM BrdU for 30 min, washed twice with PBS, and fixed in cold 70% ethanol for 30 min. BrdU detection was performed by using the BrdU staining kit according to the manufacturer’s instructions. A total population of over 400 cells was analyzed in several arbitrarily chosen microscopic fields to determine the BrdU labeling index (percentage of cells synthesizing DNA).

Measurement of apoptosis
Apoptotic cell death was measured by fluorescent microscopic analysis of cell DNA staining patterns with Hoechst 33258. MCF-MUT and MCF-hGH cells were trypsinized with 0.5% trypsin and washed twice with serum-free medium. The cells were then seeded onto glass coverslips in six-well plates and incubated in serum-free medium. After a culture period of 24 h in the serum-free medium, the cells were fixed for 20 min in 4% paraformaldehyde in PBS, pH 7.4, at room temperature. The cells were then rinsed twice in PBS and then stained with the karyophilic dye Hoechst 33258 (20 µg/ml) for 10 min at room temperature. Following washing with PBS, nuclear morphology was examined under an utlraviolet-visible fluorescence microscope (Carl Zeiss Axioplan). Apoptotic cells were distinguished from viable cells by their nuclear morphology characterized by nuclear condensation and fragmentation as well as the higher intensity of blue fluorescence of the nuclei. For statistical analysis, 200 cells were counted in eight random microscopic fields at 400x magnification. To verify the methodology above we also used the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. TUNEL was performed on cells plated on glass coverslips using the in situ death detection kit according to the manufacturer’s instructions. The percentage of TUNEL-positive MCF-MUT or MCF-hGH cells (relative to total cells) was determined by counting 400 cells in 15–20 randomly chosen fields per coverslip on each of three coverslips for each experiment.

Transient transfection and luciferase assays
STAT5 reporter assays were performed as previously described (12). For CHOP and Elk-1 reporter assays, MCF-MUT and MCF-hGH cells were cultured to 60–80% confluence for transfection in six-well plates. One microgram of reporter plasmid pFR-Luc was transfected together with 20 ng of the respective fusion trans-activator plasmid (pFA-CHOP, pFA-Elk-1 or pFC2-dbd) and 2 µg of PCMVß (ß-galactosidase expression vector). For each well, 4 µg of DOTAP for each microgram of DNA was used as per the manufacturer’s instructions. DNA and the DOTAP reagents were diluted separately in 100 µl of serum-free medium, mixed and incubated at room temperature for 30 min. The DNA-lipid complex was diluted to a final volume of 6 ml (for triplicate samples) with serum-free medium. Cells in each well were rinsed once with 2 ml serum-free medium after which 2 ml of diluted DNA-lipid complex was overlaid in each well and incubated for 6 h. After incubation, serum free medium containing 2% FBS was added to each well so as to incubate the cells in 0.5% serum for 12–16 h. The cells were washed in PBS and lysed by a freeze-thaw cycle with 300 µl of 1x lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100), and the resulting cell lysate was collected by centrifugation at 14,000 x g for 15 min. The supernatant was used for the assay of luciferase and ß-galactosidase activity. The luciferase activities were normalized on the basis of protein content as well as on the ß-galactosidase activity of pCMVß vector. The ß-galactosidase assay was performed with 20 µl of precleared cell lysate according to a standard protocol (31). Mean and standard deviations of at least three independent experiments are shown in the figures.

Cell-substrate attachment assay
Attachment, operationally defined as the number of single isolated cells or small cell aggregates resistant to shear forces, was measured by a modification of a method previously described (32). In brief, MCF-hGH and MCF-MUT cell lines were maintained in 10% FBS-supplemented RPMI before being serum-deprived for 12 h. Cells were then washed in 1x PBS, detached in Ca2+- and Mg2+-free PBS-based cell-dissociation buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4) and collected by centrifugation at 300 x g for 10 min. Cells were resuspended in serum-free media (SFM) and plated in a cross-wise mixing movement on rat-tail tendon collagen-coated dishes (5 µg/cm2, according to manufacturer’s instructions) to a final concentration of 1 x 104 cells in a total volume of 2 ml, and incubated for various time periods. Media was then gently aspirated, the dishes rinsed twice in fresh SFM to remove unattached cells, and the number of attached cells were counted under a microscope eyepiece grid. Data are expressed as the mean number of cells per grid field (± SD). Ten grid fields were counted for each cell line at each experimental condition.

