From the Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore, the
Department of Medicine, National University of Singapore,
Singapore 119074, Republic of Singapore, and ** CNRS UMR 5578, Physiologies Energetiques Cellulaires et Moléculaires,
Université Claude Bernard, Lyon-1, France
Received for publication, January 17, 2001, and in revised form, March 26, 2001
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ABSTRACT |
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By use of cDNA array technology we have
screened 588 genes to determine the effect of autocrine production of
human growth hormone (hGH) on gene expression in human mammary
carcinoma cells. We have used a previously described cellular model to
study autocrine hGH function in which the hGH gene or a
translation-deficient hGH gene was stably transfected into MCF-7 cells.
Fifty two of the screened genes were regulated, either positively (24)
or negatively (28), by autocrine production of hGH. We have now characterized the role of one of the up-regulated genes,
chop (gadd153), in the effect of autocrine
production of hGH on mammary carcinoma cell number. The effect of
autocrine production of hGH on the level of CHOP mRNA was exerted
at the transcriptional level as autocrine hGH increased chloramphenicol
acetyltransferase production from a reporter plasmid containing a
1-kilobase pair fragment of the chop promoter. The
autocrine hGH-stimulated increase in CHOP mRNA also resulted in an
increase in CHOP protein. As a consequence, autocrine hGH stimulation
of CHOP-mediated transcriptional activation was increased. Stable
transfection of human CHOP cDNA into mammary carcinoma cells
demonstrated that CHOP functioned not as a mediator of hGH-stimulated
mitogenesis but rather enhanced the protection from apoptosis afforded
by hGH in a p38 MAPK-dependent manner. Thus transcriptional
up-regulation of chop is one mechanism by which hGH
regulates mammary carcinoma cell number.
The growth hormone
(GH)1 gene is expressed in
the normal and neoplastic mammary gland of the cat and dog (1). In
human mammary gland, hGH mRNA identical to pituitary hGH is also
expressed by normal tissue and by benign and malignant tumoral tissue,
immunoreactive hGH being restricted to epithelial cells (2). We have
also recently localized hGH mRNA to the epithelial cell component
of normal mammary gland and in various proliferative disorders of the
mammary gland.2 The pituitary
and mammary gland GH gene transcripts originate from the same
transcription start site but are regulated differentially, since
mammary gland GH gene transcription does not require Pit-1 (3). GH
receptor mRNA and protein have also been detected in the mammary
gland epithelia of murine and rabbit (4-6), bovine (7), and human
species (8-10). Both endocrine GH and autocrine/paracrine-produced GH
therefore possess the capacity to exert a direct effect on the
development and differentiation of mammary epithelia in
vitro (11) and in vivo (12). 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 mammary carcinoma (MCF-7) cells
(13). The autocrine hGH-producing cells display a marked insulin-like
growth factor-1-independent increase in cell number in both serum-free
and serum-containing conditions as well as a specific increase in
STAT5-mediated transcription (13). The increase in mammary carcinoma
cell number as a consequence of autocrine production of hGH is a result
of both increased mitogenesis and decreased apoptosis and is dependent
on the activities of both p44/42 and p38 MAP kinases (14). Also,
autocrine hGH production results in enhancement of the rate of mammary
carcinoma cell spreading on a collagen substrate (15). All of the
studied effects of autocrine hGH on mammary carcinoma cell behavior are
mediated via the hGH receptor (14). Thus, autocrine production of hGH by mammary carcinoma cells may direct mammary carcinoma cell behavior to impact on the final clinical prognosis.
One major mechanism by which GH affects cellular function is by
regulating the level of specific mRNA species (16). It is therefore
likely that many of the effects of autocrine hGH on mammary carcinoma
cell function are mediated by specific regulation of certain genes.
Here we have used a cDNA microarray to identify some hGH-regulated
genes that may be of importance in mediating the apparently pleiotropic
effects of hGH on mammary carcinoma cell function. One gene that was
observed to be up-regulated by the autocrine production of hGH was
chop (C/EBP homologous protein) otherwise known as growth
arrest and DNA damage-inducible protein 153 (gadd153).
gadd153/chop encodes a small nuclear protein that dimerizes
with members of the C/EBP family of transcription factors (17). It has
been observed that CHOP protein influences gene expression as both a
dominant negative regulator of C/EBP binding to one class of DNA
targets and positively by directing CHOP-C/EBP heterodimers to
alternate sequences (18). CHOP has also been demonstrated to tether to
members of the immediate-early response, growth-promoting transcription
factor family, JunD, c-Jun, and c-Fos resulting in transactivation of
AP-1 target genes (19). CHOP-mediated transcriptional activation in
response to stress requires phosphorylation of CHOP by p38 MAP kinase
(20), and we have recently demonstrated that GH stimulates
CHOP-mediated transcription in a p38 MAP kinase-dependent
manner in Chinese hamster ovary cells stably transfected with GH
receptor cDNA (21). Studies utilizing cells and animals with a
targeted deletion of the chop gene have implicated CHOP in
programmed cell death in response to endoplasmic reticulum (ER) stress
(22), and overexpression of CHOP in growth factor-dependent
32D myeloid precursor cells (23) also results in apoptosis. CHOP
protein has recently been demonstrated to be up-regulated by
erythropoietin (24), resulting in erythroid differentiation (25).
We have therefore proceeded to characterize the role of CHOP in the
regulation of mammary carcinoma cell number in response to hGH. We
first demonstrate that autocrine production of hGH up-regulates
cellular CHOP mRNA and protein levels resulting in enhanced
CHOP-mediated transcription in a p38 MAP kinase-dependent manner. Finally, we demonstrate that forced expression of CHOP offers
dramatic protection from apoptotic cell death in mammary carcinoma
cells stimulated with hGH. Thus transcriptional up-regulation of
chop is one mechanism by which hGH regulates mammary
carcinoma cell number.
Materials--
DOTAP transfection reagent was obtained from
Roche Molecular Biochemicals. The Cell Titer 96 cell proliferation kit
was obtained from Promega (Madison, WI). Geneticin® (G418) was
purchased from Life Technologies, Inc. All other tissue culture
materials were obtained from HyClone Laboratories (Logan, UT).
Peroxidase-conjugated anti-mouse IgG was obtained from Pierce. ECL
detection reagents and HybondTM-N Nylon membranes were
purchased from Amersham Pharmacia Biotech. Effectene transfection
reagent, the RNAeasy total RNA kit, and the One Step RT-PCR kit were
purchased from Qiagen (Santa Clarita, CA). The BrdUrd staining kit was
obtained from Zymed Laboratories Inc. (South San
Francisco, CA). The Oligolabeling Kit was obtained from Amersham
Pharmacia Biotech. CHOP and
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)
(26) under the control of the metallothionein 1a promoter (designated
MCF-hGH) (13). 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 (13). 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
published previously (13). Neither MCF-7 nor MCF-MUT cells produce
detectable amounts of hGH protein under serum-free conditions, whereas
MCF-hGH cells secrete ~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 (13), and cell spreading
(15).
Human CHOP/GADD 153 cDNA cloned into the XhoI site
of the ampicillin-resistant pCMV-neo vector and the p5W1 construct
containing the promoter sequence spanning the region Cell Culture--
MCF-hGH and MCF-MUT cells (13) and MCF-CMV and
MCF-CHOP cells (see below) were cultured at 37 °C in 5%
CO2 in RPMI supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin,
and 2 mM L-glutamine.
