(1999) J. Biol. Chem. 274, 27529 -27535). In the present investigation, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti-apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit of PI 3-kinase, PDK1, and Akt. These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3-kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing Akt by real-time quantitative reverse transcriptionpolymerase chain reaction using the TaqMan TM fluorogenic probe system. We observed a 2.1-fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survival.The serine threonine kinase Akt/protein kinase B is an important mediator of metabolic as well as survival responses to insulin and growth factors (1). Akt is activated by translocation to plasma membrane when the PI 3-kinase-generated 3-phosphoinositides bind to its pleckstrin homology domain (2). For its full activation it needs to be further phosphorylated by 3-phosphoinositide-dependent kinase1 (PDK1) 1 at Thr-308 and by PDK2 at Ser-473. The metabolic actions of insulin mediated by Akt include stimulation of GLUT4 translocation and activation of glycogen synthase and the glycolytic enzyme 6-phosphofructose-

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.

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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/EBP␣ and decreased their binding as well as the binding of CBP and HDAC2 to the bcl-2 promoters. Mutation of Sp1 and C/EBP␣ 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.The cytogenetic hallmark of most follicular B-cell lymphomas is the chromosomal translocation of the antiapoptotic bcl-2 gene from 18q21 to the immunoglobulin heavy chain (IgH) locus at 14q32 (9, 54, 55). This t(14;18)(q32;q21) translocation constitutes the most common chromosomal translocation in human lymphoid malignancies. Approximately 85% of follicular and 20% of diffuse B-cell lymphomas possess this translocation. The t(14;18) translocation places bcl-2 in the same transcriptional orientation as IgH and results in deregulated overexpression of bcl-2 (15). Increased cell survival due to bcl-2 overexpression has been shown to contribute to the development of many B-cell lymphomas and confer resistance to a variety of anticancer therapies (12,26,43,50).Two promoters mediate transcriptional control of the bcl-2 gene (52). The 5Ј promoter (P1) is located 1,386 to 1,423 bp upstream of the bcl-2 translational start site, and it is GC-rich with multiple Sp1 sites. The start sites of the 3Ј promoter (P2) are located 1.3 kb downstream of the P1 promoter. P2 has a classic TATA and CAAT box and a simian virus 40 (SV40) decamer/Ig octamer motif. Important cis elements and associated trans-acting factors participating in the deregulation of bcl-2 have been characterized within the promoter regions. A major positive regulator of P1 activity is a cyclic AMP (cAMP) response element (CRE). CREB (CRE-binding protein) binds to this site and is essential for bcl-2 expression during B-cell development and for bcl-2 deregulation in t(14;18) lymphomas (27, 58). In addition, NF-B activates bcl-2 in t(14;18) lymphoma cells through interactions with the CRE and Sp1 binding sit...

Insulin-like growth factor-I (IGF-I

The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the eect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wildtype p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were de®ned. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas. Oncogene (2001) 20, 240 ± 251.

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. Con¯icting evidence exists on the e ects of NF-kB expression on Bcl-2 levels in di erent cell types. Lymphoma cells with the t(14;18) translocation show high levels of nuclear NF-kB proteins. We observed decreased levels of endogenous Bcl-2 when the IkBa-super-repressor was expressed in a t(14;18) cell line. Deletion analysis of the bcl-2 promoter indicated that the repressive e ect of the IkBa-super-repressor occurred through a region that contained no NF-kB consensus sequences. This highly active region contained a c-AMP response element (CRE) and several Sp1 binding sites. Chromatin immunoprecipitation assays with antibodies speci®c for the NF-kB and CREB/ATF family members, as well as Sp1, resulted in the isolation of this IkBa-super-repressor responsive region of the bcl-2 promoter. Mutation of the CRE and the two Sp1 sites in di erent combinations in bcl-2 reporter constructs resulted in the loss of bcl-2 promoter repression by the IkBa-super-repressor. We therefore conclude that the activation of bcl-2 by NF-kB in t(14;18) lymphoma cells is mediated through the CRE and Sp1 binding sites.