BAG-1 Expression Research Articles

Nuclear or cytoplasmic localization of Bag-1 distinctly correlates with pathologic behavior and outcome of gastric carcinomas

Bag-1 is an antiapoptotic protein with its altered expression and localization in malignancies. To clarify the role of Bag-1 in gastric carcinogenesis, its expression was examined by immunohistochemistry and in situ hybridization on a tissue microarray containing gastric carcinomas, adjacent nonneoplastic mucosa (NNM), adenomas, intestinal metaplasia (IM), or gastritis. Gastric carcinoma tissue and cell lines were studied for Bag-1 expression by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). The results demonstrated that Bag-1 proteins were differentially expressed in the nucleus or cytosol of MKN28, AGS, MKN45, KATO-III, or HGC-27 cell lines, despite similar levels of messenger RNA (mRNA) expression. The Bag-1 mRNA overexpression was detectable in 73.3% of 15 gastric carcinomas without significant difference in its encoding products' levels. The nuclear Bag-1 expression gradually decreased from gastritis, IM, adenoma to carcinoma (P < .05), and negatively correlated with lymphatic invasion or lymph node metastasis, cytoplasmic Bag-1 expression, negative parafibromin expression, and poor prognosis (P < .05). Cytoplasmic Bag-1 was weakly immunoreactive in carcinomas, compared with gastritis (P < .05), and positively associated with invasive depth and poor prognosis of the carcinoma (P < .05). The positive rate of Bag-1 mRNA expression was higher in adjacent IMs than carcinomas or adjacent NNM (P < .05). Bag-1 mRNA was expressed more in carcinomas from female patients than the male counterparts (P < .05). There was a positive correlation of Bag-1 mRNA expression with invasive depth and venous invasion (P < .05). Our study indicated that aberrant expression and subcellular distribution of Bag-1 might play an important role in the malignant transformation of gastric epithelial cells and should be considered as a biomarker for gastric carcinogenesis, subsequent progression, and prognosis.

Expression and Significance of bag-1, bcl-2 in Non-small Cell Lung Cancer and the Correlation with Multi-drug Resistance.

bag-1, bcl-2 and bax are all apoptosis-related proteins. They play a role in the diagnosis, progress, metastasis and prognosis of tumor. The aim of the study was to investigate the expression of bag-1, bcl-2 and bax in non-small cell lung cancer, and to study the relationship between their expression levels and the clinical pathological characteristics, furthermore, to evaluate their correlation with multi-drug resistance. The expressions of bag-1, bcl-2 and bax in 140 non-small cell lung cancer tissues (40 of 140 were processed neoadjuvant chemotherapy) and 15 lung benign lesion tissues were examined with SP immuno-histochemical stain. The positive expression rates of bag-1 and bcl-2 protein in non-small cell lung cancer were significantly higher than those in pulmonary benign lesion tissues (P<0.05), but the positive expression rate of bax in non-small cell lung cancer was significantly lower than that in pulmonary benign lesion tissues (P<0.05). The expressions of bag-1, bcl-2 and bax protein were not related to the age and sex of patients, histological classification, P-TNM stage and lymph node involvement of the cancer (P>0.05), but bag-1 was related to the differentiation degree of the tumor. The lower the differentiation was, the higher the levels of expression of bag-1 were. bcl-2 protein expression was highly positive correlated with the bag-1 protein expression in non-small cell lung cancer (r =0.371, P<0.01), and bcl-2 protein was highly negative correlated with bax protein expression (r=-0.225, P<0.01). The positive expression rates of bag-1 and bcl-2 showed increasing trends from the patients without neoadjuvant therapy to those with neoadjuvant therapy, but the difference had no statistic significance (P>0.05). The high expression of bag-1, bcl-2 protein and the low expression of bax protein exist in nonsmall cell lung cancer. The expression level of bag-1 protein is closely related to the differentiation degree of non-small cell lung cancer. A highly positive correlation exists between bag-1 and bcl-2 expression, and a highly negative correlation is observed between bcl-2 and bax expression. The study doesn't provide the evidence that there is a close correlation between the expression levels of bag-1, bcl-2, bax and the multi-drug resistance in non-small cell lung cancer.

