Clinical interpretation and implications of whole-genome sequencing.

IMPORTANCE: Whole-genome sequencing (WGS) is increasingly applied in clinical medicine and is expected to uncover clinically significant findings regardless of sequencing indication. OBJECTIVES: To examine coverage and concordance of clinically relevant genetic variation provided by WGS technologies; to quantitate inherited disease risk and pharmacogenomic findings in WGS data and resources required for their discovery and interpretation; and to evaluate clinical action prompted by WGS findings. DESIGN, SETTING, AND PARTICIPANTS: An exploratory study of 12 adult participants recruited at Stanford University Medical Center who underwent WGS between November 2011 and March 2012. A multidisciplinary team reviewed all potentially reportable genetic findings. Five physicians proposed initial clinical follow-up based on the genetic findings. MAIN OUTCOMES AND MEASURES: Genome coverage and sequencing platform concordance in different categories of genetic disease risk, person-hours spent curating candidate disease-risk variants, interpretation agreement between trained curators and disease genetics databases, burden of inherited disease risk and pharmacogenomic findings, and burden and interrater agreement of proposed clinical follow-up. RESULTS: Depending on sequencing platform, 10% to 19% of inherited disease genes were not covered to accepted standards for single nucleotide variant discovery. Genotype concordance was high for previously described single nucleotide genetic variants (99%-100%) but low for small insertion/deletion variants (53%-59%). Curation of 90 to 127 genetic variants in each participant required a median of 54 minutes (range, 5-223 minutes) per genetic variant, resulted in moderate classification agreement between professionals (Gross κ, 0.52; 95% CI, 0.40-0.64), and reclassified 69% of genetic variants cataloged as disease causing in mutation databases to variants of uncertain or lesser significance. Two to 6 personal disease-risk findings were discovered in each participant, including 1 frameshift deletion in the BRCA1 gene implicated in hereditary breast and ovarian cancer. Physician review of sequencing findings prompted consideration of a median of 1 to 3 initial diagnostic tests and referrals per participant, with fair interrater agreement about the suitability of WGS findings for clinical follow-up (Fleiss κ, 0.24; P < 001). CONCLUSIONS AND RELEVANCE: In this exploratory study of 12 volunteer adults, the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of detection of genetic variation with the highest potential clinical effects, and uncertainty about clinically reportable findings. In certain cases, WGS will identify clinically actionable genetic variants warranting early medical intervention. These issues should be considered when determining the role of WGS in clinical medicine.

Role of the cyclic AMP response element in the bcl-2 promoter in the regulation of endogenous Bcl-2 expression and apoptosis in murine B cells.

We have previously shown for B-cell lines that the cyclic AMP response element (CRE) is a major positive regulatory site in the bcl-2 promoter. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutated CRE was introduced into the bcl-2 promoter region (mutCRE-bcl2 mice). Quantitative chromatin immunoprecipitation assays revealed that mutation of the CRE abolished the binding of CREB/ATF and CBP transcription factors to the bcl-2 promoter and greatly diminished the binding of NF-kappaB factors. The mutant CRE significantly reduced the expression of Bcl-2 in B cells and rendered them susceptible to surface immunoglobulin- and chemotherapeutic agent-induced apoptosis. The low levels of Bcl-2 were not changed with activation of the cells. The numbers of pre-B, immature B, and mature B cells in the bone marrow were decreased, as were the numbers of splenic B cells in mutCRE-bcl2 mice. Our findings indicate that the CRE in the bcl-2 promoter has an important functional role in the regulation of endogenous Bcl-2 expression and plays a critical role in the coordination of signals that regulate B-cell survival.

Protein/DNA arrays identify nitric oxide-regulated cis-element and trans-factor activities some of which govern neuroblastoma cell viability.

