BCL2/adenovirus E1B 19-kDa Interacting Protein 3 Expression Research Articles

Expression Analysis of caspase-6, caspase-9 and BNIP3 in Prostate Cancer:

Altered regulation of cell death is a feature of human cancer. The aim of this study was to explore whether the expression of the proapoptotic proteins caspase-6, caspase-9, and Bcl-2/adenovirus E1B-19kDa-interacting protein3 (BNIP3) is altered in prostate cancers. We analyzed the expression of caspase-6, caspase-9, and BNIP3 in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. Normal glandular cells expressed caspase-6 and BNIP3 proteins in 10 (9.3%) and 9 (8.4%) prostate tissues, respectively. By contrast, the prostate cancers expressed caspase-6 and BNIP3 in 65 (60.7%) and 69 (64.5%) cases, respectively. Prostate intraepithelial neoplasia (PIN) showed caspase-6 and BNIP3 expression in 65% and 65% of cases, respectively. We observed caspase-9 expression in 40 (37.4%) normal, 8 (40%) PIN, and 45 (42.1%) cancer tissues. None of the expression of caspase-6, caspase-9 or BNIP3 was associated with pathological characteristics such as tumor size, patient age, Gleason score, or tumor stage. Our data showed that prostate cancer and PIN cells display higher expression of the proapoptotic proteins caspase-6 and BNIP3 than normal cells. Neo-expression of these proteins from the PIN stage suggests that apoptosis deregulation might occur in the early stage of prostate carcinogenesis, and that altered expression of proapoptotic proteins may be a feature of prostate cancer.

Expression and Functional Characterization of the BNIP3 Protein in Renal Cell Carcinomas

BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) is a BH3-only protein that regulates apoptosis and autophagy. BNIP3 plays also an important role in hypoxia-induced cell response and is regulated by HIF1. Here, we studied a possible association of BNIP3 expression and the prognosis of patients with renal cell carcinomas (RCCs) and examined the functional relevance of BNIP3 in the regulation of cell survival and apoptosis of renal carcinoma cells. BNIP3 expression was determined by immunohistochemistry in RCC tumor tissue samples of 569 patients using a tissue microarray. Functional characterization of BNIP3 in renal carcinoma cells indicates prosurvival effects. In human RCC tumor samples, high cytoplasmic BNIP3 expression was associated with high-grade RCCs and regional lymph node metastasis. BNIP3 expression correlated negatively with disease-specific survival. Multivariate Cox regression analysis retained BNIP3 expression as an independent prognostic factor in patients without distant metastasis. Together, our studies imply that BNIP3 regulates cell survival in RCCs and its expression is an independent prognostic marker in patients with localized RCCs.

Nutrition deficiency promotes apoptosis of cartilage endplate stem cells in a caspase-independent manner partially through upregulating BNIP3.

Nutrition deficiency is reported to induce apoptosis of chondrocytes and degeneration of cartilage endplate (CEP) in rabbit. Cartilage endplate stem cells (CESCs) are important for the integrity of structure and function of CEP. Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) has been reported to regulate apoptosis, autophagy, and cytoprotection. In this study, we aimed to determine whether nutrition deficiency induces apoptosis of CESCs, and whether or not the BNIP3-related pathway is activated in CESCs during nutrition deficiency. CESCs isolated from degenerated human CEP were cultured under normal or nutrition-deficient condition. Then, apoptosis was analyzed by flow cytometry. The expression and intracellular localization of BNIP3 were detected by quantitative real-time polymerase chain reaction, western blot analysis, and immunofluorescence assay, respectively. Mitochondrial membrane potential (MMP) and caspase-3 activity were measured by JC-1 staining and caspase-3 activity assay. Our results showed that nutrition deficiency promotes apoptosis and BNIP3 expression in CESCs. Notably, knockdown of BNIP3 could partially decrease nutrition deficiency-induced apoptosis of CESCs. In addition, nutrition deficiency could also induce upregulation of BNIP3, resulting in mitochondrial translocation of BNIP3 and loss of MMP in CESCs in a time-dependent manner. However, nutrition deficiency showed no effects on caspase-3 activity in CESCs. In summary, nutrition deficiency may promote CESC apoptosis partially through upregulating BNIP3, which might lead to activation of the BNIP3-related pathway and apoptosis of CESCs in a caspase-independent manner.

