The BAG-1 cochaperone is a negative regulator of p73-dependent transcription.

High-level expression of Bcl-2 associated athanogene (BAG-1) protects cancer cells from stress-induced cell death and growth inhibition. These protective effects of BAG-1 are dependent on interactions with the HSC70 and HSP70 chaperones. However, the key stress-response molecules that are regulated by a BAG-1/chaperone mechanism have not been identified. In this study, we investigated the effects of BAG-1 overexpression on the function of p53 family proteins, p53, p63 and p73. Overexpression of BAG-1 isoforms interfered with the transactivating activity of p73 and p63, but had modest and variable effects on p53-dependent transcription. p73 and BAG-1 interacted in intact cells and overexpression of BAG-1 decreased the expression of p73. siRNA-mediated ablation of endogenous BAG-1 increased the activity of a p73-responsive promoter and this was reversed by knock-down of p73. The ability of BAG-1 to modulate p73 activity and expression, and to interact with p73 were dependent on amino acid residues required for the interaction of BAG-1 with HSC70 and HSP70. These results show that BAG-1 inhibits the transactivating functions of p73 and provide new insight into the mechanisms that control the expression of p73. Inhibition of p73 function may be one mechanism that contributes to the pro-survival activity of BAG-1.

BAG-1 expression and function in human cancer.

BAG-1 is a multifunctional protein that interacts with a wide range of target molecules to regulate apoptosis, proliferation, transcription, metastasis and motility. Interaction with chaperone molecules may mediate many of the effects of BAG-1. The pathways regulated by BAG-1 play key roles in the development and progression of cancer and determining response to therapy, and there has been considerable interest in determining the clinical significance of BAG-1 expression in malignant cells. There is an emerging picture that BAG-1 expression is frequently altered in a range of human cancers relative to normal cells and a recent report suggests the exciting possibility that BAG-1 expression may have clinical utility as a prognostic marker in early breast cancer. However, other studies of BAG-1 expression in breast cancer and other cancer types have yielded differing results. It is important to view these findings in the context of current knowledge of BAG-1 expression and function. This review summarises recent progress in understanding the clinical significance of BAG-1 expression in cancer in light of our understanding of BAG-1 function.

Identification of a novel human BCL-X promoter and exon.

BCL-XL is a key anti-apoptotic BCL-2 family protein that is widely expressed in human cancer cells and is induced in response to diverse survival signals. The translation initiation codon for BCL-XL is located in BCL-X exon II and previous analyses have indicated that BCL-XL RNAs initiate close to the start of exon II or additionally contain a non-coding first exon (exon IA) spliced to exon II. Using 5' RACE we have now identified a novel BCL-X non-coding exon (exon IB) which is spliced directly to exon II in place of exon IA. Exon IB-containing RNAs encoded BCL-XL and were detected in non-malignant lymphocytes and lymphoma cells from lymph node biopsies and were expressed at significant levels in cell lines derived from ovarian, colon and breast cancers. We identified two TATA-box sequences upstream of exon IB and demonstrated that surrounding genomic sequences contained strong promoter activity in lymphoma cells (approximately 300-fold active relative to controls). We have therefore identified a powerful new BCL-X promoter and a novel exon that contributes to BCL-XL expression.

High level expression of differentially localized BAG-1 isoforms in some oestrogen receptor-positive human breast cancers.

Sensitivity to oestrogens and apoptosis are critical determinants of the development and progression of breast cancer and reflect closely linked pathways in breast epithelial cells. For example, induction of BCL-2 oncoprotein expression by oestrogen contributes to suppression of apoptosis and BCL-2 and oestrogen receptor (ER) are frequently co-expressed in tumours. BAG-1/HAP is a multifunctional protein which complexes with BCL-2 and steroid hormone receptors (including the ER), and can suppress apoptosis and influence steroid hormone-dependent transcription. Therefore, analysis of expression of BAG-1 in human breast cancer is of considerable interest. BAG-1 was readily detected by immunostaining in normal breast epithelial cells and most ER-positive tumours, but was undetectable or weakly expressed in ER-negative tumours. BAG-1 positive cells showed a predominantly cytoplasmic or cytoplasmic plus nuclear distribution of staining. A correlation between ER and BAG-1 was also evident in breast cancer derived cell lines, as all lines examined with functional ER expression also expressed high levels of BAG-1. In addition to the prototypical 36 kDa BAG-1 isoform, breast cancer cells expressed higher molecular weight isoforms and, in contrast to BCL-2, BAG-1 expression was independent of oestrogens. BAG-1 isoforms were differentially localized to the nucleus or cytoplasm and this was also independent of oestrogens. These results demonstrate a close association between BAG-1 and functional ER expression and suggest BAG-1 may be useful as a therapeutic target or prognostic marker in breast cancer.

