Growth Regulation via Insulin-Like Growth Factor Binding Protein-4 and −2 in Association with Mutant K-ras in Lung Epithelia
2006; Elsevier BV; Volume: 169; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2006.051068
ISSN1525-2191
AutoresHanako Sato, Takuya Yazawa, Takehisa Suzuki, Hiroaki Shimoyamada, Koji Okudela, Masaichi Ikeda, Kenji Hamada, Hisafumi Yamada‐Okabe, Masayuki Yao, Yoshinobu Kubota, Takashi Takahashi, Hiroshi Kamma, Hitoshi Kitamura,
Tópico(s)Lung Cancer Treatments and Mutations
ResumoGain-of-function point mutations in K-ras affect early events in pulmonary bronchioloalveolar carcinoma. We investigated altered mRNA expression on K-Ras activation in human peripheral lung epithelial cells (HPL1A) using oligonucleotide microarrays. Mutated K-Ras stably expressed in HPL1A accelerated cell growth and induced the expression of insulin-like growth factor (IGF)-binding protein (IGFBP)-4 and IGFBP-2, which modulate cell growth via IGF. Other lung epithelial cell lines (NHBE and HPL1D) revealed the same phenomena as HPL1A by mutated K-ras transgene. Lung cancer cell growth was also accelerated by mutated K-ras gene transduction, whereas IGFBP-4/2 induction was weaker compared with mutated K-Ras-expressing lung epithelial cells. To understand the differences in IGFBP-4/2 inducibility via K-Ras-activated signaling between nonneoplastic lung epithelia and lung carcinoma, we addressed the mechanisms of IGFBP-4/2 transcriptional activation. Our results revealed that Egr-1, which is induced on activation of Ras-mitogen-activated protein kinase signaling, is crucial for transactivation of IGFBP-4/2. Furthermore, IGFBP-4 and IGFBP-2 promoters were often hypermethylated in lung carcinoma, yielding low basal expression/weak induction of IGFBP-4/2. These findings suggest that continuous K-Ras activation accelerates cell growth and evokes a feedback system through IGFBP-4/2 to prevent excessive growth. Moreover, this growth regulation is disrupted in lung cancers because of promoter hypermethylation of IGFBP-4/2 genes. Gain-of-function point mutations in K-ras affect early events in pulmonary bronchioloalveolar carcinoma. We investigated altered mRNA expression on K-Ras activation in human peripheral lung epithelial cells (HPL1A) using oligonucleotide microarrays. Mutated K-Ras stably expressed in HPL1A accelerated cell growth and induced the expression of insulin-like growth factor (IGF)-binding protein (IGFBP)-4 and IGFBP-2, which modulate cell growth via IGF. Other lung epithelial cell lines (NHBE and HPL1D) revealed the same phenomena as HPL1A by mutated K-ras transgene. Lung cancer cell growth was also accelerated by mutated K-ras gene transduction, whereas IGFBP-4/2 induction was weaker compared with mutated K-Ras-expressing lung epithelial cells. To understand the differences in IGFBP-4/2 inducibility via K-Ras-activated signaling between nonneoplastic lung epithelia and lung carcinoma, we addressed the mechanisms of IGFBP-4/2 transcriptional activation. Our results revealed that Egr-1, which is induced on activation of Ras-mitogen-activated protein kinase signaling, is crucial for transactivation of IGFBP-4/2. Furthermore, IGFBP-4 and IGFBP-2 promoters were often hypermethylated in lung carcinoma, yielding low basal expression/weak induction of IGFBP-4/2. These findings suggest that continuous K-Ras activation accelerates cell growth and evokes a feedback system through IGFBP-4/2 to prevent excessive growth. Moreover, this growth regulation is disrupted in lung cancers because of promoter hypermethylation of IGFBP-4/2 genes. Lung cancer is a leading cause of cancer-related death in many countries. Among the various histological types of lung cancer, adenocarcinomas are significantly more prevalent than squamous cell carcinoma.1Travis WD Travis LB Devesa SS Lung cancer.Cancer. 