Bcl-2-associated Athanogene-1 Expression Research Articles

Supramolecular nanodrug targeting CDK4/6 overcomes BAG1 mediated cisplatin resistance in oral squamous cell carcinoma

Chemoresistance to cisplatin remains a significant challenge affecting the prognosis of advanced oral squamous cell carcinoma (OSCC). However, the specific biomarkers and underlying mechanisms responsible for cisplatin resistance remain elusive. Through comprehensive bioinformatic analyses, we identified a potential biomarker, BCL2 associated athanogene-1 (BAG1), showing elevated expression in head and neck squamous cell carcinoma (HNSCC). Since OSCC represents the primary pathological type of HNSCC, we investigated BAG1 expression in human tumor tissues and cisplatin resistant OSCC cell lines, revealing that silencing BAG1 induced apoptosis in cisplatin-resistant cells both in vitro and in vivo. This effect led to impaired cell viability of cisplatin resistant OSCC cells and indicated a positive correlation between BAG1 expression and the G1/S transition during cell proliferation. Based on these insights, the administration of a CDK4/6 inhibitor in combination with cisplatin effectively overcame cisplatin resistance in OSCC through the CDK4/6-BAG1 axis. Additionally, to enable simultaneous drug delivery and enhance synergistic antitumor efficacy, we developed a novel supramolecular nanodrug LEE011-FFERGD/CDDP, which was validated in an OSCC orthotopic mouse model. In summary, our study highlights the potential of a combined administration of CDK4/6 inhibitor and cisplatin as a promising therapeutic regimen for treating advanced or cisplatin resistant OSCC.

An increase in BAG-1 by PD-L1 confers resistance to tyrosine kinase inhibitor in non–small cell lung cancer via persistent activation of ERK signalling

High programmed cell death 1 ligand 1 (PD-L1) expression in tumour tissues was associated with poor outcomes in non–small cell lung cancer (NSCLC) due to evasion of tumour immune surveillance. However, the role of PD-L1 in tumour invasion and resistance to tyrosine kinase inhibitor (TKI) treatments is not fully understood. Here, we provide evidence to support the involvement of PD-L1 expression in the invasiveness and TKI resistance in NSCLC cells by increased Bcl-2-associated athanogene-1 (BAG-1) expression. The upregulation of BAG-1 transcription by PD-L1 was verified by constructing the BAG-1 promoters using the polymerase chain reaction (PCR) and deletion mutations for luciferase reporter assays. The results indicated that C/EBPβ phosphorylation by extracellular signal-regulated kinase (ERK) signalling was responsible for PD-L1-mediated BAG-1 transcription. Mechanistically, the PD-L1-induced BAG-1 expression reciprocally increased PD-L1 expression due to persistent activation of ERK signalling, and it consequently conferred TKI resistance in NSCLC cells. The mechanistic action of this cell model was further confirmed by an animal model, affirming that PD-L1 conferred tumour invasiveness and TKI resistance via persistent activation of ERK signalling by the PD-L1/BAG-1 axis. We therefore suggest a combination of an ERK inhibitor with a TKI as a potential strategy for conquering PD-L1-mediated tumour invasion and TKI resistance in NSCLC patients whose tumours harbour high PD-L1/high BAG-1 expression.

Over-expression of BAG-1 in head and neck squamous cell carcinomas (HNSCC) is associated with cisplatin-resistance

