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• W2057979382 abstract "Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis. Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis. From the beginning of its description, apoptosis has been considered as an active process requiring RNA and protein synthesis, especially in neurons. Blockade by macromolecular synthesis inhibitors has long been used as a criterion to establish the apoptotic nature of a particular cell death (discussed in Ref. 1Weil M. Jacobson M.D. Coles H.S. Davies T.J. Gardner R.L. Raff K.D. Raff M.C. J. Cell Biol. 1996; 133: 1053-1059Crossref PubMed Scopus (356) Google Scholar). Although identification of death receptor-induced pathways that do not require de novo protein synthesis has slightly restrained this notion (2Martin S.J. Trends Cell Biol. 1993; 3: 141-144Abstract Full Text PDF PubMed Scopus (122) Google Scholar), the question of the transcriptional control of apoptosis, the role it plays, and the nature of newly expressed genes still remains. The first studies on the transcriptional regulation of apoptosis were orientated by the concept of a preset rheostat based on the observation that the anti-apoptotic effect of Bcl-2 could be counteracted by pro-apoptotic Bax (3Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar). Many studies were undertaken to examine possible expression variations of these two proteins. In fact, transcription of selective members of the Bcl-2 family was repeatedly reported during different forms of apoptosis, in particular in neurons. These data suggested that differential expression of certain genes may be essential to the initiation of apoptotic neuronal death. However, the number of genes whose differential expression could be essential to the initiation of apoptosis has become difficult to estimate. At least 20 Bcl-2 family members (4Cory S. Huang D.C. Adams J.M. Oncogene. 2003; 22: 8590-8607Crossref PubMed Scopus (1305) Google Scholar) and an increasing number of other components of the core apoptotic machinery (5Reed J.C. Doctor K. Rojas A. Zapata J.M. Stehlik C. Fiorentino L. Damiano J. Roth W. Matsuzawa S. Newman R. Takayama S. Marusawa H. Xu F. Salvesen G. Godzik A. Genome Res. 2003; 13: 1376-1388Crossref PubMed Scopus (101) Google Scholar) have been identified in mammals to date. Moreover, much less is known about early mechanisms leading to the activation of the apoptotic machinery, and they could also be regulated at the transcriptional level. The expression of all these components cannot be simultaneously detected by classical methods used to evaluate mRNA (reverse transcription (RT) 1The abbreviations used are: RT, reverse transcription; CGN, cerebellar granule neuron(s); FDR, false discovery rate; GO, Gene Ontology; ER, endoplasmic reticulum; UPR, unfolded protein response; PI, phosphatidylinositol.-PCR, Northern blotting, and differential display), which can give only a biased view of transcriptional regulations associated with apoptosis. The recent development of genome-scale methods of gene expression analysis now offers new perspectives (6Chiang L.W. Grenier J.M. Ettwiller L. Jenkins L.P. Ficenec D. Martin J. Jin F. DiStefano P.S. Wood A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2814-2819Crossref PubMed Scopus (97) Google Scholar). Primary cultures of cerebellar granule neurons (CGN) are particularly suited to conduct this kind of study. CGN are one of the best characterized in vitro models of neuronal apoptosis (7Miller T.M. Johnson Jr., E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar), which depends on the synthesis of new RNA and proteins (8D'Mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (853) Google Scholar). Moreover, CGN constitute the most abundant neuronal population in the central nervous system of mammals and can be cultured in vitro up to 98% homogeneity. When dissociated from early postnatal mice, CGN can survive and differentiate in culture in the presence of serum and depolarizing levels of extracellular KCl ([KCl]o = 25 mm) (7Miller T.M. Johnson Jr., E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar). Depolarization is presumed to mimic the endogenous excitatory activity that is required for survival during cerebellar development in vivo (9Ikonomidou C. Bosch F. Miksa M. Bittigau P. Vo ̈ckler J. Dikranian K. Tenkova T.I. Stefovska V. Turski L. Olney J.W. Science. 1999; 283: 70-74Crossref PubMed Scopus (1738) Google Scholar). Lowering [KCl]o to 5 mm in the absence of serum triggers apoptosis (8D'Mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (853) Google Scholar). This presumably mimics the naturally occurring death that takes place in the external granular layer of newborn rat cerebellum (10Wood K.A. Dipasquale B. Youle R.J. Neuron. 