Advanced Osteoarthritis in Humans Is Associated With Altered Collagen VI Expression and Upregulation of ER-stress Markers Grp78 and Bag-1

To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo- or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2-associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cell-associated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patient-matched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen.

Expression and Clinical Role of Antiapoptotic Proteins of the Bag, Heat Shock, and Bcl-2 Families in Effusions, Primary Tumors, and Solid Metastases in Ovarian Carcinoma

Cancer progression is associated with reduced apoptosis and increased proliferation. We hypothesized that upregulation of the Bag family of survival cochaperones and its molecular partners of the Bcl-2 and heat shock protein (HSP) families would correlate with disease progression and survival in ovarian cancer. Bag-1, Bag-4, HSP27, HSP70, Bcl-2, and Bcl-X(L) expression was immunohistochemically analyzed in effusions (188) and patient-matched solid tumors (43 primary carcinomas, 81 solid metastases). Results were analyzed for anatomic site-related differences, and association with clinicopathologic parameters and survival. Bag-1, Bag-4, and HSP70 were detected in the tumor cell nuclei and cytoplasm, whereas HSP27, Bcl-2, and Bcl-X(L) had exclusively cytoplasmic localization. Antiapoptotic protein expression in effusions differed significantly from primary tumors and metastases. Cytoplasmic Bag-1 (P=0.002), nuclear and cytoplasmic HSP70 (P<0.001), and Bcl-2 (P=0.001) expression was higher in primary carcinomas and solid metastases compared with effusions, whereas Bcl-X(L) (P=0.01), nuclear Bag-1 (P<0.001), nuclear Bag-4 (P=0.01), and cytoplasmic Bag-4 (P=0.002) were upregulated in effusions. Bcl-X(L) expression was associated with poor response to chemotherapy at diagnosis (P=0.02) and HSP27 expression was associated with high-grade tumors (P=0.01). Increased cytoplasmic HSP70 staining in effusions correlated with poor overall survival for the entire cohort (P=0.01). In primary carcinomas, higher Bcl-2 expression correlated with worse overall (P=0.04) and progression-free (P=0.02) survival. Antiapoptotic proteins are differentially expressed in effusions compared with solid tumors, whereas primary carcinomas and solid metastases have comparable expression patterns. HSP70 expression in effusions may be a prognostic marker of poor survival, with a similar role for Bcl-2 in primary carcinomas.

Induction of heat shock response by curcumin in human leukemia cells

Heat shock response is an adaptive response, which helps the cells to regulate their physiological homeostasis under stress. Here we show that the natural compound curcumin induces nuclear translocation of the heat shock transcription factor (HSF)-1, its binding to a heat shock regulatory element (HSE), and the subsequent activation of the hsp70 promoter through the extracellular regulated kinase (ERK)/mitogen activated protein (MAP) ERK (MEK) and c-jun N-terminal kinase (JNK) pathways, but not through p38. We observe that curcumin activates hsp70A and hsp70B mRNA transcription, increases HSP protein expression but decreases the expression of Bag-1, a Hsp70 co-chaperone in K562 cells. This induction Hsp70 protein expression goes in line with the anti-inflammatory and anti-proliferative properties of curcumin in chronic myelogenous leukemia.

CD24 expression has a prognostic impact in breast carcinoma

We investigated the prognostic significance of BAG-1 and CD24 in invasive breast carcinomas. Seventy cases of invasive breast carcinoma were studied immunocytochemically for the expression of BAG-1 and CD24. The results were correlated with several prognostic parameters, including 5-year survival. Univariate analysis showed a significant correlation of BAG-1 and CD24 overall positive staining with several adverse prognostic parameters, such as increased stage ( p<0.0001), tumor grade 3 ( p=0.016 and p=0.02, respectively), positive lymph nodes ( p<0.0001), and increased tumor size ( p<0.0001). Similar results were found for BAG-1 nuclear staining, as well as for positive cytoplasmic CD24 expression. Both of our markers studied had a significant, negative effect on survival. Multivariate analysis further revealed an independent prognostic impact for CD24 overall staining. The results of our study showed that overall cytoplasmic and especially nuclear BAG-1 expression, as well as overall and cytoplasmic CD24 expression, correlates with adverse prognostic parameters. An independent prognostic value for overall CD24 staining was also demonstrated.