Toxic nitric oxide (NO) levels can regulate gene expression. Using a novel protein/DNA array, we show that toxic NO levels regulate the binding of trans-factors to various cis-elements in neuroblastoma cells, including CRE and those recognized by the transcription factors AP1, AP2, Brn-3a, EGR, E2F1 and SP1. Functionality of some of the cis-elements was confirmed by electro mobility shift and reporter assays. Interestingly, CREB, AP-1, Brn-3a, EGR and E2F1 can control mammalian cell viability. NO induced the anti-apoptotic Bcl-2 protein and its mRNA prior to the onset of death of 30-60% of the cells. Promoter analysis of the bcl-2 gene confirmed the involvement of a CRE in NO-dependent bcl-2 transcription. Neuroblastoma cells over-expressing bcl-2 became much more resistant to NO-induced apoptosis; conversely, Bcl-2 knockdown cells were rendered markedly more sensitive to NO. Together these results suggest that Bcl-2 counteracts NO-induced apoptosis in a fraction of the cell population. Thus, NO stimulates the binding of many trans-factors to their cognate cis-elements, some of which can regulate cell viability through transcriptional activation of target genes. Our results emphasize that a DNA/protein array approach can reveal novel, global transcription factor activities stimulated by cell death-regulating molecules.

Histone deacetylase inhibitors down-regulate bcl-2 expression and induce apoptosis in t(14;18) lymphomas.

Histone deacetylase (HDAC) inhibitors are promising antitumor agents, but they have not been extensively explored in B-cell lymphomas. Many of these lymphomas have the t(14;18) translocation, which results in increased bcl-2 expression and resistance to apoptosis. In this study, we examined the effects of two structurally different HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on the cell cycle, apoptosis, and bcl-2 expression in t(14;18) lymphoma cells. We found that in addition to potent cell cycle arrest, TSA and NaB also dramatically induced apoptosis and down-regulated bcl-2 expression, and overexpression of bcl-2 inhibited TSA-induced apoptosis. The repression of bcl-2 by TSA occurred at the transcriptional level. Western blot analysis and quantitative chromatin immunoprecipitation (ChIP) assay showed that even though HDAC inhibitors increased overall acetylation of histones, localized histone H3 deacetylation occurred at both bcl-2 promoters. TSA treatment increased the acetylation of the transcription factors Sp1 and C/EBPalpha and decreased their binding as well as the binding of CBP and HDAC2 to the bcl-2 promoters. Mutation of Sp1 and C/EBPalpha binding sites reduced the TSA-induced repression of bcl-2 promoter activity. This study provides a mechanistic rationale for the use of HDAC inhibitors in the treatment of human t(14;18) lymphomas.

Transcription factor gata4 regulates cardiac BCL2 gene expression in vitro and in vivo.

The transcription factor GATA-4 protects cardiomyocytes against doxorubicin-induced cardiotoxicity. Here, we report the identification of Bcl2 as a direct target gene of GATA4 that may mediate the prosurvival function of GATA4 in cardiomyocytes. Bcl2 transcript and protein levels were reduced by doxorubicin in neonatal rat ventricular cardiomyocytes (NRVC) and in mouse heart as determined by RT-PCR and Western blot analysis. The reduction in Bcl2 was prevented by overexpression of GATA4 in NRVC and in transgenic mouse heart. Also, expression of GATA4 increased baseline Bcl2 levels by 30% in NRVC and 2.7-fold in transgenic heart, indicating the sufficiency of GATA4 to up-regulate Bcl2 gene expression. GATA4 knockdown by siRNA reduced Bcl2 levels by 48% in NRVC, suggesting that GATA4 is required for Bcl2 constitutive gene expression. Transfection of HEK cells with GATA4 plasmids activated Bcl2 promoter and elevated Bcl2 protein levels. Deletion and mutagenesis analysis revealed that a consensus GATA motif at base -266 on the promoter conserved across multiple species is partially responsible for the promoter activity. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrate that GATA4 directly bound to this GATA site. Together, these results indicate that GATA4 positively regulates cardiac Bcl2 gene expression in vitro and in vivo.

A genome-wide view of the in vitro response to l-asparaginase in acute lymphoblastic leukemia.

