Repression Of Bcl-2 Research Articles

MicroRNA-1 Regulates Cardiomyocyte Apoptosis by Targeting Bcl-2

MicroRNA-1 (miR-1) is preferentially expressed in cardiac muscles, and the expression has been demonstrated to be involved in cardiac development and cardiovascular diseases. Here we report that miR-1 is closely related with ischemia/reperfusion injury in a rat model. The level of miR-1 is inversely correlated with Bcl-2 protein expression in cardiomyocytes of the I/R rat model. In vitro, the level of miR-1 was dramatically increased in response to H(2)O(2). Overexpression of miR-1 facilitated H(2)O(2)-induced apoptosis in cardiomyocytes. Inhibition of miR-1 by antisense inhibitory oligonucleotides caused marked resistance to H(2)O(2). Through bioinformatics, we identified the potential target sites for miR-1 on the 3' UTR of Bcl-2. miR-1 significantly reduced the expression of Bcl-2 in the levels of mRNA and protein. The post-transcriptional repression of Bcl-2 was further confirmed by luciferase reporter experiments. These data demonstrated that miR-1 plays an important role in the regulation of cardiomyocyte apoptosis, which is involved in post-transcriptional repression of Bcl-2.

Direct Transcriptional Repression of BCL2 by BCL6 in Germinal Centre B Cells and Its Disruption in B Cell Lymphomas with BCL2 Locus Alterations

The human proto-oncogene BCL6 encodes a BTB/POZ-zinc finger transcriptional repressor that is required for germinal centre (GC) development and is expressed in the majority of normal GC B cells and in the majority of B cell lymphoma (B-NHL), including follicular lymphoma (FL) and a subset of diffuse large B cell lymphomas (DLBCLs). Deregulation of BCL6, by chromosomal translocation or somatic hypermutation, is implicated in the pathogenesis of B-NHL. The precise function of BCL6 in GC development and lymphomagenesis is still unclear also due to the very few direct BCL6 target genes that have been identified. In order to identify physiologically relevant BCL6 direct target genes, we used a novel approach combining high-throughput biochemical (ChIP-on-chip), bioinformatics (ARACNe) (Basso et al., Nature Genetics, 2005) and gene expression profile analysis (Klein et al., Proc. Natl. Acad. Sci., 2004) of normal GC B cells. The results (see abstract by Saito et al.) have identified a core of bona fide target genes whose promoter is bound by BCL6, whose transcription is dynamically linked to BCL6, and whose expression is down-regulated in GC B cells. Among the genes meeting these stringent criteria, we found BCL2, encoding the anti-apoptotic molecule whose expression is deregulated by chromosomal translocations or gene amplification in the majority of FL and in a subset of DLBCL. Further investigations showed that BCL6 represses BCL2 transcription by binding specific DNA sites within the BCL2 promoter region. Suppression of BCL6 expression via specific siRNAs leads to increased levels of BCL2, and, conversely, constitutive BCL6 expression prevents B cell receptor (BCR)-induced upregulation of BCL2 in B cells. Consistent with a physiological role for BCL6- mediated BCL2 suppression, immunohistochemical analysis shows that BCL2 expression is absent in GC B cells where BCL6 is highly expressed. Notably, the comparative analysis of BCL6 and BCL2 expression in B-NHL cell lines showed a conserved inverse correlation between BCL6 and BCL2 levels in cell lines carrying a normal BCL2 locus. However, cell lines carrying chromosomal translocations or amplifications affecting the BCL2 gene displayed the pathologic co-expression of both proteins, suggesting that the alterations affecting the BCL2 locus prevent BCL6-mediated suppression. These results indicate that one critical role of BCL6 is the modulation of antiapoptotic function in GC B cells via BCL2 transcriptional repression, suggesting a mechanism by which B cells may die in the GC if not rescued by BCR and CD40 engagement, both of which downregulate BCL6 while inducing BCL2 expression. Alterations of the BCL2 locus may contribute to lymphomagenesis by making the gene resistant to suppression by BCL6, and therefore allowing B cells to avoid apoptosis in the absence of antigen- and T-cell-mediated rescue.