Single-cell spreading assays
Cell spreading in single cell preparations was measured by counting the number of cells that possessed a spread morphology (epitheliod, phase-dark with lamelloid extensions) in each microscope field (33). Spread cells were expressed as the proportion of total cells attached in each respective microscope field (± SD). Ten grid fields were counted for each experimental condition.

Statistics
All experiments were repeated at least three to five times. All numerical data are expressed as mean ± SD. Data were analyzed using the two-tailed t test or ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of B2036 on autocrine hGH stimulated JAK2 tyrosine phosphorylation
We have previously demonstrated that autocrine production of hGH in mammary carcinoma cells increases the phosphotyrosine content of JAK2 upon attachment to a collagen matrix (13). To determine if JAK2 is differentially tyrosine-phosphorylated in MCF-hGH and MCF-MUT cells cultured under serum-free conditions, we immunoprecipitated JAK2 from cell extracts prepared from MCF-hGH and MCF-MUT cells after a 12-h incubation in serum-free medium. SDS-PAGE and subsequent western blotting for phosphotyrosine demonstrated an increased phosphotyrosine content of JAK2 in MCF-hGH, compared with MCF-MUT cells (Fig. 1AGo). JAK2 protein was identified as a single band at 130 kDa. Addition of 600 nM B2036 to the medium during the 12 h incubation of MCF-hGH cells in serum-free medium resulted in an equivalent level of JAK2 tyrosine phosphorylation to that observed with MCF-MUT cells in the absence or presence of 600 nM B2036 in serum-free medium. Expression of JAK2 has previously been demonstrated to be equivalent between MCF-MUT and MCF-hGH cells (12). Equal amounts of JAK2 protein were immunoprecipitated at all points as indicated by the loading control (Fig. 1BGo). Thus, autocrine production of hGH in mammary carcinoma cells increases the phosphotyrosine content of JAK2 via interaction with the hGH receptor.



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  		Figure 1. Effect of B2036 on autocrine hGH-stimulated JAK2 tyrosine phosphorylation. JAK2 tyrosine phosphorylation (A) in MCF-7 cells stably transfected with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or in MCF-7 cells stably transfected with the hGH gene (MCF-hGH) in the presence or absence of 600 nM B2036. Cell lysates were prepared and equalized for protein content and JAK2 was immunoprecipitated with rabbit polyclonal anti-JAK2 as described in Materials and Methods. Immunoprecipitates were subjected to SDS-PAGE and Western blot analysis for phosphotyrosine using PY20 monoclonal antibody. The loading control for JAK2 is shown in (B). Phosphorylated-JAK2 and JAK2 were detected as bands migrating at 130 kDa. The data are representative of at least three separate experiments.

 
Autocrine hGH stimulated mammary carcinoma cell proliferation is inhibited by B2036
We have previously demonstrated that autocrine production of hGH in MCF-hGH cells results in increased cell number under both serum-deficient and serum containing conditions in comparison to MCF-MUT cells (12). To determine if the autocrine hGH stimulated increase in cell number was mediated by autocrine hGH interaction with the hGH receptor we estimated total MCF-MUT and MCF-hGH cell number in the absence or presence of increasing concentrations of B2036 in serum-free medium after 24 (Fig. 2AGo) and 48 h (Fig. 2BGo). Concentrations of B2036 ranging from 100 to 1000 nM did not affect MCF-MUT total cell number in comparison to MCF-MUT cells cultured in the absence of B2036 after either 24 (Fig. 2AGo) or 48 h (Fig. 2BGo). In contrast, B2036 prevented the autocrine hGH stimulated increase in cell number observed in MCF-hGH cells cultured in serum-free media after both 24 and 48 h (Fig. 2CGo) The minimal concentration of B2036 required to effect full inhibition of the autocrine hGH-stimulated increases in cell number was observed at 600 nM. This same concentration of B2036 was also able to completely inhibit the increase in cell number due to the synergistic effect of autocrine hGH with heterologous factors in serum (Fig. 2DGo). Therefore, increases in cell number observed in MCF-hGH cells compared with MCF-MUT cells, due to autocrine production of hGH, were completely inhibited by B2036 in both serum deficient and serum containing conditions.