To investigate the effect of l-asparaginase on acute lymphoblastic leukemia (ALL), we used cDNA microarrays to obtain a genome-wide view of gene expression both at baseline and after in vitro exposure to l-asparaginase in cell lines and pediatric ALL samples. In 16 cell lines, a baseline gene expression pattern distinguished l-asparaginase sensitivity from resistance. However, for 28 pediatric ALL samples, no consistent baseline expression pattern was associated with sensitivity to l-asparaginase. In particular, baseline expression of asparagine synthetase (ASNS) was not predictive of response to l-asparaginase. After exposure to l-asparaginase, 5 cell lines and 10 clinical samples exhibited very similar changes in the expression of a large number of genes. However, the gene expression changes occurred more slowly in the clinical samples. These changes included a consistent increase in expression of tRNA synthetases and solute transporters and activating transcription factor and CCAAT/enhancer binding protein family members, a response similar to that observed with amino acid starvation. There was also a consistent decrease in many genes associated with proliferation. Taken together, the changes seem to reflect a consistent coordinated response to asparagine starvation in both cell lines and clinical samples. Importantly, in the clinical samples, increased expression of ASNS after l-asparaginase exposure was not associated with in vitro resistance to l-asparaginase, indicating that ASNS-independent mechanisms of in vitro l-asparaginase resistance are common in ALL. These results suggest that targeting particular genes involved in the response to amino acid starvation in ALL cells may provide a novel way to overcome l-asparaginase resistance.

CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPalpha myeloid oncoproteins induce bcl-2 via interaction of their basic regions with nuclear factor-kappaB p50.

The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases. We find that CEBPA and Bcl-2 RNA levels correlate highly in low-risk human AMLs, suggesting that inhibition of apoptosis via induction of bcl-2 by CCAAT/enhancer binding protein alpha (C/EBPalpha) or its mutant variants contributes to transformation. C/EBPalphap30, lacking a NH2-terminal transactivation domain, or C/EBPalphaLZ, carrying in-frame mutations in the leucine zipper that prevent DNA binding, induced bcl-2 in hematopoietic cell lines, and C/EBPalpha induced bcl-2 in normal murine myeloid progenitors and in the splenocytes of H2K-C/EBPalpha-Emu transgenic mice. C/EBPalpha protected Ba/F3 cells from apoptosis on interleukin-3 withdrawal but not if bcl-2 was knocked down. Remarkably, C/EBPalphaLZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA binding, and C/EBPalphap30 also activated the promoter. C/EBPalpha and the C/EBPalpha oncoproteins cooperated with nuclear factor-kappaB (NF-kappaB) p50, but not p65, to induce bcl-2 transcription. Endogenous C/EBPalpha preferentially coimmunoprecipitated with p50 versus p65 in myeloid cell extracts. Mutation of residues 297 to 302 in the C/EBPalpha basic region prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65. These findings suggest that C/EBPalpha or its mutant variants tether to a subset of NF-kappaB target genes, including Bcl-2, via p50 to facilitate gene activation and offer an explanation for preferential in-frame rather than out-of-frame mutation of the leucine zipper with sparing of the basic region in C/EBPalphaLZ oncoproteins. Targeting interaction between C/EBPalpha basic region and NF-kappaB p50 may contribute to the therapy of AML and other malignancies expressing C/EBPs.

Critical elements of the immunoglobulin heavy chain gene enhancers for deregulated expression of bcl-2.

Translocation of the bcl-2 gene to the immunoglobulin heavy chain gene is the most common alteration in follicular lymphoma. The result is the deregulated expression of bcl-2 and increased resistance to cell death. Regulation of the immunoglobulin heavy chain gene is controlled in part by four DNase I-hypersensitive regions located 3' of the gene. Here, we show that these four enhancer regions also contribute to bcl-2 up-regulation in t(14;18) cells. The enhancers are able to individually or in combination activate bcl-2 promoter activity. The HS4 enhancer region was found to impart the largest positive effect on the bcl-2 promoter, activating it by 6-fold, whereas addition of the HS1,2 region with HS4 increased promoter activity by approximately 9-fold. Nuclear factor kappaB binding sites were shown to be primarily responsible for the positive activity contributed by the HS1,2 and HS4 regions, and we observed the in vivo interaction of these factors with the human immunoglobulin heavy chain gene enhancer regions in t(14;18) cells. In addition, two Sp1 binding sites in HS4 were also found to positively influence bcl-2 activity, and Sp1 was observed to interact with the human HS4 enhancer in vivo. These results suggest that the interactions of the nuclear factor kappaB and Sp1 transcription factors with the immunoglobulin heavy chain enhancer region are important for bcl-2 deregulation in t(14;18) cells.

NF-kappaB activates Bcl-2 expression in t(14;18) lymphoma cells.

