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Autocrine Human Growth Hormone (hGH) Regulation of Human Mammary Carcinoma Cell Gene Expression

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m100437200

ISSN

1083-351X

Autores

Hichem C. Mertani, Tao Zhu, EyleenL.K. Goh, Kok‐Onn Lee, Gérard Morel, Peter E. Lobie,

Tópico(s)

Pancreatic function and diabetes

Resumo

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 (24Coutts M. Cui K. Davis K.L. Keutzer J.C. Sytkowski A.J. Blood. 1999; 93: 3369-3378Crossref PubMed Google Scholar) or negatively (28Wood T.J. Sliva D. Lobie P.E. Pircher T.J. Gouilleux F. Wakao H. Gustafsson J.A. Groner B. Norstedt G. Haldosen L.A. J. Biol. Chem. 1995; 270: 9448-9453Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), 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. 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 (24Coutts M. Cui K. Davis K.L. Keutzer J.C. Sytkowski A.J. Blood. 1999; 93: 3369-3378Crossref PubMed Google Scholar) or negatively (28Wood T.J. Sliva D. Lobie P.E. Pircher T.J. Gouilleux F. Wakao H. Gustafsson J.A. Groner B. Norstedt G. Haldosen L.A. J. Biol. Chem. 1995; 270: 9448-9453Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), 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. growth hormone human growth hormone chloramphenicol acetyltransferase mitogen-activated protein kinase reverse transcriptase-polymerase chain reaction 5′-bromo-2′-deoxyuridine N-[1-(2,3-dioleoylloxy)propyl]-N,N,N-trimethylammonium methyl sulfate endoplasmic reticulum fetal bovine serum base pair phosphate-buffered saline bovine serum albumin confocal laser scanning microscopy cytomegalovirus transforming growth factor-β adenomatous polyposis coli (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetetrazdium, inner salt The growth hormone (GH)1 gene is expressed in the normal and neoplastic mammary gland of the cat and dog (1Mol J.A. Garderen E.V. Selman P.J. Wolfswinkel J. Rijnberk A. Rutteman G.R. J. Clin. Invest. 1995; 95: 2028-2034Crossref PubMed Scopus (109) Google Scholar). 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 (2Mol J.A. Henzen-Logman S.C. Hageman P. Misdorp W. Blankestein M.A. Rijnberk A. J. Clin. Endocrinol. & Metab. 1995; 80: 3094-3096Crossref PubMed Google Scholar). 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. 2M. Raccurt, E. Moudilou, S. Recher, T. Garcia-Caballero, L. Frappart, R. Dante, G. Morel, P. E. Lobie, and H. C. Mertani, manuscript in preparation. 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 (3Lantinga-van Leeuwen I.S. Oudshoorn M. Mol J.A. Mol. Cell. Endocrinol. 1999; 150: 121-128Crossref PubMed Scopus (22) Google Scholar). GH receptor mRNA and protein have also been detected in the mammary gland epithelia of murine and rabbit (4Lincoln D.T. Waters M.J. Breiphol W. Sinowatz F. Lobie P.E. Acta Histochem. 1990; 40: 47-49Google Scholar, 5Jammes H. Gaye P. Belair L. Djiane J. Mol. Cell. Endocrinol. 1991; 75: 27-35Crossref PubMed Scopus (43) Google Scholar, 6Ilkbahar Y. Wu K. Thodarson G. Talamentes F. Endocrinology. 1995; 136: 386-392Crossref PubMed Scopus (55) Google Scholar), bovine (7Glimm D.R. Baracos V.E. Kennely J.J. J. Endocrinol. 1990; 126: R5-R8Crossref PubMed Scopus (69) Google Scholar), and human species (8Sobrier M.L. Duquesnoy P. Duriez B. Amselem S. Goossens M. FEBS Lett. 1993; 319: 16-20Crossref PubMed Scopus (113) Google Scholar, 9Mertani H.C. Delehaye-Zervas M.C. Martini J.F. Postel-Vinay M.C. Morel G. Endocrine. 1995; 3: 135-142Crossref PubMed Scopus (64) Google Scholar, 10Mertani H.C. Garcia-Caballero T. Lambert A. Gerard F. Palayer C. Boutin J.M. Vonderhaar B.K. Waters M.J. Lobie P.E. Morel G. Int. J. Cancer. 1998; 79: 202-211Crossref PubMed Scopus (141) Google Scholar). 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 (11Plaut K. Ikeda M. Vonderhaar B.K. Endocrinology. 1993; 133: 1843-1848Crossref PubMed Scopus (56) Google Scholar) and in vivo (12Feldman M. Ruan W. Cunningham B.C. Wells J.A. Kleinberg D.L. Endocrinology. 1993; 133: 1602-1608Crossref PubMed Scopus (77) Google Scholar). 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 (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar). 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 (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar). 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 (14Kaulsay K.K. Zhu T. Bennett W.F. Lee K.O. Lobie P.E. Endocrinology. 2001; 142: 767-777Crossref PubMed Scopus (81) Google Scholar). Also, autocrine hGH production results in enhancement of the rate of mammary carcinoma cell spreading on a collagen substrate (15Kaulsay K.K. Mertani H.C. Lee K.O. Lobie P.E. Endocrinology. 2000; 141: 1571-1584Crossref PubMed Scopus (30) Google Scholar). All of the studied effects of autocrine hGH on mammary carcinoma cell behavior are mediated via the hGH receptor (14Kaulsay K.K. Zhu T. Bennett W.F. Lee K.O. Lobie P.E. Endocrinology. 2001; 142: 767-777Crossref PubMed Scopus (81) Google Scholar). 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 (16Isaksson O.G. Eden S. Jansson J.O. Annu. Rev. Physiol. 1985; 47: 483-499Crossref PubMed Google Scholar). 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 waschop (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 (17Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (993) Google Scholar). 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 (18Ubeda M. Wang X.Z. Zinszner H. Wu I. Habener J.F. Ron D. Mol. Cell. Biol. 1996; 16: 1479-1489Crossref PubMed Google Scholar). 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 (19Ubeda M. Vallejo M. Habener J.F. Mol. Cell. Biol. 1999; 19: 7589-7599Crossref PubMed Scopus (119) Google Scholar). CHOP-mediated transcriptional activation in response to stress requires phosphorylation of CHOP by p38 MAP kinase (20Wang X.Z. Ron D. Science. 1996; 272: 1347-1349Crossref PubMed Scopus (753) Google Scholar), 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 (21Zhu T. Lobie P.E. J. Biol. Chem. 2000; 275: 2103-2114Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). 