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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
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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. 1B). 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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) and 48 h (Fig. 2B).
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. 2A) or 48 h
(Fig. 2B). 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. 2C) 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. 2D).
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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). 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. 3) or 5'-bromo-2'-deoxyuridine (BrdU)
incorporation (Fig. 4). MCF-hGH cells
cultured in serum-free medium incorporated significantly more
[3H]-thymidine than MCF-MUT cells cultured
under the same conditions (Fig. 3). 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. 3). Six hundred
nanomolar B2036 did not reduce the level of
[3H]-thymidine incorporation in MCF-MUT cells
(Fig. 3). Similarly, the percentage of MCF-hGH cells (Fig. 4A) in
S-phase as determined by the incorporation of BrdU was significantly
higher than that observed in MCF-MUT cells (Fig. 4A). Treatment of
MCF-hGH cells with 600 nM B2036 in serum-free media (Fig. 4A) also reduced the percentage of cells with BrdU incorporation to
that observed in MCF-MUT cells in the absence of B2036 (Fig. 4).
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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. 5). 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. 5A).
Treatment of MCF-hGH cells with 600 nM B2036 reversed the
inhibition of apoptosis afforded by the autocrine production of hGH
(Fig. 5A). 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. 5B). Thus, protection of
mammary carcinoma cells from apoptotic cell death by autocrine hGH is
mediated via the hGH receptor.
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), CHOP
(Fig. 6B) and Elk-1 (Fig. 6C) transcription was mediated via the hGH
receptor. To measure the transcriptional response mediated by STAT5
(Fig. 6A), 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. 6A). 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. 6A).
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. 6B) or Elk-1 (Fig. 6C).
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. 6B) 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. 6B). 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. 6B). We also used a trans-activation reporter assay specific
for Elk-1 (Fig. 6C) to determine whether autocrine hGH can stimulate
Elk-1-dependent transcription. We demonstrated an autocrine
hGH-dependent transcriptional activation of Elk-1 (Fig. 6C) 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. 6C).
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. 6C). Thus, autocrine hGH
stimulated increases in STAT5, CHOP and Elk-1 mediated transcription
are all mediated via the hGH receptor.
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), 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. 7A) and cell spreading (Fig. 7B) 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. 7B). 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. 7B) 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Footnotes |
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2 These authors contributed equally to this work.
Received August 7, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 D. L. Kleinberg, T. L. Wood, P. A. Furth, and A. V. Lee Growth Hormone and Insulin-Like Growth Factor-I in the Transition from Normal Mammary Development to Preneoplastic Mammary Lesions Endocr. Rev., February 1, 2009; 30(1): 51 - 74. [Abstract] [Full Text] [PDF]