1993; 11: 621-632Abstract Full Text PDF PubMed Scopus (318) Google Scholar). In this study, we used genome-scale, long oligonucleotide microarrays to discover sets of genes associated with CGN apoptosis induction. We focused data analysis on components of the core apoptotic machinery. We identified several of these genes whose up-regulation has not been previously reported during neuronal apoptosis. However, genes involved in the execution of apoptosis were not significantly over-represented among differentially expressed genes. Primary Cultures of CGN—CGN cultures were prepared from 7-day-old murine pups (C57Bl/6J mice, Charles River Laboratories) as described by Miller and Johnson (7Miller T.M. Johnson Jr., E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar) with slight modifications. Briefly, freshly dissected cerebella were incubated for 10 min at 37 °C with 0.25 mg/ml trypsin, and cells were dissociated in Hanks' balanced salt solution without Ca2+ and Mg2+ in the presence of 0.5 mg/ml trypsin inhibitor and 0.1 mg/ml DNase I by several steps of mechanical disruption. The resulting cell suspension was centrifuged and resuspended in basal Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 10 mm HEPES, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 20 mm KCl to achieve a final concentration of 25 mm. The cell suspension was filtered through a Falcon 40-μm cell strainer and plated in a coated dish for 25 min to allow attachment of non-neuronal cells. Neurons were then resuspended, counted, and seeded at a density of 25 × 104 cells/cm2 in culture dishes previously coated with poly-d-lysine (BD Biosciences). The granule neurons were cultured at 37 °C in a humidified incubator with 6% CO2 and 94% air for 6 days. To prevent proliferation of remaining non-neuronal cells, 10 μm cytosine β-d-arabinofuranoside was added to the culture medium 24 h after plating. At 6 days in vitro, granule neurons represented >98% of the cultured cells (data not shown). KCl Deprivation, Survival, and Apoptotic Assays—CGN were washed and incubated for the indicated times in serum-free basal Eagle's medium supplemented with l-Gln, HEPES, antibiotics, and the N-methyl-d-aspartate antagonist (+)-MK-801 (1 μm) and containing either 25 mm KCl (K25 medium) or 5 mm KCl (K5 medium). We chose to use K25 medium as a control instead of the initial culture medium to exclude gene expression differences resulting from serum deprivation. Serum deprivation has been shown to induce the death of a small proportion of cultured CGN (7Miller T.M. Johnson Jr., E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar). However, this neuronal death proceeds mainly through necrosis (11Villalba M. Bockaert J. Journot L. Neuroreport. 1997; 8: 981-985Crossref PubMed Scopus (48) Google Scholar). Moreover, we added 1 μm MK-801 to both the K25 and K5 media to avoid any change in gene expression due to uncontrolled N-methyl-d-aspartate receptor stimulation by endogenously released glutamate. Neuronal survival was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the culture medium was replaced with K25 medium containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and neurons were returned to the incubator for 2 h. The medium was removed, and blue formazan produced by living cells was solubilized in Me2SO. The absorbance of blue formazan was estimated at 570 nm. To assess apoptosis, cytochrome c subcellular localization was detected by immunocytochemistry using an antibody from Pharmingen; caspase-3 activation was estimated by immunostaining with an antibody that specifically recognizes cleaved caspase-3 (Cell Signaling Technology); and nuclear condensation was visualized by Hoechst 33258 staining. Immunocytochemistry and Western blotting were performed as described previously (12Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.-C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1093) Google Scholar). RNA Preparation and Real-time Quantitative RT-PCR—Total RNA was extracted using the RNAqueous® kit and treated with the DNase I from the DNA-free™ kit (Ambion, Inc.) according to the manufacturer's instructions. Poly(A)+ RNA was isolated from total RNA with Oligotex™ (Qiagen Inc.). Two rounds of hybridization with oligo(dT) beads were carried out successively to improve mRNA purity. RNAs were used to perform a two-step RT-PCR. Briefly, either 1 μg of total RNA or 50 ng of mRNA were reverse-transcribed using 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) in the presence of 2.5 μm N6 random primers and 0.5 mm dNTP. Either 10 or 0.5 ng of the resulting cDNA were used as template for real-time PCR using ABI Prism 7000 with the SYBR® Green PCR Master Mix (Applied Biosystems). Primers were designed with Primer Express™ software (Applied Biosystems). The sequences of all the primers used are provided in Supplemental Table 1. PCR was performed in a volume of 10 μl in the presence of 300 nm specific primers. Thermal cycling parameters were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Data were analyzed with ABI Prism 7000 SDS software. The relative amounts of specifically amplified cDNAs were calculated with the comparative threshold cycle (ΔΔCt) method (13Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (127160) Google Scholar). The β2-microglobulin amplicon was used as a reference. Long Oligonucleotide Microarray Fabrication—The long oligonucleotides of the Mouse Genome Oligo Set Version 2 (Operon) were used. Operon