Caspase-Dependent and Caspase-Independent Pathways Are Involved in Cadmium-Induced Apoptosis in Primary Rat Proximal Tubular Cell Culture.

We designed this study to investigate whether cadmium induces caspase-independent apoptosis and to investigate the relationship between the caspase-dependent and caspase-independent apoptotic pathways. Cadmium (1.25–2.5 μM) induced oxidative stress in rat proximal tubular (rPT) cells, as seen in the reactive oxygen species levels; N-acetylcysteine prevented this. Cyclosporin A (CsA) prevented mitochondrial permeability transition pore opening and apoptosis; there was mitochondrial ultrastructural disruption, mitochondrial cytochrome c (cyt c) translocation to the cytoplasm, and subsequent caspase-9 and caspase-3 activation. Z-VAD-FMK prevented caspase-3 activation and apoptosis and decreased BNIP-3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) expression levels and apoptosis-inducing factor/endonuclease G (AIF/Endo G) translocation. Simultaneously, cadmium induced prominent BNIP-3 expression in the mitochondria and cytoplasmic AIF/Endo G translocation to the nucleus. BNIP-3 silencing significantly prevented AIF and Endo G translocation and decreased the apoptosis rate, cyt c release, and caspase-9 and caspase-3 activation. These results suggest that BNIP-3 is involved in the caspase-independent apoptotic pathway and is located upstream of AIF/Endo G; both the caspase-dependent and caspase-independent pathways are involved in cadmium-induced rPT cell apoptosis and act synergistically.

BNIP3 Protein Suppresses PINK1 Kinase Proteolytic Cleavage to Promote Mitophagy

Mutations in PINK1 (PTEN-induced putative kinase 1) cause early onset familial Parkinson's disease (PD). PINK1 accumulates on the outer membrane of damaged mitochondria followed by recruiting parkin to promote mitophagy. Here, we demonstrate that BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a mitochondrial BH3-only protein, interacts with PINK1 to promote the accumulation of full-length PINK1 on the outer membrane of mitochondria, which facilitates parkin recruitment and PINK1/parkin-mediated mitophagy. Inactivation of BNIP3 in mammalian cells promotes PINK1 proteolytic processing and suppresses PINK1/parkin-mediated mitophagy. Hypoxia-induced BNIP3 expression results in increased expression of full-length PINK1 and mitophagy. Consistently, expression of BNIP3 in Drosophila suppresses muscle degeneration and the mitochondrial abnormality caused by PINK1 inactivation. Together, the results suggest that BNIP3 plays a vital role in regulating PINK1 mitochondrial outer membrane localization, the proteolytic process of PINK1 and PINK1/parkin-mediated mitophagy under physiological conditions. Functional up-regulation of BNIP3 may represent a novel therapeutic strategy to suppress the progression of PD.

Expression of BNIP3 and its correlations to hypoxia-induced autophagy and clinicopathological features in salivary adenoid cystic carcinoma.

