Bcl-2 Promoter Research Articles

Association of the CC genotype of the regulatory BCL2 promoter polymorphism (−938C>A) with better 2-year survival in patients with glioblastoma multiforme

Bcl-2 plays a key role in the downregulation of apoptosis and proliferation and leads to increased chemoresistance in glioblastoma multiforme (GBM). The authors investigated the role of a common regulatory single-nucleotide polymorphism (-938C>A), which is located in the inhibitory P2 promoter of BCL2. Data from 160 patients suffering from GBM were retrospectively evaluated. Study inclusion criteria consisted of available DNA and, in patients still alive, a follow-up of at least 24 months. Results were analyzed with respect to the basic clinical data, type of surgical intervention (gross-total resection [GTR] versus stereotactic biopsy [SB]), adjuvant therapy, MGMT promoter methylation, and survival at the 2-year follow-up. At the 2-year follow-up, 127 (79.4%) of the 160 patients had died. Kaplan-Meier curves revealed a significantly higher rate of survival for homo- and heterozygous C-allele carriers (p = 0.031). In the GTR group, the survival rate was 47.1% for homozygous C-allele carriers, 32.0% for heterozygous C-allele carriers, and only 21.4% for homozygous A-allele carriers (p = 0.024). The SB group showed no genotype-dependent differences. Multivariable Cox regression revealed that the BCL2 (-938AA) genotype was an independent negative prognostic factor for 2-year survival in the GTR group according to the BCL2 (-938CC) genotype reference group (hazard ratio 2.50, 95% CI 1.14-5.48, p = 0.022). These results suggested that the (-938C>A) polymorphism is a survival prognosticator as well as a marker for a high-risk group among patients with GBM who underwent GTR.

TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex

TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca(2+)-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.

The distinct role of guanine nucleotide exchange factor Vav1 in Bcl-2 transcription and apoptosis inhibition in Jurkat leukemia T cells

To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells. Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (ΔΨ(m)) was measured using DiOC(6)(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays. Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (ΔΨ(m)) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression. Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity.

Impact of MTHFR gene C677T polymorphism on Bcl-2 gene methylation and protein expression in colorectal cancer

Objective. To investigate the impact of MTHFR C677T polymorphism on Bcl-2 gene promoter CpG island (CGI) methylation and Bcl-2 protein expression. Material and methods. MTHFR polymorphisms of 86 sporadic colorectal cancer (CRC) patients and 100 healthy volunteers were analyzed by PCR-based restriction fragment length polymorphism, and Bcl-2 promoter CGI methylation in 86 CRC tissues and 86 paired nonneoplastic adjacent tissues was determined by methylation-specific PCR. Bcl-2 oncoprotein expression in 70 CRC tissues and paired nonneoplastic adjacent tissues was detected by immunohistochemistry. Results. The frequency of MTHFR 677 T allele and combined variant genotypes (677CT + TT) in CRC patients was significantly higher than that in healthy controls (p = 0.023 and p = 0.035, respectively), and there is a significant association between 677TT or 677(CT + TT) genotypes and CRC (OR = 2.534, p = 0.045 and OR = 1.888, p = 0.035, respectively). The frequency of methylated Bcl-2 promoter CGI in tumor tissues was significantly lower than that in nonneoplastic adjacent tissues (p = 0.014). The frequency of methylated Bcl-2 promoter CGI in CRC tissues of the individuals with CC genotype was significantly higher than that of those with CT/TT genotypes (p = 0.018), there was significant distribution difference of C and T alleles between individuals with methylated and unmethylated Bcl-2 promoter CGI in colorectal cancer tissues (p = 0.023). Bcl-2 promoter hypomethylation was significantly correlated with Bcl-2 oncoprotein expression in colorectal cancer tissues (r = 0.558, p < 0.001). Conclusion. Bcl-2 promoter is hypomethylated in colorectal cancer tissue, and there is a significant correlation between MTHFR 677 TT or CT/TT genotypes and CRC or Bcl-2 promoter CGI methylation/oncoprotein expression in CRC.

C/EBPα, C/EBPα Oncoproteins, or C/EBPβ Preferentially Bind NF-κB p50 Compared with p65 Via Conserved Residues In the C/EBP Basic Region