BackgroundIn order to improve therapy for head and neck squamous cell carcinoma (HNSCC), biomarkers associated with local and/or distant tumor relapses and cancer drug resistance are urgently needed. This study identified a potential biomarker, Bcl-2 associated athanogene-1 (BAG-1), that is implicated in HNSCC insensitive to cisplatin and tumor progression.MethodsPrimary and advanced (relapsed from parental) University of Michigan squamous cell carcinoma cell lines were tested for sensitivity to cisplatin and gene expression profiles were compared between primary (cisplatin sensitive) and the relapsed (cisplatin resistant) cell lines by using Agilent microarrays. Additionally, differentially expressed genes phosphorylated AKT, and BAG-1, and BCL-xL were evaluated for expression using HNSCC tissue arrays.ResultsAdvanced HNSCC cells revealed resistant to cisplatin accompanied by increased expression of BAG-1 protein. siRNA knockdown of BAG-1 expression resulted in significant improvement of HNSCC sensitivity to cisplatin. BAG-1 expression enhanced stability of BCL-xL and conferred cisplatin resistant to the HNSCC cells. In addition, high levels of expression of phosphorylated AKT, BAG-1, and BCL-xL were observed in advanced HNSCC compared to in that of primary HNSCC.ConclusionIncreased expression of BAG-1 was associated with cisplatin resistance and tumor progression in HNSCC patients and warrants further validation in larger independent studies. Over expression of BAG-1 may be a biomarker for cisplatin resistance in patients with primary or recurrent HNSCCs and targeting BAG-1 could be helpful in overcoming cisplatin resistance.

BAG-1 as a biomarker in early breast cancer prognosis: a systematic review with meta-analyses

Background:The co-chaperone protein Bcl-2-associated athanogene-1 (BAG-1) is overexpressed in breast cancer and has been incorporated in the oncotype DX and PAM50 breast cancer prognostic assays. Bcl-2-associated athanogene-1 exists as multiple protein isoforms that interact with diverse partners, including chaperones Hsc70/Hsp70, Ser/Thr kinase Raf-1 and Bcl-2, to promote cancer cell survival. The BAG-1L isoform specifically binds to and increases the transcriptional activity of oestrogen receptor in cells, and in some, but not all studies, BAG-1 expression is predictive of clinical outcome in breast cancer.Methods:A systematic review of published studies reporting BAG-1 (mRNA and/or protein) expression and clinical outcome in early breast cancer. The REporting Recommendations for Tumour MARKer and Prognostic Studies (REMARK) criteria were used as a template against which data were assessed. Meta-analyses were performed for studies that provided a hazard ratio and 95% confidence intervals for clinical outcomes including disease-free survival or breast cancer-specific survival from univariate analysis.Results:Eighteen studies used differing methodologies and reported on differing outcomes. Meta-analyses were only possible on results from a subset of reported studies. Meta-analyses suggested improved outcome with high BAG-1 mRNA and high BAG-1 nuclear expression by immunohistochemisty.Conclusions:Increased levels of BAG-1 are associated with better breast cancer outcomes.

