Functional Study Of RbAp46, A Novel Tumor Suppressor Gene

The retinoblastoma suppressor (Rb) associated protein 46 (RbAp46) is a nuclear protein of the WD-repeat protein family and a component of the histone deacetylase complex that physically interacts with Rb. Previously, we demonstrated before that RbAp46 is a gene up-regulated by the Wilms' tumor suppressor (WTl) and functions as a negative regulator of cell growth. In this study, we investigated the ability of RbAp46 to inhibit transformed phenotype of adenovirus-transformed human embryonic kidney (HEK) 293 cells in tumorigenic assays. We have found that expression of RbAp46 suppressed clonal growth of HEK 293 cells in soft-agar and inhibited tumor growth of these cells in nude mice. Expression of RbAp46 resulted in an increase of cells in the G2/M fraction of cell cycle and augmented apoptosis in serum starved cells. The results suggest that high levels of RbAp46 expression inhibit the transformation of tumor cells through interfering with normal cell cycle and/or enhancing apoptosis of cells.To explore the molecular mechanisms of RbAp46 function, we then used RbAp46 as bait in a yeast two-hybrid screening and found that RbAp46 interacts specifically with the C-terminal region of BRCA1 (the BRCT domain), a domain involved in the transact!vation activity of BRCA1. Co-immunoprecipitation assays demonstrated that the interaction of RbAp46 with BRCA1 requires the first two of the four Trp-Asp (WD)-repeats of RbAp46. We also showed that expression of RbAp46 represses the transactivation activity mediated by the BRCT/Gal4 fusion protein and inhibits the transactivation of the p21 promoter mediated by the full-length BRCA1. Interestingly, the association of BRCA1 and RbAp46 is disrupted in cells treated with DNA-damaging agents. These important results suggest that RbAp46 may specifically interact with BRCA1 and modulate its transactivation activity hi response to DNA damage.Our previous work demonstrated that RbAp46 might be a novel tumor suppressor gene that function to inhibit abnormal cell growth and it may be involved in cell cycle regulation in cells with damaged DNA. We finally investigated the expression profiles of RbAp46 in cells treated with DNA-damagmg agents and its functions in cell apoptosis induced by DNA-damaging agents. We have found that adriamycin (ADR), capmtothecin (CPT) and gamma radiation induced significant decrease of RbAp46 mRNA levels in MCF-7 cells. The down-regulation of RbAp46 mRNA was dose-and time-dependent; significant down-regulation was first observed at 6 h after exposure to 5 uM ADR or 10 uM CPT. Gamma irradiation induced a dose-dependent down-regulation of RbAp46 mRNA, which qualitatively similar to that of ADR and CPT. The expression pattern of RbAp46 in DNA damaged cells exhibited a strong similarity to those of tumor suppressor BRCA1 and BRCA2. ADR and CPT induced distinct dose- and time-dependent alterations in cell cycle distribution; but these alterations did not correlate well with corresponding changes in RbAp46 mRNA levels.Using TG3B cells-transfected with RbAp46 expression vector, we tested the effect of RbAp46 on apoptosis induced by adriamycin. Compared with TG3B control cells, RbAp46-expressing TG3B cells exhibited a delay in apoptosis in cells treated with 5 uM ADR. These results suggest that RbAp46 may play roles in the cellular response to DNA-damaging agents and in coordinating cell cycle arrest and DNA repair. Its interaction with BRCA1 further highlights its importance in maintaining genomic integrity and prevention from tumorigenesis.Taken together, these results indicate that RbAp46 is an important protein that function as a tumor suppressor and plays an important role in cell growth, proliferation and apoptosis. However, the molecular mechanism by which RbAp46 functions during development needs more extensive investigation.