AbstractAbstract 708Nuclear Factor-κB (NF-κB) is a key mediator of the inflammatory response and also inhibits apoptosis. Activation of NF-κB is prevalent in AML, Hodgkin's lymphoma and subsets of non-Hodgkin's lymphoma, as well as in multiple solid tumors. NF-κB p50 (NF-κB1) and p65 (Rel A) are the most prevalent NF-κB subunits. NF-κB p65 has 30–50 fold higher affinity than p50 for the cytoplasmic protein IκB, and so under basal conditions p50:p65 heterodimers are held in the cytoplasm whereas p50:p50 homodimers are found in the nucleus bound to chromatin where they repress NF-κB target genes via association with HDACs. Canonical NF-κB activation signals lead to IκB proteosome-mediated degradation and translocation of p50:p65 heterodimers into the nucleus to replace inhibitory p50:p50 dimers with activating complexes on chromatin. We postulate an alternative means to activate NF-κB target genes wherein induction or activation of C/EBP proteins allows them to bind chromatin-bound p50 and displace HDACs in cells lacking canonical NF-κB activation. We found that C/EBPα induces bcl-2 and FLIP RNA expression via promoter interaction dependent upon the presence of NF-κB p50, that C/EBPα present in two myeloid leukemia cell lines preferentially binds p50 compared with p65, and that mutation of the C/EBPα basic region prevents bcl-2 induction and weakens interaction with p50 but not p65 (Paz-Priel et al 2005; 2009; Wang et al 2009). Using p50 and p65 variants containing the FLAG epitope at either their N- or C-termini we now demonstrate using reciprocal co-immunoprecipitation that p50 has much higher affinity than p65 for C/EBPα, for N-terminal or leucine zipper mutants of C/EBPα expressed in a subset of AML cases, and for the longer LAP or shorter LIP isoforms of C/EBPβ. The FLAG-tagged p65 proteins retained the ability to bind DNA in a gel shift assay and to activate a reporter containing two κB sites as effectively as untagged p65. Deletion of the p65 trans-activation domain (TAD) did not increase p65 affinity for C/EBPα, indicating that an inhibitory effect of the TAD does not account for specificity of C/EBPα:p50 interaction. Moreover, the increased affinity of the p50 rel homolog domain (RHD) for C/EBPα compared with the p65 RHD suggests that residues that differ between these RHDs rather than those in common make key contacts wih the C/EBP basic region. Germline deletion of the gene encoding p65, by injecting p65(flox/flox); Mx1-CRE mice with pIpC, did not reduce C/EBPα expression, prevent interaction of C/EBPα with the bcl-2 promoter in a ChIP assay, or reduce bcl-2 RNA expression, in contrast to our prior findings with p50 gene deletion. A C/EBPα variant harboring a single point mutation in the basic region, C/EBPα(R300A), has minimal affinity for p50, and further saturating mutagenesis of the C/EBPα basic region identifies an additional block of six amino acids, RERNNI, identical in C/EBPβ and critical for interaction with NF-κB p50. In conclusion, increased affinity of C/EBP proteins for p50 compared with p65 lends support to the hypothesis that C/EBP interaction with p50:p50 complexes provides an alternative means for normal cells or cancer cells to activate NF-κB target genes, for example in response to C/EBP induction or activation. C/EBP interaction with p50:p65 in activated cells may also occur, via p50, to further activate genes regulated by NF-κB. In addition, these findings suggest that targeting the C/EBP:p50 interface rather than C/EBP:p65 interaction will prove more effective in the therapy of inflammatory or malignant conditions, and our mapping of relevant basic region residues and the finding that the p50 RHD retains increased affinity for C/EBPα compared wih the p65 RHD provides a first step towards rational design of such inhibitors. Disclosures:No relevant conflicts of interest to declare.

A novel SATB1 binding site in the BCL2 promoter region possesses transcriptional regulatory function

Abstract BCL2 is a key regulator of apoptosis. Our previous work has demonstrated that special AT-rich sequence-binding protein 1 (SATB1) is positively correlated with BCL2 expression. In the present study, we report a new SATB1 binding site located between P1 and P2 promoters of the BCL2 gene. The candidate SATB1 binding sequence predicted by bioinformatic analysis was investigated in vitro and in vivo by electrophoretic gel mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). One 25-bp sequence, named SB1, was confirmed to be SATB1 binding site. The regulatory function of SB1 and its relevance to SATB1 were further examed with dual-luciferase reporter assay system in Jurkat cells. We found that SB1 could negatively regulate reporter gene activity. Mutation of SATB1 binding site further repressed the activity. Knockdown of SATB1 also enhanced this negative effect of SB1. Our data indicate that the SB1 sequence possesses negative transcriptional regulatory function and this function can be antagonized by SATB1.