Correlation between the expression of BAG-1, p53 and platinum-based chemotherapeutic sensitivity in NSCLC

Objective To detect the expression of BAG-1 and p53 in postoperative patients paraffin section of non-snmll cell lung cancer (NSCLC), and retrospectively analyze the relationships of clinical feature and platinum-based chemotherapeutic sensitivity in NSCLC, to further investigate the resistance mechanism of platinum. Methods The expression of BAG-1 and p53 was measured by immunohistochemistry in paraffin sections of 125 NSCLC postoperative patients. The existence and prognosis were analyzed after follow-up. Results BAG-1 and p53 showed high expression in NSCLC (positive rate 64.8 %, 46.7 %); BAG-1 positive or p53 negative expression had survival advantage(P<0.05); The group of BAG-1, p53 negative expression could benefit from platinum-based chemotherapy (P<0.05). Conclusion Expressions of BAG-1, p53 correlates with platinum-based chemotherapeutic sensitivity, and maybe become new types of clinical prognostic indicators and contribute to individualized treatment options for NSCLC. Key words: BAG-1; Genes, p53; Carcinoma, non-small-cell-lung; Platinum; Chemotherapeutic sensitivity

BAG-1 is preferentially expressed in neuronal precursor cells of the adult mouse brain and regulates their proliferation in vitro

BAG-1 protein has been well characterized as necessary for proper neuronal development. However, little is known about the function of BAG-1 in the adult brain. In this work, the expression and localization of BAG-1 in the mature mouse brain was studied. The levels of both BAG-1 isoforms decrease significantly in the brain during development. BAG-1 was found preferentially expressed in Neuronal Precursor Cells (NPCs) in the two major niches of neurogenesis. Lentiviral mediated overexpression of BAG-1 increased the proliferation rate of cultured NPCs. In addition, depletion of BAG-1 from NPCs induced a decrease in NPCs proliferation in the presence of a stress hormone, corticosterone. These data suggest a role for BAG-1 in mechanisms of neurogenesis in the adult mouse brain.

BAG-1 predicts patient outcome and tamoxifen responsiveness in ER-positive invasive ductal carcinoma of the breast

BAG-1 (bcl-2-associated athanogene) enhances oestrogen receptor (ER) function and may influence outcome and response to endocrine therapy in breast cancer. We determined relationships between BAG-1 expression, molecular phenotype, response to tamoxifen therapy and outcome in a cohort of breast cancer patients and its influence on tamoxifen sensitivity in MCF-7 breast cancer cells in vitro. Publically available gene expression data sets were analysed to identify relationships between BAG-1 mRNA expression and patient outcome. BAG-1 protein expression was assessed using immunohistochemistry in 292 patients with invasive ductal carcinoma and correlated with clinicopathological variables, therapeutic response and disease outcome. BAG-1-overexpressing MCF-7 cells were treated with antioestrogens to assess its effects on cell proliferation. Gene expression data demonstrated a consistent association between high BAG-1 mRNA and improved survival. In ER+ cancer (n=189), a high nuclear BAG-1 expression independently predicted improved outcome for local recurrence (P=0.0464), distant metastases (P=0.0435), death from breast cancer (P=0.009, hazards ratio 0.29, 95% CI: 0.114–0.735) and improved outcome in tamoxifen-treated patients (n=107; P=0.0191). BAG-1 overexpression in MCF-7 cells augmented antioestrogen-induced growth arrest. A high BAG-1 expression predicts improved patient outcome in ER+ breast carcinoma. This may reflect both a better definition of the hormone-responsive phenotype and a concurrent increased sensitivity to tamoxifen.

