Regulation of BAG-1 IRES-mediated translation following chemotoxic stress

Abstract

There are three major isoforms of BAG-1 in mammalian cells, termed BAG-1L (p50), BAG-1M (p46) and BAG-1S (p36) that function as pro-survival proteins and are associated with tumorigenesis and chemoresistance. Initiation of BAG-1 protein synthesis can occur by both cap-dependent and cap-independent mechanisms and it has been shown that synthesis of BAG-1S is dependent upon the presence of an internal ribosome entry segment (IRES) in the 5'-UTR of BAG-1 mRNA. We have shown previously that BAG-1 IRES-meditated initiation of translation requires two trans-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB) for function. The former protein allows BAG-1 IRES RNA to attain a structure that permits binding of the ribosome, while the latter protein appears to be involved in ribosome recruitment. Here, we show that the BAG-1 IRES maintains synthesis of BAG-1 protein following exposure of cells to the chemotoxic drug vincristine but not to cisplatin and that this is brought about, in part, by the relocalization of PTB and PCBP1 from the nucleus to the cytoplasm.

Figure 1. Protein synthesis is inhibited in the presence of cisplatin and vincristine

A HeLa cells were treated with the doses of vincristine or cisplatin shown. Protein synthesis rates following exposure of cells to chemotoxic agents (after 24 hours) were determined as follows; cells were seeded at concentration of 5×10⁵/ml then incubated with/without the drug in the presence of ³⁵S methionine. At the end of the labelling period (20 min) cells were washed twice in PBS and then re-suspended in a final volume of 1ml PBS. Cell suspensions were then spotted in triplicates of 20μl on to a piece of 3MM filter paper (Whatman) divided into squares, and the filter paper dried. Filters were washed for 3×15 mins in 5% TCA and then 1×15 min in methanol before drying and cutting into squares. Radioactivity was measured by scintillation counting. There was an inhibition of protein synthesis to 70% of the control level in cisplatin treated cells and to 65% in vincristine treated cells at the highest doses used. B HeLa cells were treated with the doses of vincristine or cisplatin shown (for 24 hours) and immunoblotting was performed to determine the levels and phosphorylation states of eukaryotic initiation factors as described previously (West et al 1998). Rabbit polyclonal antibodies used to detect phosphorylated eIF4E, eIF4E, eIF2α, phosphorylated eIF2 α, eIF2α, 4EBP1 and eIF4G were kind gifts from Dr S.Morley (PeIF4E, eIF4E and eIF4G), Prof. R. Denton (4E-BP1), and Prof. C. Proud (eIF2α) respectively, and were used at dilutions of 1:7000, 1: 2000, 1:1000, and 1:2000, and 1:2000, respectively. Blots were then stripped and re-probed with actin to ensure equal loading. Blots were then incubated with peroxidase-conjugated secondary antibodies raised against rabbit immunoglobulins and developed using the chemiluminescence reagent ECL (GE Healthcare) and the resulting X-ray film was then scanned.

