Oligonucleotide-based Microarray Analysis Research Articles

RNA-Seq reveals complex genetic response to deepwater horizon oil release in Fundulus grandis

BackgroundThe release of oil resulting from the blowout of the Deepwater Horizon (DH) drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq) in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future.ResultsOur de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set.ConclusionRNA-Seq may be successfully applied to feral and extremely polymorphic organisms that do not have an underlying genome sequence assembly to address timely environmental problems. Additionally, the observed changes in a large set of transcript expression levels are indicative of a complex response to the varied petroleum components to which the fish were exposed.

The paracrine effect of exogenous growth hormone alleviates dysmorphogenesis caused by tbx5 deficiency in zebrafish (Danio rerio) embryos.

BackgroundDysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome.MethodsUsing an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5 morphants, we demonstrated that GH and some GH-related genes were markedly downregulated. Zebrafish embryos microinjected with tbx5-morpholino (MO) antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group.ResultsThe attenuating effects of GH in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors (erk1, akt2), and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes (amhc, vmhc, and cmlc2).ConclusionsBased on our results, tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner.

Gene expression profiling of grade II oligodendrogliomas and ependymomas

Grade II gliomas are morphologically and clinically heterogeneous tumors for which histopathological typing remains the major tool for clinical classification. To what extent the major histological subtypes (astrocytic, oligodendroglial, oligoastrocytic and ependymal tumors) constitute is largely unresolved. Morphological classification can often be ambiguous and would be facilitated by specific subtype markers. Gene expression profiling is becoming an established method to characterize different types of cancers, and it has been used extensively for glioblastoma analysis and for low-grade glioma to glioblastoma comparison. However, few studies of low-grade oligodendrogliomas and ependymomas have been reported. Using oligonucleotide-based microarray analysis, we compared the transcriptional profiles of grade II oligodendrogliomas and grade II ependymomas (two types of grade II gliomas). An essential initial step toward more objective tumor classification is the establishment of taxonomy for tumors based on their gene expression profiles. We aimed to identify expression profiles that differentiate these two types of grade II gliomas. After comparing the expression signature of a tumor to that of normal tissue, differentially expressed genes were used to perform hierarchal clustering, principal component analysis and prediction analysis. Hierarchal clustering and principal component analysis divided nine different tumor tissue samples and four normal brain tissue samples into categories that corresponded well with clinical pathology analysis, grade II oligodendrogliomas, grade II ependymomas and normal tissues. By using expression data from the most informative gene clusters, we constructed a tumor-clustering model. Class prediction using nearest shrunken centroid analysis withcross-validation identified 53 differentially expressed genes between the two types of grade II gliomas. Key words: Grade II ependymoma, grade II oligodendrogliomas, microarray, differentially expressed genes, clustering, classification, prediction.

Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis.

BackgroundCytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence in situ hybridization (FISH).ResultsUsing a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23.ConclusionsOur results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future.

Severe lysosomal storage disease of liver in del(1)(p36): A new presentation

1p36 deletion is the most common terminal deletion syndrome with an estimated occurrence of 1:5000 live births. The deletion is of variable size. It usually involves less than 10 Mb in the 1pter-1p36.23 interval. Variability of the phenotype is partially related to the extent of the deletion. Some children with a 1p36 deletion were reported with obesity and hyperphagia, raising the question of possible phenotypic overlap with Prader–Willi syndrome. Correlation between presence of obesity and the size of the deletion has only been documented in one case. We report a 11-year-old girl with 1p36 deletion and the classical dysmorphological features. In late infancy, she developed an uncontrolled voracious appetite, overweight, truncal obesity and elevated serum transaminases. Liver biopsy disclosed severe steatosis. The hepatocytes contained accumulation of lipofuscins. Lipolysosomes were abnormally numerous and extremely enlarged. These features have not been previously reported in 1p36 deletion. Oligonucleotide-based microarray analysis showed a subtelomeric 2.2 Mb deletion at 1p36.33p36.32. This suggests that this chromosome segment is a critical region for obesity and hyperphagia. The accumulation in the liver with abnormal ultrastructure may be an additional feature of this form of syndromal obesity. 1p36 deletion syndrome should be considered in patients with obesity, hyperphagia and liver fat accumulation.