Regulation of ERα-mediated transcription of Bcl-2 by PI3K-AKT crosstalk: Implications for breast cancer cell survival

Both estrogen, through the estrogen receptor (ER), and growth factors, through the phosphatidylinositol-3-kinase (PI3K)-AKT pathway, have been shown to independently promote cell survival. Here, we investigated the role of ER/PI3K-AKT crosstalk in the regulation of cell survival in MCF-7 breast carcinoma cells. The ER inhibitor ICI 182,780 was used to determine the requirement of the ER for estrogen in the suppression of tumor necrosis factor-alpha (TNFalpha) induced apoptosis. Gene reporter assays and Western blot analyses were used to determine the involvement of the pro-survival factor Bcl-2 and the coactivator GRIP1 in this survival crosstalk. We demonstrated that an intact ER signaling pathway was required for estrogen to suppress apoptosis induced by TNFalpha. Our gene reporter assays revealed that ERalpha, not ERbeta, was targeted by AKT, resulting in transcriptional potentiation of the full-length Bcl-2 promoter, ultimately leading to increased Bcl-2 protein levels. AKT targeted both activation function (AF) domains of the ERalpha for maximal induction of Bcl-2 reporter activity, although the AF-II domain was predominately targeted. In addition, AKT also caused an upregulation of GRIP1 protein levels. Finally, AKT and GRIP1 cooperated to increase Bcl-2 protein expression to a greater level than either factor alone. Collectively, our study suggests a role for ER/PI3K-AKT crosstalk in cell survival and documents the ability of AKT to regulate Bcl-2 expression via differential activation of ERalpha and ERbeta as well as regulation of GRIP1.

High-Mobility Group A1 Proteins Regulate p53-Mediated Transcription of Bcl-2 Gene

We have previously described a mechanism through which the high-mobility group A1 (HMGA1) proteins inhibit p53-mediated apoptosis by delocalizing the p53 proapoptotic activator homeodomain-interacting protein kinase 2 from the nucleus to the cytoplasm. By this mechanism, HMGA1 modulates the transcription of p53 target genes such as Mdm2, p21(waf1), and Bax, inhibiting apoptosis. Here, we report that HMGA1 antagonizes the p53-mediated transcriptional repression of another apoptosis-related gene, Bcl-2, suggesting a novel mechanism by which HMGA1 counteracts apoptosis. Moreover, HMGA1 overexpression promotes the reduction of Brn-3a binding to the Bcl-2 promoter, thereby blocking the Brn-3a corepressor function on Bcl-2 expression following p53 activation. Consistently, a significant direct correlation between HMGA1 and Bcl-2 overexpression has been observed in human breast carcinomas harboring wild-type p53. Therefore, this study suggests a novel mechanism, based on Bcl-2 induction, by which HMGA1 overexpression contributes to the escape from apoptosis leading to neoplastic transformation.

Low Frequency of Epigenetic Events in Urothelial Tumors in Young Patients

Bladder urothelial cell carcinoma is uncommon in young patients. We recently reported a series of tumors in patients younger than 20 years at diagnosis and performed exhaustive genetic screening for molecular alterations. Few events typical of bladder urothelial cell carcinoma were detected. Since many carcinogenic events occur at the epigenetic rather than the genetic level, we analyzed the same tumors for alterations in DNA hypermethylation. We compared our findings with those in tumors in older patients with similar pathological profiles. We analyzed 76 bladder urothelial cell carcinomas from 3 groups stratified by age at diagnosis, including less than 19, 20 to 45 and greater than 46 years (median 78), and matched for low grade and nonmuscle invasive stage. We used quantified methyl specific polymerase chain reaction to investigate promoter methylation for 8 tumor suppressor genes implicated in urothelial carcinogenesis. Tumors in the youngest age group had the lowest incidence of global hypermethylation compared to the other tumors with a methyl index of 37.5% vs 62.5% and 50%, respectively (ANOVA p = 0.009). When individual loci were analyzed, younger patients had a significantly lower rate and concentration of methylation at APC, Bcl2, MGMT and E-cadherin promoters than in the older groups (p <0.05). Few differences were present between the 2 older cohorts but the APC and MGMT methylation concentration increased with age. Urothelial tumors in patients younger than 19 years have a low rate of epigenetic alteration. Tumors in patients older than 20 years have epigenetic profiles similar to those of tumors in patients within the typical bladder urothelial cell carcinoma age range.

BCL6promoter interacts with far upstream sequences with greatly enhanced activating histone modifications in germinal center B cells

BCL6 encodes a transcriptional repressor that is essential for the germinal center (GC) reaction and important in lymphomagenesis. Although its promoter has been well studied, little is known concerning its possible regulation by more distal elements. To gain such information, we mapped critical histone modifications associated with active transcription within BCL6 as well as far upstream sequences at nucleosomal resolution in B-cell lines and in normal naive and GC B cells. Promoter-associated and intronic CpG islands (CGIs) in BCL6 showed a reciprocal pattern of histone modifications. Gene expression correlated with a paradoxical loss from the intronic CGI of histone H3 lysine-4 trimethylation, normally associated with transcription, suggesting that the intronic CGI may interfere with transcription. In an approximately 110-kb region extending 150-260 kb upstream of BCL6, highly active histone modifications were present only in normal GC B cells and a GC B-cell line; this region overlaps with an alternative breakpoint region for chromosomal translocations and contains a GC-specific noncoding RNA gene. By chromosome conformation capture, we determined that the BCL6 promoter interacts with this distant upstream region. It is likely that transcriptional enhancers in this region activate BCL6 and overcome strong autorepression in GC B cells.