The expression of Bcl-2/adenovirus E1B 19 kDa-interacting protein3 (BNIP3) has been explored in many human malignancies, but not in adenoid cystic carcinoma (ACC). This study investigated the clinical significance of expression of BNIP3 in ACC tissues and cells and elucidated its correlations to hypoxia-induced autophagy. Immunohistochemical and immunofluorescence staining were used to explore BNIP3, HIF-1α and LC3 expression. BNIP3 was positively expressed in 41 cases (63.1%), and was significantly correlated with histological grade (P= 0.001). HIF-1α was positively expressed in 52 cases (80.0%) and was significantly correlated with TNM stage (P= 0.023) and histological grade (P= 0.024). LC3 was positively expressed in 37 cases (56.9%) and was significantly correlated with TNM stage (P= 0.019). The expression of BNIP3 was correlated with HIF-1α expression (P= 0.011). The overall survival in the negative BNIP3 expression group tended to be better than in the positive BNIP3 expression (P= 0.011). In vitro experiment, BNIP3 immunofluorescence staining was detected in cells treated with CoCl2 (for hypoxic condition). The data indicated that BNIP3 plays a vital role in the tumorigenesis of adenoid cystic carcinoma and could be a new target for gene therapy of adenoid cystic carcinoma.

MiR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs.

Oxidative stress plays a critical role in cardiovascular diseases. Salidroside, a glycoside from Rhodiola rosea, has been used as an antioxidative therapy for oxidative injury in cardiac diseases. However, the mechanism underlying its antioxidant effect needs to be elucidated. Treatment of HUVECs with H2O2 significantly decreased the expression of miR-103 in a dose- and time-dependent manner, whereas pretreatment with salidroside significantly inhibited this decrease. Subsequent analysis showed that overexpression of miR-103 abrogated cell activity and ROS production induced by H2O2. Bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) was determined to be a novel miR-103 target in HUVECs. Interestingly, H2O2 treatment upregulated BNIP3 expression; in turn, this effect was inhibited by pretreatment with salidroside. Further studies confirmed that the knockdown of BNIP3 enhanced cell activity and suppressed the ROS production induced by H2O2. These results demonstrated for the first time that salidroside protects HUVECs in part by upregulating the expression of miR-103, which mediates BNIP3 downregulation and plays an important role in the cytoprotective actions.

The interplay between epigenetic silencing, oncogenic KRas and HIF-1 regulatory pathways in control of BNIP3 expression in human colorectal cancer cells

Bcl-2/adenovirus E1B-19kDa-interacting protein 3 (BNIP3) is an important mediator of cell survival and a member of the Bcl-2 family of proteins that regulate programmed cell death and autophagy. We have previously established a link between the expression of oncogenic HRas and up-regulation of BNIP3 and the control of autophagy in cancer cells. However, in view of varied expression of BNIP3 in different tumor types and emerging uncertainties as to the role of epigenetic silencing, oncogenic regulation and the role of BNIP3 in cancer are still poorly understood.In the present study we describe profound effect of KRas on the expression of methylated BNIP3 in colorectal cancer cells and explore the interplay between HIF-1, hypoxia pathway and oncogenic KRas in this context. We observed that BNIP3 mRNA remains undetectable in aggressive DLD-1 cells harboring G13D mutant KRAS and HT-29 colorectal cancer cells unless the cells are exposed to demethylating agents such as 5-aza-2′-deoxycytidine. Following this treatment BNIP3 expression remains uniquely dependent on the Ras activity. We found that hypoxia or pharmacological activation of HIF-1 alone contributes to, but is not sufficient for efficient induction of BNIP3 mRNA transcription in cells lacking mutant KRas activity. The up-regulation of BNIP3 by KRas in this setting is mediated by the MAPK pathway, and is attenuated by the respective inhibitors (PD98059, U0126). Thus, we demonstrate the novel mechanism where activity of Ras is essential for 5-aza-2′-deoxycytidine-mediated BNIP3 expression. Moreover, we found that 5-aza-2′-deoxycytidine-mediated or enforced up-regulation of BNIP3 in DLD-1 cells results in KRas-dependent resistance to 5-Fluorouracil.

