The Molecular Biology of Colorectal Cancer

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

N.K. Clemo, N.J. Arhel, J.D. Barnes, J. Baker, M. Moorghen, G.K. Packham, C. Paraskeva, A.C. Williams

Abstract

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 γ-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.

  • retinoblastoma protein (Rb)
  • BAG-1
  • colorectal
  • adenoma
  • apoptosis
  • radiation

Introduction

The retinoblastoma tumour suppressor gene (RB1) encodes a 110 kDa nuclear phosphoprotein that is inactivated in a wide range of human tumours, including retinoblastomas, breast carcinomas, osteosarcomas, and small cell lung carcinomas (reviewed in [1]). By contrast, in the majority of colorectal carcinomas, the RB1 gene is frequently amplified and overexpressed (reviewed in [2]), suggesting that Rb (retinoblastoma protein) expression may be selected for in colorectal carcinogenesis. Recently, it has been shown that, in addition to its role as a negative regulator of the cell cycle, Rb can suppress apoptosis [3]. However, the mechanisms by which Rb can regulate apoptosis are yet to be fully elucidated.

We have previously shown that Rb interacts with BAG-1 (Bcl-2 associated athanogene-1) using in vitro translated protein in colorectal tumour cells and that this interaction controls the subcellular distribution of BAG-1 proteins ([4], Figure 1). BAG-1 is a cell survival protein that interferes with apoptosis induced by a wide range of factors, including Fas, chemotherapeutic agents, growth factor withdrawal and heat-shock [5]. Although interaction and regulation of the 70 kDa heat-shock proteins is considered to be key for BAG-1 function, BAG-1 also interacts with several apoptosis regulators, including Bcl-2 and the Raf-1 kinase (reviewed in [5]). Human BAG-1 exists as three distinct isoforms that share a common C-terminus [p50 (BAG-1L), p46 (BAG-1M) and p36 (BAG-1S)] generated by alternative translation initiation, each being differentially localized in the cell [6,7]. p50 BAG-1 localizes predominantly to the nucleus while p46 and p36 BAG-1 localize in both nuclear and cytoplasmic fractions, with p36 BAG-1 exhibiting preferential cytoplasmic localization [68]. Nuclear localization of p50 BAG-1 is thought to be conferred by a nuclear localization signal situated at the N-terminus of this isoform [9] but absent from p36 BAG-1, and present only in a truncated form in p46 BAG-1. Subcellular localization of BAG-1 may be important for its activity since some functions of BAG-1 are specific for individual BAG-1 proteins [9,10].

Figure 1 Rb/BAG-1 interaction

(A) Rb/BAG-1 interaction as shown by GST pull-down assay. Purified GST-fusion proteins were used to pull-down interacting proteins from adenoma-derived S/RG/C2 cell lysates. Protein complexes were analysed by Western blotting using anti-Rb mouse monoclonal antibody (G3 245). (B) Rb and BAG-1 protein interaction in colonic epithelial cells. (i) and (ii) Adenoma-derived S/RG/C2 cell lysates were immunoprecipitated with anti-BAG-1 G3E2, anti-Rb G3 245, anti-MDM2 (murine double minute clone 2 oncoprotein) (4B2) monoclonal antibodies or no antibody (Con), and immune complexes were analysed by Western blotting using anti-Rb G3 245 (i) or anti-BAG-1 G3E2 (ii) monoclonal antibodies. (C) Confocal immunofluorescence microscopy showing relocalization of the BAG-1 protein upon E7 expression in the adenoma-derived RG/C2 cell line. E7-expressing S/RG/RE7 and vector control S/RG/Neo cells were stained with mouse anti-Rb (G3 245) and rabbit anti-BAG-1 antibodies (TB2), followed by anti-mouse-FITC and anti-rabbit TRITC (tetramethylrhodamine β-isothiocyanate) antibodies (Sigma), and mounted using Vectashield mounting medium with DAPI (4,6-diamidino-2-phenylindole) (1.5 μg/ml, Vector Laboratories). Cells were viewed under the Leica TCS-NT confocal laser scanning microscope.