Studies on the Site and Mode of TMPyP4 Interactions with Bcl-2 Promoter Sequence G-Quadruplexes

TMPyP4 (Mesotetra(N-methyl-4-pyridyl)porphine) is known to have a high affinity for G-quadruplex DNA. However, there is still some controversy over the exact site(s) and mode(s) of TMPyP4 binding to G-quadruplex DNA. We examined TMPyP4 interactions with seven G-quadruplex forming oligonucleotides. The parent oligonucleotide is a 27-mer with a wild-type (WT) G-rich sequence of the Bcl-2 P1 promoter mid-region (5′-d(CGG GCG CGG GAG GAA GGG GGC GGG AGC-3′)). This sequence folds into at least three unique loop isomer quadruplexes. The two mutant oligonucleotides used in this study are shorter (23-mer) sequences in which nonquadruplex core bases were eliminated and two different (-G-G-) → (-T-T-) substitutions were made to restrict the folding complexity. The four additional mutant oligonucleotides were labeled by substituting a 2-aminopurine (2-AP) base for an A or G in either the first three-base lateral loop or the second five- or seven-base lateral loop (depending on the G→T mutation positions). Spectroscopic and microcalorimetric studies indicate that four molecules of TMPyP4 can be bound to a single G-quadruplex. Binding of the first two moles of TMPyP4 appears to occur by an end or exterior mode (K ≈ 1 × 107 M−1), whereas binding of the third and fourth moles of TMPyP4 appears to occur by a weaker, intercalative binding mode (K ≈ 1 × 105 M−1). As the mid-loop size decreases from seven to five bases, end binding occurs with significantly increased affinity. 2-AP-labeled Bcl-2 promoter region quadruplexes show increased fluorescence of the 2-AP base on addition of TMPyP4. The change in fluorescence for 2-AP bases in the second half of the TMPyP4 titration lends support to our previous speculation regarding the intercalative nature of the weaker binding mode.

Turning off Transcription of the bcl-2 Gene by Stabilizing the bcl-2 Promoter Quadruplex with Quindoline Derivatives

Human bcl-2 gene is an apoptosis-related oncogene containing a GC-rich sequence which is located upstream from P1 promoter and has the potential to form G-quadruplex structures. However, the regulatory role of the quadruplex and the effect of its ligands on bcl-2 have not been clarified. Here, we demonstrated that the G-quadruplex structure was disrupted when partial mutation of G --> A was made, resulting in a 2-fold increase in basal transcriptional activity of bcl-2 promoter. Quindoline derivatives, the highly active G-quadruplex ligands developed by our group, could significantly suppress bcl-2 transcriptional activation but had less effect on mutated bcl-2 transcription. These results provided direct evidence that G-quadruplex structure formed in bcl-2 promoter region could function as a transcriptional repressor element, and G-quadruplex specific ligands could regulate the transcription of bcl-2 through stabilization of quadruplex structure. The results further indicated that quindoline derivatives could induce apoptosis of HL-60 tumor cells.

Abstract 3898: Octamer Binding Protein-2 (Oct-2): A non-oncogene addiction in germinal center derived lymphomas and a promising therapeutic target

Abstract To identify genes required for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B Cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic shRNAs in this screen targeted Oct2, encoding a POU domain transcriptional activator, primarily lymphoid restricted. It was identified due its ability to bind the DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific coactivator OCA-B interacts with Oct-2 enhancing its transactivation potential. Although Oct2 and OCA-B are dipensable for Ig transcription, they are essential for germinal center (GC) development. To understand the apoptotic cell death of DLBCL cells following shRNA Oct2 induction we investigated the genetic pathways controlled by Oct2. We profiled gene expression changes in DLBCL cells upon Oct2 knocked down and merged this data with genome-wide assessment of Oct2 and OCA-B binding sites, coupling chromatin immunoprecipitation (ChiP) with high-throughput sequencing (ChIPSeq). More than 60% of the Oct2 target genes also showed OCA-B biding. The Oct2/OCA-B overlapping set of targets was enriched for genes selectively expressed in GC B cells. We found that Oct2/OCA-B lie upstream many transcription factors known to play critical roles in GC development such as BCL6, MTA3, PU.1, IRF8 and SpiB. Oct2 regulates these genes in DLBCL cells and in centroblasts. Among Oct-2 downstream effectors, BCL6 could rescue DLBCL cells from the Oct2 shRNA lethal effect. The Oct2/OCA-B binding of BCL6 promoter was confirmed by single locus ChIP in primary GC B-cells and derived lymphomas. Gel shifts experiments showed that Oct2 binds a non canonical octamer site at the BCL6 transcription start site. Our findings uncovered a novel aspect of the Oct2 biology believed to regulate the activity of octamer containing promoters. Interestingly, Oct2 controls the expression of GC specific genes that do not harbor a canonical octamer motif. By array CGH, amplification of Oct2 and OCA-B was found in less than 1% of non Hodgkin lymphoma (NHL) patient samples. Nonetheless, lymphoma cells become dependent on the Oct2 controlled network to survive, being an example of non oncogene addiction. This turns Oct2 into an attractive therapeutic target for NHL treatment. Oct2 and OCA-B lie upstream of BCL6, widely considered a master regulator of the GC response, suggesting that Oct2 directed therapy should kill the same DLBCLs as BCL6 Peptide Inhibitor. Furthermore, all GC and Post-GC B cells tested require Oct2 for survival, indicating that Oct2 directed therapy might have a broder activity spectrum than the BCL6 directed therapy. The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity since this interaction is exclusively required in GC B cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3898.