Protective Effect of Adeno-Mediated Human Bcl-xL Gene Transfer to the Mouse Liver in a Partial Ischemia/Reperfusion Model

The protective effect of heat preconditioning has been ascribed to the induction of heat shock proteins (HSP) in the liver. We detected an increase in Bcl-xL expression prior to HSP 70 expression in the rat liver after heat preconditioning. The net effect of overexpression of human Bcl-xL with a recombinant adenovector was estimated in a partial ischemia/reperfusion model of the mouse liver. The time courses of the expression of HSP, Bcl-xL, Bcl-2, Bax, and Bag-1 in the SD rat liver after heat preconditioning were studied by Western blotting. The localizations of Bcl-xL, Bcl-2, and Bax at 6 h after preconditioning were examined by immunostaining. The expression of Bcl-xL in the C57/BL mouse liver after intravenous injection of the recombinant adenovector was assessed by Western blotting and immunostaining. The protective effect of overexpression of Bcl-xL was estimated in a 60-min partial ischemia/reperfusion model of the mouse liver. The expression of Bcl-xL peaked 12 h after heat preconditioning. The overexpression of Bcl-xL decreased enzyme release, histological cell injury, and the number of TUNEL-positive cells. Transfer of the human Bcl-xL gene to the liver had a protective effect against ischemia/reperfusion injury in a mouse model.

Induction of BAG2 protein during proteasome inhibitor‐induced apoptosis in thyroid carcinoma cells

Proteasome inhibitors exhibit cytotoxic against tumours of different histology. However, the mechanism of apoptosis induction by these compounds remains unclear and is likely to be a complex cascade of events. Bcl-2-associated athanogene (BAG) family proteins are characterized by their property of interaction with a variety of partners involved in modulating the proliferation/death balance, including heat shock proteins (HSP), Bcl-2, Raf-1. The role of BAG family proteins in proteasome inhibition has not been elucidated. Effects of proteasome inhibitors on BAG2 expression were evaluated using real-time reverse transcription-polymerase chain reaction (RT-PCR). BAG2 expression was knocked down by small interfering RNAs (siRNA). Cell death was evaluated using Annexin V/propidium iodide staining and subsequent FACS. The proteasome inhibitors, MG132, PSI, lactacystin and epoxomicin, induced BAG2 at the transcriptional level. MG132-induced apoptosis was significantly suppressed by BAG2 knockdown using RNA interference. Our results suggest that BAG2 is a novel molecule induced by proteasome inhibition, which exhibits a pro-apoptotic property in death of thyroid cancer cells induced by proteasome inhibition.

The expression of BAG-1 and its clinical significance in human lung cancer.

BAG-1 is a multifunctional anti-apoptotic protein that binds to a variety of cellular proteins and affects their functions. It has been proven that overexpression of BAG-1 was observed in a number of human maligancies and correlated with their prognosis. The aim of this study is to determine BAG-1 mRNA expression in human lung cancer tissues and its clinical significance. The expression of BAG-1 was detected by real-time PCR in formalin-fixed, paraffin-embedded human lung cancer tissues of 156 cases. (1) The median survival time (MST, 33.5 months) in BAG-1 low-expression group was significantly higher than that (20.5 months) in BAG-1 highexpression group (P <0.005); (2) The 3-year and 5-year survival rates (44.74%; 26.32%) in BAG-1 low-expression group were dramatically higher than that in BAG-1 high-expression group (32.5%; 21.25%)(P <0.05), respectively. The survival curves in BAG-1 low-expression group was also significantly better than that in BAG-1 high-expression group (P =0.045); (3) The further analysis indicated that the survical curves in BAG-1 low-expression group were much better than that in BAG-1 high-expression group in the lung cancer patient with TNM stage I, or with squamous cell cacrinoma, or without any metastasis, respectively (P <0.05). BAG-1 may be a biomarker for the prognosis of lung cancer patients, especially for the lung cancer patients with stage I or squamous cell carcinoma.

The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured Neospora caninum

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.

