(hGH) Regulation of Human Mammary Carcinoma Cell Gene Expression

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 24, Issue of June 15, pp. 21464 –21475, 2001 Printed in U.S.A.

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* Received for publication, January 17, 2001, and in revised form, March 26, 2001 Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M100437200

Hichem C. Mertani‡§¶, Tao Zhu‡§, Eyleen L. K. Goh‡, Kok-Onn Lee储, Ge´rard Morel**, and Peter E. Lobie‡储‡‡ From the ‡Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore, the 储Department of Medicine, National University of Singapore, Singapore 119074, Republic of Singapore, and **CNRS UMR 5578, Physiologies Energetiques Cellulaires et Mole´culaires, Universite´ Claude Bernard, Lyon-1, France

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 translationdeficient 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

* 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. This 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 Mole´culaires, Universite´ 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. 1 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;

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 autocrineproduced 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 hGHproducing cells display a marked insulin-like growth factor-1independent 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 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-(4sulfophenyl)-2H-tetetrazdium, inner salt. 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.

21464

This paper is available on line at http://www.jbc.org

CHOP Is a Mediator of GH-stimulated Cell Survival 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. EXPERIMENTAL PROCEDURES

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). Peroxidaseconjugated 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 ␤-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. 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

21465

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 ⫺951 to ⫹91 of the human CHOP gene were a generous gift from Dr. Nikki J. Holbrook (26). 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 ␮g/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 pyrocarbonatetreated 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 ⫺70 °C for further analysis. 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 [␣-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. 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 [␣-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 ␮l of reagent mix (containing dATP, dGTP, and dTTP and random hexanucleotides) and 50 ␮Ci of [␣-32P]dCTP was added to the denatured DNA, and the volume was adjusted to 49 ␮l with distilled water. 1 ␮l 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. 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 ␮g of the CHOP cDNA fragment labeled to a specific activity of 1–2 ⫻ 109 dpm/␮g was added, and the membrane was incubated at 68 °C overnight. The membrane was washed three times with prewarmed 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⫻

21466

CHOP Is a Mediator of GH-stimulated Cell Survival

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 ␤-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. Reverse Transcriptase-Polymerase Chain Reaction—RT-PCR was performed in a final volume of 50 ␮l containing 0.2 ␮g of mRNA template, 0.6 ␮M primers, 2.0 ␮l of enzyme mix, 400 ␮M 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 ␤-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. Sequences of the primers are as follows: CHOP primers (sense) 5⬘-GCACCTCCCAGAGCCCTCACTCTCC-3⬘ and (antisense) 5⬘-GTCTACTCCAAGCCTTCCCCCTGCG-3⬘; ␤-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). 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 ␮g of reporter plasmid (p5W1) and 1.0 ␮g 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 serumfree 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 ␮l 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 ␮M 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 ␮l 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 ␤-actin was detected with a 1:500 dilution of ␤-actin monoclonal antibody. 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.

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. Transient Transfection Procedure and Luciferase Assays—MCFMUT 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 ␮g of pCMV␤ and 1 ␮g of reporter plasmid pFR-Luc were transfected together with 20 ng of the respective fusion transactivator plasmid (pFA-CHOP or pFC2-dbd). For each well, 4 ␮g of DOTAP for each ␮g of DNA was used as per the manufacturer’s instructions. DNA and the DOTAP reagents were diluted separately in 100 ␮l 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 ␮l 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 ␮l of precleared cell lysate according to a standard protocol (21).


21467

TABLE I Identification by cDNA array of genes positively regulated by the autocrine production of hGH in mammary carcinoma cells Genes were considered positively regulated when they exhibited a 2-fold or greater increase in the presence of autocrine hGH (MCF-hGH cells) compared to the absence of autocrine hGH (MCF-MUT cells) in three independently performed experiments. Fold regulation is calculated as the ratio of the level of gene expression in MCF-hGH cells to that in MCF-MUT cells. GeneBank™ number

M15024 X15218 X03484 D10924 M74816 M31899 M32865 M36089 M74524 M29971 K00065 U07418 D21090 D21235 M60974 S40706 M97287 U10421 X67951 U14722 X52425 M21097 Y00796 X87838

