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• W2034260252 abstract "Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of human cancer. The core members of this pathway include the tumor suppressor protein, pRB, which through binding to a number of cellular proteins, most notably members of the E2F transcription factor family, regulates progression through the cell division cycle. With the aim of identifying transcriptional changes provoked by deregulation of the pRB pathway, we have used cell lines that conditionally express a constitutively active phosphorylation site mutant of pRB (pRBΔCDK) or p16INK4A (p16). The expression of pRBΔCDK and p16 resulted in significant repression and activation of a large number of genes as measured by high density oligonucleotide array analysis. Transcriptional changes were found in genes that are essential for DNA replication and cell proliferation. In agreement with previous results, we found a high degree of overlap between genes regulated by p16 and pRB. Data we have obtained previously for E2F family members showed that 74 of the genes repressed by pRB and p16 were induced by the E2Fs and 23 genes that were induced by pRB and p16 were repressed by the E2Fs. Thus, we have identified 97 genes as physiological targets of the pRB pathway, and the further characterization of these genes should provide insights into how this pathway controls proliferation. We show that Gibbs sampling detects enrichment of several sequence motifs, including E2F consensus binding sites, in the upstream regions of these genes and use this enrichment in an in silico filtering process to refine microarray derived gene lists. Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of human cancer. The core members of this pathway include the tumor suppressor protein, pRB, which through binding to a number of cellular proteins, most notably members of the E2F transcription factor family, regulates progression through the cell division cycle. With the aim of identifying transcriptional changes provoked by deregulation of the pRB pathway, we have used cell lines that conditionally express a constitutively active phosphorylation site mutant of pRB (pRBΔCDK) or p16INK4A (p16). The expression of pRBΔCDK and p16 resulted in significant repression and activation of a large number of genes as measured by high density oligonucleotide array analysis. Transcriptional changes were found in genes that are essential for DNA replication and cell proliferation. In agreement with previous results, we found a high degree of overlap between genes regulated by p16 and pRB. Data we have obtained previously for E2F family members showed that 74 of the genes repressed by pRB and p16 were induced by the E2Fs and 23 genes that were induced by pRB and p16 were repressed by the E2Fs. Thus, we have identified 97 genes as physiological targets of the pRB pathway, and the further characterization of these genes should provide insights into how this pathway controls proliferation. We show that Gibbs sampling detects enrichment of several sequence motifs, including E2F consensus binding sites, in the upstream regions of these genes and use this enrichment in an in silico filtering process to refine microarray derived gene lists. Data generated in the last decade have pointed to a central role for the retinoblastoma protein (pRB) pathway in regulating the progression through the G1 phase of the mammalian cell cycle (1Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4944) Google Scholar). The core members of this pathway include, in addition to pRB (and its family members p107 and p130), the D-type cyclins that, in association with CDK4 and CDK6, promote proliferation of the cell cycle, in part through the phosphorylation of the pRB family members, and the INK4 family of cyclin-dependent kinase inhibitors that specifically bind and inhibit the activity of CDK4 and CDK6. The high frequency by which alterations have been identified in the pRB pathway in human cancer taken together with the understanding of the central role of its core members in regulating cell proliferation have led several researchers to suggest that the deregulation of the pRB pathway is an obligatory event in human cancer (e.g. see Ref. 2Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21908) Google Scholar).The biochemical mechanism by which pRB is restricting cell proliferation is widely believed to involve protein-protein interactions. Several hundred proteins have been reported to bind to pRB (3Morris E.J. Dyson N.J. Adv. Cancer Res. 2001; 82: 1-54Crossref PubMed Scopus (296) Google Scholar), however, the relevance of most of these interactions is poorly understood. The most studied and the best