DNA Methyltransferase 1 and 3B Activate BAG-1 Expression via Recruitment of CTCFL/BORIS and Modulation of Promoter Histone Methylation

In a previous genomic analysis, using somatic methyltransferase (DNMT) knockout cells, we showed that hypomethylation decreased the expression of as many genes as were observed to increase, suggesting a previously unknown mechanism for epigenetic regulation. To address this idea, the expression of the BAG family genes was used as a model. These genes were used because their expression was decreased in DNMT1(-/-), DNMT3B(-/-), and double knockout cells and increased in DNMT1-overexpressing and DNMT3B-overexpressing cells. Chromatin immunoprecipitation analysis of the BAG-1 promoter in DNMT1-overexpressing or DNMT3B-overexpressing cells showed a permissive dimethyl-H3-K4/dimethyl-H3-K9 chromatin status associated with DNA-binding of CTCFL/BORIS, as well as increased BAG-1 expression. In contrast, a nonpermissive dimethyl-H3-K4/dimethyl-H3-K9 chromatin status was associated with CTCF DNA-binding and decreased BAG-1 expression in the single and double DNMT knockout cells. BORIS short hairpin RNA knockdown decreased both promoter DNA-binding, as well as BAG-1 expression, and changed the dimethyl-H3-K4/dimethyl-H3-K9 ratio to that characteristic of a nonpermissive chromatin state. These results suggest that DNMT1 and DNMT3B regulate BAG-1 expression via insulator protein DNA-binding and chromatin dynamics by regulating histone dimethylation.

Expression patterns and prognostic value of Bag-1 and Bcl-2 in breast cancer

IntroductionBcl-2 antanogene-1 (Bag-1) binds the anti-apoptotic mediator Bcl-2, and enhances its activity. Bcl-2 and Bag-1 are associated with chemotherapy resistance in cancer cells. Drugs that target Bcl-2 are currently in clinical development. The purpose of the present study was to examine expression patterns of Bag-1 in a large cohort of breast tumors and to assess the association with Bcl-2, estrogen receptor, progesterone receptor and Her2/neu, and other clinical/pathological variables.MethodsTissue microarrays containing primary specimens from 638 patients with 10-year follow-up were employed, and the expression of Bag-1, Bcl-2, estrogen receptor, progesterone receptor and Her2/neu was assessed using our automated quantitative analysis method. We used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and we measured biomarker expression within the mask using Cy5 conjugated antibodies.ResultsHigh Bcl-2 expression was associated with improved survival in the entire cohort and in the node-positive subset (P = 0.008 and P = 0.002, respectively). High Bag-1 expression was associated with improved survival in the node-positive subset (P = 0.006). On multivariable analysis, neither Bcl-2 nor Bag-1 retained their independence as prognostic markers. Strong associations were found between Bag-1, Bcl-2, estrogen receptor and progesterone receptor.ConclusionBag-1 and Bcl-2 expression in breast tumors is associated with improved outcome and steroid receptor positivity. Evaluation of Bcl-2 and Bag-1 expression in breast cancer may identify a subset of patients with a favorable prognosis, who might not benefit from chemotherapy or who might benefit from Bcl-2 targeting agents in addition to antihormonal therapy.

Subcellular distribution affects BAG1 function

BAG1 is a potent neuroprotectant as well as a marker of differentiation in neuronal cells. It is known that BAG1 mainly localizes to the nucleus during neuronal development, whereas BAG1 shifts to the cytosol upon neuronal differentiation suggesting that distinct BAG1 functions depend on its subcellular localization. Here, we show that forced BAG1 expression within the nucleus when compared to full-length BAG1 expression and to control cells completely abolishes the neuroprotective effects of BAG1 supporting the notion that cytosolic interaction with Hsp70 is mandatory for BAG1 mediated neuroprotection. At the same time, we observed that cells can no longer differentiate into post-mitotic neurons when BAG1 is only present in the nucleus. In addition, phospho-Erk levels are decreased in those cells indicating that BAG1 has to translocate to the cytosol for Raf-dependent MAPK activation.