Control of cell cycle entry and apoptosis in B lymphocytes infected by Epstein-Barr virus.

Infection of human B cells with Epstein-Barr virus (EBV) results in activation of the cell cycle and cell growth. To interpret the mechanisms by which EBV activates the cell, we have assayed many proteins involved in control of the G0 and G1 phases of the cell cycle and regulation of apoptosis. In EBV infection most of the changes, including the early induction of cyclin D2, are dependent on expression of EBV genes, but an alteration in the E2F-4 profile was partly independent of viral gene expression, presumably occurring in response to signal transduction activated when the virus binds to its receptor, CD21. By comparing the expression of genes controlling apoptosis, including those encoding several members of the BCL-2 family of proteins, the known relative resistance of EBV-immortalized B-cell lines to apoptosis induced by low serum was found to correlate with expression of both BCL-2 and A20. A20 can be regulated by the NF-kappaB transcription factor, which is known to be activated by the EBV LMP-1 protein. Quantitative assays demonstrated a direct temporal relationship between LMP-1 protein levels and active NF-kappaB during the time course of infection.

BAX frameshift mutations in cell lines derived from human haemopoietic malignancies are associated with resistance to apoptosis and microsatellite instability.

Bax suppresses tumorigenesis in a mouse model system and Bax-deficient mice exhibit lymphoid hyperplasia suggesting that BAX functions as a tumour suppressor in human haemopoietic cells. We examined BAX expression in 20 cell lines derived from human haemopoietic malignancies and consistent with a potential tumour suppressor function, identified two cell lines, DG75 (a Burkitt lymphoma cell line) and Jurkat (a T-cell leukaemia line), which lacked detectable BAX expression. Apoptosis of DG75 cells induced by low serum or ionomycin was significantly delayed relative to similar Burkitt lymphoma cell lines with normal BAX levels. Although DG75 and Jurkat cells expressed several BAX RNA species including the prototypical BAX alpha RNA, the absence of BAX protein was due to single base deletions and additions in a polyguanine tract within the BAX open reading frame. These frameshift mutations result in premature termination of translation and have recently also been identified in some colon cancers with microsatellite instability. Although mismatch repair defects are not considered a common feature of haemopoietic malignancies, DG75 and Jurkat cells had widespread microsatellite instability and did not express detectable levels of MSH2. In Jurkat cells, lack of MSH2 expression was due to a point mutation in exon 13 of MSH2 resulting in premature termination of translation. Our results suggest that a pathway linking mismatch repair defects, BAX tumour suppressor frameshift mutations and resistance to apoptosis may be a key feature of some lymphomas and leukaemias.

Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation.

The Bcl-2 oncoprotein is a key regulator of apoptosis and the Bag-1 protein interacts with Bcl-2 and cooperates with Bcl-2 to suppress apoptosis. The human Bag-1 cDNA is essentially identical with a previously described cDNA encoding RAP46, which interacts with activated steroid hormone receptors. However, there is considerable confusion over the structure of Bag-1/RAP46 proteins and their relationship to endogenous Bag-1 proteins. Here we have characterized Bag-1 expression in mammalian cells. We demonstrate that, in addition to the previously identified 32 kDa murine and 36 kDa human Bag-1 proteins, cells express a second 50 kDa Bag-1 isoform. In some murine cell lines p50 is expressed at the same level as p32 Bag-1, and p50 and p32 Bag-1 proteins have distinct subcellular localizations, suggesting that they are functionally distinct. The published mouse Bag-1 cDNA is partial, and sequencing of additional murine Bag-1 RNA 5' sequences demonstrated that human and murine Bag-1 cDNAs contain longer open reading frames than originally suspected. We determined which open reading frames gave rise to the Bag-1 isoforms in human cells. Surprisingly, translation of neither protein initiated at the first in-frame methionine, and cells do not express Bag-1/RAP46 proteins with the previously proposed structures; p50 Bag-1 initiates at an upstream CUG codon, whereas p36 Bag-1 initiates at a downstream AUG codon. Therefore, cells express two differently localized Bag-1 isoforms generated by alternative translation initiation, and Bag-1 proteins may play a dual role in regulating apoptosis and steroid hormone-dependent transcription.

Reduced signal transduction through glucocorticoid receptor in Burkitt's lymphoma cell lines.