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  		Figure 2. Autocrine hGH-stimulated increase in mammary carcinoma cell number is inhibited by B2036. Increase in total cell number of MCF-7 cells stably transfected with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or in MCF-7 cells stably transfected with the hGH gene (MCF-hGH) in serum-free media in the presence of increasing concentrations of B2036 after 24 (A) and 48 (B) h. The increase in MCF-MUT and MCF-hGH cell number when grown in serum-free media (C) or in media containing 10% FBS (D) ± 600 nM B2036 after both 24 and 48 h was also estimated. The effect of B2036 on exogenous hGH stimulated increase in total MCF-MUT cell number is presented in (E). Total cell number was estimated as described in Materials and Methods. Results represent means ± SD of triplicate determinations. Results presented are representative of at least three (usually five to seven) independent experiments. c, P < 0.001 between paired values. ***, P < 0.001 between cell spreading at 24 and 48 h vs. the beginning of the experiment. **, P < 0.001 between MCF-hGH and MCF-MUT for the corresponding time intervals. 2+, P < 0.001 between MCF-hGH in either SFM vs. MCF-hGH in B2036-supplemented medium for the corresponding concentration.

 
We also examined the ability of B2036 to inhibit the increase in cell number stimulated by treatment of MCF-MUT cells with 100 nM exogenous hGH (Fig. 2EGo). Exogenous hGH increased MCF-MUT cell number as previously described (12). B2036 inhibited the hGH stimulated increase in cell number in a dose-dependent manner with 50% inhibition of the exogenous hGH stimulated increase in cell number observed at approximately 100 nM B2036. Complete inhibition of the exogenous hGH stimulated increase in cell number was achieved at 200 nM B2036.

Because the increase in cell number observed in MCF-hGH compared with MCF-MUT cells could also be due to prevention of apoptosis (see below), we therefore examined the effect of autocrine production of hGH in mammary carcinoma cells on the number of cells in S-phase using assays measuring either [3H]-thymidine (Fig. 3Go) or 5'-bromo-2'-deoxyuridine (BrdU) incorporation (Fig. 4Go). MCF-hGH cells cultured in serum-free medium incorporated significantly more [3H]-thymidine than MCF-MUT cells cultured under the same conditions (Fig. 3Go). Treatment of MCF-hGH cells with 600 nM B2036 reduced the level of [3H]-thymidine incorporation to that observed in MCF-MUT cells in the absence of B2036 (Fig. 3Go). Six hundred nanomolar B2036 did not reduce the level of [3H]-thymidine incorporation in MCF-MUT cells (Fig. 3Go). Similarly, the percentage of MCF-hGH cells (Fig. 4AGo) in S-phase as determined by the incorporation of BrdU was significantly higher than that observed in MCF-MUT cells (Fig. 4AGo). Treatment of MCF-hGH cells with 600 nM B2036 in serum-free media (Fig. 4AGo) also reduced the percentage of cells with BrdU incorporation to that observed in MCF-MUT cells in the absence of B2036 (Fig. 4Go).



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  		Figure 3. Effect of B2036 on autocrine hGH stimulated [3H]-thymidine incorporation. [3H]-thymidine incorporation in MCF-7 cells stably transfected with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or in MCF-7 cells stably transfected with the hGH gene (MCF-hGH) in serum-free media ± 600 nM B2036. [3H]-thymidine incorporation assays were performed as described in Materials and Methods. Results represent means ± SD of triplicate determinations. Results presented are representative of at least three (usually five to seven) independent experiments. c, P < 0.001 between paired values.