미만형 위암에서 임상병리학적 인자와 Hsp70, BAG1과 Raf-1 발현간의 상관성

본 연구에서는 미만형 위암조직에서의 임상병리학적 인자와 HSP70, BAG1, Raf-1의 발현간의 상관관계를 평가하고자 하였다. Hsp70은 열충격단백질(heat shock protein)의 환경적 스트레스로부터 세포를 보호하는 분자생물학적 chaperone으로 작용하는 것뿐 아니라 anti-apoptotic 작용을 통하여 암세포의 생존과 전이를 촉진시키는 것으로 알려져 있다. Proto-oncogene의 일종인 Raf는 사람을 포함한 진핵세포의 생장에 관여하는 중요한 신호전달경로인 mitogen-activated protein kinase (MAPK)경로의 중심인자로서 다양한 암에서 그 발현이 증가한다고 알려져 있다. Bcl-2에 결합하여 세포사멸을 저해하는 것으로 처음 보고된 Bcl-2-associated athanogene-1 (BAG-1)은 다양한 암에서 그 발현이 증가된다고 보고되었다. 또한 BAG-1은 HSP70과 상호작용하여 Raf-1-ERK signal pathway와 cell growth를 조절하는 것으로 보고되었다. Hsp70의 발현은 임파절 전이가 있는 경우 24례(64.9%)에서 양성, 13례(35.1%)에서 음성을 보였으며, 임파절 전이가 없는 경우 6례(26.1%)에서 양성, 17례(73.9%)에서 음성을 보여 임파절의 전이가 있는 경우가 임파절 전이가 없는 경우에 비해 유의하게 높은 발현율을 보였다(p=0.007). N-stage에 따른 발현은 N-stage가 증가함에 따라 발현율이 유의하게 증가하였다(p=0.006). BAG1의발현은 전체 대상 환자 중 43례(71.7%)에서 양성, 17례(28.3%)에서 음성소견을 보였으며, 65세 미만에서 65세 이상에 비해 유의하게 높은 발현율을 보였다(p=0.035). RAF1의 경우 전체 대상 환자 중 45례(75.0%)에서 양성, 15례(25.0%)에서 음성소견을 보였으며, BAG1 양성환자에서 RAF1의 발현이 유의하게 증가 하였다(p=0.021). 본연구 결과 Hsp70은 종양의 진행과 상관성이 있을 것으로 생각되어지며, BAG1과 Raf-1은 미만형위 암의 진단적 마커로 유용할 것으로 사료된다. The aim of this study was to evaluate the relationships between the expression of Heat shock protein70 (HSP70), Raf-1 and Bcl-2-associated athanogene-1 (BAG1) protein in diffuse type gastric carcinoma and examine association of HSP70, Raf-1 and BAG1 expression with various clinic-pathological factors and survival. Heat shock protein70 is induced in the cells in response to various stress conditions, including carcinogens. Overexpression of heat shock protein 70 has been observed in many types of cancer. The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. Overexpression of BAG1 protein has been documented in some type of human cancer. BAG1 has been reported to interact with protein involved with a variety of signal pathway, and regulation of cell differentiation, survival and apoptosis. These interaction partners include HSP70 and Raf-1. The percentage of tumors exhibiting HSP70 positivity was significantly in cases of positive lymph node metastasis (64.9%) compared to cases without lymph node metastasis (35.1%, p=0.007). HS70 expression was correlated with pathological N-stage (p=0.006). Expression of BAG1 was detected in the majority of diffuse type gastric carcinoma tissues (71.7%), especially in younger patients (80% vs 52.6%, p=0.035). Furthermore BAG1 expression was correlated with tumor size (p=0.020). Raf-1 expression was found to be significantly associated with tumor size (p=0.005). The result indicate that HSP70 was significantly correlated the progression of diffuse type gastric cancer. Expression of BAG1 and Raf-1 may be used as diagnostic markers for gastric carcinoma.

Expression and role of BAG-1 in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps.

The pathogenesis of human eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP and nECRSwNP) remains undetermined. We aimed to investigate the expression and role of Bcl-2-associated athanogene-1 (BAG-1) and heat shock protein 70 (Hsp70) in ECRSwNP and nECRSwNP. BAG-1 protein expression was investigated by immunohistochemistry and western blotting, and messenger RNA (mRNA) expression of BAG-1 and Hsp70 was assessed by real-time polymerase chain reaction (PCR) in 27 subjects with ECRSwNP, 28 subjects with nECRSwNP, and 23 control subjects. Moreover, the effects of various stimulators with different concentrations and time on BAG-1 were evaluated on a nasal explant culture. Results showed that significant elevations in total visual analog scale (VAS) score, endoscopy score, CT score, and rate of positive skin prick test (SPT) were found in the ECRSwNP group compared with the nECRSwNP group. BAG-1 and Hsp70 were overexpressed in CRSwNP, especially in ECRSwNP, and BAG-1 expression was closely related to Hsp70. BAG-1 mRNA was augmented by IL-4, IL-17A, and IL-1β, but suppressed by IFN-γ, respectively. In conclusion, eosinophils triggered a more severe form of inflammation in CRSwNP compared with non-eosinophilic inflammatory cells. The expressions of BAG-1 and Hsp70 were upregulated in CRSwNP, especially in ECRSwNP. IL-4, IL-17A, and IL-1β may be critical for BAG-1 gene expression.