1995; 75: 191-202Crossref PubMed Scopus (737) Google Scholar, 2Colby TV Koss MK Travis WD Tumors of the lower respiratory tract.in: Colby TV Koss MK Travis WD Atlas of Tumor Pathology, series 3, fascicle 13. 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IARC Press, Lyon2004: 73-75Google Scholar Like adenocarcinomas, atypical adenomatous hyperplasia cells often carry a mutated K-ras gene, and thus K-ras gene mutations are considered to be involved in early-stage tumorigenesis of lung adenocarcinoma.20Kobayashi T Tsuda H Noguchi M Hirohashi S Shimosato Y Goya T Hayata Y Association of point mutation in c-K-ras oncogene in lung adenocarcinoma with particular reference to cytologic subtypes.Cancer. 1990; 66: 289-294Crossref PubMed Scopus (81) Google Scholar, 21Marchetti A Buttitta F Pellegrini S Chella A Bertacca G Filardo A Tognoni V Ferreli F Signorini E Angeletti CA Bevilacqua G Bronchioloalveolar lung carcinomas: K-ras mutations are constant events in the mucinous subtype.J Pathol. 1996; 179: 254-259Crossref PubMed Scopus (108) Google Scholar, 22Westra WH Baas IO Hruban RH Askin FB Wilson K Offerhaus GJ Slebos RJ K-ras oncogene activation in atypical alveolar hyperplasias of the human lung.Cancer Res. 1996; 56: 2224-2228PubMed Google Scholar, 23Cooper CA Carey FA Bubb VJ Lamb D Kerr KM Wyllie AH The pattern of K-ras mutation in pulmonary adenocarcinoma defines a new pathway of tumour development in the human lung.J Pathol. 1997; 181: 401-404Crossref PubMed Scopus (77) Google Scholar, 24Yoshida Y Shibata T Kokubu A Tsuta K Matsuno Y Kanai Y Asamura H Tsuchiya R Horohashi S Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung.Lung Cancer. 2005; 50: 1-8Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar However, it is unclear which kinds of genes are up-regulated in lung airway epithelial cells in response to the continuous activation of the mutated K-ras gene. Here, we show that K-ras gene activation in lung airway epithelia, including lung cancer cells, not only accelerates cell growth but also induces two kinds of growth-modulating factors, IGFBP-4 and IGFBP-2, through the MEK-MAPK-Egr-1 pathway. Furthermore, IGFBP-4 and IGFBP-2 expression/induction levels are substantially lower in lung cancer cells because of hypermethylation of IGFBP-4 and IGFBP-2 gene promoters. These results suggest that induction of IGFBP-4 and IGFBP-2 in lung airway epithelia is one of the feedback mechanisms controlling excessive growth via K-ras gene activation and that neo-plastic airway epithelia might gradually lose these growth-modulating systems as tumor aggressiveness increases. Human peripheral lung epithelial cell lines (HPL1A and HPL1D; donated from Aichi Cancer Center Research Institute, Nagoya, Japan), a human bronchial epithelial cell line (NHBE, which was immortalized by simian virus 40), and non-small-cell lung cancer cell lines (A549, H820, TKB6, TKB14, TKB1, and TKB5) were used in this study.10Okudela K Hayashi H Ito T Yazawa T Suzuki T Nakane Y Sato H Ishi H KeQin X Masuda A Takahashi T Kitamura H K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation (possible contribution to non-invasive expansion of lung adenocarcinoma).Am J Pathol. 