Reply:

We thank Dr. Bortolami and colleagues for their interest in our recent article showing perturbation of hepatocytic transforming growth factor β (TGF-β) signaling during progression of hepatitis C virus (HCV)-related chronic liver disorders.1 In parallel with emergence of epithelial-to-mesenchymal transition paradigm in fibrocarcinogenic steps, a large body of work has established roles for epithelial cells as important mediators of progressive fibrosis and carcinogenesis.2 Our data indicate that hepatocytes undergo transition from the tumor-suppressive TGF-β pathway, which is characteristic of mature epithelial cells, to the fibrogenic TGF-β pathway, which appears to favor the state of flux shown by the activated hepatic stellate cells (HSC), accelerating liver fibrosis while increasing risk of cancer. Our finding of hepatocytes involvement in fibrogenesis confirms the recent reports from other laboratories that mature hepatocytes can acquire a mesenchymal phenotype and undertake the activated HSC functions.3, 4 Chronic inflammation associated with persistent HCV infection is clearly the primary inducer of liver fibrosis and cancer. Fibrotic stages and necroinflammatory activities of HCV-related liver diseases therefore correlate closely with the risk of hepatocellular carcinoma (HCC). As suggested by Bortolami and colleagues, TGF-β, interleukin-1β, and DNA oxidative damage are increased in chronic hepatitis C and cirrhotic livers. These findings suggest that elevated interleukin-1β and DNA oxidative damage can alter hepatocytic TGF-β signaling in HCV-related preneoplastic lesions. TGF-β signaling of hepatocytes affected by chronic inflammation acts as a key regulatory element that offers a general framework for understanding the origins of HCV-related HCC. Reversible shifting of Smad-dependent signaling between tumor suppression and oncogenesis in hyperactive Ras-expressing cells indicates that oncogenic activities such as cell proliferation and invasion are promoted by Smad3 phosphorylated at the linker region (pSmad3L) pathway.5 Linker phosphorylation of Smad3 indirectly inhibits C-terminal phosphorylation, minimizing Smad3 phosphorylated at the C-terminal region (pSmad3C) signaling.5 In the liver, a major function of TGF-β is to limit regenerative growth of hepatocytes in response to injury by inhibiting cell growth and inducing apoptosis. pSmad3C-mediated signaling appears to take part in cytostatic function of TGF-β such as growth inhibition and apoptosis of hepatocytes. For example, Bcl-2 repression by TGF-β requires C-terminal phosphorylation of Smad3.6 To evade apoptosis, HCC might acquire Bcl-2 overexpression induced by pSmad3L pathway. Escaping apoptosis is a critical step in the progression to full malignancy of cancers, which overcome multiple fail-safe genetic controls. From the viewpoint of TGF-β signaling, a key therapeutic aim in cancer would be restoration of the lost tumor suppressor function observed in normal hepatocytes at the expense of effects promoting aggressive behavior in HCC. Our model suggests that specific inhibitors of the pSmad3L-mediated oncogenic/fibrogenic signaling should inhibit progression of HCC. Koichi Matsuzaki*, Iwata Ozaki , * Departments of Gastroenterology and Hepatology, Kansai Medical University, Osaka, Japan, Health Administration Center, Saga Medical School, Saga University, Saga, Japan.

Eradication of p53-Mutated Head and Neck Squamous Cell Carcinoma Xenografts Using Nonviral p53 Gene Therapy and Photochemical Internalization

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.

Apoptosis Induction by Activator Protein 2α Involves Transcriptional Repression of Bcl-2

Activator protein 2alpha (AP-2alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2alpha. We found that AP-2alpha induced apoptosis efficiently in cells treated with benzyloxycar-bonyl-IETD-fluoromethyl ketone or FADD-silenced cells but failed to do so in benzyloxycarbonyl-LEHD-fluoromethyl ketone-treated or apoptosis protease activation factor-1 (Apaf1)-silenced cells, suggesting the central role of mitochondria in AP-2alpha-induced apoptosis. In good correlation, cells overexpressing AP-2alpha showed a reduction in mitochondrial membrane potential (Deltapsi(m)), cytochrome c and Smac/DIABLO release into cytosol, and Bax translocation into mitochondria. We found that the pro-apoptotic protein Bax is important for AP-2alpha-induced apoptosis as adenovirus AP2 failed to induce apoptosis in HCT116 Bax(-/-) cells. However, we found the IAP (inhibitor of apoptosis) inhibitor Smac/DIABLO may have a limited role in AP-2alpha-induced apoptosis as we found the IAP member Survivin down-regulated by AP-2alpha. Although the total Bax level remains unaltered, we found a time-dependent increase in the activated form of Bax in adenovirus AP2-infected cells. In addition, we show that AP-2alpha transcriptionally represses Bcl-2 by binding to its promoter both in vitro and in vivo and that this is essential for AP-2alpha-induced apoptosis as ectopic expression of Bcl-2 efficiently inhibited apoptosis induced by AP-2alpha. Furthermore, we show that chemotherapy-induced endogenous AP-2alpha down-regulates Bcl-2 and induces apoptosis in an AP-2alpha-dependent manner. Moreover, we demonstrate that inhibition of okadaic acid or staurosporine-sensitive pathways in AP-2alpha overexpressing breast cancer cells resulted in AP-2alpha-dependent apoptosis induction. These results suggest that AP-2alpha induces apoptosis by down-regulating Bcl-2 and utilizing a bax/cytochrome c/Apaf1/caspase 9-dependent mitochondrial pathway.