Overexpressed nuclear BAG‐1 in human hepatocellular carcinoma is associated with poor prognosis and resistance to doxorubicin

Bcl-2-associated athanogene-1 (BAG-1) is a multifunctional anti-apoptotic protein which regulates an array of cellular processes, including apoptosis, signaling, proliferation, transcription, and cell motility and has been reported to be over-expressed in a number of human malignancies. To investigate the possible involvement of BAG-1 in tumorigenesis of hepatocellular carcinoma (HCC), we performed Western blot analysis in eight paired samples of HCC and adjacent peritumoral tissues and immunohistochemistry in 65 paraffin sections of HCC, which both showed an enhanced expression of nuclear BAG-1 isoform in HCC tissues. Statistical analysis confirmed that overexpression of nuclear BAG-1 in HCC tissues was significantly associated with histological grading (P < 0.001), poor prognosis (P = 0.004), and was found to be an independent prognostic indicator for HCC (P = 0.023). We also noted that BAG-1 was overexpressed in four HCC cell lines compared with a normal hepatocyte cell line, and BAG-1 overexpression increased resistance of HCC cells to doxorubicin, a common chemotherapeutic agent for HCC. Furthermore, we observed that knock down of BAG-1 with siRNA in HepG2 cells increased the chemosensitivity of cells, a process mediated through inhibition of doxorubicin-triggered NF-κB activation; and knock down of BAG-1 suppressed proliferation and cell cycle transition of HepG2 cells. In consequence, our results for the first time indicated that BAG-1 was dysregulated in HCC and suppression of BAG-1 expression which resulted in inhibiting of NF-κB signaling might be developed into a new strategy in HCC therapy.

Role of BCL2-Associated Athanogene 1 in Differential Sensitivity of Human Endothelial Cells to Glucocorticoids

Chronic therapy with synthetic glucocorticoids has been associated with cardiovascular side effects, although differential interindividual susceptibility to glucocorticoids has been observed. The objective of this study was to identify the molecular mechanisms leading to differential glucocorticoid responses in endothelial cells. We tested the sensitivity of 42 human umbilical vein endothelial cells (HUVECs) to dexamethasone as determined by changes in gene expression, promoter transactivation, and procoagulant activity. We identified that 16 HUVECs were sensitive in every test, 14 HUVECs were sensitive in at least 1 test and 12 HUVECs were resistant in every test to dexamethasone. Nuclear translocation assays revealed that Dex-sensitive HUVECs have higher basal and Dex-stimulated levels of nuclear glucocorticoid receptor compared with Dex-resistant HUVECs. Cycloheximide assays revealed that Dex-resistant HUVECs have significantly shorter glucocorticoid receptor protein half-lives than Dex-sensitive HUVECs. Dex-resistant HUVECs have a stronger interaction of glucocorticoid receptor with the proteasomal recruiting protein, BCL2-associated athanogene 1 (BAG1), as shown by immunoprecipitation assays. Silencing BAG1 expression increased Dex-sensitivity in resistant HUVECs, whereas BAG1 overexpression decreased Dex-sensitivity in sensitive HUVECs. Finally, Dex-resistant HUVECs presented higher BAG1 expression than Dex-sensitive HUVECs. In vitro endothelial sensitivity to Dex varies within individuals and is inversely proportional to BAG1 protein expression and glucocorticoid receptor protein turnover.

BAG1 Overexpression Restrains the Anti-Apoptotic BCL2, MCL1 and HSP70 Proteins in Acute Myeloid Leukemia.

