E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

Abstract

Background: PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells.

Methods: PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit.

Results: Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway.

Conclusion: Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling pathways provided here will further enhance insights on the mechanisms of E2F-1-induced cancer cell apoptosis as a strategy for cancer therapy.

Figure 1

A. Up-regulation of PUMA at the transcription level after Ad-E2F-1 expression in SK-MEL-2 cells. SK-MEL-2 cells were infected with Ad-E2F-1 at MOI 100. Ad-LacZ was used as control. Cells were harvested after infection at 8 hours, 12 hours, and 16 hours. Real-time PCR was performed as described in Materials and Methods. β-actin was used as an internal control. The bar graphs are expressed as fold increase relative to Ad-LacZ-infected cells adjusted for β-actin. Each of the experiments is a representation of two independent experiments performed in duplicate. After adjustment for β-actin mRNA expression, real-time PCR results showed a 2.6-, 5-, and 10-fold induction at the PUMA mRNA level after 8 hours, 12 hours and 16 hours of Ad-E2F-1 infection, respectively. B. Up-regulation of PUMA at the protein level after Ad-E2F-1 expression in SK-MEL-2 cells. Western blot analysis of PUMA at indicated time points after Ad-LacZ or Ad-E2F-1 infection. Anti-actin was used to show the equal loading and transfer in each lane. E2F-1 expression was also shown at the indicated time points to correlate with PUMA expression. PUMA/actin showed the ratio of the scanned optical density of each protein band. In SK-MEL-2 cells, PUMA protein level increased at 8 hours post-infection with Ad-E2F-1 and was still pronounced at 16 hours and 18 hours post-infection with Ad-E2F-1.

Figure 2

A. E2F-1 expression up-regulates the activity of the PUMA promoter in SK-MEL-2 cells. SK-MEL-2 cells were co-transfected with a total of 0.88 μg of DNA, including 0.8 μg of PUMA promoter reporter plasmid and 0.08 μg phRL-CMV plasmid (as an internal control) in a 12-well plate by using Lipofectamine 2000 transfection reagent. After 6 hours of transfection, the cells were infected with Ad-LacZ (as control) and Ad-E2F-1. Luciferase activity was examined 16 hours later as described in the manufacturer's protocol using the Dual Luciferase Reporter Assay System (Promega). The bar graphs depict the fold of activation in the luciferase assay after normalization for Renilla luciferase readout values. Each of the experiments is a representation of at least three independent experiments performed in duplicate. B. Transactivation domain of E2F-1 was required for the up-regulation of the PUMA promoter in SK-MEL-2 cells. SK-MEL-2 cells were co-transfected as in Fig. 2A. After 6 hours of transfection, the cells were infected with Ad/T-E2FD (expressing truncated E2F-1) at MOI 100 plus AdTet-off at MOI 5 (using as a helper virus), Ad/T-E2FD at MOI 100 plus AdTet-off at MOI 5, and doxycycline 1 μg/ml or Ad-LacZ at MOI 100. Luciferase activity was examined 16 hours later as in Fig. 2A. The bar graphs depict the fold of activation in the luciferase assay after normalization for Renilla luciferase readout values. Each of the experiments is a representation of at least three independent experiments performed in duplicate.

Figure 3

Resistance in response to E2F-1 expression in the HCT116 PUMA-/- cell line compared to the HCT116 PUMA+/+ cell line by cell viability assay. Both floating and adherent cells were harvested, counted, and scored at indicated time points. Viability was assessed using the trypan blue exclusion method. Results are expressed as the percent viable cells at each point and values represent the means ± SD (bars) of three independent experiments. A. HCT116 PUMA-/- and HCT116 PUMA+/+ cell lines were treated by infection with Ad-LacZ or Ad-E2F-1 at indicated MOI. The viability was assessed at day 4 after virus infection. B. Time course of cell viability of HCT116 PUMA-/- and HCT116 PUMA+/+ cells in response to Ad-LacZ or Ad-E2F-1 infection at MOI of 50.

Figure 4

A. E2F-1 expression up-regulates the activity of the PUMA promoter in HCT116 PUMA+/+ cells. HCT116 PUMA+/+ cells were co-transfected with a total of 0.82 μg of DNA, including 0.8 μg of PUMA promoter reporter plasmid and 0.02 μg phRL-CMV plasmid (as an internal control) in a 12-well plate by using Lipofectamine 2000 transfection reagent, and then processed as in Fig. 2A. 4B Up-regulation of PUMA at the protein level after Ad-E2F-1 expression in HCT116 PUMA+/+ cells. Western blot analysis of PUMA was performed in HCT116 PUMA+/+ cells at indicated time points as in Fig. 1B. PUMA/actin showed the ratio of the scanned optical density of each protein band. PUMA protein level was increased at 12 hours and 16 hours of infection with Ad-E2F-1.

Figure 5

Bax mitochondrial translocation after Ad-E2F-1 expression in SK-MEL-2 cells and HCT116 PUMA+/+ cells. SK-MEL-2 cells (a,b) and HCT116PUMA+/+ cells (c,d) were infected with Ad-LacZ and Ad-E2F-1. After 24 hours of infection, cells were stained with Mitotracker Red 580 and Bax (green), then counterstained with DAPI (blue) as described in the Methods. After 24 hours of Ad-E2F-1 infection in SK-MEL-2 cells (b) and HCT116PUMA+/+ cells (d), Bax clustered and translocated to mitochondria (an overlay of the three stains is given in yellow, which shows co-localization of Bax in mitochondria).

Figure 6

Activation of caspase-9 was accompanied by PUMA-mediated E2F-1-induced apoptosis. 2 × 10⁶ of SK-MEL-2 (A), HCT116 PUMA+/+ cells (B), and HCT116PUMA-/- cells (C) were infected with Ad-E2F-1 or Ad-LacZ and lysed at indicated time points to collect their intracellular contents. The cell lysates were assayed for caspase-9 activity by measuring the digestion of p-nitroanaline (pNA) as a substrate. The OD405 values were measured, and the percentage values were compared with Ad-LacZ infected cells as indicated. Caspase-9 activity was induced 3.6 fold, 3.0 fold, and 1.5 fold after 48 hours of Ad-E2F-1 infection in SK-MEL-2 cells, HCT116 PUMA+/+ cells, and HCT116PUMA-/- cells, respectively.