Preparation of Total RNA--
Total RNA was isolated from
MCF-MUT and MCF-hGH cells using the RNAeasy total RNA kit according to
manufacturer's instructions and resuspended in diethyl
pyrocarbonate-treated water. Quantification and purity of the RNA was
assessed by A260/A280
absorption, and RNA quality was assessed by agarose gel
electrophoresis. RNA samples with ratios greater than 1.6 were stored
at Analysis of Differential Gene Expression by Use of cDNA
Microarray--
Poly(A)+ RNA was isolated from total RNA
using oligotex resin as described (27). Three independently derived
poly(A)+ RNA samples from the respective cell lines were
pooled before labeling for hybridization to the cDNA microarray.
Poly(A)+ RNA was reverse-transcribed with Moloney murine
leukemia virus reverse transcriptase in the presence of
[ Probe Labeling for Northern Blot Analysis--
The CHOP cDNA
fragment was derived from MCF-hGH cells by RT-PCR as described below
and labeled with [ RNA Gel Electrophoresis and Northern Blot Analysis--
800 ng
of poly(A)+ RNA (mRNA) was fractionated by 0.7%
formaldehyde-agarose gel electrophoresis. The mRNA in the gel was
transferred to a HybondTM-N Nylon membrane by Vacuum
blotter (Bio-Rad) at the pressure of 5 inches of Hg in 10× SSC (for 90 min). The nylon membrane was rinsed with 2× SSC and allowed to
air-dry. The RNA was immobilized onto the membrane by UV light
cross-linking. The membrane was prehybridized in Expresshyb
Hybridization Solution with 0.1 mg/ml heat-denatured salmon testes DNA
at 68 °C for 30 min. Approximately 50 µg of the CHOP cDNA
fragment labeled to a specific activity of 1-2 × 109
dpm/µg was added, and the membrane was incubated at 68 °C
overnight. The membrane was washed three times with pre-warmed wash
solution 1 (2 × SSC, 1% SDS) at 68 °C for 30 min, followed by
one washing step with pre-warmed wash solution 2 (0.1× SSC, 0.5% SDS)
at 68 °C for 30 min. The membrane was then washed in 2× SSC at room temperature for 5 min, and the radioactivity was detected by
autoradiography. For re-probing to detect Reverse Transcriptase-Polymerase Chain Reaction--
RT-PCR was
performed in a final volume of 50 µl containing 0.2 µg of mRNA
template, 0.6 µM primers, 2.0 µl of enzyme mix, 400 µM of each dNTP, 1× reaction buffer, and 1× Q-Solution
by use of the Qiagen OneStep RT-PCR Kit. Briefly, RNA template was reverse-transcribed into cDNA for 30 min at 50 °C; Hotstart
Taq DNA polymerase was activated by heating for 15 min at
95 °C; the denatured cDNA templates were amplified by the
following cycles: 94 °C/30 s, 55 °C/30 s, and 72 °C/60 s. A
final extension was performed for 10 min at 72 °C. In order to
compare the PCR products semi-quantitatively, 15-40 cycles of PCR
(annealing temperature 55 °C) were performed to determine the
linearity of the PCR amplification, and the amplified
Sequences of the primers are as follows: CHOP primers (sense)
5'-GCACCTCCCAGAGCCCTCACTCTCC-3' and (antisense)
5'-GTCTACTCCAAGCCTTCCCCCTGCG-3'; CAT Reporter Assay for CHOP Promoter Construct--
MCF cells
were cultured to 80% confluence in 6-well plates. Transient
transfection was performed in serum-free RPMI with DOTAP according to
the manufacturer's instructions. 1 µg of reporter plasmid (p5W1) and
1.0 µg of pSV2-LUC were transfected per well in serum-free RPMI
medium for 12 h before the medium was changed to fresh serum-free
RPMI with or without 100 nM hGH or 10% FBS. After a
further 24 h cells were washed with PBS and scraped into lysis
buffer (250 mM Tris-HCl, pH 8.0, 1 mM
dithiothreitol). The protein content of the samples was normalized, and
chloramphenicol acetyltransferase and luciferase assays were performed
as described previously (28). Results were normalized to the level of
luciferase activity to control for transfection efficiency.
Western Blot Analysis--
MCF-MUT and MCF-hGH were grown to
confluence and serum-deprived for 3 h before being incubated in
serum-free medium or serum-free medium supplemented with 100 nM hGH or 10% FBS for 24 h. MCF-CMV and MCF-CHOP were
grown to confluence and serum-deprived for 12 h. Crude nuclear
extracts were prepared from MCF cells according to the protocol
described (29). Briefly, cells were rinsed twice with ice-cold
phosphate-buffered saline (PBS) and collected by centrifugation, and
the cell pellet was resuspended in 500 µl of Nonidet P-40 lysis
buffer (10 mM Tris, pH 7.4, 6.6 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 500 µM phenylmethylsulfonyl fluoride). After a 15-min
incubation on ice, the suspension was homogenized, and cell debris was
removed by centrifugation. The crude nuclei were washed in 500 µl of
the same buffer, collected by microcentrifugation, lysed in Laemmli
sample buffer, and boiled for 10 min. Proteins were resolved by
SDS-polyacrylamide (12%) gel electrophoresis and transferred to
nitrocellulose membrane. The membranes were blocked with 5% non-fat
dry milk in phosphate-buffered saline with 0.1% Tween 20 (PBST) for
1 h at 22 °C. The blots were then treated with the primary
antibody in PBST containing 1% non-fat dry milk at 4 °C overnight.
After three washes with PBST, immunolabeling was detected by ECL
according to the manufacturer's instructions. CHOP protein was
detected with a 1:500 dilution of CHOP monoclonal antibody, and
Confocal Laser Scanning Microscopy--
MCF cells were cultured
on glass coverslips as described previously (13). At the end of the
respective treatment period, cells were rinsed with ice-cold
phosphate-buffered saline (PBS), fixed in ice-cold 4%
paraformaldehyde/PBS (pH 7.4), permeabilized for 10 min with 0.1%
Triton X-100, blocked in 2% BSA, and incubated with a monoclonal
antibody against CHOP (dilution 1:150 in 1% BSA/PBS, pH 7.4) followed
by anti-mouse IgG conjugated to tetramethylrhodamine B isothiocyanate
at room temperature (dilution 1:300 in 1% BSA/PBS, pH 7.4). After 5 washes in PBS the coverslips were mounted and labeled cells visualized
with a Carl Zeiss Axioplan microscope equipped with epifluorescence
optics and a Bio-Rad MRC1024 confocal laser system. Images were
converted to the tagged information file format and processed with the
Adobe Photoshop program.