MiR-210 suppresses BNIP3 to protect against the apoptosis of neural progenitor cells

MiR-210 is a hypoxia-inducible factor (HIF)-1 target gene and is the most consistently and predominantly upregulated miRNA in response to hypoxia in various cancer cell lines. Our recent study shows that hypoxia increased miR-210 expression in neural progenitor cells (NPCs) in a time-dependent manner. However, the role of miR-210 in NPCs remains unknown. Following the identification of the miR-210 putative target genes, we demonstrated that the Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3), which is regulated by HIF-1 and activates cell death, is regulated by miR-210 in NPCs under hypoxia. Moreover, the over-expression of miR-210 decreased apoptosis in NPCs, and the inhibition of miR-210 expression remarkably increased the number of TUNEL-positive NPCs by 30% in response to hypoxia. Importantly, miR-210 mimics reduced both BNIP3 protein expression and the translocation of AIF into the nucleus, which reduced cell death, whereas miR-210 inhibitors reversed this process, leading to cell death during hypoxia. Taken together, we report a novel feedback loop of BNIP3 regulation in NPCs under hypoxia. HIF-1 is activated under hypoxia and then induces the expression of both BNIP3 and miR-210. The upregulation of miR-210 then directly suppresses BNIP3 expression to maintain the survival of NPCs under hypoxia. This negative feedback regulation might partially contribute to protection against hypoxia-induced cell death via the inhibition of AIF nuclear translocation.

Different molecular mechanisms involved in spontaneous and oxidative stress-induced mitochondrial fragmentation in tripeptidyl peptidase-1 (TPP-1)-deficient fibroblasts

NCLs (neuronal ceroid lipofuscinoses) form a group of eight inherited autosomal recessive diseases characterized by the intralysosomal accumulation of autofluorescent pigments, called ceroids. Recent data suggest that the pathogenesis of NCL is associated with the appearance of fragmented mitochondria with altered functions. However, even if an impairement in the autophagic pathway has often been evoked, the molecular mechanisms leading to mitochondrial fragmentation in response to a lysosomal dysfunction are still poorly understood. In this study, we show that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form) also exhibit a fragmented mitochondrial network. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 (Bcl2/adenovirus E1B 19 kDa interacting protein 3) as well as a decrease in the abundance of mitofusins 1 and 2, two proteins involved in mitochondrial fusion. Using RNAi (RNA interference) and quantitative analysis of the mitochondrial morphology, we show that the inhibition of BNIP3 expression does not result in an increase in the reticulation of the mitochondrial population in LINCL cells. However, this protein seems to play a key role in cell response to mitochondrial oxidative stress as it sensitizes mitochondria to antimycin A-induced fragmentation. To our knowledge, our results bring the first evidence of a mechanism that links TPP-1 deficiency and oxidative stress-induced changes in mitochondrial morphology.

Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death

Hypoxia-inducible pro-death protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3), provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of autophagy accelerates BNIP3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating autophagy will attenuate BNIP3-induced cardiomyocyte death. Neonatal rat cardiac myocytes (NRCMs) were adenovirally transduced with BNIP3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100 nM). Cell death was assessed at 48 h. BNIP3 expression increased autophagosome abundance 8-fold and caused a 3.6-fold increase in cardiomyocyte death as compared with control. Rapamycin treatment of BNIP3-expressing cells led to further increase in autophagosome number without affecting cell death. BNIP3 expression led to accumulation of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression). BNIP3, but not the transmembrane deletion variant, interacted with LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular fractionation, provoke lysosome permeabilization or alter lysosome pH. Rather, BNIP3-induced autophagy caused a decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes, prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced cell death.