Name

-Fold stimulation

Myb proto-oncogene protein SKI oncogene raf proto-oncogene HM89 (probable G protein-coupled receptor LCR1 homolog) Clusterin DNA repair helicase [ERCC3] ATP-dependent DNA helicase II DNA repair protein XRCC1 Ubiquitin-conjugating enzyme E2 Methylated DNA protein cysteine methyltransferase Superoxide dismutase DNA mismatch repair protein hMLH1 UV excision repair protein RAD23 (HHR23B) UV excision repair protein RAD23 (HHR23A) Growth arrest and DNA damage-inducible protein GADD45 Growth arrest and DNA damage-inducible protein GADD153 DNA-binding protein SATB1 Homeobox protein HOXA1 Proliferation-associated protein PAG Activin type 1 receptor Interleukin-4 receptor ␣ chain CD19 B-lymphocyte antigen Integrin ␣ L ␤-Catenin

3.05 4.10 2.00 3.45 2.25 12.50 6.10 5.55 3.50 2.85 13.45 2.30 2.45 3.75 3.95 5.10 3.20 8.20 2.15 4.10 2.15 4.90 3.70 3.60

Generation of MCF-7 Cells Stably Transfected with Human CHOP cDNA—MCF-7 cells grown in serum-free RPMI medium were transfected with 1 ␮g 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 ␮g/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 serumdeprived 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 ␮l/well, according to the indicated serum conditions and times. At the end of the 24-h period, 20 ␮l/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 ␮g/ml) for 10 min at room temperature. Following washing with PBS, nuclear morphology was examined under a UVvisible 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. RESULTS

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 damageinducible protein (GADD)45 and GADD153 (otherwise known as CHOP). Other up-regulated genes of particular interest include

21468


TABLE II Identification by cDNA array of genes negatively regulated by the autocrine production of hGH in mammary carcinoma cells Genes were considered negatively regulated when they exhibited a 2-fold or greater 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. Fold regulation is calculated as the ratio of the level of gene expression in MCF-hGH cells to that in MCF-MUT cells. GeneBank™ number

J04111 L25080 X02751 M26708 M25753 U10564 U41816 M33294 J03746 M68891 M59079 X69111 M95809 M97796 X69391 M28372 L12579 D26156 D90209 L05515 L14922 D28118 M36711 M76541 U30504 M29366 S59184 M14200

Name

-Fold stimulation

Transcription factor AP-1 (c-jun proto-oncogene) Transforming protein RhoA Transforming protein p21 (n-Ras) Prothymosin-␣ Cyclin B1 CDK tyrosine 15-kinase; wee1Hu C-1 Tumor necrosis factor receptor 1 Glutathione S-transferase microsomal Endothelial transcription factor GATA-2 CCAAT-binding transcription factor subunit DNA-binding protein inhibitor ID-3 Basic transcription factor (62-kDa subunit; BTF-2) DNA-binding protein inhibitor ID-2 60 S ribosomal protein L6 (DNA-binding protein TAX) Sterol regulatory element-binding protein CCAAT displacement protein Transcriptional activator hSNF2b cAMP-dependent transcription factor ATF-4 cAMP-response element-binding protein CRE-BPA Activator 1 140-kDa subunit DB1 (zinc finger protein 161) Transcription factor AP-2 Transcriptional repressor protein YY1 Transcription initiation factor TFIID subunit TAFII31 Protein-tyrosine kinase receptor ERBB-3 Related to receptor tyrosine kinase (RYK) Acyl-CoA-binding protein

0.10 0.20 0.25 0.40 0.30 0.30 0.40 0.50 0.40 0.15 0.10 0.15 0.30 0.05 0.40 0.10 0.20 0.30 0.10 0.05 0.40 0.20 0.40 0.50 0.20 0.45 0.45 0.35

the ski oncogene, homeobox protein HOXA1, and ␤-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. 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 ␤-actin mRNA did not differ between the two cell lines and was used as loading control (Fig. 2B). 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 ␤-actin mRNA remained constant between MCFMUT and MCF-hGH cells under the different experimental conditions (Fig. 2D). 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 ⫺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 MCFhGH 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. 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


21469

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 5ⴕ start site of the chop gene. MCF-MUT and MCF-hGH cells in serum-free media or in serumfree media supplemented with either 100 nM hGH or 10% FBS were transiently transfected with the respective plasmids (1 ␮g of p5W1 and 1 ␮g 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.