 


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  		Figure 4. Effect of B2036 on autocrine hGH stimulated 5'-bromo-2'-deoxyuridine incorporation. 5'-bromo-2'-deoxyuridine (BrdU) incorporation in MCF-7 cells stably transfected with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or in MCF-7 cells stably transfected with the hGH gene (MCF-hGH) in serum-free media in the absence and presence of 600 nM B2036 (A). The effect of B2036 on the exogenous hGH stimulated increase in BrdU incorporation in MCF-MUT cells was also examined (B). BrdU incorporation assays were performed as described in Materials and Methods. For statistical analysis, approximately 400 cells were counted in each of eight random microscopic fields. Results represent means ± SD of triplicate determinations of the percentage of cell nuclei incorporating BrdU. Results presented are representative of at least three (usually five to seven) independent experiments. c, P < 0.001 between paired values. SF, Serum-free.

 
We also examined the ability of B2036 to inhibit the increase in BrdU incorporation stimulated by treatment of MCF-MUT cells with 100 nM exogenous hGH. Exogenous hGH increased the nuclear incorporation of BrdU in MCF-MUT cells as may be observed in Fig. 4BGo. B2036 inhibited the hGH-stimulated increase in BrdU incorporation in a dose-dependent manner with 50% inhibition of the exogenous hGH stimulated increase in BrdU incorporation observed at approximately 100 nM B2036. Complete inhibition (loss of statistical significance) of the exogenous hGH stimulated increase in BrdU incorporation was achieved at 200 nM B2036.

Effect of B2036 on the protection from apoptosis afforded by autocrine production of hGH
One mechanism by which autocrine hGH may also contribute to an increase in total MCF-hGH cell number is by offering protection from apoptotic cell death. GH has previously been demonstrated to be protective against apoptosis (34). We therefore first examined the ability of both exogenous and autocrine hGH to protect MCF cells against apoptosis induced by serum withdrawal (Fig. 5Go). MCF-MUT cells were therefore incubated for 24 h in serum-free media or in serum-free media containing 100 nM hGH and the level of apoptosis compared with that of MCF-hGH cells in serum-free media. Addition of 100 nM exogenous hGH only marginally reduced the level of apoptosis of MCF-MUT cells in serum-free media (from 36% in SFM to 31% in SFM supplemented with 100 nM hGH). However, autocrine production of hGH in MCF-hGH cells afforded dramatic protection from apoptosis in comparison to MCF-MUT cells in the presence or absence of 100 nM exogenous hGH (Fig. 5AGo). Treatment of MCF-hGH cells with 600 nM B2036 reversed the inhibition of apoptosis afforded by the autocrine production of hGH (Fig. 5AGo). We also verified these results by use of the TUNEL assay which labels DNA strand breaks with biotin-dUTP by use of exogenous terminal deoxynucleotidyl transferase (Fig. 5BGo). Thus, protection of mammary carcinoma cells from apoptotic cell death by autocrine hGH is mediated via the hGH receptor.



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  		Figure 5. Effect of B2036 on the protection from apoptosis afforded by autocrine production of hGH. Apoptotic cell death was quantified in MCF-7 cells stably transfected with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or in MCF-7 cells stably transfected with the hGH gene (MCF-hGH) in serum-free media ± 600 nM B2036. Apoptotic cell death was determined by fluorescent microscopic analysis of cell DNA staining patterns with Hoechst 33258 (A) or by TUNEL (B) as described in Materials and Methods. The results are presented as means ± SD. Results presented are representative of at least three (usually five to seven) independent experiments. c, P < 0.001 between paired values.

