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• W2024375726 abstract "The EGR1 transactivator is overexpressed in prostate cancer, and its expression pattern suggests that EGR1 could potentially regulate a number of steps involved in initiation and progression of prostate cancer, such as mitogenesis, invasiveness, angiogenesis, and metastasis. To identify potential EGR1 target genes in an unbiased manner, we have utilized adenovirus-mediated expression of EGR1 in a prostate cancer cell line to identify specific genes that are induced by EGR1. Using oligonucleotide arrays, a number of EGR1-regulated genes were identified and their regulation was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. One of the largest gene classes identified in this screen includes several neuroendocrine-associated genes (neuron-specific enolase, neurogranin), suggesting that EGR1 overexpression may contribute to the neuroendocrine differentiation that often accompanies prostate cancer progression. This screen also identified several growth factors such as insulin-like growth factor-II, platelet-derived growth factor-A, and transforming growth factor-β1, which have previously been implicated in enhancing tumor progression. The insulin-like growth factor-II gene lies within the 11p15.5 chromosomal locus, which contains a number of other imprinted genes, and EGR1 expression was found to induce at least two other genes in this locus (IPL, p57KIP2). Based on our results, coupling adenoviral overexpression with microarray and quantitative reverse transcription-polymerase chain reaction analyses could be a versatile strategy for identifying target genes of transactivators. The EGR1 transactivator is overexpressed in prostate cancer, and its expression pattern suggests that EGR1 could potentially regulate a number of steps involved in initiation and progression of prostate cancer, such as mitogenesis, invasiveness, angiogenesis, and metastasis. To identify potential EGR1 target genes in an unbiased manner, we have utilized adenovirus-mediated expression of EGR1 in a prostate cancer cell line to identify specific genes that are induced by EGR1. Using oligonucleotide arrays, a number of EGR1-regulated genes were identified and their regulation was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. One of the largest gene classes identified in this screen includes several neuroendocrine-associated genes (neuron-specific enolase, neurogranin), suggesting that EGR1 overexpression may contribute to the neuroendocrine differentiation that often accompanies prostate cancer progression. This screen also identified several growth factors such as insulin-like growth factor-II, platelet-derived growth factor-A, and transforming growth factor-β1, which have previously been implicated in enhancing tumor progression. The insulin-like growth factor-II gene lies within the 11p15.5 chromosomal locus, which contains a number of other imprinted genes, and EGR1 expression was found to induce at least two other genes in this locus (IPL, p57KIP2). Based on our results, coupling adenoviral overexpression with microarray and quantitative reverse transcription-polymerase chain reaction analyses could be a versatile strategy for identifying target genes of transactivators. platelet-derived growth factor polymerase chain reaction reverse transcription insulin-like growth factor transforming growth factor green fluorescent protein nerve growth factor core-binding factor Beckwith-Wiedemann syndrome basic fibroblast growth factor Cancer initiation and progression depends upon altered expression of whole networks of genes. Therefore, transcriptional regulators constitute one of the most important classes of differentially expressed genes in cancer, as they are uniquely poised to coordinately regulate gene networks. For example, the protooncogene c-myccan be activated through gene amplification or dysregulation of tumor suppressor pathways, and is able to promote proliferation by activating the expression of many genes involved in cell cycle progression (1Grandori C. Eisenman R.N. Trends Biochem. Sci. 1997; 22: 177-181Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 2Dang C.V. Mol. Cell. Biol. 1999; 19: 1-11Crossref PubMed Scopus (1395) Google Scholar). To understand the physiological significance