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 lefthand 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.

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 MCFhGH 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 MCFhGH 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 ␤-actin protein under the different experimental conditions (Fig. 4A). 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 MCFhGH 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␤ 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 MCFMUT 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 CHOPmediated 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. 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 ␮M SB203580 (Fig. 5B). Thus autocrine hGH production by mammary carcinoma cells resulted in enhancement of CHOP-mediated transcription in a p38 MAP kinase-

21470


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.

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 ␮M) (Fig. 6C). The basal level of CHOPmediated 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 ␮M 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 MCFCMV cells (Fig. 7A). Stimulation of MCF-CMV and MCFCHOP cells with 10% FBS resulted in equivalent increases in cell number after 48 h. Since the increase in cell number


21471

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 serumfree 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 vectortransfected 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 ␮M SB203580) abrogated the protection from apoptosis afforded by hGH stimulation of CHOP-overexpressing mammary carcinoma cells (MCF-CHOP). DISCUSSION

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 ␮M) 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.

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 hGHstimulated increase in cell number but is not involved in hGHstimulated 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 MCFhGH 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

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 MCFMUT 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-␤-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

3

H. C. Mertani, E. L. K. Goh, and P. E. Lobie, unpublished data.

21472


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. CHOPmediated 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 ␮M 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.

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. We have observed here that the ␤-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 4 R. Graichen, Y. Sun, K. O. Lee, and P. E. Lobie, manuscript in preparation.

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. 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 ␥-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 reg-


FIG. 7. Effect of CHOP overexpression in mammary carcinoma cells on the exogenous hGH and FBS-stimulated increase in total cell number and 5ⴕ-bromo-2ⴕ-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 MCFCMV 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.

ulated by exogenously applied or “endocrine” hGH. 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 adipo-

21473

FIG. 8. A, effect of CHOP overexpression in mammary carcinoma cells stably transfected with CHOP cDNA on the exogenous hGH and FBSstimulated protection from apoptotic cell death. MCF-CMV and MCFCHOP cells were cultured in serum-free medium for 24 h or in serumfree 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 ␮M where indicated. The results are presented as mean ⫾ S.D. *, p ⬍ 0.01.

cytic differentiation in response to metabolic stress by interfering with the accumulation of adipogenic C/EBP isoforms (C/ EBP␣ 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

21474


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). 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