 
Effect of B2036 on autocrine hGH stimulated transcriptional activation mediated via STAT5, CHOP, or Elk-1
We have previously demonstrated that autocrine production of hGH in mammary carcinoma cells results in increased levels of transcription mediated via STAT5 (12). GH has also been demonstrated to use the p44/42 MAP kinase-dependent transcription factor Elk-1 (35, 36) and the p38 MAP kinase-dependent transcription factor CHOP (35). We therefore examined if autocrine hGH stimulation of STAT5 (Fig. 6AGo), CHOP (Fig. 6BGo) and Elk-1 (Fig. 6CGo) transcription was mediated via the hGH receptor. To measure the transcriptional response mediated by STAT5 (Fig. 6AGo), we used a luciferase reporter plasmid containing three copies of the GAS-like element from the serine protease inhibitor 2.1 gene promoter (SPI-GLE1-LUC) (37). This GAS-like element utilizes either STAT5a or STAT5b to mediate transcriptional activation (28). MCF-hGH cells possessed a significantly higher level of STAT5 mediated transcription compared with MCF-MUT cells when grown in serum free medium (Fig. 6AGo). Treatment of MCF-hGH cells with 600 nM B2036 in serum-free medium largely abrogated the ability of autocrine hGH to stimulate STAT5 mediated transcription (Fig. 6AGo). To determine if autocrine production of hGH resulted in CHOP or Elk-1-dependent transcription we used a trans-activation reporter assay specific for either CHOP (Fig. 6BGo) or Elk-1 (Fig. 6CGo). We therefore first transiently transfected both MCF-MUT and MCF-hGH cells with the fusion trans-activator plasmid pFA-CHOP consisting of the DNA binding domain of GAL4 (residue 1–147) and the transactivation domain of CHOP together with the luciferase reporter plasmid (pFA-Luc) and pCMVß vector, respectively. The luciferase activities were measured and normalized on the basis of protein content as well as on the ß-galactosidase activity of the pCMVß vector. Autocrine hGH production by MCF-hGH cells resulted in higher CHOP (Fig. 6BGo) mediated transcriptional activation compared with MCF-MUT cells. Autocrine hGH failed to stimulate CHOP mediated reporter expression in cells transfected with a plasmid encoding the GAL4 DNA binding domain (residue 1–147) lacking an activation domain, indicative that the CHOP transcriptional activation domain is required for hGH-stimulated reporter expression (Fig. 6BGo). Treatment of MCF-hGH cells with 600 nM B2036 in serum-free medium abrogated the ability of autocrine hGH to stimulate CHOP mediated transcription (Fig. 6BGo). We also used a trans-activation reporter assay specific for Elk-1 (Fig. 6CGo) to determine whether autocrine hGH can stimulate Elk-1-dependent transcription. We demonstrated an autocrine hGH-dependent transcriptional activation of Elk-1 (Fig. 6CGo) in MCF-hGH cells compared with MCF-MUT cells. hGH failed to stimulate Elk-1 mediated reporter expression in cells transfected with a plasmid encoding the GAL4 DNA binding domain (residue 1–147) lacking an activation domain, indicative that the Elk-1 transcriptional activation domain is required for hGH-stimulated reporter expression (Fig. 6CGo). Treatment of MCF-hGH cells with 600 nM B2036 in serum-free medium also abrogated the ability of autocrine hGH to stimulate Elk-1 mediated transcription (Fig. 6CGo). Thus, autocrine hGH stimulated increases in STAT5, CHOP and Elk-1 mediated transcription are all mediated via the hGH receptor.



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  		Figure 6. Effect of B2036 on autocrine hGH-stimulated transcriptional activation mediated via STAT5, CHOP or Elk-1. Transcriptional response mediated through either STAT5 (A), CHOP (B) or Elk-1 (C) in MCF-7 cells stably transfected with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or in MCF-7 cells stably transfected with the hGH gene (MCF-hGH) in the presence and absence of 600 nM B2036. A comparison of the efficacy of inhibition by B2036 of STAT5 mediated transcription by exogenous vs. autocrine produced hGH is presented in (D). Cells were cultured to confluency and transiently transfected with the respective plasmids and luciferase assays performed as described in Materials and Methods. Results are presented as the relative luciferase activity normalized to constitutive ß-galactosidase expression and are given as means ± SD of triplicate determinations. Results presented are representative of at least three (usually five to seven) independent experiments. c, P < 0.001 between paired values. SF, Serum-free.