Abstract LB-46: Chemo-sensitization by modulation of Bcl-2 expression: The first example of an i-motif interactive small molecule that modulates gene expression

Abstract For the first time we provide evidence that not only illustrates the i-motif DNA secondary structure has potential to serve as a molecular target, but that within the Bcl-2 promoter this targeting can result in the modulation of gene expression and increase in chemo-sensitivity. The Bcl-2 oncoprotein is a key regulator and inhibitor of the intrinsic apoptotic pathway. Evasion of apoptosis is a well-known hallmark of cancer and is also recognized to play an important role in chemo-resistance in a variety of cancers particularly of lymphocytic origin. The significant over-expression of Bcl-2 common in low-grade lymphomas and lymphocytic leukemias has been associated with prolonged cell survival despite apoptotic stimuli and therefore is an attractive target for chemo-sensitization. Transcriptional regulation of Bcl-2 involves two promoter sites, P1 and P2, with P1 exerting the majority of control. Directly upstream of the P1 promoter is a GC-rich element capable of transitioning to DNA secondary structures. Previously we have shown the G-rich promoter element forms a mixed parallel/antiparallel G-quadruplex that has potential to adopt three different G-quadruplex structures. In recently published studies we have demonstrated that the complementary C-rich sequence has potential to form a stable i-motif structure. We now show that Bcl-2 expression can be modulated at the transcriptional level through small molecule targeting and destabilization of the i-motif. A high-throughput fluorescence resonance energy transfer (FRET) screening assay yielded a steroid derivative (compound 59) from the NCI diversity set that increased the i-motif FRET probe fluorescence by 2.5 fold indicative of substantial destabilization of the i-motif structure. The destabilization of the Bcl-2 i-motif was then confirmed with CD spectral and thermal stability analyses. As proof of principle we demonstrated that following treatment with compound 59, Bcl-2 gene expression is significantly decreased as detected by Real-Time RT-PCR in MCF-7, MCF-7 tamoxifen resistant breast cancer cell lines and the GRANTA-519 mantle cell lymphoma cell line. In addition, the GRANTA-519 cell line displays resistance to etoposide-induced apoptosis; however, in the presence of compound 59 the cells exhibited an increase in capase activity in comparison to cells that were treated with etoposide alone. These studies have been expanded to include a chemo-sensitization lymphoma cell line model in which two cell lines differ in Bcl-2 expression, low versus high, and sensitivity to etoposide-induced apoptosis, sensitive versus resistant, respectively. We anticipate from preliminary data that in response to compound 59 treatment the etoposide-resistant cell line that over-expresses Bcl-2 will have an increased apoptotic response similar to its etoposide sensitive counter-part. A Bcl-2 i-motif non-interactive compound is used as a negative control. Small molecules that target the Bcl-2 i-motif and result in the down-regulation of Bcl-2 expression have the potential to act as chemo-sensitizing agents to improve current chemotherapy in refractory patients by an alternative mechanism to ABT-737, which is in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-46.