provided the following information about the oligonucleotides. The 16,463 70-mers were designed from representative sequences of the UniGene Database Build Mm.102 (February 2002) and the Mouse Reference Sequence (RefSeq) Database, each oligonucleotide representing a unique gene. An amino linker was attached to the 5′-end of each oligonucleotide. Oligonucleotides were designed to have melting temperatures of 78 ± 5 °C. More than 98% of the oligonucleotides were within 1000 bases from the 3′-end of the available gene sequence. Oligonucleotides were selected to limit secondary structure. BLAST alignments were performed to exclude oligonucleotides that could cross-hybridize with other sequences of the Mouse UniGene Database. Each oligonucleotide had ≤70% of overall identity to any other gene and could not have >20 contiguous bases common to any other gene. Oligonucleotides were dissolved in Tris/EDTA and diluted twice in 2× spotting solution (ArrayIt, TeleChem International, Inc.) to a final concentration of 10 μm. Oligonucleotides were spotted using Amersham Generation III Array spotter on aminosilane-coated mirror glass slides (7-Star, Amersham Biosciences). Prior to hybridization, DNA was cross-linked to the slides by UV irradiation and washed twice with 0.2% SDS solution and twice with distilled water. cDNA Labeling—Poly(A)+ RNA was reverse-transcribed, and cDNA was labeled using the CyScribe cDNA post-labeling kit (Amersham Biosciences) in a two-step procedure according to the manufacturer's protocol. In the first step, aminoallyl-dUTP was incorporated during first-strand cDNA synthesis. A mixture of random nonamers and anchored oligo(dT) was used for priming the synthesis catalyzed by CyScript™ reverse transcriptase. For each reaction, 500 ng of mRNA were used as template, and incubation was carried out for 2 h at 42 °C. Following alkaline hydrolysis of the RNA template, the cDNA was purified on QIAquick PCR purification columns (Qiagen Inc.) according to the manufacturer's instructions, except that QIAquick wash buffer was replaced with 5 mm potassium phosphate buffer (pH 8.5) containing 80% ethanol, and cDNA was eluted with 4 mm potassium phosphate buffer (pH 8.5). In the second step, the aminoallyl-modified cDNA was chemically coupled to Cy3 and Cy5 N-hydroxysuccinimidyl esters. The coupling reactions were performed separately with Cy3 and Cy5 so that the cDNA derived from neurons incubated in K25 or K5 medium was labeled with a different dye. The coupling reaction was terminated by the addition of hydroxylamine. The fluorescent cDNAs were subsequently purified using the CyScribe GFX purification kit (Amersham Biosciences). Dual Color Microarray Hybridization—Of the cDNA derived from each condition (K5 or K25 medium), one-half was labeled with Cy3 and the other half with Cy5 to perform dye swap hybridizations: K25-Cy3 + K5-Cy5 and K25-Cy5 + K5-Cy3. This enabled us to account for any bias in dye coupling or emission efficiency of Cy dyes. The two labeled cDNA preparations (0.9–1.2 μg/condition) were combined together with 10 μg of oligonucleotide A80 and denatured at 95 °C for 5 min. They were subsequently added to microarray hybridization buffer (Amersham Biosciences) and applied to the microarrays in individual chambers of an automated slide processor (Amersham Biosciences). Hybridization was carried out at 37 °C for 12 h. Hybridized slides were washed at 37 °C successively with 1× SSC and 0.2% SDS for 10 min, twice with 0.1× SSC and 0.2% SDS for 10 min, with 0.1× SSC for 1 min, and with isopropyl alcohol before air drying. Microarrays were immediately scanned in both Cy3 and Cy5 channels with an Amersham Generation III Array scanner with 10-μm resolution. ArrayVision™ software (Imaging Research Inc.) was used for image analysis. A local background was calculated for each spot as the median value of the fluorescence intensities of four squares surrounding the spot. This background was subtracted from the foreground fluorescence intensity. Detection limit was calculated as (average + (3 × S.D.)) of the fluorescence intensities of three different negative controls spotted 12 times along each slide. Intensity values under this detection limit were replaced with the threshold value before any normalization and statistical analysis. Microarray Data Analyses—Fluorescence intensity ratios from each microarray were normalized using the R package SMA (statistical microarray analysis) by the local weight regression (lowess) algorithm called print-tip (14Yang Y.H. Dudoit S. Luu P. Lin D.M. Peng V. Ngai J. Speed T.P. Nucleic Acids Res. 2002; 30: e15Crossref PubMed Scopus (2834) Google Scholar). In this procedure, spotting variation is taken into account, and the normalizing quotient varies with the intensity of the spot. Once the two channels had been normalized for each microarray, normalization was performed between array data by creating a matrix of normalized log intensity ratios across different slides. We then scaled the matrix such that each column had the same median absolute deviation as described by Yang et al. (14Yang Y.H. Dudoit S. Luu P. Lin