immune response promote HIF-1a transcription [8–10], whereas some autocrine growth factors enhance translation of the HIF-1a protein [1]. Indeed, the loss of function of tumour suppressors and the gain of function of oncogenes also regulate different steps that lead to HIF-1 activation [1,11]. In this context we also found that overexpression of the anti-apoptotic and pro-survival protein bcl- 2, in human melanoma and breast carcinoma cells, under hypoxia, enhances HIF-1a protein expression and HIF-1 activity consequently leading to angiogenesis through vascular endothelial growth factor (VEGF) [12,13]. Moreover, the treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide exterts antiangiogenic activity [14]. We also demonstrated that bcl-2 plays a role, in cooperation to hypoxia, in cell migration and invasion, contributing to tumour progression [15,16]. Indeed, a significant positive correlation between the expression levels of HIF-1a and bcl-2 was found in neuroblastoma [17]. This study thoroughly investigated the mechanism by which bcl-2 regulates HIF-1 in tumour cells exposed to hypoxic conditions. It identified the stabilization of HIF-1a protein as a mechanism by which bcl-2 induces the activation of HIF-1 in hypoxic melanoma cells, through the impairment of ubiquitin- dependent HIF-1a degradation with the involvement of the b isoform of the molecular chaperone HSP90. Results bcl-2 modulation regulates HIF-1a protein expression in conditions strictly dependent on oxygen availability We have previously reported that bcl-2 overexpression in human breast carcinoma and melanoma cell lines increases HIF-1 expression and activity and VEGF secretion under hypoxic conditions [12,13,18]. The ability of bcl-2 to modulate VEGF expression under hypoxia has been also extended to several other human melanoma cell lines (Figure S1A,B). The relevance of HIF-1a as the main mediator of bcl-2 induced VEGF secretion under hypoxic conditions has been demonstrated using siRNA directed to HIF-1a in M14 cells stably transfected with bcl-2 expression vector (Figure S1C). In fact, the down-regulation of HIF-1a protein reduced VEGF expression both in control cells and bcl-2 overexpressing clones. Interestingly, after HIF-1a reduction, VEGF levels secreted by bcl-2 transfectants were similar to those ones of control cells (Figure S1D). To evaluate whether down-regulation of bcl-2 shows opposite effect of bcl-2 overexpression in terms of HIF-1a protein expression, we silenced the endogenous expression of bcl-2 gene transfecting M14 cells with siRNA-targeting bcl-2 mRNA (si-bcl-2) and then exposing them to normoxia or hypoxia for 24 h. Western blot analysis demonstrated that the delivery of si-bcl-2 reduced expression of bcl-2 protein (Figure 1A) while, as expected, the transfection of a scrambled si-RNA (si-contr) did not have any effect on bcl-2 protein expression when compared to untransfected parental cell line (data not shown). Then, we evaluated the impact of reduced bcl-2 expression on HIF-1a protein expression. As expected, HIF- 1a protein was undetectable in all cells under normoxic conditions, while an increased HIF-1a protein expression was observed in the cells exposed to si-contr under hypoxia, but not in the cells after down-regulation of the bcl-2 protein expression (Figure 1A). To further characterize the impact of bcl-2 on HIF-1a expression, we evaluated whether bcl-2 overexpression was able to cooperate with other stimuli, beyond hypoxia, known to modulate HIF-1 a expression [1]. Firstly, we verified if increased cell density affected the level of HIF-1a protein in M14 cells stably transfected with empty vector (puro) and in their two derivative HIF-1a Stabilization by Bcl-2 stably bcl-2 overexpressing clones (Bcl2/5, Bcl2/37). As shown in Figure 1B, while HIF-1a protein is detectable at same extent in all cell lines plated at low density (sparse), regardless of bcl-2 expression, an increased HIF-1a protein expression was observed in bcl-2 transfectants, compared to the control line, either when they were plated at high density (dense) or when they reached high cell density (4 days of culture) and, as expected and previously reported [12,18], in hypoxic conditions. HIF-1b was constitutively expressed in the cells, and none of those stimuli modulated its expression. Nuclear translocation of HIF-1a subunit is a necessary step for HIF-1 transcriptional activity through its association with HIF-1b, which is constitutively localized in the nucleus [1]. In our experimental model, high cell density conditions induced the nuclear expression of HIF-1a in bcl-2 overexpressing clones while its expression was undetectable in control cells (Figure S2A). In parallel, control cells and bcl-2 overexpressing clones exhibited density-dependent induction of the HIF-1-dependent transcrip- tional activity under normoxic conditions of about 2.3 fold (p = 0.039) while HRE-dependent transcriptional activity was not found to be significantly changed in control cells (p = 0.49) (Figure S2B). To further investigate the induction of HIF-1a protein observed in bcl-2 transfectants under high cell density conditions, we evaluated whether the creation of a local hypoxic microenviron- ment could be responsible for HIF-1a induction. Hence, the cells were cultured at high density and gently shaked to disrupt any potential oxygen gradient due to the inter-cellular environment and to ensure a homogenous oxygen concentration within the cell culture medium. As depicted in Figure 1C, the gentle shaking drastically reduced the high density-dependent HIF-1a induction in bcl-2 transfectants, thus indicating that oxygen pericellular gradient is an important factor contributing in the increase of HIF- 1a expression by bcl-2 in high cell density conditions. To confirm these results, we plated cells in high density conditions with decreasing volumes of medium, to enhance the oxygen exchange rate. As shown in Figure 1D, the decrease of culture medium volume from 4 to 1 ml determined a medium volume-dependent reduction of HIF-1a protein expression in both bcl-2 transfectants. Next, we evaluated whether any differences existed between control cells and bcl-2 overexpressing clones in terms of HIF-1a induction in response to growth-factor stimulation, another condition that induces hypoxia-independent HIF-1a expression even in normoxia [19]. As shown in Figure 1E, both insulin and the Epidermal Growth Factor (EGF) induced HIF-1a protein expression in all the cells under normoxia but more importantly no difference in the levels of HIF-1a protein was observed in bcl-2 transfectants compared to control cells. bcl-2 promotes HIF-1a protein stability preventing its ubiquitin-mediated degradation Since bcl-2 overexpression in melanoma cells under hypoxia did not alter HIF-1a mRNA levels [12], we investigated the impact of bcl-2 overexpression on HIF-1a protein stabilization under hypoxia. Firstly, we performed time course experiments to study the kinetics of HIF-1a protein induction in control cells and bcl-2 overexpressing clones. As shown in Figure 2A (left and right panels), exposure of cells to hypoxia determined a HIF-1a protein induction, at a greater extent in bcl-2 transfectants compared to control cells, as previously reported. In particular, HIF-1a protein level reached the maximum value at 24 h of hypoxia in all cell lines, but it decreased at later time point of 48 h, slower in bcl-2 overexpressing clones than in control cells. To verify whether bcl-2 enhances HIF-1a protein expression by affecting its translational rate, we determined the possible PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11772