of this overexpression, it is imperative to identify target genes that become activated in response to such overexpression. Until recently, identifying target genes of specific transactivators has been impeded by the scarcity of promoter sequence data. However, even comprehensive genome sequence is insufficient to unambiguously identify target promoters, since many transactivators bind to cognate sequences that deviate considerably from consensus binding sites. Techniques such as DNA footprinting and reporter assays have been extremely useful in the analysis of suspected target promoters, but these techniques do not provide proof that a transactivator activates a given promoter in vivo. In addition, these methods are time-consuming and are not suited for efficient identification of novel target genes. Recent studies have demonstrated that at least two transcription factors, ETS2 and EGR1, are overexpressed in prostate cancer (3Liu A.Y. Corey E. Vessella R.L. Lange P.H. True L.D. Huang G.M. Nelson P.S. Hood L. Prostate. 1997; 30: 145-153Crossref PubMed Scopus (56) Google Scholar, 4Eid M.A. Kumar M.V. Iczkowski K.A. Bostwick D.G. Tindall D.J. Cancer Res. 1998; 58: 2461-2468PubMed Google Scholar, 5Thigpen A.E. Cala K.M. Guileyardo J.M. Molberg K.H. McConnell J.D. Russell D.W. J. Urol. 1996; 155: 975-981Crossref PubMed Scopus (59) Google Scholar). The EGR1 transactivator was originally identified as an immediate-early gene that is rapidly induced in response to a variety of stimuli. More recently, several studies have focused attention on the role of EGR1 in coordinating responses to hypoxia and vascular injury. In these systems, EGR1 activates expression of tissue factor (which eventually triggers vascular fibrin deposition) and several growth factors such as PDGF-A,1 PDGF-B, TGF-β1, IGF-II, and bFGF (6Yan S.F. Zou Y.S. Gao Y. Zhai C. Mackman N. Lee S.L. Milbrandt J. Pinsky D. Kisiel W. Stern D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8298-8303Crossref PubMed Scopus (168) Google Scholar, 7Bae S.K. Bae M.H. Ahn M.Y. Son M.J. Lee Y.M. Bae M.K. Lee O.H. Park B.C. Kim K.W. Cancer Res. 1999; 59: 5989-5994PubMed Google Scholar, 8Silverman E.S. Collins T. Am. J. Pathol. 1999; 154: 665-670Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 9Liu C. Calogero A. Ragona G. Adamson E. Mercola D. Crit. Rev. Oncog. 1996; 7: 101-125Crossref PubMed Google Scholar). Interestingly, many of these same factors have also been implicated in various stages of prostate tumor progression (e.g. angiogenesis, metastasis), adding further evidence that at least some steps of tumor progression are mechanistically related to wound healing and hypoxic responses (10Battegay E.J. J. Mol. Med. 1995; 73: 333-346Crossref PubMed Scopus (500) Google Scholar). Although increased expression of several of these genes has been implicated in development and progression of prostate cancer (11Gold L.I. Crit. Rev. Oncog. 1999; 10: 303-360PubMed Google Scholar, 12Culig Z. Hobisch A. Cronauer M.V. Radmayr C. Hittmair A. Zhang J. Thurnher M. Bartsch G. Klocker H. Prostate. 1996; 28: 392-405Crossref PubMed Scopus (281) Google Scholar), it has not yet been established whether their up-regulation in this context is functionally linked to increased levels of transactivators such as EGR1. To determine the physiological significance of EGR1 overexpression in prostate cancer, we have developed a high-throughput screen for genes that are induced by EGR1. This strategy employs a recombinant adenovirus that expresses EGR1 in the LAPC4 prostate cancer cell line. Changes in gene expression are then analyzed using microarray technology, which has made it possible to simultaneously track changes in expression levels of thousands of genes. Finally, expression of the candidate target genes in primary prostate tumor specimens is rapidly determined using quantitative RT-PCR analysis. Compared with reporter-based assays, a major advantage of this approach is that it measures the response of endogenous promoters in their native chromatin context. Using this strategy, we have identified several genes that are regulated by EGR1 overexpression. These include signaling proteins, transcription regulators, neuroendocrine proteins, and membrane-associated proteins involved in adhesion and signaling. These results not