Transient Transfection Procedure and Luciferase
Assays--
MCF-MUT and MCF-hGH cells or MCF-CMV and MCF-CHOP cells
(see below) were cultured to 80% confluence for transfection
experiments in 6-well plates (21). 1 µg of pCMV Generation of MCF-7 Cells Stably Transfected with Human CHOP
cDNA--
MCF-7 cells grown in serum-free RPMI medium were
transfected with 1 µg of either the human CHOP (GADD 153) cDNA
expression plasmid or the corresponding empty vector (pCMV-Neo) using
DOTAP according to the manufacturer's instructions. Stable
transfectants were selected with 600 µg/ml G418 for 14 days as
described previously (13, 30). Expression of CHOP was subsequently
determined by Western blot analysis and confocal laser scanning
microscopy (as above). MCF-7 cells transfected with CHOP cDNA were
designated as MCF-CHOP, whereas the vector transfected control cells
were designated as MCF-CMV.
Cell Proliferation and 5'-Bromo-2'-deoxyuridine Incorporation
Assays--
Total cell number was estimated by use of the Cell Titer
96 kit as described previously (13). Briefly, MCF-CMV and MCF-CHOP cell
lines were maintained in 10% FBS-supplemented RPMI before being
serum-deprived for 12 h. Cells were then washed three times in
serum-free medium by centrifugation at 300 × g for 10 min. All three cell lines were resuspended in SFM and plated to a final concentration of 1 × 104 cells/well (25% confluence)
in a total volume of 100 µl/well, according to the indicated serum
conditions and times. At the end of the 24-h period, 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, phenazine
methosulfate. 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
enzyme-linked immunosorbent assay 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 measuring incorporation of
BrdUrd (14). For incorporation of BrdUrd, subconfluent MCF-CMV and
MCF-CHOP 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 lines were
pulse-labeled with 20 mM BrdUrd for 30 min, washed twice
with PBS, and fixed in cold 70% ethanol for 30 min. BrdUrd detection
was performed by using the BrdUrd 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 BrdUrd 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
Hoescht 33258 (31). MCF-MUT and MCF-hGH cells were trypsinized with
0.5% trypsin and washed twice with serum-free RPMI medium. The cells
were then seeded onto glass coverslips in 6-well plates and incubated
in serum-free RPMI medium. After a culture period of 24 h in the serum-free RPMI 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
Hoescht 33258 (20 µg/ml) for 10 min at room temperature. Following washing with PBS, nuclear morphology was examined under a UV-visible fluorescence microscope (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 the blue fluorescence of the nuclei. For
statistical analysis, 200 cells were counted in eight random
microscopic fields at × 400 magnification.
Statistics--
All experiments were repeated at least three to
five times. All numerical data are expressed as mean ± S.D. Data
were analyzed using the two-tailed t test or analysis of variance.
Identification of Genes Regulated by the Autocrine
Production of hGH in Human Mammary Carcinoma Cells--
To identify
genes regulated by autocrine production of hGH in mammary carcinoma
cells, we screened a high density cDNA array with labeled cDNA
derived from either 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). We have
previously detailed the extensive characterization of these two cell
lines (13, 15; see "Experimental Procedures"). Of 588 screened
genes, 24 exhibited a 2-fold or greater increase in their expression in
the presence of autocrine hGH (MCF-hGH cells) (range
2-13.5-fold) compared with the absence of autocrine hGH (MCF-MUT
cells) (Fig. 1 and Table
I). Surprisingly most of the
up-regulated genes were to be found among those grouped as DNA
synthesis/repair/recombination proteins. Of note is the dramatic effects of autocrine hGH on the expression of DNA repair helicase (ERCC3), ATP-dependent helicase II, DNA repair protein
XRCC1, superoxide dismutase, UV excision repair protein RAD23, growth arrest, and DNA damage-inducible protein (GADD)45 and GADD153 (otherwise known as CHOP). Other up-regulated genes of particular interest include the ski oncogene, homeobox protein
HOXA1, and Northern Blot and Semi-quantitative RT-PCR Analysis of
the Effect of Autocrine hGH, Exogenous hGH, and FBS on the Level of
CHOP mRNA in Mammary Carcinoma Cells--
To verify the
up-regulation of CHOP mRNA observed by cDNA array screening, we
first examined the level of CHOP mRNA in MCF-MUT and MCF-hGH cells
by Northern blot analysis. CHOP mRNA of the appropriate size (0.9 kilobase pairs) was detected in both MCF-MUT and MCF-hGH cells with an
increased level of CHOP mRNA observed in MCF-hGH (Fig.
2A) cells as observed in the
cDNA array. The level of
To verify further that autocrine hGH production in mammary carcinoma
cells resulted in an increased level of CHOP mRNA, we resorted to
semiquantitative RT-PCR. The validation that the conditions of RT-PCR
used here yielded semiquantitative estimates of mRNA is described
under "Experimental Procedures." One amplified fragment of the
predicted size (422 bp) appropriate for CHOP mRNA was detected in
MCF-MUT and MCF-hGH cells (Fig. 2C). Autocrine production of hGH by MCF-hGH cells resulted in an increased level of CHOP mRNA in
comparison to MCF-MUT cells. Stimulation of MCF-MUT cells with 100 nM hGH for 24 h slightly increased CHOP mRNA, but
stimulation of MCF-hGH cells with 100 nM hGH did not
further increase the level of CHOP mRNA compared with cells grown
in serum-free media. Interestingly, growth of either MCF-MUT or MCF-hGH
cells in 10% FBS for 24 h dramatically reduced the level of CHOP
mRNA. The expression of Effect of Autocrine hGH, Exogenous hGH, and FBS on CAT Activity
from a Reporter Plasmid Containing the Promoter Region 1-Kilobase Pairs
Proximal to the 5' Start Site of the CHOP Gene--
To determine if
the autocrine hGH-stimulated increase in CHOP mRNA was due to an
increase of chop gene transcription, we utilized a
chloramphenicol acetyltransferase (CAT) reporter plasmid containing the
promoter region Effect of Autocrine hGH, Exogenous hGH, and FBS on the Level of
CHOP Protein in Mammary Carcinoma Cells--
We next determined the
level of CHOP protein in MCF-MUT and MCF-hGH cells cultured in
serum-free media by Western blot analysis. CHOP was identified as a
protein with a molecular mass of 28 kDa. Autocrine production of
hGH in MCF-hGH cells resulted in an increased expression of CHOP
protein as compared with MCF-MUT cells (Fig. 4A). We also determined the
level of CHOP protein in both MCF-MUT and MCF-hGH cells stimulated with
either 100 nM hGH or 10% FBS. Exogenous hGH minimally
enhanced the expression of CHOP in MCF-MUT cells but interacted with
autocrine hGH in MCF-hGH cells to increase CHOP protein levels above
that observed in MCF-hGH cells in serum-free media. Similarly 10% FBS
stimulated a slight increase in CHOP protein expression in MCF-MUT
cells but interacted with autocrine hGH in MCF-hGH cells to increase
CHOP protein levels above that observed in MCF-hGH cells grown in
serum-free media or serum-free media supplemented with 100 nM hGH. To verify equal loading of proteins the membranes
were stripped and reblotted to demonstrate equivalent levels of
We also verified the autocrine hGH-stimulated increase in CHOP protein
observed by Western blot analysis by use of confocal laser scanning
microscopy (CLSM). CHOP protein was primarily localized to the nucleus
(Fig. 4B). In serum-free medium, MCF-hGH cells demonstrated
a higher level of CHOP protein expression compared with MCF-MUT cells
in confirmation of the results obtained by Western blot analysis.
