The Bmi-1 helix-turn and ring finger domains are required for Bmi-1 antagonism of (-) epigallocatechin-3-gallate suppression of skin cancer cell survival

Abstract

The Bmi-1 Polycomb group (PcG) protein is an important epigenetic regulator of chromatin status. Elevated Bmi-1 expression is observed in skin cancer and contributes to cancer cell survival. (-) Epigallocatechin-3-gallate (EGCG), an important green tea-derived cancer prevention agent, reduces Bmi-1 level resulting in reduced skin cancer cell survival. This is associated with increased p21(Cip1) and p27(Kip1) expression, reduced cyclin, and cyclin dependent kinase expression, and increased cleavage of apoptotic markers. These EGCG-dependent changes are attenuated by vector-mediated maintenance of Bmi-1 expression. In the present study, we identify Bmi-1 functional domains that are required for this response. Bmi-1 expression reverses the EGCG-dependent reduction in SCC-13 cell survival, but Bmi-1 mutants lacking the helix-turn-helix-turn-helix-turn (Bmi-1ΔHT) or ring finger (Bmi-1ΔRF) domains do not reverse the EGCG impact. The reduction in Ring1B ubiquitin ligase activity, observed in the presence of mutant Bmi-1, is associated with reduced ability of these mutants to interact with and activate Ring1B ubiquitin ligase, the major ligase responsible for the ubiquitination of histone H2A during chromatin condensation. This results in less chromatin condensation leading to increased tumor suppressor gene expression and reduced cell survival; thereby making the cells more susceptible to the anti-survival action of EGCG. We further show that these mutants act in a dominant-negative manner to inhibit the action of endogenous Bmi-1. Our results suggest that the HT and RF domains are required for Bmi-1 ability to maintain skin cancer cell survival in response to cancer preventive agents.

Keywords: Chemoprevention; EGCG; Ezh2; Histone methylation; Polycomb group proteins; Skin cancer.

Fig. 1

Wild-type and mutant Bmi-1 impact on EGCG suppression of SCC-13 cell survival. A) Schematic of Bmi-1 showing the ring finger (RF, amino acids 18–56), helix–turn–helix–turn–helix–turn–helix (HT, amino acids 165–220) domains, nuclear localization signal (NL, amino acids 232–235) and proline/serine-rich sequence (PS, amino acids 248–324). Bmi-1 is a 326 amino acid protein. B) SCC-13 cells, growing at low confluence, were infected with 5.0 MOI of indicated FLAG-Bmi-1 adenovirus in the presence of 5.0 MOI of tAd5-TA helper virus. At one day post-infection, cells were treated with 0 or 60 μM EGCG for two days and the cells were harvested and counted. The values are mean ± SEM, n = 3. Asterisks indicate a significant increase over control (day zero, open bar), p < 0.05. C) Expression of FLAG-Bmi-1 proteins. SCC-13 cells were infected with 5.0 MOI of indicated FLAG-Bmi-1 adenovirus in the presence of 5.0 MOI of tAd5-TA helper virus. At one day post-infection, cells were treated with 0 or 60 μM EGCG for two days and the cells were then harvested and extracts were prepared for anti-FLAG detection of FLAG-Bmi-1. Similar blots were obtained in each of three experiments.

Fig. 2

Impact of EGCG and Bmi-1 on cell morphology. SCC-13 cells, growing at low confluence, were infected with 5.0 MOI of indicated FLAG-Bmi-1 adenovirus in the presence of 5.0 MOI of tAd5-TA helper virus. At one day post-infection, cells were treated with 0 or 60 μM EGCG for two days and the cells were photographed. Only wild-type Bmi-1 prevents the EGCG-associated morphological changes. Similar results were observed in each of three independent experiments.

Fig. 3

Immunolocalization of FLAG-Bmi-1. Sub-confluent SCC-13 cells were grown on glass cover slips, and then infected with 5 MOI of the indicated virus. After 24 h, the cells were treated with 0 or 60 μM EGCG for an additional 48 h. At 72 h post-infection, the cells were washed twice in PBS, fixed and stained with anti-FLAG-Cy3 (red) and the nuclei with Hoechst (blue). The arrows indicate nuclear Bmi-1 staining. Similar results were observed in each of the three separate experiments.

Fig. 4

Impact of Bmi-1 mutants on EGCG suppression of PcG action. SCC-13 cells were infected with 5 MOI of the indicated virus and after 24 h the cells were treated with 0 or 60 μM EGCG for an additional 48 h. A) Protein extracts were prepared for immunoblot detection of the indicated proteins. B) The values EGCG challenge experiments are plotted. The values indicate percent reduction in endpoint in EGCG-treated cells versus control as assessed by densitometry. The values represent mean + SEM, n = 3. The tAd5-hBmi-1 values are significantly elevated (p < 0.05) as compared to the other values.

Fig. 5

Effect of Bmi-1 proteins on EGCG-dependent regulation of downstream targets. Subconfluent cultures of SCC-13 cells were infected with 5 MOI the indicated virus and 5.0 MOI of Ad5-TA helper virus. After 24 h, the cells incubated with 0 or 60 μM EGCG and after an additional 48 h and total cell lysates were prepared for immunoblot detection of the indicated epitopes. β-Actin was used as a protein loading control. The additional arrows in the procaspase 9, procaspase 8 and PARP panels indicate the apoptosis-activated cleaved forms of these proteins. Similar results were observed in three separate experiments.

Fig. 6

Reduced interaction of Bmi-1 mutants with Ring1B. SCC-13 cells were infected with 5 MOI of empty virus (EV) or viruses encoding FLAG-tagged wild-type or mutant Bmi-1. After 24 h total cell extract was prepared for immunoprecipitation with anti-FLAG. Total protein (100 μg) was incubated with 2 μg of anti-FLAG for 2 h at 4 C followed by overnight treatment with 25 μl of protein-agarose A/B beads, and the precipitates were electrophoresed prior to immunoblot detection of Ring1B (top panel). Total extract was electrophoresed in parallel for detection of FLAG-Bmi-1 and β-actin (bottom two panels). Similar results were observed in each of three independent experiments. Anti-FLAG was used to detect each of the Bmi-1 proteins including wild-type Bmi-1, Bmi-1(ΔRF) and Bmi-1(ΔHT).

Fig. 7

Mutant Bmi-1 proteins function as inhibitors of wild-type Bmi-1. A) Subconfluent SCC-13 cell cultures were infected with 5 MOI of each indicated adenovirus. When two viruses are used, each virus is delivered at 5 MOI. At 4 d post-infection, the cells were harvested and counted. The open bar is the starting cell number at day = 0 and the closed bars are after 4 days of proliferation. The values are mean ± SEM, n = 3. B) Mutant Bmi-1 proteins inhibit Bmi-1 suppression of p21^Cip1 level. Cells were treated with virus as described above and extracts were prepared for immunoblot to detect p21^Cip1 and β-actin.