Figure 2. The BAG-1 IRES is functional in vincristine-treated cells

A Immunoprecipitation of BAG-1 isoforms during chemotoxic stress was performed as described previously (Stoneley et al., 2000) cells were labelled with ³⁵ S methionine for 30 min before harvesting and the BAG-1 isoforms were immunoprecipitated over night at 4°C using a BAG-1 monoclonal antibody mix (3.9F1E11 + 3.10G3E2, Neomarkers). The data suggest that 24 hours following treatment of cells with vincristine (i) but not cisplatin (ii) there is continued synthesis of the p36 isoform of BAG-1 when compared to the p50 isoform. B Schematic representation of the constructs used to measure IRES-activity of BAG-1. The generation of these plasmids is described in detail in Coldwell et al 2001. C HeLa cells were transfected with the monocistronic hairpin vector phpBL (closed squares) or control vectors phpL (closed circles) or pGL3 (closed triangles) and 24 hours post-transfection, vincristine (i) or cisplatin (ii) were applied to cells at the doses shown and 24 hours later, cells were lysed and assayed for enzyme activity. Luciferase expression was assayed using the luciferase assay system (Promega) and normalised to the transfection control of β-galactosidase (cells were transfected with pcDNA3.1/HisB/lacZ from Invitrogen), which was assayed using the Galactolight plus assay system (Tropix). All light emissions were measured over 10 seconds using an Optocomp1 Luminometer (MGM instruments). Relative luciferase activity was calculated as the average of a proportion of the activity in untreated cells (firefly luciferase expression/β-Gal expression), and efficiency expressed as (luciferase activity in treated cells)/(luciferase activity in untreated control cells). Errors were calculated as the standard deviation of the three calculated IRES activities, and expressed as a percentage of the average activity. All experiments were performed in triplicate on at least three independent occasions. There is continuation of luciferase production mediated by translation initiation through the BAG-1 IRES in vincristine but not cisplatin treated cells. Closed squares, phpBL, closed circles pGL3, closed triangles phpL.

Figure 3. BAG-1 IRES does not function during mitosis or apoptosis

Ai HeLa cells were transfected with the monocistronic hairpin vector phpBL (grey bars) or control vectors phpL (white bars) or pGL3 (black bars). 40 hours post transfection cell populations were synchronised by treatment with nocodazole (2μg/ml) that arrests cells undergoing mitosis. The mitotic cells (found in suspension) were harvested and half of these cells were lysed and luciferase activity determined (M), whilst the remaining cells were re-plated in fresh media and grown for a further 2 hours (G1). Transfected cells were also treated with aphidicoline (5μg/ml) which prevents exit from S phase by inhibiting DNA synthesis. Again a proportion of these cells were harvested and assayed immediately following treatment (S) whilst the remainder were re-grown in fresh media for a further 8 hours to allow progression into G2 when they were harvested. Cell samples were sorted by FACS to show arrest in the specific stages of the cell cycle (Aii). Luciferase activity was measured for each population of cells, normalised to the transfection control (as described in the legend to figure 2) and expressed relative to cells maintained in an asynchronous state. The data shows that there is no significant increase in BAG-1-IRES-mediated translation during the cell cycle. Aiii HeLa cells were transfected with the monocistronic hairpin vector phpBL (closed squares) or control vectors phpL (closed circle) or pGL3 (closed triangle). 40 hours post-transfection the medium was supplemented with His-tagged TRAIL to a final concentration of 0.25 μg/ml. Cells were harvested at 0, 2, 4, 6, 8 and 10hours after the addition of TRAIL and luciferase activity determined. The BAG-1 IRES does not function in TRAIL-mediated apoptosis. Closed squares, phpBL, closed circles pGL3, closed triangles phpL. Bi and ii: HeLa cells were treated with the doses of vincristine or cisplatin shown and after 24 hours immunoblotting was performed to determine the levels of PTB-1 and PCBP1. The PTB polyclonal rabbit antibodies were used at a concentration of 1:2000 and were a gift from Chris Smith. The data show that there was no change in the levels of these proteins.

Figure 4. There is a change in sub-cellular localisation of PTB and PCBP1 in cells treated with vincristine but not cisplatin

Monoclonal PTB antibodies and polyclonal PCBP1 and La antibodies were used in indirect immunofluorescence on control cells (A) and cells that had been treated with vincristine (B) and cisplatin (C) in order to assess the localisation during chemotoxic stress of the BAG-1 IRES trans-acting factors (PTB and PCBP1) when compared to an unrelated ITAF (La). Approximately 1 × 10⁵ HeLa cells were plated into chamber slides and treated with 4 nM vincristine, 4μM of cisplatin or maintained under control conditions for 24 hours. Cells were fixed using 1:1 acetone: methanol and proteins bound by addition of primary antibody. The localisation of the proteins was determined using fluorescently labelled secondary antibodies and fluorescence microscopy.