PP52 Amplification of the chromosome 17 q22 amplicon containing TOP2A gene is correlated with better survival in HER2 amplified/hormone receptors negative breast cancers

Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma (WHO grade III) and/or glioblastoma (WHO grade IV). The molecular basis of astrocytoma progression is still poorly understood, in particular with respect to the progression-associated changes at the mRNA level. Therefore, we compared the transcriptional profile of approximately 6800 genes in primary WHO grade II gliomas and corresponding recurrent high-grade (WHO grade III or IV) gliomas from eight patients using oligonucleotide-based microarray analysis. We identified 66 genes whose mRNA levels differed significantly (P < 0.01, ≥2-fold change) between the primary and recurrent tumors. The microarray data were corroborated by real-time reverse transcription-polymerase chain reaction analysis of 12 selected genes, including 7 genes with increased expression and 5 genes with reduced expression on progression. In addition, the expression of these 12 genes was determined in an independent series of 43 astrocytic gliomas (9 diffuse astrocytomas, 10 anaplastic astrocytomas, 17 primary, and 7 secondary glioblastomas). These analyses confirmed that the transcript levels of nine of the selected genes (COL4A2, FOXM1, MGP, TOP2A, CENPF, IGFBP4, VEGFA, ADD3, and CAMK2G) differed significantly in WHO grade II astrocytomas as compared to anaplastic astrocytomas and/or glioblastomas. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in astrocytoma progression.

Effects of 5-aza-2′-deoxycytidine on the Gene Expression Profile During Embryogenesis of the Ascidian Ciona intestinalis: A Microarray Analysis

DNA methylation is an important epigenetic factor that participates in silencing genes. Genomic approaches to studying DNA methylation promise to be particularly fruitful, since DNA methylation is involved in global control of gene expression in many organisms. With its draft genome completed and a large quantity of available cDNA data, Ciona intestinalis is newly emerging as an invaluable model organism for investigating genome-wide gene expression and function. Here we examine the effects of 5-aza-2'-deoxycytidine (5-aza-CdR), a chemical that blocks CpG methylation, on the gene expression profile of early C. intestinalis embryos, using oligonucleotide-based microarray analysis. Embryos treated with 5-aza-CdR show delayed gastrulation and are developmentally arrested at the neurula stage. They subsequently lose cellular adhesion and finally die. Apoptosis was not detected in these embryos by TUNEL staining at 12 h, indicating that the defects observed did not result from 5-aza-CdR-induced apoptosis. Gene expression profiles of 12-h-old 5-aza-CdR-treated embryos compared to wild-type revealed 91 upregulated genes and 168 downregulated genes. Although nearly half of these encoded proteins with unknown functions, several encoded cell-signaling molecules and transcription factors. In addition, genes associated with the stress response and cell defense were upregulated, whereas genes involved in cell adhesion were downregulated.

Disruption of ZAS3 in Mice Alters NF-κB and AP-1 DNA Binding and T-Cell Development

The large zinc finger proteins, ZAS, regulate the transcription of a variety of genes involved in cell growth, development, and metastasis. They also function in the signal transduction of the TGF-beta and TNF-alpha pathways. However, the endogenous protein of a representative member, ZAS3, is rapidly degraded in primary lymphocytes, which limits the determination of its physiological function in vitro. Therefore, we have generated mice with targeted disruption of ZAS3. Oligonucleotide-based microarray analyses revealed subtle but consistent differences in the expression of genes, many of which are associated with receptor or signal transduction activities between ZAS3+/+ and ZAS3-/- thymi. Gel mobility shift assays showed altered DNA binding activities of NF-kappaB and AP-1 proteins in ZAS3-deficient tissues, including the thymus. Lymphocyte analysis suggested a subtle but broad function of ZAS3 in regulating T-cell development and activation. In CD3+ ZAS3-/- thymocytes, the CD4/ CD8 ratio was decreased and CD69 expression was decreased. In peripheral CD4+ ZAS3-/- lymphocytes we observed an increased number of memory T cells.