2004; 164: 91-100Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 25Masuda A Kondo M Saito T Yatabe Y Kobayashi T Okamoto M Suyama M Takahashi T Establishment of human peripheral cell lines (HPL1) retaining of differentiated characteristics and responsiveness to epidermal growth factor, hepatocyte growth factor, and transforming growth factor β1.Cancer Res. 1997; 57: 4898-4904PubMed Google Scholar, 26Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Katoh H Fujimoto M Yokoyama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (76) Google Scholar Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin and were maintained at 37°C in 5% CO2. Subconfluent cells were used for the following experiments. To examine cell growth rate, cells were seeded at 5 × 104 cells/60-mm dish in 3 ml of medium and cultivated for up to 96 hours. After washing with phosphate-buffered saline (PBS) (pH 7.4), the cells were harvested by trypsinization and counted. Each examination was performed in triplicate. Statistical analysis was performed with the paired t-test, and differences between values were considered statistically significant at P < 0.05. Total RNA was extracted using Isogen (Nippon Gene, Tokyo, Japan), and cDNA synthesis and subsequent PCR reactions were performed using an RNA-PCR kit (Takara, Shiga, Japan) according to the manufacturer's instructions. The alteration of mRNA levels was investigated by semiquantitative RT-PCR. β-Actin served as an internal control. The cycle number of PCR was set in the range that the PCR product was exponentially increasing and was determined as 25 cycles for IGFBP-4 and IGFBP-2, 30 cycles for egr-1, and 22 cycles for β-actin. Densitometry scanning was conducted using National Institutes of Health (Rockville, MD) image computer software. The forward and reverse primer sequences specific for IGFBP-4, IGFBP-2, egr-1, and β-actin are detailed in Table 1.Table 1PCR Primers Used in This StudyTargetForward primer sequenceIGFBP-4 cDNA5′-TTCATCCCCATCCCCAACTGC-3′IGFBP-2 cDNA5′-TTGCAGACAATGGCGATGACC-3′egr-1 cDNA5′-GCAGCAGCAGCACCTTCAACC-3′β-Actin cDNA5′-GCGGGAAATCGTGCGTGACAT-3′Wild-type K-ras cDNA5′-GACGGCCGAAGCTTGCTGAAAATGACTGAATATAAACTTG-3′Mutated K-ras cDNA5′-GACGGCCGAAGCTTGCTGAAAATGACTGAATATAAACTTG-3′IGFBP-4 cDNA containing full coding region5′-GCGGTCATCCTGCCCCTCTGC-3′IGFBP-2 cDNA containing full coding region5′-GGGCGAGGGAGGAGGAAGAAG-3′IGFBP-4 promoter −1298 to +297 (for Luc)*Luciferase assay;5′-CACGCCCGGCTAACTT-3′ −855 to +297 (for Luc)5′-TTCAACGCAAAACTT-3′ −185 to +297 (for Luc)5′-ACTGCGCCGCTTCCTTCTTCG-3 −125 to +297 (for Luc)5′-CGACTCAGGACAGCGCC-3′ +11 to +297 (for Luc)5′-GCAGCCGCTCAGCCCCC-3′ −565 to −174 (for ChIP)†chromatin immunoprecipitation assay;5′-TGCGGGGGCGGGAGAGGT-3′ −317 to +131 (for ChIP)5′-GGCGGGGCGGGACGAGAC-3′ −695 to −43 (for BS)‡bisulfite sequencing.5′-TGAATGGGTTTGAGAGGTAAATATT-3′Egr-1-binding site-mutated IGFBP-4 promoter Site 1 mutant (for Luc)5′-CAAATGCGCCCTGGGGAGATTGCTAGGTCGGGAGAGGTTGCAAGGGGCAAGTG-3′ Site 2 mutant (for