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 (22Zinszner H. Kuroda M. Wang X. Batchvarova N. Lightfoot R.T. Remotti H. Stevens J.L. Ron D. Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1712) Google Scholar), and overexpression of CHOP in growth factor-dependent 32D myeloid precursor cells (23Friedman A.D. Cancer Res. 1996; 56: 3250-3256PubMed Google Scholar) also results in apoptosis. CHOP protein has recently been demonstrated to be up-regulated by erythropoietin (24Coutts M. Cui K. Davis K.L. Keutzer J.C. Sytkowski A.J. Blood. 1999; 93: 3369-3378Crossref PubMed Google Scholar), resulting in erythroid differentiation (25Cui K. Coutts M. Stahl J. Sytkowski A.J. J. Biol. Chem. 2000; 275: 7591-7596Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). 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 ofchop is one mechanism by which hGH regulates mammary carcinoma cell number. DOTAP transfection reagent was obtained fromRoche 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 β-actin monoclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The fusiontrans-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 fromCLONTECH 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) (26Outinen P.A. Sood S.K. Liaw P.C. Sarge K.D. Maeda N. Hirsh J. Ribau J. Podor T.J. Weitz J.I. Austin R.C. Biochem. J. 1998; 332: 213-221Crossref PubMed Scopus (214) Google Scholar) under the control of the metallothionein 1a promoter (designated MCF-hGH) (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar). 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 (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar). 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 (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar). 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 (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar), and cell spreading (15Kaulsay K.K. Mertani H.C. Lee K.O. Lobie P.E. Endocrinology. 2000; 141: 1571-1584Crossref PubMed Scopus (30) Google Scholar). 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 (26Outinen P.A. Sood S.K. Liaw P.C. Sarge K.D. Maeda N. Hirsh J. Ribau J. Podor T.J. Weitz J.I. Austin R.C. Biochem. J. 1998; 332: 213-221Crossref PubMed Scopus (214) Google Scholar). MCF-hGH and MCF-MUT cells (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar) 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 mml-glutamine. 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 A 260/A 280absorption, 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. Poly(A)+ RNA was isolated from total RNA using oligotex resin as described (27Park J.S. Luethy J.D. Wang M.G. Fargnoli J. Fornace Jr., A.J. McBride O.W. Holbrook N.J. Gene ( Amst. ). 1992; 116: 259-267Crossref PubMed Scopus (119) Google Scholar). 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 [α-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, β-actin, α-tubulin, 23-kDa highly basic protein, ribosomal protein S9) did not differ by more than 10% between MCF-MUT and MCF-hGH cells. The CHOP cDNA fragment was derived from MCF-hGH cells by RT-PCR as described below and labeled with [α-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 [α-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. 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 × 109dpm/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 β-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 β-actin DNA fragment was labeled and hybridized to the stripped membrane and subjected to autoradiography as described above. 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; HotstartTaq 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 β-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′; β-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; β-actin 581 bp). 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 mmdithiothreitol). The protein content of the samples was normalized, and chloramphenicol acetyltransferase and luciferase assays were performed as described previously (28Wood T.J. Sliva D. Lobie P.E. Pircher T.J. Gouilleux F. Wakao H. Gustafsson J.A. Groner B. Norstedt G. Haldosen L.A. J. Biol. Chem. 1995; 270: 9448-9453Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Results were normalized to the level of luciferase activity to control for transfection efficiency. 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 (29Adelmant G. Gilbert J.D. Freytag S.O. J. Biol. Chem. 1998; 273: 15574-15581Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 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 β-actin was detected with a 1:500 dilution of β-actin monoclonal antibody. MCF cells were cultured on glass coverslips as described previously (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar). 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. 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 (21Zhu T. Lobie P.E. J. Biol. Chem. 2000; 275: 2103-2114Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). 1 μg of pCMVβ 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 mmEDTA, 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 β-galactosidase activity. The luciferase activities were normalized on the basis of protein content as well as on the β-galactosidase activity of pCMVβ vector. The β-galactosidase assay was performed with 20 μl of precleared cell lysate according to a standard protocol (21Zhu T. Lobie P.E. J. Biol. Chem. 2000; 275: 2103-2114Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). 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 (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar, 30Moller C. Hansson A. Enberg B. Lobie P.E. Norstedt G. J. Biol. Chem. 1992; 267: 23403-23408Abstract Full Text PDF PubMed Google Scholar). 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. Total cell number was estimated by use of the Cell Titer 96 kit as described previously (13Kaulsay K.K. Mertani H.C. Tornell J. Morel G. Lee K.O. Lobie P.E. Exp. Cell Res. 1999; 250: 35-50Crossref PubMed Scopus (104) Google Scholar). 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 enzym

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