AbstractAbstract 2492 BACKGROUND:Childhood Acute Myeloid Leukemia (AML) is a heterogeneous disease characterized by recurrent genetic aberrations; impaired apoptosis of leukemia cells can also contribute to disease development. BCL2-associated AthanoGene1 (BAG1) is a multifunctional protein preferentially supporting cell survival. Its aberrant expression was demonstrated in diverse cancer types. We previously reported high expression of BAG1 protein in leukemia cell lines. We showed that leukemic cell survival was impaired after silencing BAG1. Additionally, we confirmed that, in vitro, BAG1 acted in synergy with BCL2 and influenced MCL1 expression, both being important anti-apoptotic players in AML (Aveic et al. PlosOne 2011). AIM:We investigated BAG1 expression and role in pediatric AML samples collected at diagnosis, as well as its partner molecules BCL2, MCL1 and HSP70. We then evaluated whether BAG1 may affect patient survival. METHODS:Expression of BAG1 was studied at mRNA and protein levels, using RQ-PCR and Western Blot (WB). Initially, 10 patients with AML whose samples were collected at diagnosis and during therapy while they were in disease remission, were included. Subsequently, wide protein expression was performed by Reverse-phase protein array (RPPA) on a further cohort of 66 newly diagnosed AML patients enrolled into the AIEOP-LAM 2002 protocol. RESULTS:BAG1 mRNA resulted to be heterogeneously expressed in AML cases. Furthermore, BAG1 protein was highly expressed in leukemia samples when compared with healthy bone marrow (hbm), and with patient bm in remission, where BAG1 was undetectable by WB analysis. By RPPA, we confirmed the overexpression of BAG1 protein in 56/66 AML patients (84.8 %) with respect to hbm (113616 ± 66721 Arbitrary Unit (AU); mean fold increase: 2.36). Then, we studied its anti-apoptotic co-partner BCL2, whose expression was found to correlate with BAG1 over-expression (110033 ± 89117 AU, p<0.001). We hypothesized that high BAG1 and BCL2 expression may sustain anti-apoptotic signal in AML. We demonstrated that the active phosphorylated form of BCL2, as well as MCL1, resulted to be overexpressed in AML pediatric patients (BCL2S70 = 70387 ± 40864 AU; p<0.001; MCL1 = 112309 ± 49254 AU; p<0.001). BAG1 has a more specialized role in regulating protein degradation; such a specialized function is accomplished together with HSP70 that co-localized with BAG1 in complexes. HSP70 accumulates inside the cells after exposure to stress, promoting their survival, and, in contrast to normal cells, it is expressed abundantly in most cancers. We showed a positive correlation between HSP70 protein expression and BAG1, BCL2 and MCL1 overexpression (112420 ± 4289 AU; p<0.001). We subdivided AML patients according to BAG1 protein expression into quartiles and studied its relation with biological and clinical features. No significant correlation was found with karyotype, molecular genetics, FAB-morphology, age and white blood count. We highlighted a higher frequency of t(8;21) AML1-ETO translocated patients (who are stratified in standard risk group with a better prognosis in the AIEOP AML 2002 protocol) with low BAG1 expression [9/14 patients (64.3 %) of first quartile; p<0.001]. Studying the event-free survival through the Kaplan Meier method of patients with low (quartile 1+2) and high BAG1 expression (quartiles 3+4), we found a better outcome for patients belonging to the former group, as compared to the latter one (72 % EFS vs. 54% at 3y, p= 0.08, SR= 0.1). CONCLUSIONS:We demonstrate that BAG1 is overexpressed in the majority of childhood AML, and that this correlated with high levels of anti-apoptotic proteins such as BCL2, MCL1 and HSP70. We propose that an aberrant activation of the anti-apoptotic pathway in AML may play a crucial role in leukemia development, and it deserves further investigations. Disclosures:No relevant conflicts of interest to declare.

Changes in the BAG1 Expression of Schwann Cells After Sciatic Nerve Crush

Bcl-2-associated athanogene-1 (BAG1), a co-chaperone for Hsp70/Hsc70, is a multifunctional protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. However, the expression and roles of BAG1 in peripheral system lesions and repair are still unknown. In this study, we investigated the dynamic changes in BAG1 expression in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that BAG1 was expressed in normal sciatic nerves. BAG1 expression increased progressively after sciatic nerve crush, reached a peak 2 weeks post-injury, and then returned to the normal level 4 weeks post-injury. Spatially, we observed that BAG1 was mainly expressed in Schwann cells and that BAG1 expression increased in Schwann cells after injury. In vitro, we found that BAG1 expression increased during the cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation process. BAG1-specific siRNA inhibited cAMP-induced Schwann cell differentiation. In conclusion, we speculated that BAG1 was upregulated in the sciatic nerve after crush, which was associated with Schwann cell differentiation.

