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.2013 Jul 22;44(1):61.
doi: 10.1186/1297-9716-44-61.

BAG3 protects bovine papillomavirus type 1-transformed equine fibroblasts against pro-death signals

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BAG3 protects bovine papillomavirus type 1-transformed equine fibroblasts against pro-death signals

Roberta Cotugno et al. Vet Res..

Abstract

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.

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Figures

Figure 1
Figure 1
BAG3 expression in equine sarcoid and normal fibroblasts. (a) Neoplastic fibroblasts from equine sarcoid show strong immunoreactivity for BAG3. Streptavidin-biotin peroxidase method. Mayer’s haematoxylin nuclear counterstain ×240.(b) Dermal fibroblasts from normal equine skin are weakly immunostained for BAG3. Streptavidin-biotin peroxidase method. Mayer’s haematoxylin nuclear counterstain ×120.
Figure 2
Figure 2
BAG3 expression and down-modulation in equine and human cell lines. (a) BAG3 expression in equine cell lines. EqS04b, E. Derm and human HeLa cell total extracts were analysed by Western blotting for BAG3 levels. The blots were also probed for tubulin as the loading control. Blot is from one experiment representative of at least two with similar results. On the right: densitometry analysis of bands.(b) BAG3 down-modulation in EqS04b and E. Derm cells. EqS04b and E. Derm cells were transfected with BAG3siRNA (siRNA) or a non-targeting siRNA (scrRNA). Non-transfected cells were included as controls (Ctrl). At the times indicated, total cell extracts were prepared and analysed by Western blotting for BAG3 levels. The blots were also probed for tubulin as the loading control. Blots are from one experiment representative of at least two with similar results.
Figure 3
Figure 3
Effect of BAG3 down-regulation on cell viability and morphology. (a) Number of viable cells in BAG3-silenced EqS04b and E. Derm cells at 48 h and 72 h following transfection. Data, expressed as a percentage of the number of scrRNA-transfected viable cells measured at the same times, are the mean values ± S.E.M. from at least three experiments (*p < 0.05, **p < 0.001 versus scrRNA-treated cells);(b) Phase contrast microscopy images of non-transfected (Ctrl), scrRNA- and BAG3siRNA (siRNA)-transfected EqS04b samples at 56 h after transfection. Images are from one analysis representative of at least three experiments with similar results.
Figure 4
Figure 4
BAG3 down-regulation induces apoptosis and affects cell cycle progression in EqS04b cells. (a) On the Y axis, the percentages of hypodiploid cells (subG0/G1) and of cells in each cell cycle phase of BAG3siRNA-transfected EqS04b cells subtracted for the corresponding percentages in scrRNA-transfected cells. Data presented are the mean values ± S.E.M. from at least three experiments (*p < 0.05, **p < 0.001 calculated on raw data of BAG3siRNA-silenced versus scrRNA-treated cells). Mean values (%) ± S.E.M. in scrRNA-transfected cells: i) at 36 h, subG0/G1 4.9 ± 1.2; G0/G1 68.59 ± 3.7; S 21.08 ± 1.8; G2/M 9.03 ± 1.0; ii) at 56 h, subG0/G1 3.5 ± 1; G0/G1 66.8 ± 2.3; S 22.4 ± 2.1; G2/M 10.6 ± 1.1. Representative histograms of cell cycle profiles of scrRNA- and BAG3siRNA-transfected cells at 36 h are on the left.(b) On the Y axis, the percentages of Annexin V positive (A+/PI-, early apoptotic cells; A+/PI+, late apoptotic cells) and necrotic cells (A-/PI+) in BAG3siRNA-silenced EqS04b cells subtracted for the corresponding percentages in scrRNA-transfected cells. Data presented are the mean values ± S.E.M. from at least three experiments (*p < 0.05, **p < 0.001 calculated on raw data of BAG3siRNA-silenced versus scrRNA-treated cells). Mean values (%) ± S.E.M. in scrRNA-transfected cells: i) at 36 h, A+/PI-, 4.2 ± 1.1; A+/PI+, 2.3 ± 0.96; A-/PI+, 3.1 ± 1; ii) at 56 h the values became comparable to those of non-transfected control cells (≤ 2% in A+/PI-, A+/PI+, A-/PI+ gated quadrants).
Figure 5
Figure 5
PEITC modulates BAG3 protein levels. Cell extracts from EqS04b cells exposed to the indicated doses of PEITC for 12 h were monitored for BAG3 levels. Actin levels were checked as the loading control. Filters were also probed for PEITC-induced tubulin degradation. Blot is from one experiment representative of at least two with similar results.
Figure 6
Figure 6
BAG3 down-modulation sensitizes equine cell lines to PEITC-induced detachment. Control, scrRNA- and BAG3siRNA-treated cells were exposed to PEITC 42 h after transfection. Control and scrRNA-treated (scrRNA) EqS04b and E. Derm cells were exposed to 20 μM and 15 μM PEITC, respectively, while EqSO4b and E. Derm BAG3 silenced cells (siRNA) were exposed to 15 μM and 12 μM PEITC, respectively, in view of the lower number of cells (about 70-80% of control and scrRNA cells).(a) Phase contrast images of control (left panels), scrRNA-transfected (middle panels), and BAG3siRNA (siRNA)-transfected (right panels) cells were acquired at the times indicated (images from one experiment representative of three with similar results).(b) Percentages of adherent cells at 8 h following PEITC treatment in non-transfected (ctrl), scrRNA-transfected, and BAG3siRNA (siRNA)-transfected cells respect to their corresponding controls exposed to vehicle only. Data presented are the mean values ± S.E.M. from at least three experiments (*p < 0.05, **p < 0.001; scrRNA-transfected versus non-transfected or siRNA versus scrRNA).
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