only illuminate the consequences of EGR1 overexpression in prostate cancer, but also provide a model for identifying target genes of specific transactivators in other types of cancer. LAPC4 human prostate carcinoma cells (13Klein K.A. Reiter R.E. Redula J. Moradi H. Zhu X.L. Brothman A.R. Lamb D.J. Marcelli M. Belldegrun A. Witte O.N. Sawyers C.L. Nat. Med. 1997; 3: 402-408Crossref PubMed Scopus (338) Google Scholar) (kindly provided by C. Sawyers, UCLA, Los Angeles, CA), were maintained in Iscove's growth medium supplemented with 10% fetal bovine serum. Adenoviral recombinants were prepared essentially as described (14Ehrengruber M.U. Lanzrein M. Xu Y. Jasek M.C. Kantor D.B. Xu Y. Schuman E.M. Lester H.A. Davidson N. Methods Enzymol. 1998; 293: 483-503Crossref PubMed Scopus (28) Google Scholar). EGR1 I293F (15Russo M.W. Matheny C. Milbrandt J. Mol. Cell. Biol. 1993; 13: 6858-6865Crossref PubMed Scopus (89) Google Scholar) was subcloned into the pAC adenoviral transfer plasmid and inserted by homologous recombination into the E1 region of adenovirus Ad5PacIGFP (14Ehrengruber M.U. Lanzrein M. Xu Y. Jasek M.C. Kantor D.B. Xu Y. Schuman E.M. Lester H.A. Davidson N. Methods Enzymol. 1998; 293: 483-503Crossref PubMed Scopus (28) Google Scholar, 16Qu Z. Wolfraim L.A. Svaren J. Ehrengruber M.U. Davidson N. Milbrandt J. J. Cell Biol. 1998; 142: 1075-1082Crossref PubMed Scopus (59) Google Scholar). As a negative control, we used an adenovirus (Ad5PacIGFP) expressing the Gal4 DNA-binding domain () fused to a mutant, nonfunctional form of the EGR1 R1 domain (EGR1 residues 269–304 with I293F mutation). LAPC4 cells were infected at a viral titer of 1 × 108plaque-forming units/ml for 2 h. Thereafter, cells were washed once with medium and then cultured for another 24 h. Examination of the cells for GFP expression revealed that each virus infected >90% of the cells. For the immunoblot analysis of Fig. 1, lysates from adenovirus-infected cells were harvested 24 h after infection, resolved on a 10% polyacrylamide gel, and blotted onto nitrocellulose. Culture and stimulation of PC12 cells with NGF was performed as described previously, and the blot was probed with the 6H10 anti-EGR1 monoclonal antibody (17Day M.L. Fahrner T.J. Ayken S. Milbrandt J. J. Biol. Chem. 1990; 265: 15253-15260Abstract Full Text PDF PubMed Google Scholar). Hybridization probes for GeneChip analysis were prepared from poly(A)+ RNA prepared from cultures of LAPC4 cells that had been infected with either adenovirus expressing EGR1 (I293F) or the control adenovirus. The poly(A)+ RNA was converted to double-stranded cDNA using an oligo(dT) primer containing the T7 promoter, and this was used to prepare biotinylated cRNA using the Bioarray HighYield kit (Enzo) according to the manufacturer's directions. The biotinylated cRNA probes were fragmented and applied as described (18Lockhart D.J. Dong H. Byrne M.C. Follettie M.T. Gallo M.V. Chee M.S. Mittmann M. Wang C. Kobayashi M. Horton H. Brown E.L. Nat. Biotechnol. 1996; 14: 1675-1680Crossref PubMed Scopus (2818) Google Scholar, 19Lipshutz R.J. Fodor S.P. Gingeras T.R. Lockhart D.J. Nat. Genet. 1999; 21 Suppl.: 20-24Crossref Scopus (1868) Google Scholar) to individual oligonucleotide HuGeneFL GeneChip arrays (Affymetrix), which contain probe sets for 5600 human genes. The signal intensities from hybridized cRNA were quantified, and the GeneChip analysis software was used to identify differentially expressed genes. Total RNA was purified, and 1 μg was used to prepare cDNA (20Lee S.L. Wang Y. Milbrandt J. Mol. Cell. Biol. 1996; 16: 4566-4572Crossref PubMed Google Scholar). Quantitative RT-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye as described (21Wittwer C.T. Herrmann M.G. Moss A.A. Rasmussen R.P. BioTechniques. 1997; 22: 130-138Crossref PubMed Scopus (1140) Google Scholar, 22Morrison T.B. Weis J.J. Wittwer C.T. BioTechniques. 