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V. Pandey, J. K. Perry, K. M. Mohankumar, X.-J. Kong, S.-M. Liu, Z.-S. Wu, M. D. Mitchell, T. Zhu, and P. E. Lobie Autocrine Human Growth Hormone Stimulates Oncogenicity of Endometrial Carcinoma Cells Endocrinology, August 1, 2008; 149(8): 3909 - 3919. [Abstract] [Full Text] [PDF]
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K. M. Mohankumar, J. K. Perry, N. Kannan, K. Kohno, P. D. Gluckman, B. S. Emerald, and P. E. Lobie Transcriptional Activation of Signal Transducer and Activator of Transcription (STAT) 3 and STAT5B Partially Mediate Homeobox A1-Stimulated Oncogenic Transformation of the Immortalized Human Mammary Epithelial Cell Endocrinology, May 1, 2008; 149(5): 2219 - 2229. [Abstract] [Full Text] [PDF]
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 G. Cao, H. Lu, J. Feng, J. Shu, D. Zheng, and Y. Hou Lung Cancer Risk Associated with Thr495Pro Polymorphism of GHR in Chinese Population Jpn. J. Clin. Oncol., April 1, 2008; 38(4): 308 - 316. [Abstract] [Full Text] [PDF]
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M. J. van den Eijnden and G. J. Strous Autocrine Growth Hormone: Effects on Growth Hormone Receptor Trafficking and Signaling Mol. Endocrinol., November 1, 2007; 21(11): 2832 - 2846. [Abstract] [Full Text] [PDF]
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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 Cell Growth via Janus Kinase-2-Signal Transducer and Activator of Transcription-5a/b Signaling Pathway Endocrinology, July 1, 2007; 148(7): 3089 - 3101. [Abstract] [Full Text] [PDF]
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 K. Pazaitou-Panayiotou, T. Kelesidis, I. Kelesidis, A. Kaprara, J. Blakeman, I. Vainas, A. Mpousoulegas, C. J Williams, and C. Mantzoros Growth hormone-binding protein is directly and IGFBP-3 is inversely associated with risk of female breast cancer Eur. J. Endocrinol., February 1, 2007; 156(2): 187 - 194. [Abstract] [Full Text] [PDF]
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 D. Yin, F. Vreeland, L. J. Schaaf, R. Millham, B. A. Duncan, and A. Sharma Clinical Pharmacodynamic Effects of the Growth Hormone Receptor Antagonist Pegvisomant: Implications for Cancer Therapy Clin. Cancer Res., February 1, 2007; 13(3): 1000 - 1009. [Abstract] [Full Text] [PDF]
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 B. S. Emerald, Y. Chen, T. Zhu, Z. Zhu, K.-O. Lee, P. D. Gluckman, and P. E. Lobie {alpha}CP1 Mediates Stabilization of hTERT mRNA by Autocrine Human Growth Hormone J. Biol. Chem., January 5, 2007; 282(1): 680 - 690. [Abstract] [Full Text] [PDF]
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S. Mukhina, D. Liu, K. Guo, M. Raccurt, S. Borges-Bendris, H. C. Mertani, and P. E. Lobie Autocrine Growth Hormone Prevents Lactogenic Differentiation of Mouse Mammary Epithelial Cells Endocrinology, April 1, 2006; 147(4): 1819 - 1829. [Abstract] [Full Text] [PDF]
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 P L Jeffery, R E Murray, A H Yeh, J F McNamara, R P Duncan, G D Francis, A C Herington, and L K Chopin Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines Endocr. Relat. Cancer, December 1, 2005; 12(4): 839 - 850. [Abstract] [Full Text] [PDF]
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K Wagner, K Hemminki, E Israelsson, E Grzybowska, R Klaes, B Chen, D Butkiewicz, J Pamula, W Pekala, and A Forsti Association of polymorphisms and haplotypes in the human growth hormone 1 (GH1) gene with breast cancer Endocr. Relat. Cancer, December 1, 2005; 12(4): 917 - 928. [Abstract] [Full Text] [PDF]
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 P. W. Parodi Dairy Product Consumption and the Risk of Breast Cancer J. Am. Coll. Nutr., December 1, 2005; 24(suppl_6): 556S - 568S. [Abstract] [Full Text] [PDF]
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X. Q. Xu, B. S. Emerald, E. L. K. Goh, N. Kannan, L. D. Miller, P. D. Gluckman, E. T. Liu, and P. E. Lobie Gene Expression Profiling to Identify Oncogenic Determinants of Autocrine Human Growth Hormone in Human Mammary Carcinoma J. Biol. Chem., June 24, 2005; 280(25): 23987 - 24003. [Abstract] [Full Text] [PDF]