understood targets for pRB are members of the E2F transcription factor family (4Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar, 5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar). The E2F transcription factors are essential for the proper transcriptional regulation of a number of genes, whose gene products control the progression through the cell cycle. These genes include CCNE1 (cyclin E1), CCNA2 (cyclin A2), and CDC25A, which are all essential for the entry into the S phase of the cell cycle, and genes that are involved in the regulation of DNA replication, such as CDC6, DHFR, and TK1 (thymidine kinase) (6Helin K. Curr. Opin. Genet. Dev. 1998; 8: 28-35Crossref PubMed Scopus (427) Google Scholar, 7Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 11-20Crossref PubMed Scopus (957) Google Scholar). When E2Fs bind to the promoters of these genes in complexes with pRB or its family members, gene expression is repressed. The repression is effectively relieved by CDK 1The abbreviations used are: CDK, cyclin-dependent kinase; HA, hemagglutinin; MES, 2-(N-morpholino)ethanesulfonic acid; PWM, position weight matrix; TGF-β, transforming growth factor-β.1The abbreviations used are: CDK, cyclin-dependent kinase; HA, hemagglutinin; MES, 2-(N-morpholino)ethanesulfonic acid; PWM, position weight matrix; TGF-β, transforming growth factor-β.-mediated phosphorylation of the pRB family members, resulting in transcription of the E2F-regulated genes and subsequent progression through the cell cycle.Because of the central role of the E2Fs in regulating the progression through the cell division cycle, we and others have used gene expression profiling and chromosomal immunoprecipitation assays to identify novel E2F target genes (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar, 8Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (494) Google Scholar, 9Ren B. Cam H. Takahashi Y. Volkert T. Terragni J. Young R.A. Dynlacht B.D. Genes Dev. 2002; 16: 245-256Crossref PubMed Scopus (900) Google Scholar, 10Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (220) Google Scholar, 11Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (391) Google Scholar, 12Markey M.P. Angus S.P. Strobeck M.W. Williams S.L. Gunawardena R.W. Aronow B.J. Knudsen E.S. Cancer Res. 2002; 62: 6587-6597PubMed Google Scholar, 13Wells J. Yan P.S. Cechvala M. Huang T. Farnham P.J. Oncogene. 2003; 22: 1445-1460Crossref PubMed Scopus (109) Google Scholar). The data obtained from these expression profiles have shown that the E2Fs, in addition to having a role in regulating the expression of genes involved in cell cycle progression and DNA replication, also regulate the expression of genes controlling differentiation, DNA repair, and apoptosis. The aim of the current study was to identify transcriptional changes provoked by the reintroduction of a functional pRB pathway in human tumors. By performing such a study we expected to identify genes whose deregulation, as a result of a non-functional pRB pathway, could contribute to transformation. Moreover, we also expected that the gene expression would give valuable therapeutic biomarkers, which could be useful when compounds aimed at restoring a functional pRB pathway in tumor cells will be tested. To perform these studies, we chose the widely used human osteosarcoma cell line U-2 OS as an experimental model because these cells do not express p16. Moreover, because of the high level of CDK activity in U-2 OS cells, the majority of pRB is in its hyperphosphorylated state. Reintroduction of p16 and a non-phosphorylatable version of pRB results in cell cycle arrest in these cells, which can be abrogated by re-expression of the E2Fs (14Zhu L. van den Heuvel S. Helin K. Fattaey A. Ewen M. Livingston D. Dyson N. Harlow E. Genes Dev. 1993; 7: 1111-1125Crossref PubMed Scopus (470) Google Scholar, 15Lukas J. Parry D. Aagaard L. Mann D.J. Bartkova J. Strauss M. Peters G. Bartek J. Nature. 1995; 375: 503-506Crossref PubMed Scopus (867) Google Scholar, 16Lukas J. Petersen B.O. Holm K. Bartek J. Helin K. Mol. Cell. Biol. 1996; 16: 1047-1057Crossref PubMed Scopus (265) Google Scholar, 17Jiang H. Chou H.S. Zhu L. Mol. Cell Biol. 1998; 18: 5284-5290Crossref PubMed Google Scholar, 18Lukas J. Sorensen C.S. Lukas C. Santoni-Rugiu E. Bartek J. Oncogene. 