 
We also compared the relative efficacy of the inhibition by B2036 on STAT5 mediated transcription stimulated by either exogenous hGH (100 nM) or autocrine produced hGH. As previously published, autocrine hGH stimulation of MCF cells resulted in a higher level of STAT5 mediated transcription than that obtained by exogenous hGH (12). STAT5 mediated gene transcription stimulated by autocrine production of hGH was also more resistant to inhibition than that observed with exogenous hGH (Fig. 6DGo), as previously demonstrated by use of another hGH receptor antagonist (hGH-G120R) (28).

Effect of B2036 on cell attachment and spreading on a collagen matrix
We have previously demonstrated that autocrine hGH dramatically enhances the rate of MCF cell spreading on, but not attachment to, a collagen matrix (13). To determine whether the autocrine hGH enhancement of cell spreading is mediated via the hGH receptor, we therefore examined the level of attachment (Fig. 7AGo) and cell spreading (Fig. 7BGo) of MCF-MUT and MCF-hGH cells in the presence of increasing concentrations of B2036. Concentrations of B2036 ranging from 0 to 1000 nM had no effect on the percentage of either MCF-MUT or MCF-hGH cells attaching to a collagen matrix. This is concordant with our observation that neither autocrine nor exogenous hGH alters the rate of attachment of MCF cells to a collagen matrix (13). In contrast, increasing concentrations of B2036 resulted in a dose-dependent inhibition of the ability of autocrine hGH to enhance the rate of cell spreading (Fig. 7BGo). Maximal inhibition of the rate of spreading of MCF-hGH cells occurred first at 600 nM B2036. Concentrations of B2036 from 600-1000 nM reduced the rate of spreading of MCF-hGH cells to that observed with MCF-MUT cells indicative that the autocrine hGH enhancement of cell spreading observed in MCF-hGH cells (Fig. 7BGo) was mediated via the hGH receptor. Concentrations of B2036 from 100 to 1000 nM did not alter the rate of cell spreading in MCF-MUT cells.