5

E. L. K. Goh and P. E. Lobie, unpublished observations.

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. REFERENCES 1. Mol, J. A., Garderen, E. V., Selman, P. J., Wolfswinkel, J., Rijnberk, A., and Rutteman, G. R. (1995) J. Clin. Invest. 95, 2028 –2034 2. Mol, J. A., Henzen-Logman, S. C., Hageman, P., Misdorp, W., Blankestein, M. A., and Rijnberk, A. (1995) J. Clin. Endocrinol. & Metab. 80, 3094 –3096 3. Lantinga-van Leeuwen, I. S., Oudshoorn, M., and Mol, J. A. (1999) Mol. Cell. Endocrinol. 150, 121–128 4. Lincoln, D. T., Waters, M. J., Breiphol, W., Sinowatz, F., and Lobie, P. E. (1990) Acta Histochem. 40, 47– 49 5. Jammes, H., Gaye, P., Belair, L., and Djiane, J. (1991) Mol. Cell. Endocrinol. 75, 27–35 6. Ilkbahar, Y., Wu, K., Thodarson, G., and Talamentes, F. (1995) Endocrinology 136, 386 –392 7. Glimm, D. R., Baracos, V. E., and Kennely, J. J. (1990) J. Endocrinol. 126, R5–R8 8. Sobrier, M. L., Duquesnoy, P., Duriez, B., Amselem, S., and Goossens, M. (1993) FEBS Lett. 319, 16 –20 9. Mertani, H. C., Delehaye-Zervas, M. C., Martini, J. F., Postel-Vinay, M. C., and Morel, G. (1995) Endocrine 3, 135–142 10. Mertani, H. C., Garcia-Caballero, T., Lambert, A., Gerard, F., Palayer, C., Boutin, J. M., Vonderhaar, B. K., Waters, M. J., Lobie, P. E., and Morel, G. (1998) Int. J. Cancer 79, 202–211 11. Plaut, K., Ikeda, M., and Vonderhaar, B. K. (1993) Endocrinology 133, 1843–1848 12. Feldman, M., Ruan, W., Cunningham, B. C., Wells, J. A., and Kleinberg, D. L. (1993) Endocrinology 133, 1602–1608 13. Kaulsay, K. K., Mertani, H. C., Tornell, J., Morel, G., Lee, K. O., and Lobie, P. E. (1999) Exp. Cell Res. 250, 35–50 14. Kaulsay, K. K., Zhu, T., Bennett, W. F., Lee, K. O., and Lobie, P. E. (2001) Endocrinology 142, 767–777 15. Kaulsay, K. K., Mertani, H. C., Lee, K. O., and Lobie, P. E. (2000) Endocrinology 141, 1571–1584 16. Isaksson, O. G., Eden, S., and Jansson, J. O. (1985) Annu. Rev. Physiol. 47, 483– 499 17. Ron, D., and Habener, J. F. (1992) Genes Dev. 6, 439 – 453 18. Ubeda, M., Wang, X. Z., Zinszner, H., Wu, I., Habener, J. F., and Ron, D. (1996) Mol. Cell. Biol. 16, 1479 –1489 19. Ubeda, M., Vallejo, M., and Habener, J. F. (1999) Mol. Cell. Biol. 19, 7589 –7599 20. Wang, X. Z., and Ron, D. (1996) Science 272, 1347–1349 21. Zhu, T., and Lobie, P. E. (2000) J. Biol. Chem. 275, 2103–2114 22. Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R. T., Remotti, H., Stevens, J. L., and Ron, D. (1998) Genes Dev. 12, 982–995 23. Friedman, A. D. (1996) Cancer Res. 56, 3250 –3256 24. Coutts, M., Cui, K., Davis, K. L., Keutzer, J. C., and Sytkowski, A. J. (1999) Blood 93, 3369 –3378 25. Cui, K., Coutts, M., Stahl, J., and Sytkowski, A. J. (2000) J. Biol. Chem. 275, 7591–7596 26. Outinen, P.A., Sood, S. K., Liaw, P. C., Sarge, K. D., Maeda, N., Hirsh, J., Ribau, J., Podor, T. J., Weitz, J. I., and Austin, R. C. (1998) Biochem. J. 332, 213–221 27. Park, J. S., Luethy, J. D., Wang, M. G., Fargnoli, J., Fornace, A. J., Jr., McBride, O. W., and Holbrook, N. J. (1992) Gene (Amst.) 116, 259 –267 28. Wood, T. J., Sliva, D., Lobie, P. E., Pircher, T. J., Gouilleux, F., Wakao, H., Gustafsson, J. A., Groner, B., Norstedt, G., and Haldosen, L. A. (1995) J. Biol. Chem. 270, 9448 –9453 29. Adelmant, G., Gilbert, J. D., and Freytag, S. O. (1998) J. Biol. Chem. 273, 15574 –15581 30. Moller, C., Hansson, A., Enberg, B., Lobie, P. E., and Norstedt, G. (1992) J. Biol. Chem. 267, 23403–23408 31. Del Bino, G., Darzynkiewicz, Z., Degraef, C., Mosselmans, R., Fokan, D., and Galand, P. (1999) Cell Proliferation 32, 25–37 32. Bruhat, A., Jousse, C., Wang, X. Z., and Ron, D. (1997) J. Biol. Chem. 272, 17588 –17593 33. Abcouwer, S. F., Schwarz, C., and Meguid, R. A. (1999) J. Biol. Chem. 274, 28645–28651 34. Goh, E. L., Pircher, T. J., and Lobie, P. E. (1998) Endocrinology 139, 4364 – 4372 35. Haeffner, A., Deas, O., Mollereau, B., Estaquier, J., Mignon, A., HaeffnerCavaillon, N., Charpentier, B., Senik, A., and Hirsch, F. (1999) Eur. J. Immunol. 29, 334 –344