1999; 18: 3930-3935Crossref PubMed Scopus (68) Google Scholar). Here, we report the identification of 74 genes, whose expression is repressed by p16 and pRB, and induced by E2F. Gibbs sampling performed in the upstream regions of these genes detects the enrichment of several sequence motifs, including E2F consensus binding sites. The enriched sequence motifs were used in a search of roughly 10,000 human upstream regulatory regions to identify novel E2F target gene candidates and to refine the microarray data.EXPERIMENTAL PROCEDURESCell Culture and Cell Cycle Analysis—U-2 OS cells expressing tetracycline responsive p16 or constitutively active pRb (pRbΔCDK) alleles were described previously (17Jiang H. Chou H.S. Zhu L. Mol. Cell Biol. 1998; 18: 5284-5290Crossref PubMed Google Scholar, 18Lukas J. Sorensen C.S. Lukas C. Santoni-Rugiu E. Bartek J. Oncogene. 1999; 18: 3930-3935Crossref PubMed Scopus (68) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (South American), 2 μg/ml puromycin. 200 μg/ml G418, and 2 μg/ml tetracycline. U-2 OS cells expressing HA-tagged ER-E2F1 (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar) were cultured in Dulbecco's modified Eagle's medium containing 10% bovine calf serum and 2.5 μg/ml puromycin. Nearly confluent cultures of U-2 OS clones were trypsinized and plated at 5 × 106 cells per 15-cm plate on the day before induction. Induction of E2F activity was accomplished by addition of 4-hydroxytamoxifen to a final concentration of 300 nm for 4 h. Exogenous p16 or pRbΔCDK proteins were induced by rinsing cells twice with phosphate-buffered saline followed by growing in tetracycline-free media. After the first hour of culture the media was changed again. RNA was isolated at 12 h for pRbΔCDK, or 16 h for p16. For cell cycle analysis, cells were pulse labeled for 30 min in 20 μm bromodeoxyuridine, harvested by trypsinzation, fixed in 1% paraformaldehyde for 5 min, and permeabilized in 70% methanol. DNA was denatured in 2 n HCl for 20 min and cells were neutralized in 0.1 m sodium borate, pH 8.5, for 2 min. Bromodeoxyuridine was detected using fluorescein isothiocyanate-labeled anti-bromodeoxyuridine monoclonal antibody (BD Biosciences), and DNA was stained in 0.5 ml of propidium iodide (10 μg/ml).RNA Preparation—RNA for analysis by microarray and quantitative real-time reverse transcriptase-PCR was isolated using the RNeasy kit (Qiagen) and integrity was determined by formaldehyde-agarose gel electrophoresis. For use in quantitative PCR, the protocol included on-column treatment with DNase I according to the manufacturer's protocol.Immunostaining—Cells were cultured in the presence or absence of tetracycline for 16 h. Expression of HA-tagged pRbΔCDK protein was detected with anti-HA (12CA5) antibody. Exogenous p16 protein was detected with anti-p16 (Santa Cruz sc-467).Quantitative Reverse Transcriptase-PCR—Changes in gene expression were confirmed using SYBR Green quantitative reverse transcriptase-PCR following the protocols from Applied Biosystems. Primers used can be found in Supplemental Materials.High Density Oligonucleotide Microarrays—We used HG-U95 A, B, C, D, and E oligonucleotide microarrays (Affymetrix) containing 62,907 probe sets. Targets for hybridization to the microarrays were prepared as described (19Lockhart 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 (2795) Google Scholar, 20Fambrough D. McClure K. Kazlauskas A. Lander E.S. Cell. 1999; 97: 727-741Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), except that hybridization was performed in 1 × MES buffer (0.1 m MES, pH 6.7, 1 m NaCl, 0.01% Triton X-100) and chips were washed in 0.1 × MES buffer. Target concentration was 30 μg of fragmented cRNA in 200 μl of hybridization solution. Images were scanned with a HewlettPackard GeneArray scanner and the images were analyzed using Affymetrix's Microarray suite 5.0 software. All targets were hybridized to two different sets of HG-U95 microarrays (chips Av2 through E). Chip replicas were analyzed as described in Ref. 5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar. The probe sets that were identified as regulated by the analysis of chip replicas were inserted into GeneSpring™ 5.1 software as gene lists for visualization, annotation, and clustering purposes. All data obtained in the array experiments will be made available. 2array.ifom-firc.it.Generation and Use of the Promoter-Exon 1 Data Base—We used the University of California Santa Cruz genome browser (assembly released 28th of June 2002) 3genome.ucsc.edu. to retrieve genomic DNA sequence for 16,031 human Reference Sequence accession numbers (NM_001234) available as of 02/11/02 including 1000 bp of upstream sequence as reported by the genome browser. Then, we blasted the exon 1 sequence as reported by the genome browser to 100 bp of the 5′-end of the cDNA as reported by the reference sequence entry. Only if this blast reported a hit with 99 or 100% identity in the correct orientation (plus/plus strand), did we consider the promoter-exon 1 sequence for further analysis. This procedure reduced the number of entries in the promoter-exon 1 data base from 16,031 to 9,652 