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  		Figure 7. Effect of B2036 on cell attachment and spreading on a collagen matrix. MCF-7 cells were stably transfected either with the hGH gene but with the start codon mutated to TTG (MCF-MUT) or with the hGH gene (MCF-hGH). Single cells were allowed to adhere to collagen-coated dishes in serum-free medium alone or in serum-free medium supplemented with the indicated concentrations of B2036. After 30 min, dishes were washed to remove nonadherent cells as described in Materials and Methods. Cell attachment (cells/field; A) and cell spreading (% total number of attached cells spread; B) were measured and are presented as the mean ± SD of triplicate determinations (n = 10). The morphology of MCF-MUT and MCF-hGH cells ± 600 nM B2036 is shown in (C). Results presented are representative of at least three (usually five to seven) independent experiments. *P < 0.01, **P < 0.001 between MCF-MUT and MCF-hGH for the corresponding concentration of B2036.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that, under the conditions used in this study, the hGH receptor specific antagonist B2036 is able to antagonize the proliferative, antiapoptotic, transcriptional and morphological effects of autocrine hGH in mammary carcinoma cells. It is therefore evident that autocrine hGH production in mammary carcinoma cells exerts its effects on cell behavior by specific interaction with the hGH and not the hPRL receptor (under conditions studied here). Interestingly, the examined effects of exogenously added hGH were also inhibited by B2036 indicative that exogenous hGH also utilizes the hGH receptor to alter mammary carcinoma cell function. How the current study (and discussion below) relates to the inability of a hGH mutant that only activates the hGH receptor (and not the PRL receptor) to stimulate STAT5 phosphorylation in T-47D human mammary carcinoma cells is not certain (16). It has, however, been conclusively demonstrated that B2036 does not bind, activate, or antagonize PRL receptors of either rat or human origin (20). It should also be noted that B2036 less effectively inhibits the cellular responses mediated by autocrine production of hGH in comparison to exogenous hGH and especially when considered on a molar basis. We have also observed this phenomenon with the use of another hGH (and hPRL) receptor antagonist, hGH-G120R (28). There are several possibilities for the relative resistance of endogenously produced hGH to inhibition by B2036 in comparison to exogenously added hGH: 1) endogenously produced hGH and hGH receptor may interact intracellularly directly after synthesis. Although, hGH receptor antagonists are internalized into the cell (38) they may be differentially compartmentalized such that they are unable to effect inhibition of the intracellular receptor. GH has been postulated to signal through an intracellular receptor as have other peptide growth factors (39); 2) the endogenously produced hGH is presumably secreted in low continuous quantities in comparison to both hGH and B2036 where they are added only once to the media. Because B2036 would be internalized into the cell and subject to degradation, the concentration of B2036 would decline over a period of time whereas endogenously produced hGH would continue to be secreted. Thus, higher effective concentrations of B2036 would be required to be added to the media to extinguish the effects of continuously produced hGH in comparison to one single addition of exogenous hGH; and 3) the secreted endogenous hGH may be delivered in closer proximity to the membrane bound receptor and at higher microenviroment concentrations than B2036, which is distributed throughout the medium. Thus, higher total concentration of B2036 would be required to compete with differing microenviroment concentrations of hGH.

The pivotal role of GH in mammary gland biology is now better defined largely due to the efforts of Kleinberg and colleagues (40, 41, 42, 43). Thus, GH is now believed to be the pituitary factor that is responsible for mammary ductal morphogenesis (43) and not PRL as previously thought (11). Also distinct roles for GH and PRL in the mammary gland have been defined by other investigators (for review see Ref. 44). It is evident, however, that the effects of GH on ductal morphogenesis are completely mediated by IGF-1 either in an endocrine or autocrine/paracrine fashion (45). It has been demonstrated that mice transgenic for hGH develop metastatic mammary adenocarcinoma (46). However, when mice were generated expressing the somatogenic specific ligand, bGH, no neoplastic alteration of the mammary gland was observed (47), indicative that in rodents specific stimulation via the PRL and not the GH receptor was required for development of mammary adenocarcinoma. Whether such receptor specific actions are relevant to the primate is not certain. It has been reported that intramuscular injection of hGH 2 days before chemotherapy in patients with advanced breast cancer induced a 2-fold increase in the proliferative activity of the tumor cells (48). Furthermore, Ng et al. (49), have demonstrated that administration of hGH to aged female rhesus monkeys increased both mammary glandular size and the epithelial proliferation index of the mammary gland (49). It remains to be determined whether the autocrine production of hGH by the primate mammary gland is of clinical consequence although autocrine production of GH has already been linked to the development of mammary carcinoma in a canine model (50).

We have demonstrated here that autocrine production of hGH offers potent protection from apoptotic cell death. GH has previously demonstrated to prevent apoptotic cell death under different experimental conditions (51, 52, 53, 54, 55). Autocrine hGH was demonstrated to be a potent inhibitor of apoptosis in mammary carcinoma, whereas exogenously administered hGH offered minimal protection. It is not clear why such differences in apoptotic protection exists between autocrine hGH and exogenously administered hGH but autocrine hGH has also been demonstrated to more potently stimulate both total increase in mammary cell number (12) and cell spreading (13) in comparison to exogenous hGH. Interestingly though, exogenous hGH stimulated BrdU incorporation in MCF cells to a similar extent to that observed with autocrine production of hGH. The observed differences between autocrine hGH and exogenous hGH in prevention of apoptosis may presumably relate to a differential ability of autocrine hGH vs. endocrine hGH to regulate the expression of genes involved in apoptotic protection. We are currently cataloging genes which are differentially regulated by autocrine production of hGH in comparison to exogenous hGH in mammary carcinoma cells (see below) to identify the basis of the differential action of autocrine vs. endocrine hGH.