CHOP Is a Mediator of GH-stimulated Cell Survival 36. Shen, J., Wu, H., and Gudas, L. (2000) Exp. Cell Res. 25, 274 –283 37. Li, H., Bartold, P. M., Zhang, C. Z., Clarkson, R. W., Young, W. G., and Waters, M. J. (1998) Endocrinology 139, 3855–3862 38. Barkas, A. E., Brodeur, D., and Stavnezer, E. (1986) Virology 151, 131–138 39. Sutrave, P., Kelly, A. M., and Hughes, S. H. (1990) Genes Dev. 4, 1462–1472 40. Sutrave, P., Copeland, T. D., Showalter, S. D., and Hughes, S. H. (1990) Mol. Cell. Biol. 10, 3137–3144 41. Engert, J. C., Servaes, S., Sutrave, P., Hughes, S. H., and Rosenthal, N. (1995) Nucleic Acids Res. 23, 2988 –2994 42. Nicol, R., and Stavnezer, E. (1998) J. Biol. Chem. 273, 3588 –3597 43. Nicol, R., Zheng, G., Sutrave, P., Foster, D. N., and Stavnezer, E. (1999) Cell Growth Differ. 10, 243–254 44. Nomura, N., Sasamoto, S., Ishii, S., Date, T., Matsui, M., and Ishizaki, R. (1989) Nucleic Acids Res. 17, 5489 –5500 45. Xu, W., Angelis, K., Danielpour, D., Haddad, M. M., Bischof, O., Campisi, J., Stavnezer, E., and Medrano, E. E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5924 –5929 46. Tan, M., Wang, Y., Guan, K., and Sun, Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 109 –114 47. Cadigan, K. M., and Nusse, R. (1997) Genes Dev. 11, 3286 –3305 48. Giarre, M., Semenov, M. V., and Brown, A. M. (1998) Ann. N. Y. Acad. Sci. 23, 43–55 49. Shimizu, H., Julius, M. A., Giarre, M., Zheng, Z., Brown, A. M., and Kitajewski, J. (1997) Cell Growth Differ. 8, 1349 –1358 50. Young, C. S., Kitamura, M., Hardy, S., and Kitajewski, J. (1998) Mol. Cell. Biol. 18, 2474 –2485 51. Kolligs, F. T., Hu, G., Dang, C. V., and Fearon, E. R. (1999) Mol. Cell. Biol. 19, 5696 –5706 52. Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, B., and Clevers, H. (1997) Science 275, 1784 –1787 53. Kinzler, K. W., and Vogelstein, B. (1996) Cell 87, 159 –170 54. Moser, A. R., Mattes, E. M., Dove, W. F., Lindstrom, M. J., Haag, J. D., and Gould, M. N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8977– 8981 55. Schlosshauer, P. W., Brown, S. A., Eisinger, K., Yan, Q., Guglielminetti, E. R., Parsons, R., Ellenson, L. H., and Kitajewski, J. (2000) Carcinogenesis 21, 1453–1456 56. Huguet, E. L., McMahon, J. A., McMahon, A. P., Bicknell, R., and Harris, A. L. (1994) Cancer Res. 54, 2615–2621 57. Lin, S. Y., Xia, W., Wang, J. C., Kwong, K. Y., Spohn, B., Wen, Y., Pestell, R. G., and Hung, M. C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4262– 4266 58. Orme, S. M., McNally, R. J., Cartwright, R. A., and Belchetz, P. E. (1998) J. Clin. Endocrinol. & Metab. 83, 2730 –2734 59. Bergad, P. L., Towle, H. C., and Berry, S. A. (2000) J. Biol. Chem. 275, 8114 – 8120 60. Meier, V. S., and Groner, B. (1994) Mol. Cell. Biol. 14, 128 –137 61. Raught, B., Khursheed, B., Kazansky, A., and Rosen, J. (1994) Mol. Cell. Biol.