entries. All position weight matrix searches using this data base were performed exactly as described by Ref. 21Kel A.E. Kel-Margoulis O.V. Farnham P.J. Bartley S.M. Wingender E. Zhang M.Q. J. Mol. Biol. 2001; 309: 99-120Crossref PubMed Scopus (156) Google Scholar.Position Weight Matrix Similarity (PWM Similarity)—The test for PWM similarity was performed in two different ways. 1) Using 50,000,000 random 25-mer oligonucleotide sequences, each sequence was presented to each PWM at the stringency indicated in Table II. A PWM score was calculated as described in Ref. 21Kel A.E. Kel-Margoulis O.V. Farnham P.J. Bartley S.M. Wingender E. Zhang M.Q. J. Mol. Biol. 2001; 309: 99-120Crossref PubMed Scopus (156) Google Scholar. If the PWM score for that sequence was equal or higher than the stringency indicated in Table II, the oligonucleotide sequence was scored as a hit. In this test, similar matrices have the tendency to recognize similar sequences as a hit. The degree of similarity was quantified using the binomial distribution where the number of successes is the number of hits that two PWMs have in common, the number of trials is the number of hits of one PWM, and the probability of success is the number of hits of the second PWM divided by 50,000,000. The cumulative p value was then calculated. PWMs were judged similar if this value fell between 0.99999 and 1 (i.e. the number of successes lies far out in the tail of the distribution). The relatively high number of random oligonucleotide sequences was necessary because half of the PWMs would recognize only 1 hit in roughly 10,000 sequences. 2) To visualize the similarity of PWMs, the same random sequences were presented to the PWMs at a stringency that would recognize 5% of the sequences as a hit. By doing so, each sequence was assigned a pattern of 0s and 1s, where 0 stands for no hit with a given PWM and 1 stands for a hit with a given PWM. These patterns were analyzed by cluster analysis using the GeneSpring 5.1. software (Silicon Genetics).Table IIMotifs enriched in E2F target gene promotersScoring Scheme for in Silico E2F Target Gene Filter—Based on the test for PWM similarity, each PWM was assigned to a PWM family: 1) E2F site (M00024, M00050, M00180, motif 5, motif “Kel et al.” (21Kel A.E. Kel-Margoulis O.V. Farnham P.J. Bartley S.M. Wingender E. Zhang M.Q. J. Mol. Biol. 2001; 309: 99-120Crossref PubMed Scopus (156) Google Scholar), and motif “Farnham”); 2) GC-rich (motif 7, motif 20, motif 33, motif 63, and motif 71); 3) CCAAT box (motif 4); and 4) CCAAT-like (motif 45). If within a PWM family more than one PWM recognizes a binding site in a promoter, only the PWM with the highest enrichment factor is considered. The enrichment factors of the PWMs that recognize a binding site in a promoter are multiplied to give the final score. Multiplication of enrichment factors is justified because the probability to find two or more motifs in the same promoter is calculated by multiplying the probabilities of finding each motif in the promoter.RESULTSPreviously, we have described the identification of several hundred genes whose expression changes significantly upon activation of E2F1, E2F2, or E2F3 (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar). In total, between 7 and 10% of all genes tested in this screen showed changes in expression level. Computer-assisted motif searches in the promoters of these genes did not show a significant enrichment for the consensus E2F DNA binding site (TTTSSCGC). 4H. Muller, unpublished data. There are several possibilities that could account for this observation. Although the more obvious explanation is that many of the genes are false positives, and several of them are not direct targets of the E2Fs, we do not believe that this is the case, because Northern blotting or quantitative PCR confirmed the microarray results for more than 95% of the genes tested (Ref. 5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar, and data not shown). Moreover, our current understanding of the E2F binding site consensus and more generally the mechanism of E2F mediated regulation of transcription is limited. Several E2F-regulated genes have been described (e.g. CCNE1, CCNA2, and MYBL2) that do not contain perfect consensus E2F DNA binding sites (22Geng Y. Eaton E.N. Picon M. Roberts J.M. Lundberg A.S. Gifford A. Sardet C. Weinberg R.A. Oncogene. 