The Rb:BAG-1 interaction was detected by yeast two-hybrid assay [4] and GST pull-down assay. GST-p36 BAG-1 and two mutants of p36 BAG-1 lacking essential amino acids within the C-terminal BAG domain required for HSC70 (heat-shock cognate 70 stress protein) binding, were used to show that the interaction between Rb and BAG-1 is not mediated via heat-shock binding proteins (Figure 1A). In addition, co-immunoprecipitation demonstrated that endogenous BAG-1 and Rb proteins interact in human colorectal epithelial cells. Cell lysates were prepared from the adenoma-derived S/RG/C2 cells, immunoprecipitated using Rb- or BAG-1-specific antibodies and analysed by immunoblotting (Figure 1B)[4]. Furthermore, the Rb:BAG-1 complex was disrupted by the viral oncoprotein E7, an inhibitor of Rb interactions, providing evidence that the Rb:BAG-1 interaction detected in cells is specific and confirming the role of the large pocket of Rb in the binding [4]. Interestingly, expression of E7 resulted in a pronounced relocalization of BAG-1, with a decrease in nuclear expression and increase in cytoplasmic expression, suggesting that the interaction with Rb may play a role in maintaining the predominant nuclear localization in colorectal tumour cells (Figure 1C).

Although reported as deregulated in a number of different cancers including colorectal cancer [11,12], we have found that BAG-1 expression is also altered in colorectal adenomas (Figure 2). In the normal colorectal epithelium, BAG-1 is found to be differentially expressed, with predominant nuclear staining at the bottom of the crypt, and an increase in cytoplasmic staining towards the top of the crypt [Figure 2A(i)]. However, in small adenomas, the staining for BAG-1 is more heterogeneous; the cytoplasmic localization of BAG-1 is lost, and the nuclear staining variable, with some areas of strong nuclear positivity [Figure 2A(ii)]. In addition, there is an increase in the BAG-1 staining with increasing size of adenoma, with large adenomas showing strong BAG-1 expression in both the nucleus and cytoplasm, as previously reported for colorectal carcinomas [Figure 2A(iii)].

Figure 2 Expression of BAG-1 in human adenoma cells inhibits radiation-induced apoptosis

(A) Expression and localization of BAG-1 (Bcl-2-associated athanogene-1) in colon tissue. BAG-1 expression was detected using conventional immunohistochemical techniques and visualized using diaminobenzidine (brown) staining, in (i) normal tissue, (ii) small adenoma, (iii) medium adenoma and (iv) large adenoma. Magnification ×40. (B) Induction of apoptosis in the S/RG/C2 cell line and the stably transfected clones expressing either pcDNA3-BAG-1L or pcDNA vector only after 72 h of 5 Gy γ-irradiation. The results are the mean values from triplicate experiments with standard deviation and Dunnett's t test comparing the difference in irradiated apoptosis levels of the stably transfected cells to the parental S/RG/C2 cell line (***P=0.001).

As predominant BAG-1 nuclear localization was observed both in colorectal adenoma and carcinoma derived cells in vitro as well as in adenoma tissue, the role of nuclear BAG-1 in cell survival after DNA damage was examined [13]. The S/RG/C2 adenoma-derived cell line was stably transfected with the nuclear-localized BAG-1L isoform and overexpression was confirmed by Western blotting. These cells were then exposed to 5 Gy γ-radiation (a dose previously shown to induced apoptosis), and apoptosis was assessed 72 h after irradiation (results are summarized in Figure 2B). Although irradiation-induced apoptosis was detected in the parental and vector control cells, there was a highly significant decrease in γ-radiation-induced apoptosis in the cells overexpressing the BAG-1L isoform (P<0.001). This demonstrates that an increase in nuclear-localized BAG-1 protein confers a survival advantage in colorectal adenoma-derived cells in vitro, and suggests that the nuclear localization of BAG-1 in adenoma tissue could potentially be important for the survival of colorectal tumour cells in vivo.

In summary, 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 in the nucleus may promote colorectal tumour cell survival by making colonic epithelial cells less sensitive to DNA damage.

Acknowledgments

This work was funded by Cancer Research UK and the Citrina Foundation. We thank the Medical Research Council for providing an Infrastructure Award to establish the School of Medical Sciences Cell Imaging Facility, and Dr Mark Jepson and Alan Leard for their assistance.

Footnotes

  • The Molecular Biology of Colorectal Cancer: Focused Meeting held at the UBHT Education Centre, Bristol, U.K., 10–11 March 2005. Organized and edited by T. Corfield (Bristol, U.K.), C. Paraskeva (Bristol, U.K.) and H. Wallace (Aberdeen, U.K.).

Abbreviations: Rb, retinoblastoma protein; BAG-1, Bcl-2 associated athanogene-1; RB13, retinoblastoma tumour suppressor gene; GST, glutathione S-transferase

References

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