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

Gene Expression Profiling Identifies a Set of Transcripts That Are Up-Regulated in Human Testicular Seminoma

Seminoma constitutes one subtype of human testicular germ cell tumors and is uniformly composed of cells that are morphologically similar to the primordial germ cells and/or the cells in the carcinoma in situ. We performed a genome-wide exploration of the genes that are specifically up-regulated in seminoma by oligonucleotide-based microarray analysis. This revealed 106 genes that are significantly and consistently up-regulated in the seminomas compared to the adjacent normal tissues of the testes. The microarray data were validated by semi-quantitative RT-PCR analysis. Of the 106 genes, 42 mapped to a small number of specific chromosomal regions, namely, 1q21, 2p23, 6p21-22, 7p14-15, 12p11, 12p13, 12q13-14 and 22q12-13. This list of up-regulated genes may be useful in identifying the causative oncogene(s) and/or the origin of seminoma. Furthermore, immunohistochemical analysis revealed that the seminoma cells specifically expressed the six gene products that were selected randomly from the list. These proteins include CCND2 and DNMT3A and may be useful as molecular pathological markers of seminoma.

Platelet Endothelial Aggregation Receptor 1 (PEAR1), a Novel Epidermal Growth Factor Repeat-containing Transmembrane Receptor, Participates in Platelet Contact-induced Activation

The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.

Oligonucleotide‐based microarray analysis of retinoic acid target genes in the protochordate, Ciona intestinalis

Oligonucleotide-based microarray analyses were carried out to identify retinoic acid target genes in embryos of the ascidian Ciona intestinalis. Of 21,938 spots, 50 (corresponding to 43 genes) showed over twofold up-regulation in retinoic acid-treated tail bud embryos. In situ hybridization verified retinoic acid-induced up-regulation of 23 genes. Many of them were expressed in the anterior tail region, where a retinaldehyde dehydrogenase homolog is expressed. Homologs of vertebrate genes involved in neurogenesis and/or neuronal functions (e.g., COUP-TF, Ci-Hox1, and SCO-spondin) were expressed in the central nervous system of Ciona embryos, and activated by retinoic acid. Genes encoding transcription factors (e.g., Ci-lmx1.2, vitamin D receptor, and Hox proteins) and apoptosis-related proteins (e.g., transglutaminase and an apoptosis-inducing factor homolog) were also activated by retinoic acid. Simultaneous treatment of embryos with retinoic acid and puromycin revealed a few direct targets, including genes encoding Ci-Hox1, Ci-Cyp26, and an Rnf126-like ring finger protein.

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal (n = 9) and osteoarthritic (n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1β and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage (P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression.

A molecular description of brain trauma pathophysiology using microarray technology: an overview.

It has been estimated that 50% of human transcriptome, the collection of mRNA in a cell, is expressed in the brain, making it one of the most complex organs to understand in terms of genomic responses to injury. The availability of genome sequences for several organisms coupled with the increasing affordability of microarray technologies makes it feasible to monitor the mRNA levels of thousands of genes simultaneously. In this paper, we provide an overview of findings using both cDNA- and oligonucleotide-based microarray analyses after experimental traumatic brain injury (TBI). Specifically, the utility of this methodology as a means of cataloging the biochemical sequelae of brain trauma and elucidating novel genes or pathways for further study is discussed. Furthermore, we offer future directions for the continued evaluation of microarray results and discuss the usefulness of microarray techniques as a testing format for determining the efficacy of mechanism-based therapies.

Probing the ArcA-P Modulon of Escherichia coli by Whole Genome Transcriptional Analysis and Sequence Recognition Profiling

The ArcB/ArcA two-component signal transduction system of Escherichia coli regulates gene expression in response to the redox conditions of growth. Over the years, genetic screens have lead to the identification of about 30 ArcA-P-controlled operons that are involved in redox metabolism. However, the discovery of 3 targets that are not implicated in respiratory metabolism (the tra operon for plasmid conjugation, psi site for Xer-based recombination, and oriC site for chromosome replication) suggests that the Arc modulon may comprise additional operons that are involved in a myriad of functions. To identify these operons, we derived the ArcA-P-dependent transcription profile of E. coli using oligonucleotide-based microarray analysis. The findings indicated that 9% of all open reading frames in E. coli are affected either directly or indirectly by ArcA-P. To identify which operons are under the direct control of ArcA-P, we developed the ArcA-P recognition weight matrix from footprinting data and used it to scan the genome, yielding an ArcA-P sequence affinity map. By overlaying both methods, we identified 55 new Arc-regulated operons that are implicated in energy metabolism, transport, survival, catabolism, and transcriptional regulation. The data also suggest that the Arc response pathway, which translates into a net global downscaling of gene expression, overlaps partly with the FNR regulatory network. A conservative but reasonable assessment is that the Arc pathway recruits 100-150 operons to mediate a role in cellular adaptation that is more extensive than hitherto anticipated.