D.M. Peng V. Ngai J. Speed T.P. Nucleic Acids Res. 2002; 30: e15Crossref PubMed Scopus (2834) Google Scholar). Normalized data were analyzed using a variance estimate and a permutation technique known as SAM (significance analysis of microarrays; available at www-stat.stanford.edu/~tibs/SAM/index.html) (15Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9802) Google Scholar). SAM was used with the one-class response and 100 permutation parameters using the k-nearest neighbor approach for lost values. The delta value was obtained by fixing the false discovery rate (FDR) for both the median and 95th percentile to 1 or 5%. An FDR of 1% indicates that, on average, 1% of the genes called significantly regulated are not actually regulated (15Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9802) Google Scholar). The eGOn Version 1.0 software of the Norwegian Microarray Consortium (available at www.mikromatrise.no) was used to assign Gene Ontology (GO) terms to individual genes from the Oligo Set and the differentially expressed gene list. Annotated genes were then clustered in functional categories defined by the Gene Ontology Consortium (16Consortium Gene Ontology Nat. Genet. 2000; 25: 25-29Crossref PubMed Scopus (27692) Google Scholar). To simplify representation, only the third row of GO nodes have been considered, and redundancy was avoided when possible. Despite this, in many cases, single genes were associated with multiple GO identifiers. This reflects the biological reality that a particular protein may function in several processes, may contain domains that carry out diverse molecular functions, and may be active in multiple locations in the cell. This explains why the sum of gene numbers contained in the different categories is often greater than the total number of annotated genes. Statistical tests available on the eGOn Version 1.0 web site were performed to identify categories of genes in which differentially expressed genes were over-represented. A two-sided, one-sample binomial test was implemented, and a p value was calculated for each GO term. This test identified terms where the number of differentially expressed genes divided by the number of genes in the Oligo Set was greater than the overall proportion (the total number of differentially expressed genes divided by the total number of genes in the Oligo Set). Genomic sequences 1 kb upstream and 200 bp downstream from transcriptional start sites of genes from the Oligo Set and the differentially expressed gene list were retrieved from DBTSS (available at dbtss.hgc.jp/index.html). They were analyzed for the presence of consensus DNA-binding sites with Match™ software and the TRANSFAC Database (available at www.gene-regulation.com/) using high quality matrices to minimize the occurrence of false positive sites. A binomial test, implemented as described above, identified consensus binding sites over-represented in the promoters of differentially expressed genes compared with those of the Oligo Set. Temporal Analysis of CGN Apoptosis—CGN were completely protected from KCl deprivation-induced apoptosis by the transcription inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide (Fig. 1a). This supports the notion that de novo RNA and protein synthesis is required for neuronal apoptosis. Expression of the genes necessary for triggering apoptosis is likely to precede the point after which neurons are irreversibly committed to death. To determine this point, we performed a series of temporal analyses based on different apoptotic criteria such as cytochrome c release from mitochondria, caspase-3 activation, and chromatin condensation. The appearance of these hallmarks was approximately concomitant (Fig. 1b). We also determined the commitment points after which half of the neurons could no longer be rescued by actinomycin D, cycloheximide, or re-addition of 25 mm KCl to the medium (Fig. 1c) because, at that time, genes necessary to apoptosis induction have already been expressed. Our data were consistent with results reported by others in mouse CGN (17Nardi N. Avidan G. Daily D. Zilkha-Falb R. Barzilai A. J. Neurochem. 1997; 68: 750-759Crossref PubMed Scopus (75) Google Scholar): transcriptional and translational commitment points were ∼5 h after deprivation. The temporal curves of apoptosis (Fig. 1b) and KCl rescue (Fig. 1c) indicated that 50% of the neurons were still alive after 7 h. These results suggested that expression of determinant genes preceded first manifestations of apoptosis by ∼2 h. This was confirmed by the expression time course of genes known to be implicated in the triggering of CGN apoptosis such as bim-EL, DP5, and c-jun (18Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar, 19Putcha G.V. Moulder K.L. Golden J.P. Bouillet P. Adams J.A. Strasser A. Johnson Jr., E.M. Neuron. 