We have demonstrated here that autocrine hGH stimulation of cells results in the transcriptional activation of CHOP. chop, also known as gadd153 (growth arrest and DNA damage) is a mammalian gene that encodes for a small nuclear protein related to the CCAAT/enhancer binding protein (C/EBP) family of transcription factors (56). We have previously demonstrated that hGH stimulation of CHO cells stably transfected with GH receptor complementary DNA (CHO-GHR1–638 cells) resulted in CHOP transcriptional activation in a p38 MAP kinase-dependent manner (35). CHOP was initially proposed to be involved in cell cycle arrest and apoptosis (57, 58, 59). It is therefore interesting that we observed hGH stimulated proliferation, both in CHO-GHR1–638 cells and in autocrine hGH producing mammary carcinoma cells (12), to be p38 MAP kinase dependent. We have recently demonstrated that the chop gene is differentially regulated by autocrine production of hGH in mammary carcinoma cells in comparison to exogenously applied hGH (Mertani, H. C., T. Zhu, K. O. Lee, G. Morel, and P. Lobie, manuscript submitted). Thus autocrine production of hGH results in a dramatic up-regulation of CHOP mRNA and protein and subsequent CHOP-mediated transcription (Mertani, H. C., T. Zhu, K. O. Lee, G. Morel, and P. Lobie, manuscript submitted). We have further demonstrated that overexpression of CHOP dramatically enhances the otherwise poor ability of exogenous hGH to protect mammary carcinoma cells from apoptosis. This is concordant with a recent study in which the level of CHOP mRNA was demonstrated to be significantly higher in breast carcinoma than in normal tissue controls (60). Exogenous hGH exhibits minimal effects on transcription of the chop gene, which may offer another explanation for the relative inefficacy of apoptotic protection of exogenous hGH. In any case, up-regulation of the chop gene and subsequent increases in CHOP-mediated transcription is one mechanism by which autocrine hGH regulates mammary carcinoma cell number.

We have also demonstrated here that autocrine hGH results in an enhancement of Elk-1 mediated transcription. GH-stimulated transcriptional regulation mediated by Elk-1 has previously been demonstrated to be mediated exclusively via p44/42 MAP kinase (35, 36). We have also previously demonstrated that autocrine hGH-stimulated mammary carcinoma cell proliferation could be completely inhibited with either the inhibitor for MEK1 (PD98058) or the inhibitor for p38 MAP kinase (SB203580) (12). GH has been reported to use Elk-1 to mediate GH induced transcription of egr-1 (61), which may provide a mechanism for the p44/42 MAP kinase-dependent component of GH-stimulated mammary carcinoma cell proliferation.

In conclusion, we have demonstrated that autocrine production of hGH affects the behavior of mammary carcinoma cells including the regulation of cell number by mitogenesis and prevention of apoptosis, transcriptional activation and changes in cell morphology. We show using the hGH receptor-specific antagonist that all of the examined changes in mammary carcinoma cell behavior stimulated by the autocrine production of hGH are mediated through the hGH receptor under the conditions studied here. Should autocrine production of hGH by mammary carcinoma cells be demonstrated to negatively alter the clinical prognosis of breast cancer, then use of specific hGH receptor antagonists such as B2036 could be entertained.


   Footnotes
 
1 Supported by the National Science and Technology Board of Singapore (P.E.L.) and The National Medical Research Council (K.O.L.). Back

2 These authors contributed equally to this work. Back

Received August 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. Dagvadorj, S. Collins, J.-B. Jomain, J. Abdulghani, J. Karras, T. Zellweger, H. Li, M. Nurmi, K. Alanen, T. Mirtti, et al.
Autocrine Prolactin Promotes Prostate Cancer