1998; 24: 954-962PubMed Google Scholar) using the TaqMan 7700 sequence detection system (PerkinElmer Life Sciences). For comparison of transcript levels between samples, a standard curve of cycle thresholds for several serial dilutions of a cDNA sample was established and then used to calculate the relative abundance of each gene. Values were then normalized to the relative amounts of glyceraldehyde-3-phosphate dehydrogenase cDNA, which were obtained from a similar standard curve. All PCR reactions were performed in duplicate. Sequences of primers used for PCR analysis are available upon request. Prostate tissue specimens, derived from radical prostatectomy, were obtained from the Alvin J. Siteman Cancer Center Tissue Procurement Core Facility at Washington University. All samples had a Gleason grade of 3 + 3 (23Bostwick D.G. Bostwick D.G. Eble J.N. Urologic Surgical Pathology. Mosby, Saint Louis1997: 343-422Google Scholar). Guided by hematoxylin and eosin-stained frozen sections, the tissue blocks were grossly dissected so that, by visual estimate, the epithelial component of the isolated tissue contained at least 75% carcinoma cells. The tissues were sectioned at 50 μm on the cryostat microtome and used for RNA isolation. A serial frozen section was stained to verify that the tissue sections used for RNA preparation were predominantly carcinoma. Quantitative RT-PCR analysis was performed as described above, with the exception that 18 S rRNA was used to normalize for the amount of input cDNA. Detection of IGF-II imprinting in human samples was performed as described (24Tadokoro K. Fujii H. Inoue T. Yamada M. Nucleic Acids Res. 1991; 19: 6967Crossref PubMed Scopus (113) Google Scholar, 25Jarrard D.F. Bussemakers M.J. Bova G.S. Isaacs W.B. Clin. Cancer Res. 1995; 1: 1471-1478PubMed Google Scholar, 26Zhan S. Shapiro D. Zhang L. Hirschfeld S. Elassal J. Helman L.J. J. Biol. Chem. 1995; 270: 27983-27986Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, genomic DNA from LAPC4 cells was isolated and amplified using two primers that span an IGF-II gene segment that contains a single nucleotide polymorphism. Sequencing of both strands of the PCR fragment revealed that LAPC4 DNA is heterozygous for this polymorphism. To determine which allele is induced by EGR1 expression, cDNA from LAPC4 cells infected with adenovirus expressing EGR1 I293F was amplified with the same primers, and this product was sequenced. Previous work has shown that the EGR1 transactivator is overexpressed in a majority of prostate cancers (4Eid M.A. Kumar M.V. Iczkowski K.A. Bostwick D.G. Tindall D.J. Cancer Res. 1998; 58: 2461-2468PubMed Google Scholar, 5Thigpen A.E. Cala K.M. Guileyardo J.M. Molberg K.H. McConnell J.D. Russell D.W. J. Urol. 1996; 155: 975-981Crossref PubMed Scopus (59) Google Scholar). To identify genes that are regulated by EGR1, we wished to achieve overexpression of EGR1 in a prostate cell line without using stimuli (e.g. growth factors) that would activate signaling pathways and induce other transcription factors. Therefore, we utilized a recombinant adenovirus that expresses EGR1 (I293F), a mutant that is resistant to repression by endogenous NAB transcriptional corepressors (27Russo M.W. Sevetson B.R. Milbrandt J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6873-6877Crossref PubMed Scopus (252) Google Scholar, 28Svaren J. Sevetson B.R. Apel E.D. Zimonjic D.B. Popescu N.C. Milbrandt J. Mol. Cell. Biol. 1996; 16: 3545-3553Crossref PubMed Scopus (328) Google Scholar), which could repress any activation by wild type EGR1. The recombinant adenovirus was used to infect the LAPC4 prostate cell line. This cell line was derived as an explant of metastatic prostate cancer, and retains many of the characteristics of normal prostate cells such as prostate- specific antigen expression and androgen dependence (13Klein K.A. Reiter R.E. Redula J. Moradi H. Zhu X.L. Brothman A.R. Lamb D.J. Marcelli M. Belldegrun A. Witte O.N. Sawyers C.L. Nat. Med. 1997; 3: 402-408Crossref PubMed Scopus (338) Google Scholar). In addition to EGR1 (I293F), the adenovirus also expresses GFP from an independent transcription unit, which allows monitoring of infection. After infection with EGR1-expressing adenovirus, visualization by fluorescence microscopy confirmed that essentially all (>95%) of the cells in culture were infected. To determine if the level of EGR1 expression in adenovirus-infected cells is significantly higher than is ever observed physiologically, the expression level of EGR1 (I293F) created by the recombinant adenovirus (Fig. 1, lane 4 of inset) was compared with the induced level of EGR1 in NGF-stimulated PC12 cells (lane 2) (29Milbrandt J. Science. 