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V. Goffin, S. Bernichtein, P. Touraine, and P. A. Kelly Development and Potential Clinical Uses of Human Prolactin Receptor Antagonists Endocr. Rev., May 1, 2005; 26(3): 400 - 422. [Abstract] [Full Text] [PDF]
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 T. Zhu, B. Starling-Emerald, X. Zhang, K.-O. Lee, P. D. Gluckman, H. C. Mertani, and P. E. Lobie Oncogenic Transformation of Human Mammary Epithelial Cells by Autocrine Human Growth Hormone Cancer Res., January 1, 2005; 65(1): 317 - 324. [Abstract] [Full Text] [PDF]
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 M. J. Waters and B. L. Conway-Campbell The oncogenic potential of autocrine human growth hormone in breast cancer PNAS, October 19, 2004; 101(42): 14992 - 14993. [Full Text] [PDF]
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S. Mukhina, H. C. Mertani, K. Guo, K.-O. Lee, P. D. Gluckman, and P. E. Lobie From The Cover: Phenotypic conversion of human mammary carcinoma cells by autocrine human growth hormone PNAS, October 19, 2004; 101(42): 15166 - 15171. [Abstract] [Full Text] [PDF]
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A. Colao, D. Ferone, P. Marzullo, and G. Lombardi Systemic Complications of Acromegaly: Epidemiology, Pathogenesis, and Management Endocr. Rev., February 1, 2004; 25(1): 102 - 152. [Abstract] [Full Text] [PDF]
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 K S G Cunha, E P Barboza, and E C Da Fonseca Identification of growth hormone receptor in localised neurofibromas of patients with neurofibromatosis type 1 J. Clin. Pathol., October 1, 2003; 56(10): 758 - 763. [Abstract] [Full Text] [PDF]
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S. Bernichtein, C. Kayser, K. Dillner, S. Moulin, J. J. Kopchick, J. A. Martial, G. Norstedt, O. Isaksson, P. A. Kelly, and V. Goffin Development of Pure Prolactin Receptor Antagonists J. Biol. Chem., September 19, 2003; 278(38): 35988 - 35999. [Abstract] [Full Text] [PDF]
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X. Zhang, T. Zhu, Y. Chen, H. C. Mertani, K.-O. Lee, and P. E. Lobie Human Growth Hormone-regulated HOXA1 Is a Human Mammary Epithelial Oncogene J. Biol. Chem., February 21, 2003; 278(9): 7580 - 7590. [Abstract] [Full Text] [PDF]
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T. Zhu, L. Ling, and P. E. Lobie Identification of a JAK2-independent Pathway Regulating Growth Hormone (GH)-stimulated p44/42 Mitogen-activated Protein Kinase Activity. GH ACTIVATION OF Ral AND PHOSPHOLIPASE D IS Src-DEPENDENT J. Biol. Chem., November 15, 2002; 277(47): 45592 - 45603. [Abstract] [Full Text] [PDF]
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R. Graichen, D. Liu, Y. Sun, K.-O. Lee, and P. E. Lobie Autocrine Human Growth Hormone Inhibits Placental Transforming Growth Factor-beta Gene Transcription to Prevent Apoptosis and Allow Cell Cycle Progression of Human Mammary Carcinoma Cells J. Biol. Chem., July 12, 2002; 277(29): 26662 - 26672. [Abstract] [Full Text] [PDF]
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K. Szepeshazi, A. V. Schally, P. Armatis, K. Groot, F. Hebert, A. Feil, J. L. Varga, and G. Halmos Antagonists of GHRH Decrease Production of GH and IGF-I in MXT Mouse Mammary Cancers and Inhibit Tumor Growth Endocrinology, October 1, 2001; 142(10): 4371 - 4378. [Abstract] [Full Text] [PDF]
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H. C. Mertani, T. Zhu, E. L. K. Goh, K.-O. Lee, G. Morel, and P. E. Lobie Autocrine Human Growth Hormone (hGH) Regulation of Human Mammary Carcinoma Cell Gene Expression. IDENTIFICATION OF CHOP AS A MEDIATOR OF hGH-STIMULATED HUMAN MAMMARY CARCINOMA CELL SURVIVAL J. Biol. Chem., June 8, 2001; 276(24): 21464 - 21475. [Abstract] [Full Text] [PDF]
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