BAG1 restores formation of functional DJ-1 L166P dimers and DJ-1 chaperone activity

Mutations in the gene coding for DJ-1 protein lead to early-onset recessive forms of Parkinson's disease. It is believed that loss of DJ-1 function is causative for disease, although the function of DJ-1 still remains a matter of controversy. We show that DJ-1 is localized in the cytosol and is associated with membranes and organelles in the form of homodimers. The disease-related mutation L166P shifts its subcellular distribution to the nucleus and decreases its ability to dimerize, impairing cell survival. Using an intracellular foldase biosensor, we found that wild-type DJ-1 possesses chaperone activity, which is abolished by the L166P mutation. We observed that this aberrant phenotype can be reversed by the expression of the cochaperone BAG1 (Bcl-2-associated athanogene 1), restoring DJ-1 subcellular distribution, dimer formation, and chaperone activity and ameliorating cell survival.

Neuron-specific overexpression of the co-chaperone Bcl-2-associated athanogene-1 in superoxide dismutase 1 G93A–transgenic mice

Bcl-2-associated athanogene-1 (BAG1) binds heat-shock protein 70 (Hsp70)/Hsc70, increases intracellular chaperone activity in neurons and proved to be protective in several models for neurodegeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases. A common property shared by all mutant SOD1 (mtSOD1) species is abnormal protein folding and the propensity to form aggregates. Toxicity and aggregate formation of mutant SOD1 can be overcome by enhanced chaperone function in vitro. Moreover, expression of mtSOD1 decreases BAG1 levels in a motoneuronal cell line. Thus, several lines of evidence suggested a protective role of BAG1 in mtSOD1-mediated motoneuron degeneration. To explore the therapeutic potential of BAG1 in a model for ALS, we generated SOD1 G93A/BAG1 double transgenic mice expressing BAG1 in a neuron-specific pattern. Surprisingly, substantially increased BAG1 protein levels in spinal cord neurons did not significantly alter the phenotype of SOD1 G93A-transgenic mice. Hence, expression of BAG1 is not sufficient to protect against mtSOD1-induced motor dysfunction in vivo. Our work shows that, in contrast to the in vitro situation, modulation of multiple cellular functions in addition to enhanced expression of a single chaperone is required to protect against SOD1 toxicity, highlighting the necessity of combined treatment strategies for ALS.

Bcl-2 associated athanogene-1 overexpression in diffuse large B-cell lymphoma.

Apoptosis and cell cycle regulation play an important role in pathogenesis and tumor progression in patients with Diffuse Large B-Cell Lymphoma (DLBCL). Bcl-2 associated athanogene-1 (BAG-1) is an antiapoptotic protein as well as a regulator of cell growth. There is no conclusive evidence about BAG-1 protein expression in this disease. To investigate the expression level of BAG-1 protein in DLBCL. Thirty patients diagnosed from 1997-2004, as having DLBCL, were selected. Also 30 normal lymph nodes were included as normal counterparts in this study. BAG-1 expression was determined by immunohistochemical staining in both DLBCL and normal lymph node samples. Of the 30 DLBCLs examined, 100% were positive for nuclear and 83% were positive for cytoplasmic BAG-1 staining. Of the 30 normal lymph nodes investigated, 20% were positive for nuclear and 0% were positive for cytoplasmic BAG-1 staining. Nuclear staining in DLBCL samples was significantly higher than those of normal lymph nodes (100% versus 20%, p <0.001). Besides, cytoplasmic staining in DLBCL samples was significantly higher than those of normal lymph nodes (83% versus 0%, p <0.001). There was no association between BAG-1 staining and patients' overall survival. Our data indicated that BAG-1 protein was deregulated in this disease similar to some other malignancies such as breast and colon cancer. Overexpression of BAG-1 in DLBCL suggests that this protein probably plays an important role in the pathogenesis of DLBCL. Besides, higher nuclear BAG-1 staining might be correlated with poor prognosis.

BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-κB activity

Although expression of the anti-apoptotic protein Bcl-2-associated athanogene-1 (BAG-1) has been reported as up-regulated in a number of malignancies, we show for the first time that BAG-1 is over-expressed in medium/large-sized colorectal adenomas and carcinomas compared with normal epithelium. To investigate whether expression of BAG-1 is important for colorectal tumour cell survival, microarray analysis was carried out on the HCT116 colorectal carcinoma cell line following transfection with BAG-1 small interfering RNA (siRNA). Analysis identified altered expression of a subset of potential nuclear factor-kappaB (NF-kappaB)-regulated genes. Furthermore, knock down of BAG-1 was shown to inhibit NF-kappaB transcriptional activity. Inhibition of NF-kappaB activity using BAG-1 siRNA or the NF-kappaB inhibitor BAY-117082 suppressed HCT116 cell yield and induced apoptosis; combined treatment had no additive effect, suggesting that the decrease in cell yield associated with knock down of BAG-1 expression is mediated via inhibition of NF-kappaB. Of clinical relevance, BAG-1 siRNA sensitized colorectal carcinoma cells to apoptosis induced by potential therapeutic agent TRAIL as well as tumour necrosis factor-alpha, both inducers of NF-kappaB activity. In summary, knock down of BAG-1 leads to inhibition of NF-kappaB, identifying BAG-1 as a novel regulator of NF-kappaB. It is proposed that, by inhibiting NF-kappaB, suppression of BAG-1 could represent a novel strategy to impede colorectal cancer cell survival and as an adjuvant increase sensitivity to current therapeutic regimes.

BAG-1 expression in normal endometrium, endometrial hyperplasia and endometrial cancer

BAG-1 (Bcl-2-associated athanogene 1) is a BCL-2 binding anti-apoptotic protein that may play a role in carcinogenesis. The purpose of this study is to compare the expression rate of BAG-1 in normal endometrium, endometrial hyperplasia and endometrial cancer, and further to determine a correlation between BAG-1 expression and clinicopathological parameters, and overall survival. Tissue samples from 43 patients who were diagnosed with endometrial cancer, tissue samples from 20 patients with endometrial hyperplasia and tissue samples from 20 normal patients were included in the study. Immunohistochemical analyses were performed using a polyclonal anti-BAG-1 antibody from paraffin-embedded blocks. Cytoplasmic BAG-1 expression of the normal endometrium, endometrial hyperplasia and endometrial cancer samples was 4/20 (20%), 3/20 (15%) and 27/43 (62%), respectively. Nuclear BAG-1 expression was 17/20 (85%), 12/20 (60%) and 16/43 (37%), respectively. Cytoplasmic BAG-1 expression correlated with cancer grade (p=0.02). The mean survival of patients with positive/negative cytoplasmic BAG-1 expression and nuclear BAG-1 expression was 49.4/45.4 and 54.0/41.1 months, respectively, but there was no statistical difference for survival (log-rank p=0.31, p=0.55). Cytoplasmic BAG-1 is more frequently expressed in endometrial cancer tissues than in normal and endometrial hyperplasia tissues (p=0.0007), and its expression correlates with cancer grade. Nuclear BAG-1 is more frequently expressed in the normal endometrium and hyperplasia tissues than in endometrial cancer tissues (p=0.002). Neither cytoplasmic nor nuclear BAG-1 expression is associated with survival.

Bcl-2-associated athanogene-1 (BAG-1): A transcriptional regulator mediating chondrocyte survival and differentiation during endochondral ossification