1987; 238: 797-799Crossref PubMed Scopus (936) Google Scholar). This immunoblot reveals that use of recombinant adenovirus is an efficient means to target EGR1 overexpression to a cell line, and that the resulting level of EGR1 expression is comparable to the level of induced EGR1 observed in the NGF-treated PC12 system. The endogenous expression level of EGR1 in cultured LAPC4 cells is relatively low, at a level that is comparable to that observed in normal prostate tissue (data not shown). To test whether EGR1 (I293F) could activate potential target genes in this cell line, we measured expression levels of three EGR1 target genes that have been identified in other systems: IGF-II, PDGF-A, and TGF-β1 (30Lee Y.I. Kim S.J. DNA Cell. Biol. 1996; 15: 99-104Crossref PubMed Scopus (14) Google Scholar, 31Dey B.R. Sukhatme V.P. Roberts A.B. Sporn M.B. Rauscher 3rd, F.J. Kim S.J. Mol. Endocrinol. 1994; 8: 595-602Crossref PubMed Scopus (195) Google Scholar, 32Liu C. Adamson E. Mercola D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11831-11836Crossref PubMed Scopus (151) Google Scholar, 33Takimoto Y. Wang Z.Y. Kobler K. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1686-1690Crossref PubMed Scopus (39) Google Scholar, 34Khachigian L.M. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). After 24 h of infection, RNA was purified from these cultures and used to generate cDNA. We employed a technique for quantitative RT-PCR analysis, in which a fluorescent dye (SYBR Green) that binds double-stranded DNA is used to quantitate the amount of amplicon as it accumulates during the PCR reaction (22Morrison T.B. Weis J.J. Wittwer C.T. BioTechniques. 1998; 24: 954-962PubMed Google Scholar, 35Schneeberger C. Speiser P. Kury F. Zeillinger R. PCR Methods Appl. 1995; 4: 234-238Crossref PubMed Scopus (147) Google Scholar, 36Becker A. Reith A. Napiwotzki J. Kadenbach B. Anal. Biochem. 1996; 237: 204-207Crossref PubMed Scopus (57) Google Scholar, 37Ririe K.M. Rasmussen R.P. Wittwer C.T. Anal. Biochem. 1997; 245: 154-160Crossref PubMed Scopus (1283) Google Scholar). For each cDNA sample, we measured the cycle number at which PCR product accumulation reaches a defined threshold. Then, the relative levels of gene expression were determined using a standard curve obtained from assays of serial dilutions of a cDNA sample containing the gene of interest. To control for genes induced by adenovirus infection alone, expression levels of these three genes in EGR1-infected LAPC4 cells were compared with those obtained in LAPC4 cells infected with a control adenovirus. As shown in Fig. 1 B, IGF-II, TGF-β1, and PDGF-A were all induced in response to EGR1 expression, indicating for the first time that these genes, in their endogenous loci, are induced by EGR1 in a prostate cell type. The same cDNA samples used in Fig. 1 were used to prepare biotinylated cRNA targets, which were then hybridized to individual oligonucleotide HuGeneFL GeneChip arrays (Affymetrix), which contain probe sets for 5600 human genes. The signal intensities from hybridized cRNA were quantified as described (18Lockhart D.J. Dong H. Byrne M.C. Follettie M.T. Gallo M.V. Chee M.S. Mittmann M. Wang C. Kobayashi M. Horton H. Brown E.L. Nat. Biotechnol. 1996; 14: 1675-1680Crossref PubMed Scopus (2818) Google Scholar, 19Lipshutz R.J. Fodor S.P. Gingeras T.R. Lockhart D.J. Nat. Genet. 1999; 21 Suppl.: 20-24Crossref Scopus (1868) Google Scholar). Using the default parameters of the GeneChip analysis software, 37% and 33% of the genes on the HuGeneFL array were scored as being present (or marginal) in the control-infected and EGR1 (I293F) expressing LAPC4 cells, respectively. The normalization factor used to compare the two data sets indicated that the global levels of hybridization from the two cRNA samples were roughly equivalent. Using defined copy numbers of synthetic, biotinylated cRNA transcripts that were added to the hybridization mixture, we estimate that the detection threshold in this experiment was approximately 5–10 copies/cell. The GeneChip data was first used to identify genes that are abundantly expressed in this cell line, since this profile may provide information regarding potential diagnostic markers and therapeutic targets in prostate cancer. A list of the 50 most highly expressed genes (excluding genes for ribosomal proteins) in TableI contains several genes that have previously been associated with various types of tumors. For example, the thymosin β-10 protein binds and sequesters G-actin and is overexpressed in a wide range of tumor types (38Santelli G. Califano D. Chiappetta G. Vento M.T. Bartoli P.C. Zullo F. Trapasso F. Viglietto G. Fusco A. Am. J. Pathol. 