The t(14;18) translocation, which is characteristic of follicular lymphoma, results in the overexpression of the bcl-2 gene dependent upon regulatory elements within the bcl-2 5' flanking region and the immunoglobulin heavy chain gene enhancers. Conflicting evidence exists on the effects of NF-kappaB expression on Bcl-2 levels in different cell types. Lymphoma cells with the t(14;18) translocation show high levels of nuclear NF-kappaB proteins. We observed decreased levels of endogenous Bcl-2 when the IkappaBalpha-super-repressor was expressed in a t(14;18) cell line. Deletion analysis of the bcl-2 promoter indicated that the repressive effect of the IkappaBalpha-super-repressor occurred through a region that contained no NF-kappaB consensus sequences. This highly active region contained a c-AMP response element (CRE) and several Sp1 binding sites. Chromatin immunoprecipitation assays with antibodies specific for the NF-kappaB and CREB/ATF family members, as well as Sp1, resulted in the isolation of this IkappaBalpha-super-repressor responsive region of the bcl-2 promoter. Mutation of the CRE and the two Sp1 sites in different combinations in bcl-2 reporter constructs resulted in the loss of bcl-2 promoter repression by the IkappaBalpha-super-repressor. We therefore conclude that the activation of bcl-2 by NF-kappaB in t(14;18) lymphoma cells is mediated through the CRE and Sp1 binding sites.

Regulation of Bcl-2 expression by C/EBP in t(14;18) lymphoma cells.

In follicular lymphomas with the t(14;18) translocation, there is increased expression of the bcl-2 gene, which is dependent upon regulatory elements within the bcl-2 5' flanking region and the immunoglobulin heavy-chain gene enhancers. We found that t(14;18) lymphomas expressed C/EBPalpha, which is not normally expressed in B lymphocytes. Expression of C/EBPalpha increased bcl-2 expression, and two regions of the bcl-2 P2 promoter that mediated this effect were identified. C/EBPbeta was also able to increase bcl-2 promoter activity through these sites. The 5' site was GC-rich and did not contain a C/EBP consensus sequence; however, C/EBP was observed to interact with this site both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. The 3' region contained the Cdx site, which mediates the effect of A-Myb on the bcl-2 promoter. In vivo binding studies revealed that C/EBP interacted with this region of the bcl-2 promoter as well. Decreased expression of C/EBP factors due to targeting of their transcripts by siRNA molecules resulted in downregulation of Bcl-2 protein. We conclude that C/EBPalpha and C/EBPbeta contribute to the deregulated expression of Bcl-2 in t(14;18) lymphoma cells.

Artificial zinc(II) complexes regulate cell cycle and apoptosis-related genes in tumor cell lines.

Various proteins involved in transcriptional regulation possess highly selective DNA-binding domains, known as zinc fingers. However, little is known about small-molecule zinc(II) complexes in the regulation of gene expression and programmed cell death. A new family of zinc(II) complexes is reported, which might be useful against human cancer cells. By using template synthesis and in vitro cell-line screening, a set of zinc(II) complexes has been found to induce apoptosis of cancer cells and display single-reagent in vitro cytotoxicity. The method used to synthesize the molecules resulted in "built-in" luminescent behavior. Confocal optical imaging clearly demonstrated penetration through the cell membrane by these metal complexes. We have discovered that C3, the meso-zinc(II) complex is an extremely efficient regulator of the cell cycle and anti-apoptosis genes bcl-2 and bcl-xL. This study provides a new insight into the development of zinc(II) complexes as potential drugs.

Regulatory elements in the immunoglobulin heavy chain gene 3'-enhancers induce c-myc deregulation and lymphomagenesis in murine B cells.