MicroRNA145 Targets BNIP3 and Suppresses Prostate Cancer Progression

The putative tumor suppressor miR145 is transcriptionally regulated by TP53 and is downregulated in many tumors; however, its role in prostate cancer is unknown. On the other hand, BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) is overexpressed in various tumors, including prostate cancer, and may transcriptionally repress the apoptosis-inducing factor (AIF) gene. Although BNIP3 transcription is controlled by hypoxia-inducible factor 1alpha (also elevated in prostate cancer), we postulated the posttranscriptional regulation of BNIP3 by miR145 through bioinformatics analysis, and herein we experimentally showed that miR145 negatively regulated BNIP3 by targeting its 3'-untranslated region. Artificial overexpression of miR145 by using adenoviral vectors in prostate cancer PC-3 and DU145 cells significantly downregulated BNIP3, together with the upregulation of AIF, reduced cell growth, and increased cell death. Artificial overexpression of wild-type TP53 in PC-3 cells (which lack TP53 protein) and DU145 cells (in which mutated nonfunctioning TP53 is expressed) significantly upregulated miR145 expression with consequent effects on BNIP3 and cell behavior as with miR145 overexpression. Analysis of prostate cancer (n = 134) and benign prostate (n = 83) tissue sample showed significantly decreased miR145 and increased BNIP3 expression in prostate cancer (P < 0.001), particularly in those with tumor progression, and both molecular changes were associated with unfavorable outcome. Abnormalities of the miR145-BNIP3 pair as part of TP53-miR145-BNIP3-AIF network may play a major role in prostate cancer pathogenesis and progression.

Expression Analysis of caspase-6, caspase-9 and BNIP3 in Prostate Cancer

Altered regulation of cell death is a feature of human cancer. The aim of this study was to explore whether the expression of the proapoptotic proteins caspase-6, caspase-9, and Bcl-2/adenovirus E1B-19kDa-interacting protein3 (BNIP3) is altered in prostate cancers. We analyzed the expression of caspase-6, caspase-9, and BNIP3 in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. Normal glandular cells expressed caspase-6 and BNIP3 proteins in 10 (9.3%) and 9 (8.4%) prostate tissues, respectively. By contrast, the prostate cancers expressed caspase-6 and BNIP3 in 65 (60.7%) and 69 (64.5%) cases, respectively. Prostate intraepithelial neoplasia (PIN) showed caspase-6 and BNIP3 expression in 65% and 65% of cases, respectively. We observed caspase-9 expression in 40 (37.4%) normal, 8 (40%) PIN, and 45 (42.1%) cancer tissues. None of the expression of caspase-6, caspase-9 or BNIP3 was associated with pathological characteristics such as tumor size, patient age, Gleason score, or tumor stage. Our data showed that prostate cancer and PIN cells display higher expression of the proapoptotic proteins caspase-6 and BNIP3 than normal cells. Neo-expression of these proteins from the PIN stage suggests that apoptosis deregulation might occur in the early stage of prostate carcinogenesis, and that altered expression of proapoptotic proteins may be a feature of prostate cancer.

Upregulation of BNIP3 and translocation to mitochondria mediates cyanide-induced apoptosis in cortical cells

Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a Bcl-2 homology domain 3 (BH3) domain only protein, has been identified as a mitochondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary rat cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either increased with BNIP3 cDNA (BNIP3 +) or knocked down with small interfering RNA (RNAi). In BNIP3 + cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (Δ ψ m ), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Ala-Asp-fluoromethyl ketone (zVAD-fmk) suppressed BNIP3 +-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knockdown by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3ΔTM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3ΔTM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3 + cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of Δ ψ m and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduce Δ ψ m . This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death.

Activation of Ras Up-regulates Pro-apoptotic BNIP3 in Nitric Oxide-induced Cell Death