1999; 155: 799-804Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In addition, CD81, a cell-surface molecule involved in cell adhesion and integrin signaling (39Maecker H.T. Todd S.C. Levy S. FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (813) Google Scholar), is also expressed at a high level. Neuroleukin is a multifunctional protein that is a phosphoglucose isomerase, but also functions as a tumor-secreted cytokine that regulates invasion and metastasis (40Watanabe H. Takehana K. Date M. Shinozaki T. Raz A. Cancer Res. 1996; 56: 2960-2963PubMed Google Scholar). The significance of the high expression levels of these proteins in the LAPC4 cell line remains to be established, but similar analyses of other prostate cancer models may substantiate their overexpression as a general characteristic of prostate cancer.Table IHighly expressed genes in LAPC4 cellsAccession no.DescriptionAccession no.DescriptionX00351β-ActinM24485GlutathioneS-transferaseU49869UbiquitinJ04617Elongation factor EF-1-αD78361Ornithine decarboxylase antizymeY00486Adenine phosphoribosyltransferaseM14199Laminin receptorAB002533Karyopherin α4X15341Cytochromec oxidase subunit ViaD38583CalgizzarinM17733Thymosin β-4S54005Thymosin β-10M33680CD81/TAPA-1J04823Cytochrome c oxidase subunit VIIIU16660Peroxisomal enoyl-CoA hydrataseM19283γ-Actin geneX16064Histamine releasing factorL76200Guanylate kinase (GUK1)M24194Guanine nucleotide binding proteinK03515Neuroleukin/glucose phosphate isomeraseV00599β-TubulinD13748Eukaryotic initiation factor 4AIX95404Non-muscle type cofilinM55409Elongation factor 1γJ04173Phosphoglycerate mutaseD13413Scaffold attachment factor AD21261Transgelin 2X98482Troponin T2J03191ProfilinX56494Pyruvate kinaseU43901Laminin receptor precursorM21142Guanine nucleotide-binding proteinX02152Lactate dehydrogenase-AJ02683ADP/ATP carrier proteinX12447Aldolase AL26247Translation initiation factor SUI1X01677GAPDHD13118ATP synthase subunit cM11147Ferritin L chainD14710ATP synthase αX03689Elongation factor TUZ47055Farnesyl pyrophosphate synthetaseX52851CyclophilinL19686Macrophage migration inhibitory factorX71973Glutathione peroxidase 4AJ001421Rer1 proteinM11353H3.3 histoneY07604Nucleoside-diphosphate kinaseU15008SnRNP D2D23662Ubiquitin-like proteinThe 50 highest expressed genes (excluding ribosomal proteins) derived from GeneChip analysis of the control LAPC4 sample are ranked in order of highest expression from upper left to lower right. Open table in a new tab The 50 highest expressed genes (excluding ribosomal proteins) derived from GeneChip analysis of the control LAPC4 sample are ranked in order of highest expression from upper left to lower right. Because we had observed up-regulation of several potential EGR1 target genes (Fig. 1) after expression of EGR1 (I293F), we used the GeneChip data sets to identify other genes that become induced as a consequence of EGR1 (I293F) expression. The average hybridization intensity across all probe sets using the cRNA prepared from LAPC4 cells expressing EGR1 I293F was normalized to that obtained from LAPC4 cells infected with a control virus. Comparison of the two data sets revealed that 144 of the genes found to be “present” in both samples (2.1% of total genes represented on the array) were induced in LAPC4 cells expressing EGR1 (I293F), but only 30 of these genes (0.5%) were induced more than 3-fold. Analysis of the results indicated several genes that were significantly altered in response to EGR1 overexpression (TableII), most of which had not previously been identified as EGR1 target genes. Many of the induced genes could be grouped into functional classes of molecules. These include transcriptional regulators, signaling molecules, as well as some neuroendocrine proteins. One signaling molecule was the Rad gene, a Ras homolog that was originally identified to be overexpressed in the muscle of patients with type II diabetes (41Reynet C. Kahn C.R. Science. 