Burkitt's lymphoma is invariably associated with chromosomal translocations that juxtapose the c-myc proto-oncogene with regulatory elements of the immunoglobulin heavy (IgH) or light chain loci resulting in the deregulation of c-myc expression. However, the enhancer elements mediating c-myc deregulation in vivo remain largely unidentified. To investigate the role of the IgH 3'-enhancers in c-myc deregulation, we used gene targeting to generate knock-in mice in which four DNase I hypersensitive regions from the murine IgH 3'-region were integrated into the 5'-region of the c-myc locus. The IgH 3'-enhancers induced the up-regulation of c-myc expression specifically in B cells of IgH-3'-E-myc mice. After approximately 10 months, the mice developed a Burkitt-like B cell lymphoma with the phenotype of B220+, IgM+, and IgD(low). Analysis of immunoglobulin gene rearrangements indicated that the lymphoma cells were of clonal origin. The presence of a rapidly expanding population of B cells in the spleen and bone marrow of young knock-in mice at 2-4 months of age was observed. Premalignant splenic B cells of knock-in mice showed higher spontaneous and induced apoptosis; however, malignant B cells were more resistant to apoptosis. The p53-ARF-Mdm2 pathway was disabled in half of the lymphomas examined, in most cases through Mdm2 overexpression. Although c-myc expression was increased in premalignant B cells, the promoter shift from P2 to P1 was observed only in malignant B cells. Our studies demonstrate that the IgH 3'-enhancers play an important role in c-myc deregulation and B cell lymphomagenesis in vivo.

Allele-specific analysis of transcription factors binding to promoter regions.

In vivo footprinting techniques are useful for the identification of regulatory elements mediating transcriptional control of a gene. However, regulation of a gene can differ between the two alleles, and further steps must be taken to distinguish between the regulatory elements occupied on one allele and those used on the second allele. Many hematologic malignancies result from chromosomal translocations, which, in some cases, relocate a gene to a transcriptionally active region leading to the deregulated expression of that gene. This situation provides an example of differential expression between two alleles. In studying the t(14; 18) and t(8; 14) translocations, which involve the bcl-2 and c-myc proto-oncogenes, respectively, we have been able to identify regulatory elements important in mediating the activation of the translocated alleles and the silencing of the normal alleles. Following in vivo methylation and isolation of genomic DNA, we were able to separate the translocated and normal alleles by electrophoresis. Using the ligation-mediated polymerase chain reaction (LMPCR) technique, we could then assess protein interactions on the two different alleles. A detailed description of this methodology with examples from our studies are provided with a discussion of how these techniques may be applied to the study of other genes.

A Myc-associated zinc finger protein-related factor binding site is required for the deregulation of c-myc expression by the immunoglobulin heavy chain gene enhancers in Burkitt's lymphoma.

The deregulation of expression of the c-myc gene in Burkitt's lymphoma results from the translocation that links one c-myc allele to one of the immunoglobulin genes. This physical linkage promotes interactions between c-myc and immunoglobulin gene regulatory elements that affect c-myc transcription initiation and elongation. We have located a region in the c-myc promoter that is required for the complete activation by the immunoglobulin heavy chain gene enhancer. This regulatory element contains a core sequence, GGGAGG, similar to the GA box recognized by the transcription factor Myc-associated zinc finger protein (MAZ). UV cross-link analysis indicated that the mass of this protein did not correspond to that of MAZ, suggesting that a protein related to but distinct from MAZ bound to this site. Mutation of this regulatory element resulted in a loss of promoter activity induced by the immunoglobulin heavy chain gene enhancer. This site was also required for the c-myc promoter shift from P2 to P1. In vivo footprinting revealed that this site was occupied on the translocated c-myc allele but not on the untranslocated allele. Taken together, these findings suggest that this regulatory element is required for the full activation of c-myc promoter activity by the immunoglobulin heavy chain gene enhancer.

Impaired proliferation and survival of activated B cells in transgenic mice that express a dominant-negative cAMP-response element-binding protein transcription factor in B cells.

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on serine 133 and mediates the proliferative response to a number of different signals. A mutant CREB with a serine to alanine substitution at position 133 (CREBM1) functions as a dominant-negative inhibitor. Transgenic mice that express the dominant-negative CREB protein in B lymphocytes were developed as a means to study the effects of the inhibition of CREB function on B-cell proliferation and survival. We have shown previously that CREB up-regulates Bcl-2 expression in B cells in response to activation signals. B cells from CREBM1 transgenic mice expressed lower levels of Bcl-2 with and without stimulation. Proliferation of B cells from the transgenic mice was impaired in part by lack of induction of activator protein 1 (AP1) transcription factors. B cells from the transgenic mice were more susceptible to induction of apoptosis with several different agents, consistent with the decreased expression of Bcl-2. These studies demonstrate that B-cell activation requires phosphorylation of CREB for the proliferative response and to protect against activation-induced apoptosis.