Gene Expression Profiling Identifies a Set of Transcripts that are Up-Regulated in Human Testicular Seminoma

Seminoma constitutes one subtype of human testicular germ cell tumors and is uniformly composed of cells that are morphologically similar to the primordial germ cells and/or the cells in the carcinoma in situ. We performed a genome-wide exploration of the genes that are specifically up-regulated in seminoma by oligonucleotide-based microarray analysis. This revealed 106 genes that are significantly and consistently up-regulated in the seminomas compared to the adjacent normal tissues of the testes. The microarray data were validated by semi-quantitative RT-PCR analysis. Of the 106 genes, 42 mapped to a small number of specific chromosomal regions, namely, 1q21, 2p23, 6p21-22, 7p14-15, 12pll, 12p13, 12q13-14 and 22q12-13. This list of up-regulated genes may be useful in identifying the causative oncogene(s) and/or the origin of seminoma. Furthermore, immunohistochemical analysis revealed that the seminoma cells specifically expressed the six gene products that were selected randomly from the list. These proteins include CCND2 and DNMT3A and may be useful as molecular pathological markers of seminoma.

Characterization of Gene Expression Profiles Associated with Glioma Progression Using Oligonucleotide-Based Microarray Analysis and Real-Time Reverse Transcription-Polymerase Chain Reaction

Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma (WHO grade III) and/or glioblastoma (WHO grade IV). The molecular basis of astrocytoma progression is still poorly understood, in particular with respect to the progression-associated changes at the mRNA level. Therefore, we compared the transcriptional profile of approximately 6800 genes in primary WHO grade II gliomas and corresponding recurrent high-grade (WHO grade III or IV) gliomas from eight patients using oligonucleotide-based microarray analysis. We identified 66 genes whose mRNA levels differed significantly (P < 0.01, > or =2-fold change) between the primary and recurrent tumors. The microarray data were corroborated by real-time reverse transcription-polymerase chain reaction analysis of 12 selected genes, including 7 genes with increased expression and 5 genes with reduced expression on progression. In addition, the expression of these 12 genes was determined in an independent series of 43 astrocytic gliomas (9 diffuse astrocytomas, 10 anaplastic astrocytomas, 17 primary, and 7 secondary glioblastomas). These analyses confirmed that the transcript levels of nine of the selected genes (COL4A2, FOXM1, MGP, TOP2A, CENPF, IGFBP4, VEGFA, ADD3, and CAMK2G) differed significantly in WHO grade II astrocytomas as compared to anaplastic astrocytomas and/or glioblastomas. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in astrocytoma progression.

Bcl-X(L) affects Ca(2+) homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors.

An oligonucleotide-based microarray analysis of 9,500 genes and expressed sequence tags (ESTs) demonstrated that the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) was significantly down-regulated in Bcl-X(L)-expressing as compared with control cells. This result was confirmed at the mRNA and protein levels by Northern and Western blot analyses of two independent hematopoietic cell lines and murine primary T cells. Bcl-X(L) expression resulted in a dose-dependent decrease in IP(3)R protein. IP(3)R expression is regulated as part of a mitochondrion-to-nucleus stress-responsive pathway. The uncoupling of mitochondrial oxidative phosphorylation resulted in induction of binding of the transcription factor NFATc2 to the IP(3)R promoter and transcriptional activation of IP(3)R. Expression of Bcl-X(L) led to a decreased induction of both NFATc2 DNA binding to the IP(3)R promoter and IP(3)R expression in response to the inhibition of mitochondrial oxidative phosphorylation. The Bcl-X(L)-dependent decrease in IP(3)R expression also correlated with a reduced T cell antigen receptor ligation-induced Ca(2+) flux in Bcl-X(L) transgenic murine T cells, and microsomal vesicles prepared from Bcl-X(L)-overexpressing cells exhibited lower IP(3)-mediated Ca(2+) release capacity. Furthermore, reintroducing IP(3)R into Bcl-X(L)-transfected cells partially reversed Bcl-X(L)-dependent anti-apoptotic activity. These results suggest that even under non-apoptotic conditions, expression of Bcl-2-family proteins influences a signaling network that links changes in mitochondrial metabolism to alterations in nuclear gene expression.