Luc)5′-GAGACTGAGGCCGCCTTGGTAGGGCGGGACGAGACTC-3′ Site 3 mutant (for Luc)5′-GAAAAGGACTTTCAGATGCTACGGCGGCTACGGCGGCGACTCAGGAC-3′ Site 4 mutant (for Luc)5′-GACTCAGGACAGCGCCCTATCCCCTAACGGCCGCCTC-3′ Site 5 mutant (for Luc)5′-CTCTCCCCCTCGCCCTACCTAGCTCCCCCACCTCTGGGAAG-3′IGFBP-2 promoter −522 to +114 (for Luc)5′-TCTAGACGGGTCTGAAACTCC-3′ −444 to +114 (for Luc)5′-CCCCAGGATGGAAGG-3′ −314 to +114 (for Luc)5′-TGAGCCGACTGAAATCTACTT-3′ −206 to +114 (for Luc)5′-TCGCGAACTGAACTGAGAGCA-3′ −134 to +114 (for Luc)5′-CAGAAGAGTGCGGAGGGACGG-3′ −31 to +114 (for Luc)5′-GCCCGCAGCCAACGC-3′ −202 to +113 (for ChIP)5′-CAGAAGAGTGCGGAGGGACGG-3′ −413 to +201 (for BS)5′-GTGATATTTTAGGATGGAAGGAGTT-3′Egr-1-binding site-mutated IGFBP-2 promoter Site 1 mutant (for Luc)5′-CACTTGCCGGCGCGAGTAAGTGTCGGGGGGGAAG-3′ Site 2 mutant (for Luc)5′-GAGGGGAGAAGGCATAGGGCGGGGAGAAG-3′ Site 3 mutant (for Luc)5′-CTTTAGGACCCGGCTGCTACGGCGAGGGAGGAG-3′Egr-1 promoter −600 to −363 (for BS)5′-GTTTGGGTTTTTTTAGTTTAGTTTA-3′ −374 to −75 (for BS)5′-TTGGGTAGTATTTTATTTGGAGTGG-3′Reverse primer sequenceSize (bp)5′-CTGCTACCCCACGCTTCCTTA-3′7675′-GGGATGTGCAGGGAGTAGAGG-3′3405′-TTTCCCCTTTCCCTTTAGCAA-3′14055′-GTGGACTTGGGAGAGGACTGG-3′8635′-CAGGATCCTCATTACATAATTACACACTTTG-3′5995′-CAGGATCCTCATTACATAATTACACACTTTG-3′5995′-CACACCCATGCTCACAAACAC-3′9695′-AGCAAGAAGGAGCAGGTGTGG-3′13055′-GACCGCCCGAGGACTGG-3′15955′-GACCGCCCGAGGACTGG-3′11525′-GACCGCCCGAGGACTGG-3′4825′-GACCGCCCGAGGACTGG-3′4225′-GACCGCCCGAGGACTGG-3′2875′-AAGCGGCGCAGTTTGGAA-3′3925′-AGCGGGGGAAGTTAGCAG-3′4485′-CCTTCCCAAAAATAAAAAAACC-3′6535′-CACTTGCCCCTTGCAACCTCTCCCGACCTAGCAATCTCCCCAGGGCGCATTTG-3′58575′-GAGTCTCGTCCCGCCCTACCAAGGCGGCC TCAGTCTC-3′58575′-GTCCTGAGTCGCCGCCGTAGCCGCCGTAGCATCTGAAAGTCCTTTTC-3′58575′-GAGGCGGCCGTTAGGGGATAGGGCGCTGTCCTGAGTC-3′58575′-CTTCCCAGAGGTGGGGGAGCTAGGTAGGGC GAGGGGGAGAG-3′58575′-CTGGCGGTCGGCAGC-3′6365′-CTGGCGGTCGGCAGC-3′5885′-CTGGCGGTCGGCAGC-3′4285′- CTGGCGGTCGGCAGC-3′3205′- CTGGCGGTCGGCAGC-3′2485′- CTGGCGGTCGGCAGC-3′1455′-CTGGCGGTCGGCAGC-3′3155′-CCCAATAACAACAACAACAAC-3′6495′-CTTCCCCCCCGACACTTACTCGCGCCGGCAAGTG-3′48985′-CTTCTCCCCGCCCTATGCCTTCTCCCCTC-3′48985′-CTCCTCCCTCGCCGTAGCAGCCGGGTCCTAAAG-3′48985′-AATACTACCCAAATAAAAATTATTCC-3′2385′-CATATATAAAAAACAAAAAACCCTAATATA-3′300Mutated sequences are underlined.* Luciferase assay;† chromatin immunoprecipitation assay;‡ bisulfite sequencing. Open table in a new tab Mutated sequences are underlined. We used PCR to amplify cDNA from a wild-type K-ras-expressing cancer cell line or a mutated K-ras cDNA-inserted plasmid vector (pSW11–1 containing a K-ras gene with a point mutation, Gly→Val, in codon 12; Riken Gene Bank, Wako, Japan) and K-ras-specific primers described in Table 1. The PCR products were cloned using the pT7Blue vector (Novagen, Madison, WI) and sequenced. The inserts were excised with HindIII and BamHI and then reinserted into the expression vector pcDNA3.1 (Invitrogen, San Diego, CA). The mutated K-ras-inserted, wild-type K-ras-inserted, or empty vector was transfected into HPL1A, HPL1D, NHBE, and TKB5 using a lipofection method (Superfect; Qiagen, Hilden, Germany). After selection for 3 weeks, the stable transfectants of mutated K-ras, wild-type K-ras, and empty vector were cloned and named SW, K, and C, respectively. To construct IGFBP-4 or IGFBP-2 expression vectors, cDNAs were obtained by RT-PCR using total RNA of HPL1A-SW and specific primer sets (Table 