MiR-15 and miR-16 induce apoptosis by targeting BCL2.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia and is characterized by predominantly nondividing malignant B cells overexpressing the antiapoptotic B cell lymphoma 2 (Bcl2) protein. miR-15a and miR-16-1 are deleted or down-regulated in the majority of CLLs. Here, we demonstrate that miR-15a and miR-16-1 expression is inversely correlated to Bcl2 expression in CLL and that both microRNAs negatively regulate Bcl2 at a posttranscriptional level. BCL2 repression by these microRNAs induces apoptopsis in a leukemic cell line model. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors.

Dynein light chain rp3 acts as a nuclear matrix-associated transcriptional modulator in a dynein-independent pathway

Cytoplasmic dynein is a motor protein complex involved in microtubule-based cargo movement. Previous biochemical evidence suggests that dynein light chain subunits also exist outside the dynein complex. Here we show that the dynein light chain rp3 is present in both the cytoplasm and the nucleus. Nuclear rp3 binds to and assembles with the transcription factor SATB1 at nuclear matrix-associated structures. Dynein intermediate chain was also detected in the nucleus, but it was dispensable for the rp3-SATB1 interaction. SATB1 facilitates the nuclear localization of rp3, whereas rp3 and dynein motor activity are not essential for nuclear accumulation of SATB1. The nuclear rp3-SATB1 protein complex is assembled with a DNA element of the matrix attachment region of the Bcl2 gene. Finally, rp3 is involved in SATB1-mediated gene repression of Bcl2. Our data provide evidence that dynein subunit rp3 has functions independent of the dynein motor.

Phosphorylated and hypoacetylated mutant p53 enhances cisplatin-induced apoptosis through caspase-9 pathway in the absence of transcriptional activation or translation

It is not completely understood how certain epithelial cells harboring mutant p53 have better response to chemotherapy. We investigate the mechanism of cisplatin-induced apoptosis in two resistant cell lines (parental TCCSUP and R273L mutant p53 transfectant) and two sensitive cell lines (V143A and N247I mutant p53 transfectants). Activation of caspase 9 was demonstrated by Western blotting, and specific inhibitor for caspase 9 could inhibit apoptosis. Inhibitors for caspases 1, 2, 6, and 8 had no effect on apoptosis. Transcriptional repression of Bcl-2 occurred during apoptosis and could be reversed by the treatment of histone deacetylase inhibitor trichostatin A (TSA). The expression of Noxa, p53 inducible ribonucleotide reductase subunit 2 (p53R2), and p53 inducible death domain (PIDD) gene were not elevated with treatment of cisplatin (CDDP). Surface trafficking of Fas or Fas-L was not observed. Ser15 of wild-type p53 and mutant p53 was phosphorylated in response to cisplatin. Acetylation of wild-type p53 increased, while acetylation of mutant p53 decreased during cisplatin treatment. Both transcriptional inhibitor actinomycin D and translational inhibitor cycloheximide did not inhibit apoptosis. These results indicated that phosphorylated and hypoacetylated mutant p53 could enhance cisplatin-induced apoptosis through activation of caspase 9 independent of transcriptional activation and translation.

Interferon consensus sequence binding protein (ICSBP; IRF-8) antagonizes BCR/ABL and down-regulates bcl-2