Neurotrophin-3 and a CREB-mediated signaling pathway regulate Bcl-2 expression in oligodendrocyte progenitor cells.

Our previous results suggested that the transcription factor CREB mediates the actions of neuroligands and growth factor signals that coupled to different signaling pathways may play different roles along oligodendrocyte (OLG) development. We showed before that CREB phosphorylation in OLG progenitors is up-regulated by neurotrophin-3 (NT-3); and moreover CREB is required for NT-3 to stimulate the proliferation of these cells. We now show that treatment of OLG progenitors with NT-3 is also accompanied by an increase in the levels of the anti-apoptotic protein Bcl-2. Interestingly, the presence of a putative CREB binding site (CRE) in the Bcl-2 gene raised the possibility that CREB could also be involved in regulating Bcl-2 expression in the OLGs. Supporting this hypothesis, the NT-3 dependent increase in Bcl-2 levels is abolished by inhibition of CREB expression. In addition, transient transfection experiments using various regions of the Bcl-2 promoter and mutation of the CRE site indicate a direct role of CREB in regulating Bcl-2 gene activity in response to NT-3. Furthermore, protein-DNA binding assays show that the CREB protein from freshly isolated OLGs indeed binds to the Bcl-2 promoter CRE. Together with our previous results, these observations suggest that CREB may play an important role in linking proliferation and survival pathways in the OLG progenitors.

Gene expression patterns associated with recurrent chromosomal translocations in acute lymphoblastic leukemia.

We obtained a global view of gene expression in both cell lines and pediatric acute lymphoblastic leukemia (ALL) samples that harbor one of several selected chromosomal abnormalities. When the cell lines were studied alone, we found that these chromosomal abnormalities were associated with the predominant variation in transcriptional programs across the set of cell lines studied. When cell lines and clinical samples were studied together, we found that each chromosomal abnormality (TEL/AML1, BCR/ABL, or MLL abnormalities) was associated with a characteristic gene expression signature that was shared by both cell lines and clinical samples. However, BCR/ABL was associated with a much more heterogeneous pattern of expression than were TEL/AML1 and MLL abnormalities. This observation has important implications for the study of BCR/ABL ALL. In addition, we systematically identified genes whose expression was associated with TEL/AML1, BCR/ABL, or MLL abnormalities in both clinical samples and cell lines. Although some of these genes have previously been described, many have not previously been reported to be associated with one of these chromosomal abnormalities. Notably, we found that the erythropoietin receptor (EPOR) is consistently highly expressed in TEL/AML1 ALL compared with BCR/ABL or MLL.

Oxidative stress-mediated down-regulation of bcl-2 promoter in hippocampal neurons.

Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

Cytokine-mediated down-regulation of the transcription factor cAMP-response element-binding protein in pancreatic beta-cells.

Cytokines are known to induce apoptosis of pancreatic beta-cells. Impaired expression of the anti-apoptotic gene bcl-2 is one of the mechanisms involved. In this study, we identified a defect involving transcription factor cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic beta-cell line, MIN6 cells, to cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) led to a significant (p < 0.01) decrease in Bcl-2 protein and mRNA levels. Cytokines decreased (56%) the activity of the bcl-2 promoter that contains a cAMP-response element (CRE) site. Similar decreases were seen with a luciferase reporter gene driven by tandem repeats of CRE and a CREB-specific Gal4-luciferase reporter, suggesting a defect at the level of CREB. The active phospho form (serine 133) of CREB diminished significantly (p < 0.01) in cells exposed to cytokines. Examination of signaling pathways upstream of CREB revealed a reduction in the active form of Akt. Cytokine-induced decrease of bcl-2 promoter activity was partially restored when cells were cotransfected with a constitutively active form of Akt. Several end points of cytokine action including decreases in phospho-CREB, phospho-Akt, and BCl-2 levels and activation of caspase-9 were observed in isolated mouse islets. Overexpression of wild-type CREB in MIN6 cells by plasmid transfection and adenoviral infection led to protection against cytokine-induced apoptosis. Adenoviral transfer of dominant-negative forms of CREB, on the other hand, resulted in activation of caspase-9 and exaggeration of cytokine-induced beta-cell apoptosis. Together, these results point to CREB as a novel target for strategies aimed at improving the survival of beta-cells.