Signal transduction through the glucocorticoid receptor (GR) was shown to be significantly reduced in Epstein-Barr virus (EBV)-positive group I Burkitt's lymphoma (BL) cell lines compared to human B-lymphocytes immortalized by EBV (LCLs). On the basis of hormone binding assays, nuclear DNA binding activity, and transactivation assays the response was reduced 5- to 10-fold. Direct sequence analysis of the expressed glucocorticoid receptor mRNA in two BL cell lines indicated that the phenotype did not result from mutation of the GR gene. By preparing a high-titer polyclonal antiserum against the t-1 region of the human GR, we further showed that the deficient GR response in BLs is largely reflected in reduced GR steady-state protein levels in BL cells compared to LCLs. However, the level of GR mRNA varies less between the BL cell lines and the LCLs. The Cp promoter of EBV which normally drives expression of the EBNA gene family in EBV-immortalized LCLs contains a functional glucocorticoid response element. Transfection of GR expression constructs to group I BL cells converted the GR response to approximately LCL levels both with respect to hormone binding and glucocorticoid-dependent transcription of a glucocorticoid-dependent promoter. A modest activation of EBNA-2 expression was seen in some such cell lines, suggesting that the lower GR response contributes to the down-regulation of EBNA expression observed in BL.

Strain variation in Epstein-Barr virus immediate early genes.

RNase protection analysis of mRNAs for Epstein-Barr virus (EBV) genes activated early in the induction of the productive cycle in Akata cells has confirmed that BZLF1 and BRLF1 behave as immediate early genes. These are likely to be the only virus immediate early genes since analysis of three other transcripts previously suggested to behave as immediate early genes in this system shows that they are, in fact, early genes. Western blotting of BRLF1 and BZLF1 demonstrates expression of these proteins early in induction of the productive cycle and reveals that the BZLF1 protein of Akata virus migrates more slowly on SDS gels than the BZLF1 protein of the prototypic virus strain B95-8. The nucleotide sequence of the BZLF1 gene and the immediate early promoters of Akata EBV have been determined and compared to other EBV strains. RFLP analysis of sequence variation in the BZLF1 gene of EBV strains from many different parts of the world shows a distribution of BZLF1 types which are not simply related to the established A and B EBV types.

Host cell and EBNA-2 regulation of Epstein-Barr virus latent-cycle promoter activity in B lymphocytes.

The six latent-cycle nuclear antigens (EBNAs) of Epstein-Barr virus (EBV), whose genes share 5' leader exons and two promoters (Cp and Wp), are differentially expressed by cells of the B lineage. To examine the possibility that EBNA gene expression is regulated through selective use of Cp and Wp, we monitored the activity of promoter-chloramphenicol acetyltransferase (CAT) gene constructs transfected into EBV-positive and EBV-negative B lymphocytes and Burkitt's lymphoma cells. Wp was a much stronger promoter than Cp in EBV genome-negative B-cell lines and was used exclusively in primary B cells. When B cells were infected with transforming EBV, Cp became the stronger promoter. This switch was not observed when B cells were infected with an immortalization-deficient virus, P3HR-1, which lacks the EBNA-2 open reading frame and expresses a mutant leader protein (EBNA-LP). Cp function was transactivated when EBV-negative or P3HR-1-infected B cells were cotransfected with Cp and a 12-kb fragment of DNA (BamHI-WWYH) that spanned the P3HR-1 deletion. This activity was mapped to the EBNA-2 gene within WWYH; constructs expressing EBNA-LP did not induce Cp function, and the deletion of 405 bp from the EBNA-2 open reading frame abolished transactivation. This research demonstrates host cell and EBNA-2 regulation of latent-cycle promoter activity in B lymphocytes, a mechanism with implications for persistence of EBV-infected lymphoid cells in vivo.

Reciprocal antagonism of steroid hormones and BZLF1 in switch between Epstein-Barr virus latent and productive cycle gene expression.

BZLF1 repression of transcription from the Epstein-Barr virus BC-R2 promoter (Cp) was shown to require a glucocorticoid response element in cis and glucocorticoids in trans. The mechanism of the repression is indirect and involves up regulation of the cellular c-fos proto-oncogene. Glucocorticoids maintain Epstein-Barr virus latency, and removal of glucocorticoids from the cell culture medium results in activation of the productive cycle. This inverse regulation of the expression of latent and productive cycle genes contributes to the switch between virus latency and the productive cycle. Glucocorticoid control of BC-R2 might also provide a mechanism for EBNA promoter switching during early infection and in development of the restricted latent pattern of gene expression.

Pathways of activation of the Epstein-Barr virus productive cycle.

The promoter for the 2.8-kb RNA of Epstein-Barr virus encoding BZLF1 and BRLF1 was identified and shown to be activated by both BZLF1 and BRLF1 but not by 12-O-tetradecanoylphorbol-13-acetate. Site-directed mutagenesis suggests that two binding sites for BZLF1 within the promoter contribute to the transactivation by BZLF1. The early kinetics of induction of the 2.8- and 1.0-kb RNAs encoding BZLF1 and BRLF1 in Akata cells treated with anti-immunoglobulin indicate that both RNAs appear within 60 min. The results indicate some likely pathways of activation of Epstein-Barr virus productive cycle gene expression.