AbstractBCR/ABL is the causative genetic aberration in chronic myelogenous leukemia (CML). Mice lacking expression of the interferon (IFN) consensus sequence binding protein (ICSBP), an IFNγ-inducible transcription factor of the interferon regulatory factor (IRF) family, develop a disease similar to human CML. Mounting evidence suggests a role for ICSBP in the pathogenesis of CML. However, the underlying mechanisms are largely unknown. By stable and conditional expression of ICSBP in wild-type and BCR/ABL-transformed 32D cells (32D/wt and 32D/BA), we found that ICSBP inhibited BCR/ABL-mediated leukemogenesis in vivo. Moreover, ICSBP also overrode BCR/ABL-mediated morphology changes, chemotherapy, and imatinib resistance, as well as BCR/ABL-induced repression of differentiation. Some of these ICSBP effects may be explained in part by an ICSBP-mediated repression of bcl-2, a major antiapoptotic target of BCR/ABL, on transcriptional and protein level. Using reporter gene assays and electrophoretic mobility shift assays we identified that the bcl-2 promoter activity was inhibited by ICSBP by way of a fragment containing 2 characteristic ICSBP-responsive elements. An inverse correlation between ICSBP and bcl-2 expression was confirmed in vivo. Collectively, our findings suggest that ICSBP antagonizes BCR/ABL by down-regulation of bcl-2 and implicates a central role for ICSBP in the pathogenesis of CML, as well as a therapeutic target to overcome drug resistance in bcl-2-dependent tumors. (Blood. 2004;103:3480-3489)

The combined treatment of aspirin and radiation induces apoptosis by the regulation of bcl-2 and caspase-3 in human cervical cancer cell

The antitumor mechanisms mediated by combined treatment of aspirin and radiation on human cervical cancer cells are unclear. In this paper, we studied whether aspirin and radiation induced apoptosis and whether the sensitivity to radiation was enhanced in aspirin-pretreated HeLa TG, human cervical cancer cells. We identified the regulation of apoptosis-responsive genes, bcl-2, caspase-3 and p53 after combined treatment. To investigate the growth inhibitory effect on HeLa TG cells after treatment of various nonsteroidal anti-inflammatory drugs (NSAIDs), we performed cell proliferation assay and colony-forming assay. In the presence of aspirin, sulindac and indomethacin, cell proliferation and colony formation were decreased in a time- and dose-dependent manner. According to flow cytometry analysis and Hoechst 33342 staining, we found that aspirin increased sub-G1 population and nuclear condensation of cervical cancer cells. Remarkably, the combined treatment decreased cell proliferation compared with treatment of 1 mM aspirin or 6 Gy radiation alone. Pretreatment of aspirin followed by irradiation also elevated the population of apoptotic cells. These results revealed that sensitivity to radiation was enhanced in aspirin-pretreated HeLa TG cells, and aspirin has the additive role for amplifying the radiotherapeutic effect in cervical cancer cells. Finally, combined treatment revealed bcl-2 repression and caspase-3 induction did not detect any change but p53 expression did. We have demonstrated that combined treatment of aspirin and radiation induces the antitumor effect mediated by bcl-2 and caspase-3 pathway in cervical cancer cells.

33] Optimizing efficacy of antisense oligodeoxynucleotides targeting inhibitors of apoptosis

It is now widely accepted that defects in the regulation of a genetically controlled suicide process, also known as programmed cell death or apoptosis, can contribute to multiple pathologies including cancer. The search for gene products involved in the regulation of apoptosis has identified numerous proteins. In particular, much interest has been devoted to members of the B-cell lymphoma-2 (bcl-2) gene family of apoptosis regulators because of their contribution to oncogenesis and drug resistance. This family of structurally related proteins comprises members that exert opposing functions in apoptosis control. The bcl-2 protein functions in normal and neoplastic cells to inhibit or delay apoptosis induced by a variety of endogenous and exogenous stimuli. The related bcl-extra large (bcl-xl) protein also acts as a negative regulator of apoptosis and protects from cell death where bcl-2 is ineffective. In contrast to normal tissues, bcl-2 and bcl-xl are frequently coexpressed in tumors, where they contribute to resistance to a broad range of anticancer agents. Pro- and antiapoptotic bcl-2 family members can heterodimerize and seemingly titrate one another's function, thereby suggesting that their relative concentration may act as a rheostat for the death program. This suggests that cell survival or death after an apoptotic stimulus is determined by competitive interactions between distinct pairs of death agonists (e.g., Bax and Bad) and antagonists (e.g., bcl-2 and bcl-xl). Therefore, any approach to turn the rheostat in favor of cell death—for example, by repression of bcl-2 or bcl-xl expression—could be therapeutically useful. In addition to bcl-2 and bcl-xl, inhibitors of apoptosis proteins (IAPs) that are directly engaged in the caspase activation pathway are described in the chapter. Further investigations are necessary to determine whether x-linked IAP (XIAP), the cell-IAPs (c-IAPs), and survivin represent additional targets for the antisense approach to cancer therapy.

Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of glucocorticoids.

Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR's transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor's ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR's cytostatic action, the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). Induction of p21(Cip1) is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27(Kip1) is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids.