Bcl-2 Inhibitor Of Transcription Research Articles

Progress of the anoikis effector bcl-2 inhibitor of transcription 1

bcl-2 inhibitor of transcription 1 (Bit1) is an anoikis effector that can play a role of caspase-independent apoptosis by down-regulating bcl-2 expression. Previous studies have found that Bit1 affects cell survival and apoptosis through Erk and FAK-PI3K-Akt-NF-κB pathways. It is worth noting that the expression of Bit1 has shown the obvious tumor specificity in different tumors, and it is closely related to TNM stage, differentiation and prognosis of the tumor. This review summarizes the expression, main mechanism, and significance of Bit1 in different tumors. Key words: Apoptosis; Neoplasms; Bcl-2 inhibitor of transcription 1

Bit1 Regulates Cell Migration and Survival in Oral Squamous Cell Carcinoma

Increasing evidence has shown that Bcl-2 inhibitor of transcription 1 (Bit1) involves a variety of biological processes in the process of tumor development and progression. We hypothesized that Bit1 would be overexpressed in oral squamous cell carcinoma (OSCC); therefore, we examined Bit1 gene expression and protein production, as well as explored the effect of elevated Bit1 levels on OSCC cells. We investigated the use of quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry analysis for Bit1 messenger RNA and protein levels.We used 75 OSCC specimens, 25 tumor-adjacent dysplasia specimens, and 25 normal oral tissue samples that matched OSCC specimens in this study. We also transfected Bit1 complementary DNA into human oral cancer cells (Tca8113) to further investigate the potential role of Bit1 in OSCC. We found that Bit1 levels in OSCC tissues were significantly higher than those in tumor-adjacent dysplasia specimens and normal oral tissue (P<.05). We also confirmed that Bit1 overexpression in the cytosol of Tca8113cells induced apoptosis. Our findings suggest Bit1 overexpression may contribute to oral cancer cell survival and dissemination. In the future, Bit1 may be an important diagnostic and therapeutic target.

The anoikis effector Bit1 displays tumor suppressive function in lung cancer cells.

The mitochondrial Bit1 (Bcl-2 inhibitor of transcription 1) protein is a part of an apoptotic pathway that is uniquely regulated by integrin-mediated attachment. As an anoikis effector, Bit1 is released into the cytoplasm following loss of cell attachment and induces a caspase-independent form of apoptosis. Considering that anoikis resistance is a critical determinant of transformation, we hypothesized that cancer cells may circumvent the Bit1 apoptotic pathway to attain anchorage-independence and tumorigenic potential. Here, we provide the first evidence of the tumor suppressive effect of Bit1 through a mechanism involving anoikis induction in human lung adenocarcinoma derived A549 cells. Restitution of Bit1 in anoikis resistant A549 cells is sufficient to induce detachment induced-apoptosis despite defect in caspase activation and impairs their anchorage-independent growth. Conversely, stable downregulation of Bit1 in these cells significantly enhances their anoikis resistance and anchorage-independent growth. The Bit1 knockdown cells exhibit significantly enhanced tumorigenecity in vivo. It has been previously shown that the nuclear TLE1 corepressor is a putative oncogene in lung cancer, and we show here that TLE1 blocks Bit1 mediated anoikis in part by sequestering the pro-apoptotic partner of Bit1, the Amino-terminal Enhancer of Split (AES) protein, in the nucleus. Taken together, these findings suggest a tumor suppressive role of the caspase-independent anoikis effector Bit1 in lung cancer. Consistent with its role as a tumor suppressor, we have found that Bit1 is downregulated in human non-small cell lung cancer (NSCLC) tissues.

Induction of Apoptosis and Anoikis by Bit1 in Pancreatic Cancer Cells

Pancreatic cancer is a highly aggressive disease with a very high mortality rate among all human cancers. The poor prognosis is in part due to intrinsic resistance to the apoptosis-inducing effects of radio- and chemotherapy. To find alternative cell death pathways that can bypass the apoptotic resistance of pancreatic cancer cells, we examined the role of the novel anoikis effector Bit1 (Bcl-2 inhibitor of transcription) in the survival and apoptotic resistance of pancreatic cancer cells. Bit1 is a mitochondrial protein that induces a caspase-independent apoptosis upon its release into the cytosol following loss of integrin-mediated attachment to extracellular matrix (anoikis). In this report, we observed that ectopic expression of Bit1 in the cytosol reduced viability and induced caspase-independent apoptosis in human pancreatic cancer cell lines, Miapaca-2 and PANC-1. While increased expression of mitochondrial Bit1 in these cells did not induce apoptosis under attached conditions, detachment significantly induced higher level of apoptosis in mitochondrial Bit1-transfected cells than in control transfected cells. Conversely, downregulation of endogenous Bit1 in PANC-1 cells further enhanced their anoikis resistance. Furthermore, exogenous expression of mitochondrial Bit1 in Miapaca-2 cells inhibited their anchorage-independent growth and enhanced their sensitivity to etoposide-mediated apoptosis. Mechanistically, we found that the Bit1 apoptosis function is in part dependent on the groucho related Amino-terminal Enhancer of Split (AES) expression and is abrogated by the transcriptional corepressor TLE1 protein. Consistent with our in vitro findings that Bit1 is an effector of apoptosis in pancreatic tumor cells, we find that Bit1 is significantly downregulated in a fraction of advanced stages of human pancreatic carcinoma tissues. Taken together, these findings indicate that the Bit1-apoptotic pathway can be targeted to trigger cell death in pancreatic cancer cells and implicate Bit1 as a novel therapeutic agent in attenuating pancreatic chemoresistance.

Metastasis of Tumor Cells Is Enhanced by Downregulation of Bit1

BackgroundResistance to anoikis, which is defined as apoptosis induced by loss of integrin-mediated cell attachment to the extracellular matrix, is a determinant of tumor progression and metastasis. We have previously identified the mitochondrial Bit1 (Bcl-2 inhibitor of transcription) protein as a novel anoikis effector whose apoptotic function is independent from caspases and is uniquely controlled by integrins. In this report, we examined the possibility that Bit1 is suppressed during tumor progression and that Bit1 downregulation may play a role in tumor metastasis.Methodology/Principal FindingsUsing a human breast tumor tissue array, we found that Bit1 expression is suppressed in a significant fraction of advanced stages of breast cancer. Targeted disruption of Bit1 via shRNA technology in lowly aggressive MCF7 cells conferred enhanced anoikis resistance, adhesive and migratory potential, which correlated with an increase in active Extracellular kinase regulated (Erk) levels and a decrease in Erk-directed phosphatase activity. These pro-metastasis phenotypes were also observed following downregulation of endogenous Bit1 in Hela and B16F1 cancer cell lines. The enhanced migratory and adhesive potential of Bit1 knockdown cells is in part dependent on their high level of Erk activation since down-regulating Erk in these cells attenuated their enhanced motility and adhesive properties. The Bit1 knockdown pools also showed a statistically highly significant increase in experimental lung metastasis, with no differences in tumor growth relative to control clones in vivo using a BALB/c nude mouse model system. Importantly, the pulmonary metastases of Bit1 knockdown cells exhibited increased phospho-Erk staining.Conclusions/SignificanceThese findings indicate that downregulation of Bit1 conferred cancer cells with enhanced anoikis resistance, adhesive and migratory properties in vitro and specifically potentiated tumor metastasis in vivo. These results underscore the therapeutic importance of restoring Bit1 expression in cancer cells to circumvent metastasis at least in part through inhibition of the Erk pathway.

Abstract 135: Apoptotic cell death triggering in the prostate carcinoma cell line PC3 by the anoikis effector Bit1

Abstract Highly aggressive cancer cells often are associated with resistance to caspase-dependent apoptosis, due, in part to loss in function or mutation in caspase effectors. Such apoptotic defects in caspase signaling pathway may provide a selective growth advantage for tumor cells and potentiate their aggressiveness and resistance to chemotherapeutic agents. Here, we test the responsiveness of the highly aggressive prostate cancer cell line PC3 cells to the apoptotic function of the novel, caspase-independent anoikis effector Bcl2 inhibitor of transcription (Bit1). Overexpression of the cytoplasmic localized Bit1 in PC3 cells results in significant induction of apoptosis as evidenced by the increased levels of histone-DNA fragments. Furthermore, PC3 cells transfected with the mitochondrial localized Bit1 exhibit significant apoptosis following loss of cell attachment as compared to vector treated cells, and such induction of anoikis is associated with increased levels of Bit1 in the cytosol. In contrast, both the control and Bit1 transfected PC3 cells show the same level of spontaneous apoptosis when grown attached to a culture dish. We also tested whether Bit1 alters the chemoresistant phenotype of PC3 cells. Consistent with the induction of anoikis sensitivity, overexpression of mitochondrial Bit1 also renders PC3 cells more sensitive to etoposide-induced apoptosis. Interestingly, the Bit1-mediated apoptosis in PC3 cells is correlated with increased NFkappa B activity as evidenced by increased nuclear translocation and transcription factor activity of the p65 subunit. Taken together, these findings indicate that activation of the caspase-independent Bit1 apoptotic pathway may serve as a preferred alternate approach in triggering apoptosis in aggressive prostate cancer cells. Bit1 may thus serve as a future therapeutic target for the treatment and alleviation of the chemoresistance characteristic of prostate cancer. 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 135.

Down-regulation of the apoptosis-inducing factor or Bcl-2 inhibitor of transcription by RNA interference can alleviate TAp63γ-induced apoptosis in esophageal squamous carcinoma EC9706 cells

In this communication, the roles of apoptosis-inducing factor (AIF) and Bcl-2 inhibitor of transcription (Bit1) in TAp63gamma-induced apoptosis were investigated in human esophageal squamous cancer EC9706 cells. Positive RNA and protein expressions of AIF and Bit1 in these cells were verified. However, no TAp63gamma could be detected. Transfection of expression vector pcDNA3.1-TAp63gamma into EC9706 resulted in TAp63gamma expression, and peak level apoptosis was observed 24 h after the transfection disclosed by DNA fragmentation assay. In addition, it was found with Western blot that AIF and Bit1 were released into cytosol from mitochondria, and AIF was further translocated into nucleus, during the stage of TAp63gamma-induced cell apoptosis. Down-regulation of either AIF or Bit1 by RNA interference could, however, alleviate TAp63gamma-induced cell apoptosis. In conclusion, TAp63gamma could induce apoptosis in human esophageal squamous cancer EC9706 cells, through at least releasing AIF and Bit1 from mitochindria into cytosol and nucleus, where apoptotic cascade takes place. These findings indicate that mitochondria-released proapoptotic proteins, AIF and Bit1, are important factors in a TAp63gamma-induced EC9706 cell apoptosis pathway.

Monoclonal Antibodies Against Human Bit1, An Apoptosis-Associated Mitochondrial Protein

Bit1 (Bcl2-inhibitor of transcription 1) is a mitochondrial protein that was found eliciting caspase-independent apoptosis when released into the cytoplasm, where it forms a complex with AES (amino-terminal enhancer of split). In our research, cytosolic fraction of suspended cultured human ovarian cancer cells was taken as immunogen, and recombined protein Bit1-His was used to select hybridomas. Two hybridoma cell lines secreting monoclonal antibodies (MAbs) against Bit1 were obtained by routine murine hybridoma technique. The MAbs were characterized by indirect ELISA, Western blotting, and immunohistochemistry, and it was found that they recognized distinct epitopes. The sandwich method to detect sera Bit1 was established, and a distinct difference of sera Bit1 between the ovarian carcinoma patients and normal controls was found. To conclude, these MAbs against human Bit1 may be useful for exploring the tumor apoptosis mechanism and monitoring patients' clinical data.

Monoclonal Antibody Against Bcl2-inhibitor of Transcription 1

Monoclonal Antibody Against Bcl2-inhibitor of Transcription 1

Protein Kinase D Is a Positive Regulator of Bit1 Apoptotic Function

Bit1 (Bcl-2 inhibitor of transcription) is a mitochondrial protein that induces caspase-independent apoptosis upon its release into the cytoplasm. Bit1 is primarily associated with anoikis (cell death induced by detachment from the extracellular matrix), because the apoptotic function of Bit1 is inhibited by integrin-mediated cell attachment but not by many other antiapoptotic treatments. Here, we show that protein kinase D (PKD) regulates Bit1 apoptotic function. Overexpression of constitutively active PKD or PKD activation by treatment with phorbol 12-myristate 13-acetate results in phosphorylation of two serine residues (Ser5 and Ser87) in a form of Bit1 that is confined to the cytoplasm and concomitantly increases the apoptotic activity of cytoplasmic Bit1. Conversely, suppressing PKD activity with pharmacological inhibitors or small interfering RNA approaches attenuates apoptosis induced by cytoplasmic Bit1. Furthermore, PKD regulates induction of cell death by wild-type Bit1 following loss of cell attachment to the extracellular matrix. Activation of PKD enhances Bit1 function in anoikis, whereas inhibiting PKD function with pharmacological inhibitors or small interfering RNA compromises the ability of Bit1 to induce anoikis. The induction of Bit1-mediated apoptosis by PKD is in part attributable to the release of Bit1 from mitochondria to the cytoplasm as a consequence of phosphorylation of Ser5 in the mitochondrial localization sequence of Bit1. Consistent with the regulatory role of PKD in the anoikis function of Bit1, we found that cell attachment to fibronectin inhibits PKD activity. These studies identify the PKD serine/threonine kinase as one of the signaling molecules through which integrin-mediated cell attachment controls Bit1 activity and anoikis.

Anoikis effector Bit1 negatively regulates Erk activity

Bcl-2 inhibitor of transcription (Bit1) is a mitochondrial protein that functions as a peptidyl-tRNA hydrolase, but, when released into the cytoplasm, it elicits apoptosis. The proapoptotic function is uniquely counteracted by integrin-mediated cell attachment. We generated a conditional KO mouse of the Bit1 gene by using the Cre-LoxP recombination system. Bit1-null mice were born alive but with some developmental abnormalities. They developed a runting syndrome after birth and died within the first 2 weeks. Cultured fibroblasts from the Bit1-null embryos [mouse embryo fibroblasts (MEFs)] were more resistant to cell death induced by loss of attachment to extracellular matrix (anoikis) than cells from the wild-type or heterozygous littermates. MEFs and tissues from Bit1 KO mice displayed a marked increase in Erk phosphorylation. Knocking down Bit1 expression in cultured cells resulted in increased Erk activation, and partially knocking down Erk reversed the increased anoikis resistance of Bit1 knockdown. The enhanced Erk activation was associated with decreased Erk phosphatase activity. These studies establish the physiological significance of Bit1 activity and begin to delineate a Bit1 signaling pathway that acts through Erk regulation.

A Bit of Anoikis

Many cells undergo apoptosis (programmed cell death) after loss of attachment to the extracellular matrix. This form of apoptosis, called anoikis, is suppressed by integrin-mediated signaling pathways. One of these pathways involves enhanced transcription of the antiapoptotic protein Bcl-2. Jan et al. used a portion of integrin α5β1 to disable integrin signaling and screened a cDNA library for proteins that affected Bcl-2 expression. They identified Bit1 (Bcl-2 inhibitor of transcription), a protein that was unrelated to any other protein in the human or mouse genomes. The authors used a combination of immunostaining, fluorescent labeling, and Western analysis to show that Bit1 was localized in the mitochondria of HeLa cells. Bit1 was released from the mitochondria of cells cultured under apoptosis-promoting conditions. Furthermore, overexpression of Bit1 produced apoptosis, with apoptosis occurring at lower levels of expression if the Bit1 was mutated to prevent mitochondrial targeting. Bit1 associated with the Groucho-related protein AES and transfection experiments in cell lines that did or did not express one or the other of the two proteins, which indicated that the two proteins interacted with each other to promote apoptosis. Bit1-mediated apoptosis was suppressed by cell attachment to fibronectin through integrin α5β1; moreover, increased expression of Bit1 enhanced sensitivity to anoikis, whereas knockdown decreased sensitivity to anoikis. Bit1-mediated apoptosis did not involve caspase activity. Bit1 or AES expressed in Chinese hamster ovary cells inhibited transcription from a Bcl-2 gene reporter. Thus, Bit1 appears to represent a previously unrecognized proapoptotic mitochondrial protein whose activity is subject to regulation through integrin-mediated signaling pathways. Y. Jan, M. Matter, J.-t. Pai, Y.-L. Chen, J. Pilch, M. Komatsu, E. Ong, M. Fukuda, E. Ruoslahti, A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. Cell 116 , 751-762 (2004). [Online Journal]