Abstract LB-234: Bcl-2, a novel target of Bmi-1 (the stem cell associated factor), induced Wnt signaling: Implications on prostate cancer

Abstract In the current report we provide a novel finding about the regulation of Bcl-2 expression in prostate cancer (CaP) cells. Bcl-2 is known to be regulated by Sonic Hedgehog (SHH) signaling. Here, we provide evidence that human CaP cells exhibit SHH-independent Bcl-2 activation which is regulated by Bmi-1-induced Wnt signaling. Bmi-1 is a member of polycomb group gene family and is reported to be involved in stemness and self-renewal capability of cancer stem cells. We investigated the role of Bmi-1 in human CaP development and delineate its mechanism of action. On the basis of immunohistochemical analysis of prostatic specimens of 125 human CaP patients, we show that Bmi-1 protein levels are highly elevated in patients with advanced disease. To understand the mechanism of action of Bmi-1, we employed two-prong strategy. Firstly, Bmi-1 was knocked down in CaP cells (LNCaP, DU145 and PC-3) by employing siRNA technique. Bmi-1-silenced CaP cells exhibited decreased proliferative and clonogenic potential. Secondly, Bmi-1 was over-expressed in CaP cells by transfecting Bmi-1 overexpressing plasmid (pbabe-Bmi-1) in CaP cells. Bmi-1-overexpressing cells exhibited increased clonogenicity and rate of proliferation. We performed a micro-array analysis of Bmi-1-silenced cells and found that silencing of Bmi-1 caused a decrease in the Cyclin D1 (Wnt signaling target) and Bcl-2 (SHH signaling target), however an increase in p16 was observed. These studies were validated by performing immunoblot analysis. We generated a hypothesis that the Bmi-1 regulates the expression of Cyclin D1 and Bcl-2 by interacting with Wnt /SHH signaling in CaP cells. We show that despite blocking SHH signaling (by treating CaP cells with cyclopamine, a SHH signaling inhibitor), Bmi-1 induces the Bcl-2 expression in CaP cells suggesting the involvement of SHH-independent pathway in the regulation of Bcl-2 activation. We identified Wnt signaling as the pathway that regulates Bcl-2 independent of SHH signaling in CaP cells. We provide evidence that Bcl-2 promoter region of Bcl-2 gene has binding sites for Tcf transcriptional factor (Wnt signaling component). We found that Bmi-1 induces Wnt signaling in CaP cells. Taken together, these studies provide deep-insight into the mechanism of CaP cell proliferation (even after chemotherapy such as cyclopamine treatment). We suggest that Bmi-1 could be developed as a novel molecular and therapeutic target for human CaP treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-234.

Simvastatin stimulates production of the antiapoptotic protein Bcl-2 via endothelin-1 and NFATc3 in SH-SY5Y cells.

The use of statins for the prevention or treatment of different neurodegenerative diseases has generated considerable interest albeit with some controversy. Mechanisms of statin-induced neuroprotection are not well understood. Recently, we reported that simvastatin stimulated neuronal gene expression and protein levels of the major antiapoptotic protein Bcl-2 in vivo and in vitro; suppression of Bcl-2 in SH-SY5Y cells reduced simvastatin neuroprotection; effects were independent of cholesterol and other products of the 3-hydroxy-3-methylglutaryl-CoA reductase pathway. Endothelin-1 (ET-1) can increase Bcl-2 abundance via the transcription factor nuclear factor of activated thymocytes (NFATc), and simvastatin was reported to increase ET-1 gene expression. We tested the hypothesis that simvastatin stimulation of Bcl-2 involves up-regulation of ET-1 and binding of NFATc to Bcl-2 promoter sites in SH-SY5Y human neuroblastoma cells. Simvastatin increased both intracellular and secreted ET-1 protein levels. Exogenous ET-1 increased Bcl-2 protein abundance, which was inhibited by ET-1 receptor antagonists. Simvastatin increased translocation of NFATc3 to the nucleus while reducing nuclear NFATc1 and having no effect on NFATc4. Endothelin-1 also increased NFATc3 levels in the nucleus, and this increase was inhibited by ET-1 receptor antagonists. Treatment of cells with simvastatin stimulated binding of NFATc3 to the Bcl-2 promoter. We report novel findings showing that up-regulation of Bcl-2 by simvastatin involves ET-1 and the transcription factor NFATc3. Discovering how statins can selectively alter a specific NFATc isoform that leads to an increase in an antiapoptotic protein will provide a new approach to understanding statin-induced neuroprotection and conditions outside the brain in which apoptosis contributes to pathophysiology.

A regulatory BCL2 promoter polymorphism (-938C&amp;gt;A) is associated with outcome in neuroblastoma

A regulatory BCL2 promoter polymorphism (-938C&amp;gt;A) is associated with outcome in neuroblastoma

OCT2 (Octamer Binding Protein 2): An Essential Role in Germinal Center B Cell Differentiation and the “Achilles Heel” of Derived Lymphomas.

Abstract 362▪▪This icon denotes an abstract that is clinically relevant.To identify genes requiered for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic small hairpins RNAs (shRNAs) in this screen targeted Oct2, encoding a POU domain transcriptional activator. Unlike Oct1, which is constitutively expressed in many cell types, Oct2 is primarily lymphoid restricted. It was identified by virtue of its ability to bind the highly conserved DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific co-activator OCA-B interacts with the POU domain of the octamer binding proteins enhancing their transactivation potential.Although Oct2 and OCA-B are not essential for Ig transcription, they are required for germinal center (GC) B cell differentiation.To understand the massive apoptotic cell death of DLBCL cells following shRNA Oct2 induction we investigated the genetic pathways controlled by Oct2. We profiled gene expression changes in DLBCL cell lines after knocking down Oct2 and merged this data set with data from genome-wide assessment of Oct2 and OCA-B binding sites, coupling chromatin immunoprecipitation (ChiP) with high-throughput sequencing technologies (ChIP-Seq).ChIP-Seq uncovered an extensive network of Oct2 target genes in DLBCL cells. More than 60% of the Oct2 target genes also showed OCA-B biding. This Oct2/OCA-B overlaping set of targets was enriched for genes selectively expressed in pan-B cells and GC B cells.We found that Oct2/OCA-B lie upstream many of the main transcription factors known to play an essential role in inducing and mantaining the GC stage of B cell development such as BCL6, MTA3, PU.1, IRF8, SpiB and OCA-B, among others. Oct2/OCA-B target these genes both in DLBCL cells and in normal human primary centroblasts.Strikingly, among Oct-2 downstream effectors, BCL6 cDNA was enough to rescue both ABC and GCB DLBCL cells from the Oct2 shRNA lethal effect.The Oct2/OCA-B binding of BCL6 promoter was confirmed in vivo by single locus ChIP in different GCB-DLBCL cell lines as well as in primary centroblasts isolated from human tonsils. Gel shifts experiments showed Oct2 binding to more than one non canonical octamer motif within the BCL6 promoter. Furthermore, computational analysis of the BCL6 promoter region bound by Oct2, showed PU.1 binding sites. Knocked down of PU.1 decreased Oct2 enrichment and viceversa suggesting cooperative Oct2/PU.1 biding to BCL6 pomoter.ChIP-Seq findings opened an entire and exciting new chapter in the Oct2 biology field. Pou transcription factors were suppossed to regulate the activity of octamer containing promoters. Interestingly, Oct2 binds to and control the expression of many GC specific genes that do not harbor a “canonical octamer” motif.Eventhough Oct2 is expressed throughout the different stages of B cell maturation, both mRNA and protein levels are enhanced in centroblasts as compared to pre-GC B cells. We found Oct2 capable of inducing its own expression as well as Oct-1 and OCA-B. This autoregulatory circuit might partially account for the Oct2 predominant role in GC specific genes expression control.By array cGH, high level amplification of Oct2 and OCA-B was found in less than 10% of non Hodgkin lymphoma patients samples. Nonetheless, these lymphoma cells become addicted to the Oct2 controlled network that sustain cell survival and proliferation, wich turns Oct2 into an attractive therapeutic target for non Hodking lymphoma patients. This critical Oct2 DLBCL cells dependency, is an example of “non oncogene addiction” that we recently defined based on our RNA interference screening. Oct-2 controls a network of regulatory relationships that sustain both normal GC B cells and malignant counterparts.In summary, we showed that Oct2 and OCA-B lie upstream of BCL6, one of the critical regulators of germinal center B cell differentiation. This suggests that Oct2-directed therapy should kill the same DLBCLs as BCL6-directed therapy.Furthermore, all GC and Post-GC B cells that were tested requiere Oct-2 for survival, indicating that Oct2-directed therapy might have a broder activity spectrum than the BCL6-directed therapy.The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity, since this interaction is exclusively required in GC B cells. Disclosures:No relevant conflicts of interest to declare.

C/EBPα or Its Leukemic Mutants Directly Activate nfkb1 Transcription and Displace HDAC1 or HDAC3 From Chromatin-Associated NF-κB p50 to Induce Anti-Apoptotic Genes.

Abstract 76Mutated variants of C/EBPα are expressed in approximately 15% of AML cases. We have previously shown that C/EBPα and its AML mutants interact directly with NF-κB p50 through the basic region. Through binding to a DNA κB site they activate anti-apoptotic genes including BCL2 and FLIP, inhibiting both the extrinsic and intrinsic apoptotic pathways. These effects do not depend on DNA binding but rather on the integrity of the basic region, which is rarely altered in C/EBPα AML oncoproteins. Interaction with p50 is necessary for the C/EBPα induction of bcl-2 and FLIP but not of myeloid differentiation genes. In addition, NF-κB p50 binds and activates the CEBPA gene in myeloid cells. We now report that C/EBPα or its AML mutants, including a leukemia-derived leucine zipper mutant that cannot bind DNA directly (C/EBPαLZ), bind in vivo to the nfkb1 (p50) promoter. Using Ba/F3 cell lines expressing an estrogen receptor fusion of C/EBPα or the C/EBPαLZ mutant we demonstrate induction of p50 mRNA within 7 hrs after the addition of estradiol. A basic region mutant (C/EBPαBR) that cannot bind the p50 protein did not activate nfkb1 transcription. Importantly, the translation inhibitor cycloheximide did not prevent nfkb1 induction by C/EBPα, suggesting direct regulation of nfkb1 transcription by C/EBPα. Remarkably, C/EBPα did not induce p65 RNA expression. A minimal 200 bp nfkb1 promoter, containing κB but not C/EBP binding sites, is trans-activated by C/EBPα 9 fold, or 33 fold by the C/EBPαLZ AML oncoprotein. The variant C/EBPαBR fails to trans-activate the nfkb1 promoter. Together, these data indicate that the gene encoding NF-κB p50 is a direct target of C/EBPα. NF-κB p65 is held in the cytoplasm IκBα and enters the nucleus to bind DNA predominantly as p65:p50 heterodimers upon cell stimulation. In contrast, p50 homodimers are present in the nucleus bound to chromatin in resting cells and inhibit transcription due to the lack of a trans-activation domain and the recruitment of co-repressors and histone deacetylases (HDACs). Therefore, the induction of p50 by C/EBPα would be expected to result in diminished expression of target genes. However, our data indicates that C/EBPα and p50 synergistically activate anti-apoptotic genes. Remarkably, ChIP analysis demonstrated that C/EBPα induction from the metallothionein promoter in Ba/F3 cells diminishes the occupation of the endogenous BCL2 and FLIP promoters by HDAC1 but not HDAC3, although induction of C/EBPα expression did not alter the cellular content of either HDAC. In contrast, occupancy of Bcl-3, another p50 transcriptional cofactor, on these promoters did not change. In transiently transfected 293T cells we confirmed that p50 binds to HDAC1 in a co-immunoprecipitation reaction. Remarkably, interaction between p50 and HDAC1 was disrupted in the presence of co-expressed C/EBPα, C/EBPβ, or the AML mutants C/EBPαLZ, or C/EBPαp30. In a striking correlation with our previous findings, the C/EBPαBR basic region mutant of C/EBPα that does not bind p50 did not interrupt this interaction. As an important control, we confirmed that HDAC1 does not interact directly with C/EBPα or its mutants, indicating that C/EBPα competes with HDAC1 for interaction with p50. Interestingly, the AML-derived C/EBPαLZ or C/EBPαp30 oncoproteins, but not wild-type C/EBPα, interrupt the interaction between HDAC3 and p50. This suggests a greater capacity for these leukemia mutants to de-repress NF-κB target genes compared with C/EBPα itself. In summary, C/EBPα cooperates with NF-κB p50 to induce several anti-apoptotic genes, thereby providing a survival advantage to the leukemic blasts. C/EBPα and p50 reciprocally regulate each other's expression at the transcriptional level, establishing a positive feedback relationship. Moreover, C/EBPα or its AML mutants displaces HDAC1, and the C/EBPα oncoproteins displace HDAC3, from NF-κB p50 homodimers bound to anti-apoptotic genes, contributing to NF-κB dysregulation in leukemia. Our data underscore the concept that the C/EBPα:NF-κB p50 complex is a potential novel therapeutic target in AML, and the C/EBPβ:p50 complex may functional similarly in lymphomas and in other malignancies where C/EBPβ rather than C/EBPα expression predominates. Disclosures:No relevant conflicts of interest to declare.

The i-Motif in the bcl-2 P1 Promoter Forms an Unexpectedly Stable Structure with a Unique 8:5:7 Loop Folding Pattern

Transcriptional regulation of the bcl-2 proto-oncogene is highly complex, with the majority of transcription driven by the P1 promoter site and the interaction of multiple regulatory proteins. A guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to bcl-2 promoter activity, as deletion or mutation of this region significantly increases transcription. This GC-rich element consists of six contiguous runs of guanines and cytosines that have the potential to adopt DNA secondary structures, the G-quadruplex and i-motif, respectively. Our laboratory has previously demonstrated that the polypurine-rich strand of the bcl-2 promoter can form a mixture of three different G-quadruplex structures. In this current study, we demonstrate that the complementary polypyrimidine-rich strand is capable of forming one major intramolecular i-motif DNA secondary structure with a transition pH of 6.6. Characterization of the i-motif folding pattern using mutational studies coupled with circular dichroic spectra and thermal stability analyses revealed an 8:5:7 loop conformation as the predominant structure at pH 6.1. The folding pattern was further supported by chemical footprinting with bromine. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. The potential of the GC-rich element within the bcl-2 promoter region to form DNA secondary structures suggests that the transition from the B-DNA to non-B-DNA conformation may play an important role in bcl-2 transcriptional regulation. Furthermore, the two adjacent large lateral loops in the i-motif structure provide an unexpected opportunity for protein and small molecule recognition.