1996; 12: 1173-1180PubMed Google Scholar, 23Schulze A. Zerfass K. Spitkovsky D. Middendorp S. Berges J. Helin K. Jansen-Durr P. Henglein B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11264-11268Crossref PubMed Scopus (319) Google Scholar, 24Lam E.W. Watson R.J. EMBO J. 1993; 12: 2705-2713Crossref PubMed Scopus (317) Google Scholar). Likewise, a number of genes identified in an E2F4-specific chromatin immunoprecipitation assay do not contain a consensus E2F binding site (11Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (391) Google Scholar). Furthermore, evidence is accumulating that E2F-mediated transcription control is exerted in tight collaboration with other transcription factors such as RYBP, YY1, Max/Mga, and Smads (25Schlisio S. Halperin T. Vidal M. Nevins J.R. EMBO J. 2002; 21: 5775-5786Crossref PubMed Scopus (173) Google Scholar, 26Ogawa H. Ishiguro K. Gaubatz S. Livingston D.M. Nakatani Y. Science. 2002; 296: 1132-1136Crossref PubMed Scopus (622) Google Scholar, 27Chen C.R. Kang Y. Siegel P.M. Massague J. Cell. 2002; 110: 19-32Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Considering these observations, we sought to identify the functionally relevant E2F target genes by comparing the E2F-induced changes in gene expression to the corresponding changes induced by pRB and/or p16. Because endogenous E2F activity is repressed by the induction of pRB or p16 in U-2 OS cells, genes that in this setting show a pattern of regulation opposite to the one observed for induction of E2F activity are likely to be the relevant E2F target genes that are deregulated in cancer cells upon loss of p16 or pRB function.We took advantage of two U-2 OS-derived cell lines that overexpress either pRbΔCDK (18Lukas J. Sorensen C.S. Lukas C. Santoni-Rugiu E. Bartek J. Oncogene. 1999; 18: 3930-3935Crossref PubMed Scopus (68) Google Scholar) or p16 (17Jiang H. Chou H.S. Zhu L. Mol. Cell Biol. 1998; 18: 5284-5290Crossref PubMed Google Scholar) in a tetracycline-regulated manner. Fig. 1 shows that both cell lines uniformly express high levels of p16 or pRbΔCDK as tested by immunostaining after removal of tetracycline (Fig. 1, A and B). This observation was confirmed by Western blot analysis showing that both pRbΔCDK and p16 reach high levels of expression 20 h after removal of tetracycline from the culture medium (Fig. 1, E and F). Both proteins were undetectable at time 0 h and reached near maximum levels of expression 16 h after induction. In the p16 cell line, endogenous pRB began to accumulate in its hypophosphorylated form 12 h after induction of p16 expression, whereas the hyperphosphorylated forms began to diminish at this time (Fig. 1E). Northern blot analysis using a PAI1 specific probe, a gene previously identified as an E2F repressed and pRb-induced gene (5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar, 28Koziczak M. Krek W. Nagamine Y. Mol. Cell. Biol. 2000; 20: 2014-2022Crossref PubMed Scopus (42) Google Scholar), revealed significant accumulation of PAI1 mRNA in both cell lines 12 h after induction of pRbΔCDK and 16 h after induction of p16 expression (Fig. 1G). The accumulation of PAI1 mRNA upon induction of pRbΔCDK and p16 expression provides an example of a gene whose expression level changes in the opposite direction as observed upon induction of E2F activity. The induction of pRbΔCDK and p16 expression is capable of arresting the cell cycle (17Jiang H. Chou H.S. Zhu L. Mol. Cell Biol. 1998; 18: 5284-5290Crossref PubMed Google Scholar, 18Lukas J. Sorensen C.S. Lukas C. Santoni-Rugiu E. Bartek J. Oncogene. 1999; 18: 3930-3935Crossref PubMed Scopus (68) Google Scholar). Cell cycle arrest in turn can regulate the expression of hundreds of genes (e.g. see Ref. 29Cho R.J. Huang M. Campbell M.J. Dong H. Steinmetz L. Sapinoso L. Hampton G. Elledge S.J. Davis R.W. Lockhart D.J. Nat. Genet. 2001; 27: 48-54Crossref PubMed Scopus (371) Google Scholar). We used the induction of PAI1 expression and the repression of cyclin E1 expression as parameters to identify the earliest time point where significant changes in the expression can be observed in the absence of detectable cell cycle arrest (Fig. 1, C and D). The time points chosen for further analysis were 12 h after the induction of pRbΔCDK and 16 h after the induction of p16. The choice of early time points should ensure maximum specificity in terms of regulation of endogenous E2F activity in the absence of indirect interference because of cell cycle arrest.Fig. 1Functional characterization of U-2 OS cells expressing p16 or pRBΔCDK in a tetracyclin-regulated manner. U-2 OS osteosarcoma cells stably transfected with plasmids encoding tetracycline-repressed wild type p16 or HA-pRbΔCDK were tested for uniform expression and to determine the appropriate time points for RNA extraction. A, immunofluorescence using an antibody specific for p16 was used to determine that most cells express the endogenous gene upon removal of tetracycline. Staining with 4,6-diamidino-2-phenylindole (DAPI) was used to control for the number of cells. B, immunofluorescence using an antibody against the HA tag present on HA-pRbΔCDK demonstrated that most cells express the exogenous gene. C and D, cell cycle analysis by propidium iodide-bromodeoxyuridine fluorescence-activated cell sorter analysis. No cell cycle block can be observed at the time points utilized for microarray analysis. E, Western immunoblot analysis demonstrates that the endogenous pRB changes from the hyper- to the faster migrating, hypo-phosphorylated form (arrow) upon removal of tetracycline from the medium. F, Western immunoblot analysis demonstrating robust expression of HA-pRbΔCDK (indicated by arrow) upon removal of tetracycline. G, expression of the E2F-repressed PAI1 gene was monitored upon induction of p16 or HA-pRbΔCDK by Northern blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We used oligonucleotide gene expression microarrays to identify genes whose expression changes significantly in the two cell lines after induction of pRbΔCDK and p16. Total RNA was isolated 0 (control) and 12 h after induction of pRbΔCDK expression and 0 (control) and 16 h after p16 expression. Each target cRNA was hybridized to two microarrays to facilitate statistical analysis of the results. The data were analyzed using Microarray suite version 5.0 (Affymetrix) and GeneSpring™ (Silicon Genetics) software. We also re-analyzed the microarray data gathered for the induction of E2F1, -2, and -3 activity derived from the HU6800FL + Hu35k chipset (Affymetrix) that had been analyzed previously using Microarray suite version 4. During this procedure, around 5% of the genes considered previously were discarded as insignificant but the total number of genes found regulated was nearly doubled. These differences are entirely because of improvements in the statistical algorithm used by Microarray suite version 5.0, not to differences in the analysis settings.To compare the data that were derived from two different chipsets, we restricted the following analyses to probe sets that were present on both chipsets. In total, we analyzed 42,089 probe sets (24.600 Unigenes) in the E2F induction experiments and 62,907 probe sets (40.915 Unigenes) in the pRbΔCDK and p16 induction experiments. 23,527 probe sets (16.897 Unigenes) were found to be comparable among each other and were used for further analysis.Activation of the E2Fs (E2F1, E2F2, and E2F3) or induced expression of pRB or p16 resulted in significant changes in the expression of a number of genes. For each of the activated or induced proteins, we organized the up-regulated and the repressed genes into a total of 10 lists of regulated genes, 5 lists of up-regulated genes, and 5 lists of down-regulated genes. We asked how to combine the lists in a way that generates a high degree of specificity without excessive loss of potential candidate genes. We used published information about the identity of E2F target genes (Refs. 5Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar and 8Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (494) Google Scholar, 9Ren B. Cam H. Takahashi Y. Volkert T. Terragni J. Young R.A. Dynlacht B.D. Genes Dev. 2002; 16: 245-256Crossref PubMed Scopus (900) Google Scholar, 10Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (220) Google Scholar, 11Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (391) Google Scholar, 12Markey M.P. Angus S.P. Strobeck M.W. Williams S.L. Gunawardena R.W. Aronow B.J. Knudsen E.S. Cancer Res. 2002; 62: 6587-6597PubMed Google Scholar, 13Wells J. Yan P.S. Cechvala M. Huang T. Farnham P.J. Oncogene. 2003; 22: 1445-1460Crossref PubMed Scopus (109) Google Scholar, and references therein) and assembled a list of genes that had been studied in detail (bona fide target genes) or that had been found regulated consistently in different high throughput screens. This list contains 47 genes, 38 of which were present on both chip sets we analyzed (Fig. 2A). We reasoned that a meaningful combination of lists of regulated genes should maximize both the total number of known target genes as well as the proportion of known target genes relative to the total number of genes in the combined list. Because the activity of E2Fs is repressed by induction of pRB and/or p16, we focused our attention on combinations of lists with opposing effects on gene expression of the E2Fs on the one h" @default.
• W2034260252 created "2016-06-24" @default.
• W2034260252 creator A5011822969 @default.
• W2034260252 creator A5054225715 @default.
• W2034260252 creator A5066665224 @default.
• W2034260252 date "2003-11-01" @default.
• W2034260252 modified "2023-10-13" @default.
• W2034260252 title "Identification of Target Genes of the p16INK4A-pRB-E2F Pathway" @default.
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