1) and were inserted into pcDNA3.1. Transfections using TKB5 cells and subsequent selection were performed as above, and the established clones were named TKB5-BP4 and TKB5-BP2, respectively. Cell lines HPL1A-SW, HPL1A-K, and HPL1A-C were used for gene chip analyses. cDNAs were synthesized using the Reverse Superscript Choice System (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's instructions. Purified total RNA (5 μg) was hybridized with an oligo-dT primer containing the T7 promoter sequence and then incubated with 200 U of Super Script II reverse transcriptase at 42°C for 1 hour. The cDNA was extracted with phenol/chloroform with Phase Lock Gel (Eppendorf-5 Prime, Inc., Boulder, CO) and concentrated by ethanol precipitation. The cRNA was also synthesized using the MEGAscript T7 kit (Ambion, Austin, TX) at 37°C for 6 hours. Mononucleotides and short oligonucleotides were removed by column chromatography (CHROMA SPIN+STE-100 column; Takara), and the cRNA in the eluates was sedimented by ethanol. Gene expression analyses were conducted using high-density oligonucleotide microarrays (HuGeneFL array, HuU95; Affymetrix, Santa Clara, CA) according to the manufacturer's instructions.27Lockhart DJ Dong H Byrne MC Follettie MT Gallo MV Chee MS Mittmann M Wang C Kobayashi M Horton H Brown EL Expression monitoring by hybridization to high-density oligonucleotide arrays.Nat Biotechnol. 1996; 14: 1675-1680Crossref PubMed Scopus (2833) Google Scholar, 28Lee CK Klopp RG Weindruch R Prolla TA Gene expression profile of aging and its retardation by caloric restriction.Science. 1999; 285: 1390-1393Crossref PubMed Scopus (1292) Google Scholar For hybridization with oligonucleotides on the chips, the cRNA was fragmented at 95°C for 35 minutes, and hybridization was performed at 45°C for 12 hours. After washing the chips, the chips were incubated with a biotinylated antibody against streptavidin and stained with streptavidin R-phycoerythrin to increase the hybridization signal, as described in the manufacturer's instructions. Each pixel level was collected with a laser scanner (Affymetrix), and the expression levels were analyzed using Affymetrix Microarray Suite version 4.0 software. Clustering analyses were conducted using the EISEN cluster program.29Eisen MB Spellman PT Brown PO Botstein D Cluster analysis and display of genome-wide expression patterns.Proc Natl Acad Sci USA. 1998; 95: 14863-14868Crossref PubMed Scopus (13371) Google Scholar Total cell lysates (50 μg protein per lane) were separated by 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were blocked for 1 hour at room temperature with 1% skim milk in Tris-buffered saline (TBS) containing 0.1% (v/v) Tween 20 (TBS-T) and then incubated with diluted mouse monoclonal anti-p44/42 MAPK, mouse monoclonal anti-phosphorylated MAPK (each from Cell Signaling Technology, Beverly, MA), rabbit polyclonal anti-IGFBP-4, or goat polyclonal anti-IGFBP-2 antibody (each from Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour. After washing three times for 10 minutes with TBS-T at room temperature, membranes were incubated at room temperature for 30 minutes with a diluted peroxidase-labeled secondary antibody against mouse (Amersham Biosciences, Buckinghamshire, UK), rabbit (Amersham Biosciences), or goat (Santa Cruz Biotechnology). The membranes were then washed three times for 10 minutes with TBS-T at room temperature, and immunopositive signals were visualized using an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences). Mouse anti-β-actin antibody (Sigma, St. Louis, MO) was used for the internal control. HPL1A, HPL1D, NHBE, and TKB5 cells (1 × 105) were seeded in 100-mm dishes and cultured for 24 hours. After rinsing with PBS three times, cultures were incubated in 10 ml of medium with or without 20 μmol/L MEK inhibitor (U0126; Promega, Madison, WI) for up to 24 hours. For recombinant IGF-1 (rIGF-1), rIGFBP-4, and rIGFBP-2 supplementation experiments, HPL1A, HPL1D, NHBE, and TKB5 cells were seeded in six-well plates at 5 × 104 cells/well in 3 ml of medium containing 600 μg of rIGFBP-4 (Genzyme-Techne Corp., Minneapolis, MN) or rIGFBP-2 (Genzyme-Techne). After 30 minutes, 75 ng of rIGF-I (Genzyme-Techne) was added to the medium, and the cells were cultured for 48 hours. For rIGF-1 supplementation experiments using TKB5 derivatives, TKB5-C, TKB5-BP4 (stably IGFBP-4-transfected TKB5 clone), and TKB5-BP2 (stably IGFBP-2-transfected clone), 5 × 105 cells were seeded in 100-mm dishes in 10 ml of medium containing 0 to 50 ng/ml rIGF-1 and incubated for 48 hours. HPL1A-SW, HPL1D-SW, or NHBE-SW cells were seeded in 96-well plates at 1 × 104 cells/well in 50 μl of medium. After culture for 24 hours, 50 μl of medium supplemented with goat anti-IGFBP-4, goat anti-IGFBP-2, or nonimmunized goat antibody (final concentration, 50 μg/ml) (R&D Systems, Minneapolis, MN) was added and cultivated for up to 48 hours. Cell growth was evaluated using a MTT assay system (Seikagaku Corp., Tokyo, Japan) and a microplate reader (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Absorbance was measured at a wavelength of 450 nm. The IGFBP-4 and IGFBP-2 promoters are located in regions 1298 bp and 522 bp (EMBL accession no. Y12508 and AY398667) from the transcription start site, respectively.30Dai B Widen SG Mifflin R Singh P Cloning of the functional promoter for human insulin-like growth factor binding protein-4 gene: endogenous regulation.Endocrinology. 1997; 138: 332-343Crossref PubMed Scopus (22) Google Scholar, 31Binkert C Margot JB Landwehr J Heinrich G Schwander J Structure of the human insulin-like growth factor binding protein-2 gene.Mol Endocrinol. 1992; 6: 826-836Crossref PubMed Google Scholar Various lengths of IGFBP-4 and IGFBP-2 promoter fragment-inserted constructs, pGL4.10IGFBP4-1298, −855, −185, −125, and +11 and pGL4.10IGFBP2-522, −444, −314, −206, −134, and −31 [each number indicates the 5′ nucleotide position of insert from the transcription start site (+1)] were generated by PCR using specific primer sets (Table 1). Each amplified promoter DNA fragment was subcloned into the pT7Blue cloning vector (Novagen) and inserted into the pGL4.10 luc2 luciferase plasmid (Promega). For construction of pGL4.10IGFBP4-522, the distal region of the inserted promoter DNA fragment of pGL4.10IGFBP4-855 was cut out with KpnI and HpaI and religated using a KpnI-HpaI linker. For mutational analysis, full-length IGFBP-4 or IGFBP-2 promoter sequences with mutation in each Egr-1-binding site were constructed using a quick change site-directed mutagenesis kit (Stratagene, La Jolla, CA), specific mutagenic primer sets (Table 1), and a full-length IGFBP-4 or IGFBP-2 