2001; 29: 615-628Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 20Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar): a peak of mRNA was determined at ∼3 h after KCl deprivation for the three genes (Fig. 1d). Moreover, Western blotting showed a peak of protein levels and phosphorylation of c-Jun after 6 h and a peak of Bim-EL after 4 h, both preceding massive cleavage and activation of caspase-3 (8 h) (Fig. 1e). These data prompted us to analyze the gene expression profile of CGN 4 h after KCl withdrawal, this time appearing as optimal to identify genes involved in the induction of neuronal apoptosis. Gene Expression Profiling of Apoptotic CGN—Gene expression profiles of CGN maintained in K25 medium (control) and in K5 medium (apoptosis) for 4 h were compared using high density long oligonucleotide microarrays. Three independent experiments were performed using distinct neuronal cultures. For >50% of the oligonucleotides, fluorescence intensities were above the detection threshold, indicating a good sensitivity. Data analysis also revealed good normalization (data not shown) as well as good reproducibility (Fig. 2a). SAM statistical analysis (15Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9802) Google Scholar) was performed using an FDR of 1% to identify genes that were significantly differentially expressed. The analysis showed that 278 genes were significantly up-regulated and that 90 genes were significantly down-regulated in apoptotic neurons compared with controls. -Fold change values distributed as described in the legend to Fig. 2b. The regulations detected with microarrays were confirmed for 45 of 48 genes (94%) by real-time RT-PCR (Table I) (data not shown).Table ITranscriptional regulations of some genes potentially involved in the control of CGN survival and apoptosisView Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Transcriptional Regulation of Apoptotic Genes—To determine whether components of the core apoptotic machinery are regulated at the transcriptional level during CGN apoptosis, we examined individual microarray data for a repertoire of apoptotic genes. We used the lists of gene families defined by Reed et al. (5Reed J.C. Doctor K. Rojas A. Zapata J.M. Stehlik C. Fiorentino L. Damiano J. Roth W. Matsuzawa S. Newman R. Takayama S. Marusawa H. Xu F. Salvesen G. Godzik A. Genome Res. 2003; 13: 1376-1388Crossref PubMed Scopus (101) Google Scholar), based on evolutionarily conserved domains shared by proteins involved in apoptosis and NF-κB induction. The expression measurements of the 229 genes contained in these different categories are presented in Supplemental Table 2. Only six genes were found to be significantly up-regulated after SAM performed with 1% FDR (Supplemental Table 2): DP5, bim, caspase-3, caspase-6, Unc5h2, and Cide-A. A less stringent SAM performed with 5% FDR identified four additional regulated genes: Raidd/Cradd, mal/Tirap, Traf3, and cytochrome c (Supplemental Table 2). All of these gene expression changes were confirmed by quantitative RT-PCR (Table I). However, K5/K25 medium ratios determined by quantitative PCR were too low for mal/Tirap (1.33 ± 0.14) and Traf3 (1.72 ± 0.26) to warrant further analysis. Two additional genes that were not identified as significantly regulated by SAM but with a K5/K25 medium ratio on microarrays above 1.6 were found to be actually up-regulated during CGN apoptosis by real-time PCR: caspase-7 and p73 (Table I). In addition, fasL up-regulation that was not detected on microarrays but that had been previously reported during CGN apoptosis (21Ginham R. Harrison D.C. Facci L. Skaper S. Philpott K.L. Neurosci. Lett. 2001; 302: 113-116Crossref PubMed Scopus (26) Google Scholar, 22Le Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar) was found by quantitative PCR (Table I). Overall, only 11 genes of the 229 gene-containing list of the apoptotic machinery were found to be significantly regulated during CGN apoptosis. In addition, most of the gene expression changes previously reported during CGN apoptosis and the regulations of some genes involved in signaling pathways controlling CGN survival were also detected both by our microarray measurements and by quantitative RT-PCR (Table I). For instance, we detected an increased level for the following genes: caspase-3; the Bcl-2 family members bim and DP5; the transcription factors c-jun, c-fos, and egr-1; the secreted glycoprotein neuronal pentraxin-1; the stress-responsive inhibitor of thioredoxin, Vdup-1; the potassium channel Task-1; and the insulin-like growth factor-binding protein Igfbp5, as observed by other investigators (7Miller T.M. Johnson Jr., E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar, 20Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar, 21Ginham R. Harrison D.C. Facci L. Skaper S. Philpott K.L. Neurosci. 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• W2057979382 created "2016-06-24" @default.
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• W2057979382 date "2005-02-01" @default.
• W2057979382 modified "2023-10-16" @default.
• W2057979382 title "Genes Regulated in Neurons Undergoing Transcription-dependent Apoptosis Belong to Signaling Pathways Rather than the Apoptotic Machinery" @default.
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• W2057979382 doi "https://doi.org/10.1074/jbc.m408971200" @default.
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