BAG-1, an anti-apoptotic protein, was identified by its ability to bind to BCL-2, HSP70-family molecular chaperones and nuclear hormone receptor family members. Two BAG-1 isoforms, BAG-1L (50 kDa) and BAG-1S (32 kDa) were identified in mouse cells and BAG-1 expression was reported in murine growth plate and articular chondrocytes. The present study aimed to elucidate the role of BAG-1 in the regulation of molecular mechanisms governing chondrocyte differentiation and turnover during endochondral ossification. In long bones of skeletally immature mice, we observed expression of BAG-1 in the perichondrium, osteoblasts, osteocytes in the bone shaft, bone marrow, growth plate and articular chondrocytes. Monolayer cultures of murine chondrocytic ATDC5 cells, which exhibited robust expression of both BAG-1 isoforms and the Bag-1 transcript, were utilized as an in vitro model to delineate the roles of BAG-1. Overexpression of BAG-1L in ATDC5 cells resulted in downregulation of Col2a1 expression, a gene characteristically downregulated at the onset of hypertrophy, and an increase in transcription of Runx-2 and Alkaline phosphatase, genes normally expressed at the onset of chondrocyte hypertrophy and cartilage mineralization in the process of endochondral ossification. We also demonstrated the anti-apoptotic role of BAG-1 in chondrocytes as overexpression of BAG-1 protected ATDC5 cells, which were subjected to heat-shock at 48 °C for 30 min, against heat-shock-induced apoptosis. Overexpression of the SOX-9 protein in ATDC5 cells resulted in increased Bag-1 gene expression. To further investigate the regulation of Bag-1 gene expression by SOX-9, CHO cells were co-transfected with the human Bag-1 gene promoter– Luciferase reporter construct and the human pSox-9 expression vector. Activity of the Bag-1 promoter was significantly enhanced by the SOX-9 protein. In conclusion, a novel finding of this study is the role of BAG-1 as a transcriptional regulator of genes involved in chondrocyte hypertrophy and cartilage mineralization during the process of endochondral ossification. Additionally, we have demonstrated for the first time the regulation of Bag-1 gene expression by SOX-9 and the anti-apoptotic role of BAG-1 in chondrocytic cells. Modulation of Bag-1 expression can therefore mediate chondrocyte differentiation and turnover, and offer further insight into the molecular regulation of endochondral ossification.

A novel role for Bcl‐2 associated‐athanogene‐1 (Bag‐1) in regulation of the endoplasmic reticulum stress response in mammalian chondrocytes

BAG-1 (Bcl-2 associated athanogene-1) is a multifunctional protein, linking cell proliferation, cell death, protein folding, and cell stress. In vivo, BAG-1 is expressed in growth plate and articular cartilage, and the expression of BAG-1 is decreased with aging. Chondrocytes respond to endoplasmic reticulum (ER) stress with decreased expression of extracellular matrix proteins, and prolonged ER stress leads to chondrocyte apoptosis. Here we demonstrate for the first time that BAG-1 is involved in ER stress-induced apoptosis in chondrocytes. Induction of ER stress through multiple mechanisms all resulted in downregulation of BAG-1 expression. In addition, direct suppression of BAG-1 expression resulted in chondrocyte growth arrest and apoptosis, while stable overexpression of BAG-1 delayed the onset of ER stress-mediated apoptosis. In addition to regulating apoptosis, we also observed decreased expression of collagen type II in BAG-1 deficient chondrocytes. In contrast, overexpression of BAG-1 resulted in increased expression of collagen type II. Moreover, under ER stress conditions, the reduced expression of collagen type II was delayed in chondrocytes overexpressing BAG-1. These results suggest a novel role for BAG-1 in supporting viability and matrix expression of chondrocytes.

The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: implications for colorectal tumour cell survival

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide variety of human cancers, the retinoblastoma protein (Rb) has been shown to be overexpressed in colon cancers, which is linked to the anti-apoptotic function of the protein. However, the mechanisms by which Rb regulates apoptosis are yet to be fully elucidated. We have established that Rb interacts with the anti-apoptotic BAG-1 (Bcl-2 associated athanogene-1) protein, and that a decrease in nuclear localization of BAG-1 is detectable when the interaction between Rb and BAG-1 is disrupted by expression of the E7 viral oncoprotein. Interestingly, although reported as deregulated in colorectal cancers, we have found that BAG-1 expression is also altered in small adenomas, where its localization was found to be predominantly nuclear. In addition, we have established that maintenance of high nuclear BAG-1 in vitro increases the resistance of adenoma-derived cells to gamma-radiation-induced apoptosis. Our work suggests a novel function for Rb, involving modulation of the subcellular localization of BAG-1. We have found predominant nuclear BAG-1 localization in small adenomas, and suggest that BAG-1 may promote colorectal tumour cell survival by making colonic epithelial cells less sensitive to DNA damage.

BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCV(E) promoter and to a lesser extent the JCV(L) promoter. Mutations in the NF-1 sites in the JCV(E) promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.