Nitric oxide (NO) produced by NO synthases causes nitration and nitrosylation of cellular factors. We have shown previously that endogenously produced or exogenously added NO induces expression of BNIP3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3), leading to death of macrophages (Yook, Y.-H., Kang, K.-H., Maeng, O., Kim, T.-R., Lee, J.-O., Kang, K.-i., Kim, Y.-S., Paik, S.-G., and Lee, H. (2004) Biochem. Biophys. Res. Commun. 321, 298-305). We now provide evidence that Ras mediates NO-induced BNIP3 expression via the MEK/ERK/hypoxia-inducible factor (HIF)-1 pathway. (a) ras-Q61L, a constitutively active form of Ras, up-regulated BNIP3 protein expression by enhancing Bnip3 promoter activity, and ras-S17N, a dominant-negative form, and ras-C118S, an S-nitrosylation mutant, blocked NO-induced BNIP3 expression, suggesting that Ras acts downstream of NO and that NO activates Ras by nitrosylation. (b) U0126, a specific MEK inhibitor, completely abolished BNIP3 expression and the stimulation of promoter activity by NO and Ras, whereas 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, SB203580, and wortmannin, specific inhibitors of soluble guanylyl cyclase, p38 MAPK, and phosphatidylinositol 3-kinase, respectively, had no effect. Ras, MEK1/2, and ERK1/2 were sequentially activated by NO treatment of macrophages. (c) Mutation of the HIF-1-binding site (hypoxia-response element) in the Bnip3 promoter abolished BNIP3 induction, and HIF-1alpha was strongly induced by NO. (d) Transient expression of activated Ras promoted macrophage death, as did NO, and this Ras-mediated cell death was inhibited by silencing BNIP3 expression. These results suggest that NO-induced death of macrophages is mediated, at least in part, by BNIP3 induction.

Selective silencing of the hypoxia-inducible factor 1 target gene BNIP3 by histone deacetylation and methylation in colorectal cancer

Hypoxia, via the hypoxia-inducible factors 1 and 2 (HIF-1 and HIF-2), upregulates many genes involved in cell survival. However, proapoptotic pathways are also induced. BCL-2/adenovirus E1B-19 kDa-interacting protein 3 (BNIP3) represents a paradigm of a cell death protein that is hypoxically upregulated via HIF-1 in most cancers. We found that in contrast to many other cell types, 6/8 colorectal cancer (CRC) cell lines show little hypoxic induction of BNIP3 despite an intact HIF signalling system. Colorectal tumour tissue also loses BNIP3 expression relative to matched normal samples. Downregulation of hypoxic BNIP3 in CRC cells was independent of the expression of other BCL-2 family members, or BNIP3L. That BNIP3 plays a functional role in hypoxic survival in CRC cells was demonstrated by the fact that CRC cell lines that do not upregulate BNIP3 or have been treated with BNIP3 RNA interference were insensitive to hypoxia-induced cell death. Promoter methylation and histone deacetylation were shown to silence BNIP3 in these CRC cell lines. Of significance, hypoxic induction of BNIP3 was restored in 4/6 cell lines by trichostatin-A treatment alone. These data suggest that BNIP3 plays an important role in hypoxic cell death and epigenetic mechanisms selectively silence its expression in CRC.

Temporomandibular joint dysfunction—A new approach: Claude Pellequer, Claude Payrot and B. MercierRev. Stomatol. 39:93–100, 1981

BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) is a BH3-only protein that regulates apoptosis and autophagy. BNIP3 plays also an important role in hypoxia-induced cell response and is regulated by HIF1. Here, we studied a possible association of BNIP3 expression and the prognosis of patients with renal cell carcinomas (RCCs) and examined the functional relevance of BNIP3 in the regulation of cell survival and apoptosis of renal carcinoma cells. BNIP3 expression was determined by immunohistochemistry in RCC tumor tissue samples of 569 patients using a tissue microarray. Functional characterization of BNIP3 in renal carcinoma cells indicates prosurvival effects. In human RCC tumor samples, high cytoplasmic BNIP3 expression was associated with high-grade RCCs and regional lymph node metastasis. BNIP3 expression correlated negatively with disease-specific survival. Multivariate Cox regression analysis retained BNIP3 expression as an independent prognostic factor in patients without distant metastasis. Together, our studies imply that BNIP3 regulates cell survival in RCCs and its expression is an independent prognostic marker in patients with localized RCCs.