Similarly the results obtained by CLSM mirrored those obtained by
Western blot analyses for MCF-MUT and MCF-hGH cells when stimulated by
either 100 nM hGH or 10% FBS.
Effect of Autocrine hGH, Exogenous hGH, and FBS on the Level
of CHOP-mediated Transcriptional Activation in Mammary Carcinoma
Cells--
We have previously demonstrated that hGH stimulation of
cells results in a p38 MAP kinase-dependent activation of
CHOP-mediated transcription (21). To determine if the autocrine
hGH-stimulated increase in CHOP protein consequently resulted in
increased CHOP-mediated transcription, 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 (residues 1-147) and the transactivation domain
of CHOP together with the luciferase reporter plasmid (pFA-Luc) and
pCMV
To determine if autocrine hGH stimulated CHOP-mediated transcription
was p38 MAP kinase-dependent, we therefore utilized the specific p38 MAP kinase inhibitor SB203580. Autocrine hGH-stimulated CHOP-mediated transcription was inhibited with 10 µM
SB203580 (Fig. 5B). Thus autocrine hGH production by mammary
carcinoma cells resulted in enhancement of CHOP-mediated transcription
in a p38 MAP kinase-dependent manner.
Overexpression of CHOP Protein Upon Stable Transfection of
MCF-7 Cells with CHOP cDNA--
To examine the role of CHOP in hGH
regulation of mammary carcinoma cell number, we therefore overexpressed
CHOP protein by stable transfection of CHOP cDNA in MCF-7 cells.
Thus, an expression plasmid containing CHOP cDNA under the control
of a CMV promoter and encoding neomycin resistance was transfected in
MCF-7 cells. For control purposes, the empty vector was also
transfected in MCF-7 cells. The respective stably transfected cells
were selected by G418 resistance and pooled so as to minimize any
potential clonal selection artifact. Cells stably transfected with the
empty vector (MCF-CMV) demonstrated a low level of endogenous CHOP
expression by Western blot analysis (Fig.
6A). CLSM confirmed the
endogenous expression of CHOP confined predominantly to the nucleus
(Fig. 6B). In contrast, MCF-7 cells transfected with CHOP
cDNA displayed markedly enhanced CHOP expression as demonstrated
both by Western blot analysis and CLSM. To demonstrate that the
overexpression of CHOP also resulted in enhanced hGH-stimulated
CHOP-mediated transcription, we therefore examined CHOP-mediated
transcription in MCF-CMV and MCF-CHOP cells stimulated by 100 nM hGH as described above. hGH stimulation of MCF-CMV cells
resulted in minimal enhancement of CHOP-mediated transcription, and
both the basal level and the hGH-stimulated increase in CHOP-mediated
transcription, were inhibited with the p38 MAP kinase-specific
inhibitor SB203580 (10 µM) (Fig. 6C). The
basal level of CHOP-mediated transcription in MCF-CHOP cells was
~3-fold greater compared with MCF-CMV cells (Fig. 6C). hGH
stimulation of MCF-CHOP cells resulted in a further dramatic increase
in CHOP-mediated transcription. Treatment of MCF-CHOP cells with 10 µM SB203580 completely abrogated the hGH enhancement of
CHOP-mediated transcription and also reduced the basal level of
CHOP-mediated transcription to that observed in MCF-CMV cells.
CHOP Overexpression in Mammary Carcinoma Cells Enhances the
hGH-stimulated Increase in Total Cell Number but Not hGH-stimulated
Mitogenesis--
CHOP has been implicated previously to be involved in
the regulation of cell number (22, 23). The number of MCF-CHOP cells after 48 h of growth in serum-free media was slightly greater than
that of MCF-CMV cells, and stimulation of MCF-CHOP cells with 100 nM hGH significantly increased cell number in comparison to
MCF-CMV cells (Fig. 7A).
Stimulation of MCF-CMV and MCF-CHOP cells with 10% FBS resulted in
equivalent increases in cell number after 48 h. Since the increase
in cell number observed in MCF-CHOP compared with MCF-CMV cells could
also be due to prevention of apoptosis (see below), we therefore
examined the effect of CHOP overexpression in mammary carcinoma cells
on the number of cells in S-phase using BrdUrd incorporation. The
percentage of MCF-CMV and MCF-CHOP cells in S-phase when cultured in
serum-free media did not significantly differ between the two cell
lines (Fig. 7B). Similarly, the percentage of MCF-CHOP cells
in S-phase after hGH stimulation as determined by the incorporation of
BrdUrd did not significantly differ from that observed in MCF-CMV cells
after hGH stimulation. CHOP is therefore a mediator of hGH-stimulated increase in cell number but is not involved in hGH-stimulated mitogenesis.
Effect of CHOP Overexpression in Mammary Carcinoma Cells on the
Exogenous hGH- and FBS-stimulated Protection from Apoptotic Cell
Death--
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 been demonstrated previously to be
protective against apoptosis (14, 34, 35), and in mammary carcinoma
cells we have demonstrated that autocrine production of hGH affords
dramatic protection from apoptotic cell death (14). In comparison,
exogenous hGH only marginally reduces the level of apoptosis of MCF-MUT
cells in serum-free media (14). We therefore examined the ability of
exogenous hGH to protect MCF-CMV and MCF-CHOP cells against apoptosis
induced by serum withdrawal. MCF-CMV and MCF-CHOP cells were therefore
incubated for 24 h in serum-free media or in serum-free media
containing either 100 nM hGH or 10% FBS, and the level of
apoptosis was determined. As expected, addition of 100 nM
exogenous hGH only marginally reduced the level of apoptosis of
MCF-CMV cells in serum-free media, whereas 10% FBS offered dramatic
protection from apoptosis (Fig.
8A). CHOP overexpression (MCF-CHOP) itself resulted in a significant protection from apoptosis compared with the vector-transfected control cells (MCF-CMV). Addition
of 100 nM hGH to MCF-CHOP cells resulted in a dramatic protection from apoptosis compared with both MCF-CHOP cells in serum-free media and also to MCF-CMV cells treated with 100 nM hGH. Ten percent FBS increased apoptotic cell death in
MCF-CHOP cells compared with MCF-CMV in the presence of 10% FBS
although the change was not significant. We also examined the p38 MAP
kinase dependence of the protection offered from apoptotic cell death afforded by hGH treatment of MCF-CHOP cells. As is evidenced in Fig.
8B, pharmacological inhibition of p38 MAP kinase (10 µM SB203580) abrogated the protection from apoptosis
afforded by hGH stimulation of CHOP-overexpressing mammary carcinoma
cells (MCF-CHOP).
We have demonstrated here that autocrine production of hGH by
mammary carcinoma cells results in specific increases and decreases in
the level of different mRNA species. A number of the regulated genes may be of importance in mediating the effects of hGH on mammary
carcinoma cell behavior, and further work should delineate how these
genes integrate the response of the cell to hGH. What also needs to be
determined is the mechanism and sequential order by which these genes
are regulated by hGH. For example, we have observed here that the
retinoic acid-regulated transcription factor HOXA-1 is
dramatically up-regulated in response to autocrine production of hGH.
Several target genes of HOXA-1 have recently been
described (36) of which some have also been demonstrated to be
hGH-regulated. These include superoxide dismutase (this study),
SAP-18 (from differential display analysis of MCF-MUT
and MCF-hGH)3 and
bmp-4 (37).
We have observed that the ski oncogene is
approximately 4-fold up-regulated in response to autocrine production
of hGH. The products of the cellular (c-Ski) and retroviral (v-Ski)
ski genes are nuclear proteins that either activate or
repress transcription depending on both the cellular and promoter
context (38-43). c-ski is highly expressed in several tumor
cell lines including those derived from neuroblastomas and carcinomas
of the stomach, vulva, and prostate (44). c-Ski and v-Ski do not bind
DNA on their own but participate in a multiprotein signaling complex
resulting in transcriptional repression required for cellular
transformation (43). It has recently been demonstrated that Ski
associates with both Smad2 and Smad3 resulting in repression of
TGF- We have observed here that the We have previously demonstrated that autocrine production of hGH in
mammary carcinoma cells results in a higher level of STAT5-mediated transcription (13, 14). It is therefore interesting that we observed
the level of YY1 mRNA to be decreased in response to autocrine
production of hGH. YY1 has been demonstrated to associate with STAT5
after GH stimulation of hepatocytes (59) and mutation of the YY1 site
situated between the two Autocrine production of hGH by mammary carcinoma cells results in a
transcriptional up-regulation of chop gene expression (this
study). This increase in chop gene expression
translates into higher cellular levels of CHOP protein with a
consequent increase in CHOP-mediated transcription. CHOP has previously
been implicated in the control of cell division since its forced
overexpression in different cell types results in growth arrest (62,
63) or apoptosis (22, 23, 64). Concordantly, embryonic fibroblasts obtained from mice with a targeted disruption of the chop
gene are resistant to apoptotic stimuli (23), and the introduction of
the chop gene into gastric cancer cells increases
sensitivity to anti-neoplastic agents (65) by activation of
AP-1-associated transcription (66). Furthermore, a linear pathway
resulting in apoptotic cell death has been described (64). Thus, Fas
receptor ligation results in consecutive activation of Rac1, p38 MAP
kinase, and CHOP and subsequent apoptosis of Jurkat cells (64). It is apparent, however, that CHOP may have divergent functions dependent on
cell type. Thus, CHOP functions as an inducible inhibitor of adipocytic
differentiation in response to metabolic stress by interfering with the
accumulation of adipogenic C/EBP isoforms (C/EBP It could be argued that the autocrine hGH induction of CHOP gene
expression occurs simply as a result of ER stress. Stable transfection
of the hGH gene into Madin-Darby canine kidney cells has been observed
to result in amplification of the Golgi complex (76). In response to an
altered function of the ER, cells exhibit a modified pattern of gene
expression encoding ER resident proteins (77). In yeast, this response
has been termed the unfolded protein response and is a positive
feedback loop by which unfolded proteins in the ER result in enhanced
expression of components required for proper protein folding in the ER
(77). Many perturbations of the cell that result in ER stress (cellular
treatment with the glycosylation inhibitor tunicamycin, agents that
interfere with calcium flux across the ER membrane, and reducing agents and deprivation of nutrients such as glucose, amino acids, and oxygen)
have also been reported to result in the induction of CHOP mRNA
(67, 78-84). However, that autocrine production of hGH results in the
induction of CHOP mRNA simply as a result of ER stress is unlikely
for a number of reasons. First, the level of intracellular hGH (1 ng
per mg of cellular protein) and secreted hGH (100 pM
secreted over a 24-h period) are not excessive and are far below the
physiological concentration of hGH. Amplification of the Golgi complex
in Madin-Darby canine kidney cells secreting hGH was observed with
overexpression of hGH comprising 10% of the secretory proteins (76).
Furthermore, all changes in MCF cellular function stimulated by
autocrine production of hGH (including CHOP-mediated transcription) can
be inhibited by the exogenous application of a hGH receptor antagonist
(13-15), the addition of which would not alter any potential ER stress
resulting from forced cellular expression of hGH (76). The changes in
CHOP mRNA upon addition of exogenous hGH in MCF-MUT cells was
minimal although exogenous hGH did result in increased levels of CHOP protein in these cells. Such a minimal response of exogenous hGH in
MCF-MUT cells could be expected since we have previously demonstrated that exogenous hGH only weakly stimulates STAT5 transcriptional activation and mitogenesis and is a poor inhibitor of apoptosis in
comparison to autocrine-produced hGH (13, 14). However, in cell lines
such as 3T3-L1, which possess a robust response to exogenous hGH (85),
a marked induction of CHOP-10 (the mouse homolog of CHOP) mRNA is
also observed in response to exogenous hGH.5 It is also apparent
that ER stressors and hGH regulate the level of CHOP mRNA by
different mechanisms. For example, glutamine deprivation increases the
level of CHOP mRNA primarily by mRNA stabilization (33),
whereas autocrine production of hGH in MCF cells did not alter the
half-life of CHOP mRNA (this study). Furthermore, increased transcription of the chop gene as a result of arsenite
treatment of cells (86) and oxidative stress (87) is mediated via a C/EBP-ATF composite and AP-1 site, respectively. Preliminary evidence from analysis of the chop promoter suggests that autocrine
hGH may utilize the ETS-binding site to stimulate transcription of the
chop gene.5 Therefore, autocrine hGH induction
of CHOP mRNA is a specific response of the cell to stimulation with
hGH and not part of a generalized cellular response to ER stress.
In conclusion we have demonstrated that autocrine production of hGH
up-regulates cellular CHOP mRNA and protein levels resulting in
enhanced CHOP-mediated transcription in a p38 MAP
kinase-dependent manner. Increased expression of CHOP
offers dramatic protection from apoptotic cell death in mammary
carcinoma cells stimulated with hGH. Thus transcriptional up-regulation
of chop is one mechanism by which hGH regulates mammary
carcinoma cell number.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin monoclonal antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The fusion
trans-activator plasmids (pFA-CHOP) consisting of the DNA
binding domain of Gal4 (residue 1-147) and the transactivation domain
of CHOP 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. The Atlas human cDNA expression array and
Expresshyb hybridization solution was obtained from
CLONTECH Laboratories Inc. (Palo Alto, CA).
Anti-mouse tetramethylrhodamine B isothiocyanate-conjugated IgG,
Hoescht dye 33528, denatured salmon testis DNA, and
5'-bromo-2'deoxyuridine was obtained from Sigma.
951 to +91 of
the human CHOP gene were a generous gift from Dr. Nikki J. Holbrook
(26).
70 °C for further analysis.
-32P]dATP for generation of radiolabeled cDNA
probes. The radiolabeled cDNA probes were purified from the
unincorporated nucleotides by gel filtration in chroma-spin 200 columns
and hybridized to the cDNA microarray according to the
manufacturer's instructions (overnight at 68 °C). After a series of
high stringency washes (three 20-min washes in 2× saline/sodium
citrate (SSC), 1% SDS followed by two 20-min washes in 0.1× SSC,
0.5% SDS), at 68 °C the membranes were exposed to x-ray film and
subject to autoradiography. The relative levels of gene expression were
quantified by densitometric scanning by use of the GS-700 imaging
densitometer from Bio-Rad according to the manufacturer's
instructions. Genes were considered differentially expressed when they
exhibited a 2-fold or greater increase or decrease in the presence of
autocrine hGH (MCF-hGH cells) compared with the absence of autocrine
hGH (MCF-MUT cells) in three independently performed experiments. The
relative expression of housekeeping genes (ubiquitin, phospholipase
A2, glyceraldehyde-3-phosphate dehydrogenase,
-actin,
-tubulin, 23-kDa highly basic protein, ribosomal protein S9) did not
differ by more than 10% between MCF-MUT and MCF-hGH cells.
-32P]dCTP (3000 Ci/mmol) using the
Oligolabeling Kit. In brief, 50 ng of DNA was denatured by heating for
2-3 min at 95-100 °C and was directly transferred to ice for 2 min. 10 µl of reagent mix (containing dATP, dGTP, and dTTP and random
hexanucleotides) and 50 µCi of [
-32P]dCTP was added
to the denatured DNA, and the volume was adjusted to 49 µl with
distilled water. 1 µl of FPLCpureTM Klenow fragment
(5-10 units) was added, and the reaction mixture was incubated at
37 °C for 60 min. The labeled CHOP cDNA fragment was denatured
by heating at 95-100 °C for 2 min and cooled immediately on ice.
The labeled CHOP cDNA fragment was used directly as a hybridization probe.
-actin as loading control,
the membrane was stripped by boiling in 0.5% SDS for 10 min and rinsed
once with wash solution 1. The
-actin DNA fragment was labeled and hybridized to the stripped membrane and subjected to autoradiography as
described above.
-actin
cDNA served as an internal control for cDNA quantity and
quality. Experiments using DNase I prior to the RT-PCR were routinely
run to control for amplification of genomic DNA.
-actin primers (sense)
5'-ATGATATCGCCGCGCTCG-3' and (antisense) 5'-CGCTCGGTGAGGATCTTCA-3'.
Amplified PCR products were visualized on a 1% agarose gel.
Amplification yielded the predicted size of the amplified fragment
(CHOP 422 bp;
-actin 581 bp).
-actin was detected with a 1:500 dilution of
-actin monoclonal antibody.
and 1 µg of
reporter plasmid pFR-Luc were transfected together with 20 ng of the
respective fusion trans-activator plasmid (pFA-CHOP or
pFC2-dbd). For each well, 4 µg of DOTAP for each µg 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 together at room temperature for 30 min. 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 of
serum-free medium, and 2 ml of diluted DNA-lipid complex was overlaid
in each well and incubated for 6 h. After incubation, RPMI medium containing 2% FBS was added to each well so as to incubate the cells
in 0.5% serum for 12 h. The cells were subsequently placed in
serum-free medium ± 100 nM for 24 h. SB203580 or
vehicle (Me2SO) was added 45 min prior to the
addition of hGH, and the incubation was continued for 24 h. The
cells were finally washed in PBS and lysed with 300 µl of 1× lysis
buffer (25 mM Tris phosphate, pH 7.8, 2 mM
EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100) by a freeze-thaw cycle, and lysate was collected by centrifugation at
14,000 rpm for 15 min. The supernatant was used for the assay of
luciferase and
-galactosidase activity. The luciferase activities were normalized on the basis of protein content as well as on the
-galactosidase activity of pCMV
vector. The
-galactosidase assay was performed with 20 µl of precleared cell lysate according to
a standard protocol (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin. The autocrine hGH-stimulated up-regulation of
multiple mRNA species observed on the DNA array was verified by
semiquantitative RT-PCR (data not shown). Twenty eight genes exhibited
a 2-fold or greater decrease in their expression in the presence
of autocrine hGH (MCF-hGH cells) (range 2-20-fold) compared with the
absence of autocrine hGH (MCF-MUT cells) (Fig. 1 and Table
II). Most of the down-regulated genes
were to be found among those grouped as DNA
binding/transcription/transcription factors, and the list of
down-regulated genes is presented in Table II. The relative expression
of housekeeping genes (ubiquitin, phospholipase A2, glyceraldehyde-3-phosphate dehydrogenase,
-actin,
-tubulin, 23-kDa highly basic protein, ribosomal protein S9) did not differ by
more than 10% between MCF-MUT and MCF-hGH cells.
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Fig. 1.
Effect of autocrine production of hGH on
relative levels of gene expression in mammary carcinoma cells.
cDNA microarray analysis of the relative levels of gene expression
in MCF-7 cells stably transfected with the hGH gene but with the start
codon mutated to TTG (MCF-MUT) (A) or in MCF-7 cells stably
transfected with the hGH gene (MCF-hGH) (B) cultured in
serum-free medium. 32P-Labeled cDNA probes generated
from poly(A)+ RNA isolated from MCF-MUT and MCF-hGH cells
were hybridized to a cDNA microarray containing 588 known human
genes. The left upper box and left lower box
encase those genes grouped as oncogenes/tumor suppressors/cell cycle
control proteins and DNA binding/transcription/transcription factors,
respectively. The right upper box encases those genes
grouped as DNA synthesis/repair/recombination proteins. The position of
CHOP cDNA is indicated by the arrow. The relative
expression level of specific cDNAs was determined by comparison
with the expression of a number of housekeeping genes.
Identification by cDNA array of genes positively regulated by the
autocrine production of hGH in mammary carcinoma cells
Identification by cDNA array of genes negatively regulated by the
autocrine production of hGH in mammary carcinoma cells
-actin mRNA did not differ between
the two cell lines and was used as loading control (Fig.
2B).
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Fig. 2.
Northern blot and semi-quantitative RT-PCR
analysis of the effect of autocrine hGH, exogenous hGH, and FBS on the
level of CHOP mRNA in mammary carcinoma cells. MCF-MUT and
MCF-hGH cells were cultured in serum-free media or in serum-free media
supplemented with either 100 nM hGH or 10% FBS. RNA was
isolated, and Northern blot analysis was performed to detect CHOP
(A) or -actin (B) mRNA as described under
"Experimental Procedures." RT-PCR was performed to detect either
CHOP (C) or
-actin (D) mRNA as described
under "Experimental Procedures" to produce fragments of 422 and 581 bp, respectively. Size markers are indicated on the left-hand
side of the gel. Lanes 1 for C and
D MCF-MUT cells in serum-free medium. Lane 2, CF-MUT cells stimulated with 100 nM hGH. Lane 3, MCF-MUT cells stimulated with 10% FBS. Lane 4, MCF-hGH
cells in serum-free medium. Lane 5, MCF-hGH cells stimulated
with 100 nM hGH. Lane 6, MCF-hGH cells
stimulated with 10% FBS.
-actin mRNA remained constant
between MCF-MUT and MCF-hGH cells under the different experimental
conditions (Fig. 2D).
951 to +91 of the chop gene (27).
Autocrine hGH production by MCF-hGH cells stimulated CAT expression
2-4-fold that observed in MCF-MUT cells (Fig.
3). Exogenous hGH (100 nM) also stimulated chop gene transcription by MCF-MUT cells but
not to the extent stimulated by autocrine production of hGH in MCF-hGH cells. Exogenous hGH (100 nM) stimulation of MCF-hGH cells
did not further enhance transcription of the chop gene.
Serum stimulation of either MCF-MUT or MCF-hGH resulted in equivalent
activation of chop gene transcription. Stabilization of CHOP
mRNA has also been reported to be one major mechanism for
regulation of cellular CHOP mRNA levels (1, 10). Autocrine
production of hGH in MCF-hGH cells did not alter the half-life of CHOP
mRNA compared with MCF-MUT cells (data not shown), and therefore
autocrine hGH up-regulates cellular CHOP mRNA level by increasing
the rate of chop gene transcription.
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Fig. 3.
Effect of autocrine hGH, exogenous hGH, and
FBS on CAT activity from a reporter plasmid containing the promoter
region 1-kilobase pairs proximal to the 5' start site of the
chop gene. MCF-MUT and MCF-hGH cells in
serum-free media or in serum-free media supplemented with either 100 nM hGH or 10% FBS were transiently transfected with the
respective plasmids (1 µg of p5W1 and 1 µg of pCMV ), and CAT
assays were performed as described under "Experimental Procedures."
Results are presented as the relative CAT activity normalized to
constitutive
-galactosidase expression and are given as means ± S.D. of triplicate determinations. *, p < 0.01.
-actin protein under the different experimental conditions (Fig.
4A).
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Fig. 4.
A, Western blot analysis of the effect
of autocrine hGH, exogenous hGH, and FBS on the level of CHOP protein
in mammary carcinoma cells. MCF-MUT and MCF-hGH cells were grown to
confluence in serum-free media or in serum-free media supplemented with
either 100 nM hGH or 10% FBS. Nuclear extracts were
prepared and subjected to SDS-polyacrylamide gel electrophoresis, and
Western blot analysis was performed as described under "Experimental
Procedures." After visualization of CHOP protein the membrane was
subsequently stripped and reblotted for -actin to demonstrate
equivalent loading. Lane 1, MCF-MUT cells in serum-free
medium. Lane 2, MCF-MUT cells stimulated with 100 nM hGH. Lane 3, MCF-MUT cells stimulated with
10% FBS. Lane 4, MCF-hGH cells in serum-free medium.
Lane 5, MCF-hGH cells stimulated with 100 nM
hGH. Lane 6, MCF-hGH cells stimulated with 10% FBS.
B, confocal laser scanning microscopic analysis of the
effect of autocrine hGH, exogenous hGH, and FBS on the level of CHOP
protein in mammary carcinoma cells. MCF-MUT and MCF-hGH cells were
grown to confluence in serum-free media or in serum-free media
supplemented with either 100 nM hGH or 10% FBS. CHOP
protein was detected with a monoclonal antibody directed against CHOP,
and confocal laser scanning microscopy was performed as described under
"Experimental Procedures." C, a,
MCF-MUT cells in serum-free medium; b, MCF-MUT cells
stimulated with 100 nM hGH; c, MCF-MUT cells
stimulated with 10% FBS; d, MCF-hGH cells in serum-free
medium; e, MCF-hGH cells stimulated with 100 nM
hGH; f, MCF-hGH cells stimulated with 10% FBS.
vector, respectively. The luciferase activities were measured
and normalized on the basis of protein content as well as on the
-galactosidase activity of the pCMV
vector. Autocrine hGH
production by MCF-hGH cells resulted in significantly higher
(2-3-fold) CHOP (Fig.
5A)-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, indicating that the CHOP transcriptional
activation domain is required for hGH-stimulated reporter expression.
100 nM hGH minimally stimulated CHOP-mediated transcription
in MCF-MUT cells, whereas stimulation of MCF-MUT cells with 10% FBS
resulted in a robust activation of CHOP-mediated transcription.
Stimulation of MCF-hGH cells with 100 nM hGH did not
significantly alter the level of CHOP-mediated transcription compared
with that observed with autocrine hGH. Stimulation of MCF-hGH cells
with 10% FBS resulted in a stimulation of CHOP-mediated transcription
similar to that observed with serum stimulation of MCF-MUT cells.
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Fig. 5.
Effect of autocrine hGH, exogenous hGH, and
FBS on the level of CHOP-mediated transcriptional activation in mammary
carcinoma cells. CHOP-mediated transcriptional response in MCF-MUT
and MCF-hGH cells in serum-free media or in serum-free media
supplemented with either 100 nM hGH or 10% FBS
(A). The p38 MAP kinase dependence of CHOP-mediated
transcription was delineated by use of the p38 MAP kinase inhibitor
SB203580 (10 µM) as indicated (B). Cells were
cultured to confluency and transiently transfected with the respective
plasmids and luciferase assays performed as described under
"Experimental Procedures." Results are presented as the relative
luciferase activity normalized to constitutive -galactosidase
expression and are given as means ± S.D. of triplicate
determinations. *, p < 0.01.
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Fig. 6.
Demonstration of functional overexpression of
CHOP protein upon stable transfection of MCF-7 cells with CHOP
cDNA. A, Western blot analysis to detect
overexpression of CHOP in mammary carcinoma cells stably transfected
with CHOP cDNA. MCF-7 cells stably transfected with an expression
vector containing CHOP cDNA (MCF-CHOP) or MCF-7 cells stably
transfected with the empty vector (MCF-CMV) were grown to confluence in
serum-free media. Nuclear extracts were prepared and subjected to
SDS-polyacrylamide gel electrophoresis, and Western blot analysis was
performed as described under "Experimental Procedures." After
visualization of CHOP protein, the membrane was subsequently stripped
and reblotted for -actin to demonstrate equivalent loading.
B, confocal laser scanning microscopic analysis to detect
overexpression of CHOP in mammary carcinoma cells stably transfected
with CHOP cDNA. MCF-CMV and MCF-CHOP cells were grown to confluence
in serum-free media. CHOP protein was detected with a monoclonal
antibody directed against CHOP, and confocal laser scanning microscopy
was performed as described under "Experimental Procedures."
A, MCF-CMV cells; B, MCF-CHOP cells;
C, effect of CHOP overexpression on the level of
CHOP-mediated transcriptional activation in mammary carcinoma cells
stably transfected with CHOP cDNA. CHOP-mediated transcriptional
response in MCF-CMV or MCF-CHOP cells in serum-free media or in
serum-free media supplemented with 100 nM hGH. SB203580 was
used at 10 µM where indicated. Results are presented as
the relative luciferase activity normalized to constitutive
-galactosidase expression and are given as means ± S.D. of
triplicate determinations. *, p < 0.01.
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Fig. 7.
Effect of CHOP overexpression in mammary
carcinoma cells on the exogenous hGH and FBS-stimulated increase in
total cell number and 5'-bromo-2'-deoxyuridine incorporation.
A, increase in total cell number of MCF-CMV and MCF-CHOP
cells in serum-free media or in serum-free media supplemented with
either 100 nM hGH or 10% FBS. Total cell number was
estimated as described under "Experimental Procedures." Results
represent means ± S.D. of triplicate determinations. *,
p < 0.01. B, BrdUrd incorporation in
MCF-CMV and MCF-CHOP cells in serum-free media or in serum-free media
supplemented with either 100 nM hGH or 10% FBS. Results
represent means ± S.D. of triplicate determinations of the
percentage of cells incorporating BrdUrd. *, p < 0.01.
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Fig. 8.
A, effect of CHOP overexpression in
mammary carcinoma cells stably transfected with CHOP cDNA on the
exogenous hGH and FBS-stimulated protection from apoptotic cell death.
MCF-CMV and MCF-CHOP cells were cultured in serum-free medium for
24 h or in serum-free media supplemented with either 100 nM hGH or 10% FBS. B, CHOP enhancement of
hGH-stimulated protection from apoptotic cell death is p38 MAP
kinase-dependent. MCF-7 cells stably transfected with an
expression vector containing CHOP cDNA or MCF-7 cells stably
transfected with the empty vector were cultured in serum-free medium
for 24 h or in serum-free media supplemented with 100 nM hGH. SB203580 was used at 10 µM where
indicated. The results are presented as mean ± S.D. *,
p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-responsive promoters via the Smad-binding element (45). The
TGF-
pathway usually functions to suppress cellular proliferation
and cellular transformation. It has been proposed that the repression
of TGF-
-inducible genes (which function as negative regulators of
cell cycle function) may be pivotal to the cellular transforming
ability of Ski (45). In this regard, it is also interesting that we
have observed that the gene for PTGF-
, the product of which
functions via the TGF-
receptor (46), is down-regulated in response
to autocrine production of
hGH.4 Thus autocrine hGH
production by mammary carcinoma cells acts in concert to both induce
negative regulators and suppress positive regulators of the TGF-
pathway.
-catenin gene is approximately 4-fold
up-regulated by the autocrine production of hGH.
-Catenin is a
pivotal component of the Wnt signaling pathway and has been implicated
in embryonic development and carcinogenesis. Normally, cytosolic
-catenin is sequestered by the adenomatous polyposis coli (APC) gene
product which promotes
-catenin degradation and by cadherins which
integrate
-catenin into adherens junctions (47). Wnt signal
activation results in stabilization of
-catenin (48, 49) and
subsequent translocation of
-catenin to the nucleus where it binds
to T cell factor class transcription factors resulting in
expression of T cell factor-responsive genes (50, 51). It is now clear
that deregulation of Wnt signal transduction is associated with
tumorigenesis. Truncations of the APC protein are linked to the
familial adenomatous polyposis coli syndrome and are found in the
majority of sporadic colon carcinomas (52, 53). Similarly APCmin (a
mutation of the murine apc gene) mice have an increased
incidence not only of intestinal neoplasia but also carcinoma of the
mammary gland (54), and an APC truncation has also been reported in a
human breast cancer cell line (55). High Wnt gene expression
(Wnt-2, Wnt-4, and Wnt-7b) has also
been reported in proliferative disorders of the human breast, and
overexpression of Wnt-1 and Wnt-3 in the murine mammary gland results
in mammary hyperplasia and an increased incidence of mammary gland
tumors (56). One of the targets of
-catenin-stimulated gene
expression in the human mammary gland is cyclin D1 (57). High
-catenin activity also significantly correlated with a poor clinical
prognosis and could serve as a prognostic marker for breast cancer
(57). Whether the autocrine hGH increase in
-catenin mRNA also
results in increased
-catenin protein and transactivation is
currently under investigation. Should it do so, then hGH regulation of
-catenin transactivation would represent a novel mechanism of
hormonal signal transduction and also provide a mechanism by which hGH excess results in carcinoma. In this regard it is interesting that the
most common cancer in acromegaly is carcinoma of the colon (58) where a
potential hGH-stimulated increase in
-catenin may be a contributing factor.
-activated sequence sites (STAT5-binding
site) of the GH-responsive element of the serine protease inhibitor 2.1 promoter results in diminished transcriptional activity (59). However,
for
-casein which is also a STAT5-regulated gene, YY1
represses transcription since the YY1 site overlaps a
-activated
sequence site (60, 61). Thus, PRL (and presumably GH) relieves the
repression of YY1 to stimulate transcription. Autocrine hGH may
therefore regulate the level of YY1 to influence specifically
STAT5-mediated gene transcription-dependent on whether YY1
is acting as an enhancer or repressor of STAT5-mediated gene transcription through a particular promoter. This would allow the
possibility that autocrine hGH could regulate a specific set of genes
separate from those regulated by exogenously applied or "endocrine"
hGH.
and -
) (67).
Furthermore, an altered form of CHOP, TLS-CHOP, formed by reciprocal
translocation of the chop gene and a gene encoding an
RNA-binding protein is associated with human myxoid liposarcoma (68,
69). Erythropoietin has also been demonstrated to up-regulate CHOP to
participate in the erythroid differentiation program (24, 25). Here we
have demonstrated that overexpression of CHOP in mammary carcinoma
cells stimulated with hGH offers dramatic protection from apoptosis in
a p38 MAP kinase-dependent manner. Such apparent
dichotomous function has also been described for NF-
B which exhibits
both pro-apoptotic and anti-apoptotic functions dependent on cell type
(70). It has been observed that CHOP protein influences gene expression as both a dominant negative regulator of C/EBP binding to one class of
DNA targets and positively by directing CHOP-C/EBP heterodimers to
other sequences (18), and this observation may constitute the mechanism
of its apparent dichotomous function. The survival function of CHOP in
mammary carcinoma cells 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 (71). It was postulated
that chop overexpression may inhibit the cellular
differentiation to facilitate mammary gland tumorigenesis (71).
However, it is plausible that mammotrophic factors such as hGH in
executing their physiological roles may simply utilize CHOP to mediate
cell survival and that the subsequent failure to undergo programmed
cell death, in situations of cellular injury associated with DNA
damage, would result in carcinoma (72). It is therefore interesting
that the DNA-damaging effect of bleomycin in human lymphocytes is
enhanced by hGH (73); and hGH and the related hormone prolactin have
both been implicated in the development of mammary carcinoma (74). It
is also interesting that GH itself has also been reported to be both
mitogenic and growth inhibitory depending on the cellular context
(75).
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FOOTNOTES |
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* This work was supported by the National Science and Technology Board of Singapore (to P. E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Present address: CNRS UMR 5578, Physiologies Energetiques Cellulaires et Moléculaires, Université Claude Bernard, Lyon-1, France.
To whom correspondence should be addressed: Inst. of Molecular
and Cell Biology, 30 Medical Dr., Singapore 117609, Republic of
Singapore. Tel.: 65-8747847; Fax: 65-7791117; E-mail: mcbpel@ imcb.nus.edu.sg.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M100437200
2 M. Raccurt, E. Moudilou, S. Recher, T. Garcia-Caballero, L. Frappart, R. Dante, G. Morel, P. E. Lobie, and H. C. Mertani, manuscript in preparation.
3 H. C. Mertani, E. L. K. Goh, and P. E. Lobie, unpublished data.
4 R. Graichen, Y. Sun, K. O. Lee, and P. E. Lobie, manuscript in preparation.
5 E. L. K. Goh and P. E. Lobie, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
GH, growth hormone;
hGH, human growth hormone;
CAT, chloramphenicol acetyltransferase;
MAP, mitogen-activated protein kinase;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
BrdUrd, 5'-bromo-2'-deoxyuridine;
DOTAP, N-[1-(2,3-dioleoylloxy)propyl]-N,N,N-trimethylammonium
methyl sulfate;
ER, endoplasmic reticulum;
FBS, fetal bovine serum;
bp, base pair;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
CSLM, confocal laser scanning microscopy;
CMV, cytomegalovirus;
TGF-, transforming growth factor-
;
APC, adenomatous polyposis
coli;
MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetetrazdium,
inner salt.
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