1993; 262: 1441-1444Crossref PubMed Scopus (283) Google Scholar). More recently, Rad expression has been shown to potentiate serum-stimulated DNA synthesis in a melanoma cell line. In addition, this activity of Rad is inhibited by the nm23 gene product, a putative suppressor of tumor metastasis (42Zhu J. Tseng Y.H. Kantor J.D. Rhodes C.J. Zetter B.R. Moyers J.S. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14911-14918Crossref PubMed Scopus (105) Google Scholar). An example of the transcription factor group is CBF-β, the non-DNA-binding subunit of the heterodimeric transcription factor core-binding factor (CBF)/polyoma enhancer-binding protein 2. Chromosomal translocations involving the human CBF-β gene (CBF-β-MYH11) are associated with a large percentage of human leukemias (43Speck N.A. Terryl S. Crit. Rev. Eukaryot. Gene Exp. 1995; 5: 337-364Crossref PubMed Scopus (150) Google Scholar). Recently, CBF activity has been shown to be required for angiogenesis in an endothelial cell line, where expression of all CBF subunits is induced by angiogenic factors, such as bFGF and vascular endothelial growth factor (44Namba K. Abe M. Saito S. Satake M. Ohmoto T. Watanabe T. Sato Y. Oncogene. 2000; 19: 106-114Crossref PubMed Scopus (52) Google Scholar). The neuroendocrine genes are particularly interesting since neuroendocrine differentiation is often observed during prostate cancer progression. Neuron-specific enolase is a widely used marker for determining the extent of neuroendocrine differentiation (45Di Sant'Agnese P.A. Cockett A.T. J. Urol. 1994; 152: 1927-1931Crossref PubMed Google Scholar, 46Abrahamsson P.A. Prostate. 1999; 39: 135-148Crossref PubMed Scopus (288) Google Scholar).Table IIEGR1 target genes in LAPC4 cellsTranscriptional regulatory proteins CBF-β/PEBP2 (10.4) Id4 (3.3) Neuro-d4 (7.9) IPL/TSSC3 (6.7) SNAP45/PSE-binding PTF (5) PHOX1 (6.1) CoREST (4.2)Signaling proteins Rad (14.2) p57KIP2 (6.3) Guanylate cyclase β1 subunit (3.3)Proteases/protease inhibitors Cystatin M/CST6 (216) Protease M/zyme/neurosin (9.6)Neuroendocrine genes Neurogranin/RC3 (23.9) Neuron-specific enolase/NSE (5.6) Protease M/zyme/neurosin (9.6) Neuro-d4 (7.9) Telencephalin/(ICAM-5) (3)Membrane-associated proteins Epithelial membrane protein 2/XMP (16.9) SLC6A10—creatine transporter (24.3) SLC6A8—creatine transporter (3.6) Connexin 26/GJB2 (21.8) PCTA-1/galectin 8 (2.8)Other Elongation factor 1-α2 (3.7) Argininosuccinate synthetase (14.9)The table lists genes that were found to be up-regulated more than 2.5-fold in response to adenoviral-mediated overexpression of EGR1 in LAPC4 cells. The numbers in parentheses indicate the -fold induction of each gene as determined by the GeneChip analysis software. Some genes are listed in more than one category. Open table in a new tab The table lists genes that were found to be up-regulated more than 2.5-fold in response to adenoviral-mediated overexpression of EGR1 in LAPC4 cells. The numbers in parentheses indicate the -fold induction of each gene as determined by the GeneChip analysis software. Some genes are listed in more than one category. To independently measure the fold induction of specific EGR1 target genes, quantitative RT-PCR analysis was used to measure expression of some of the genes identified by GeneChip analysis. The induction by EGR1 (I293F) of the genes chosen for this analysis spanned a range from 2- to 50-fold. Fold induction calculated from the GeneChip data was compared with that obtained using quantitative RT-PCR analysis (Table III). Induction of specific genes was confirmed for the most part by our quantitative RT-PCR analysis. However, for some genes, the actual fold induction by quantitative RT-PCR (e.g. protease M, Rad, IGF-II, and TGF-β1) was significantly greater than that derived" @default.
• W2024375726 created "2016-06-24" @default.
• W2024375726 creator A5004205875 @default.
• W2024375726 creator A5007830810 @default.
• W2024375726 creator A5023365864 @default.
• W2024375726 creator A5038303393 @default.
• W2024375726 creator A5052732313 @default.
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• W2024375726 date "2000-12-01" @default.
• W2024375726 modified "2023-09-29" @default.
• W2024375726 title "EGR1 Target Genes in Prostate Carcinoma Cells Identified by Microarray Analysis" @default.
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