Lithium blocks stress-induced changes in depressive-like behavior and hippocampal cell fate: The role of glycogen-synthase-kinase-3β

Mood disorders are the most common psychiatric disorders. Although the mechanisms implicated in the genesis of mood disorders are still unclear, stress is known to predispose to depression, and recently, studies have related hippocampal neurogenesis and apoptosis to depression. In the present study we first examined the balance between cell birth–death in the hippocampus and subventricular zone (SVZ) of pre-pubertal and adult rats subjected to chronic-mild-stress (CMS). CMS led to increased corticosterone secretion and induced depressive-like symptoms (assessed in the forced-swimming test); these endocrine and behavioral effects were paralleled by decreased hippocampal, but not SVZ, cell proliferation/differentiation and by increased apoptotic rate. In order to determine if lithium, a known mood stabilizer with antidepressant properties, could prevent the stress-induced events, we analyzed the same parameters in a group of rats treated with lithium during the stress exposure period (CMS+Li) and observed that the hormonal, behavioral and cell turnover effects of CMS were abrogated in these animals. Subsequently, to search for possible pathways through which CMS and lithium influence behavior, cell fate and synaptic plasticity, we analyzed the expression of glycogen-synthase-kinase-3β (GSK-3β), as well as some of its downstream targets (B-cell-CLL/lymphoma2-associated athanonege (BAG-1) and synapsin-I). CMS increased GSK-3β and decreased synapsin-I and BAG-1 expression in the hippocampus. Interestingly, co-administration of lithium precluded the CMS-induced effects in GSK-3β, synapsin-I and BAG-1 expression. Our observation that specific inhibition of this kinase with AR-A014418 blocked the effects of CMS in depressive-like behavior and in BAG-1 and synapsin-I expression confirmed the involvement of the GSK-3β pathway in stress-induced effects. In summary, these results reveal that lithium, by regulating the activity of GSK-3β, prevents the deleterious effects of stress on behavior and cellular functions.

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.

POS-02.45: Bag-1 and bax expression in benign prostatic hyperplasia and prostate cancer

Apoptosis is a decisive mechanism in cell processes such as homeostasis and development. Dysregulation of apoptosis may contribute to the process of prostate tumorigenesis by reducing the rate of cell death. Bag-1 and bax are important molecules involved in the regulation of apoptosis and prostate cancer is a prevalent disease among western countries. The aim of the present study is to examine the differential expression of the above apoptotic genes in a group of patients with hyperplasia and prostate cancer.

Cytoplasmic Localization of BAG-1 in Leukoplakia and Carcinoma of the Tongue: Correlation with p53 and C-Erbb2 in Carcinoma

The present study evaluated the clinical significance of BAG-1, an antiapoptotic protein, in leukoplakia and carcinoma of the tongue. BAG-1 expression was evaluated by immunohistochemistry in paraffin-embedded tissues of leukoplakia (n=25) and carcinoma of the tongue (n=61). Cytoplasmic expression was predominantly seen in 80% and 70% of patients with leukoplakia and carcinoma, respectively. BAG-1 expression was found to be significantly lower in tobacco users than in non-tobacco users. BAG-1 expression in tobacco-using leukoplakia and carcinoma patients was compared by grouping the carcinoma patients according to lymph node status and disease stage. Carcinoma patients with tumor-positive lymph nodes had significantly lower BAG-1 expression than patients with negative lymph nodes and leukoplakia. Further, a trend towards an inverse correlation was observed with p53 and c-erbB2. In univariate and multivariate survival analysis, patient subgroups with 2+ or 3+ marker positivity (BAG-1 negativity, p53 and c-erbB2 positivity) had a reduced overall survival compared with patient subgroups with 1+ marker positivity or negativity. BAG-1 negativity in association with p53 and c-erbB2 positivity identified a subgroup of tongue cancer patients with an aggressive phenotype. Hence, an antiapoptotic protein, BAG-1, was found to be down-regulated in chewing-tobacco-mediated tongue carcinogenesis.