promoter-inserted pT7 cloning vector, according to the manufacture's instructions. In brief, PCR using a normal IGFBP-4 or IGFBP-2 promoter sequence-inserted pT7 cloning vector and a respective mutagenic primer set specific for each Egr-1-binding site was performed, and the parental DNA template in the reactant was digested with DpnI (New England BioLabs, Beverly, MA). The nicked plasmids that were generated by PCR and had mutated IGFBP-4/2 promoter sequences in each Egr-1-binding site were repaired in XL1-Blue cells (Stratagene). Then, the mutated IGFBP-4/2 promoter sequences were inserted into the pGL4.10 luc2 luciferase plasmid (Promega). HPL1A-SW cells were transiently transfected using the FuGENE6 transfection reagent according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN). Cells were seeded at 50% confluency in six-well plates. After 24 hours, cells were co-transfected with 1 μg/well of reporter plasmid vector (promoter DNA-inserted pGL4.10 luc2) and 50 ng/well of control vector (pGL4.7TK) (Promega). At 24 hours after transfection, cells were washed three times with PBS, and the lysates were subjected to the luciferase assay. Luciferase activity was measured according to the manufacturer's instructions using a luminometer (Turner Biosystems, Sunnyvale, CA). Transfections were performed in quadruplicate, and experiments were performed at least three times. For binding reaction with the putative Egr-1-binding sequence in the IGFBP-4 and IGFBP-2 promoters, 2 μl of recombinant human Egr-1 protein (Alexis Biochemicals, Lausen, Switzerland) was mixed with 1 pmol of digoxigenin-labeled double-stranded IGFBP-4 or IGFBP-2 promoter-specific DNA probes with or without methylated CpGs (Table 2) containing 20 mmol/L Tris-HCl, pH 7.9, 50 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 5% glycerol, 1 μg of poly dIdC, and 5 mmol/L dithiothreitol. The mixture was incubated 30 minutes at 20°C. Unlabeled DNA probe containing consensus Egr-1-binding sequence (5′-GCGGGGGCG-3′) or nonspecific sequence was used for the competitor (Table 2). Probes with mutation in the Egr-1-binding sites (Table 2) were also used to examine whether Egr-1 could bind to the mutated sequences used in the mutational promoter assay. These oligonucleotide probes were purchased from Greiner Japan (Atsugi, Japan). For supershift experiments, a rabbit IgG antibody against Egr-1 (Santa Cruz Biotechnology) or a nonimmunized rabbit IgG antibody (Santa Cruz Biotechnology) was added to the mixture and left 30 minutes at 4°C before gel electrophoresis in 5% acrylamide/bisacrylamide (29:1) gels with 0.25× TBE buffer for 3 hours at 150 V and 4°C with recirculating buffer. The protein-probe complexes were contact-blotted and fixed with UV for 5 minutes on positively charged nylon membranes (Perkin-Elmer, Wellesley, MA), and the digoxigenin-labeled probes were detected with a chemiluminescence detection kit (Roche Diagnostics, Mannheim, Germany).Table 2Probes for Electrophoretic Mobility Shift Assay (Sense Sequences)IGFBP-4 promoter Egr-1-binding site 15′-GGGAGATTGCGGGGGCGGGAGAGGT-3′ Egr-1-binding site 1, mutated5′-GGGAGATTGCTAGGTCGGGAGAGGT-3′ Egr-1-binding site 1, methylated5′-GGGAGATTGCmGGGGGCmGGGAGAGGT-3′ Egr-1-binding site 25′-GCCGCCTTGGCGGGGCGGGACGAGA-3′ Egr-1-binding site 2, muta
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