21475

14, 1752–1763 62. Barone, M. V., Crozat, A., Tabaee, A., Philipson, L., and Ron, D. (1994) Genes Dev. 8, 453– 464 63. Zhan, Q., Lord, K. A., Alamo, I., Jr., Hollander, M. C., Carrier, F., Ron, D., Kohn, K. W., Hoffman, B., Liebermann, D. A., and Fornace, A. J., Jr. (1994) Mol. Cell. Biol. 14, 2361–2371 64. Brenner, B., Koppenhoefer, U., Weinstock, C., Linderkamp, O., Lang, F., and Gulbins, E. (1997) J. Biol. Chem. 272, 22173–22181 65. Kim, R., Ohi, Y., Inoue, H., Aogi, K., and Toge, T. (1999) Anticancer Res. 19, 1779 –1783 66. Kim, R., Ohi, Y., Inoue, H., and Toge, T. (1999) Anticancer Res. 19, 5399 –5405 67. Batchvarova, N., Wang, X. Z., and Ron, D. (1995) EMBO J. 14, 4654 – 4661 68. Crozat, A., Aman, P., Mandahl, N., and Ron, D. (1993) Nature 363, 640 – 644 69. Rabbitts, T. H., Forster, A., Larson, R., and Nathan, P. (1993) Nat. Genet. 4, 175–180 70. Lin, B., Williams-Skipp, C., Tao, Y., Schleicher, M. S., Cano, L. L., Duke, R. C., and Scheinman, R. I. (1999) Cell Death Differ. 6, 570 –582 71. Arnal, M., Solary, E., Brunet-Lecomte, P., and Lizard-Nacol, S. (1999) Int. J. Mol. Med. 4, 545–548 72. Hartwell, L. (1992) Cell 71, 543–546 73. Cianfarani, S., Tedeschi, B., Germani, D., Prete, S. P., Rossi, P., Vernole, P., Caporossi, D., and Boscherini, B. (1998) Eur. J. Clin. Invest. 28, 41– 47 74. Wennbo, H., and Tornell, J. (2000) Oncogene 19, 1072–1076 75. Nam, S. Y., and Lobie, P. E. (2000) Obesity Rev. 1, 73– 86 76. Rudick, V. L., Rudick, M. J., and Brun-Zinkernagel, A. M. (1993) J. Cell Sci. 104, 509 –520 77. Shamu, C., Cox, J., and Walter, P. (1994) Trends Cell Biol. 4, 56 – 60 78. Bartlett, J. D., Luethy, J. D., Carlson, S. G., Sollott, S. J., and Holbrook, N. J. (1992) J. Biol. Chem. 267, 20465–20470 79. Chen, Q., Yu, K., Holbrook, N. J., and Stevens, J. L. (1992) J. Biol. Chem. 267, 8207– 8212 80. Price, B. D., and Calderwood, S. K. (1992) Cancer Res. 52, 3814 –3817 81. Carlson, S. G., Fawcett, T. W., Bartlett, J. D., Bernier, M., and Holbrook, N. J. (1993) Mol. Cell. Biol. 13, 4736 – 4744 82. Marten, N. W., Burke, E. J., Hayden, J. M., and Straus, D. S. (1994) FASEB J. 8, 538 –544 83. Wang, X. Z., Lawson, B., Brewer, J. W., Zinszner, H., Sanjay, A., Mi, L. J., Boorstein, R., Kreibich, G., Hendershot, L. M., and Ron, D. (1996) Mol. Cell. Biol. 16, 4273– 4280 84. Fawcett, T. W., Xu, Q., and Holbrook, N. J. (1997) Cell Stress Chaperones 2, 104 –109 85. Goh, E. L., Pircher, T. J., Wood, T. J., Norstedt, G., Graichen, R., and Lobie, P. E. (1997) Endocrinology 138, 3207–3215 86. Fawcett, T. W., Martindale, J. L., Guyton, K. Z., Hai, T., and Holbrook, N. J. (1999) Biochem. J. 339, 135–141 87. Guyton, K. Z., Xu, Q., and Holbrook, N. J. (1996) Biochem. J. 314, 547–554