Authors: Ashley Pretorius, Habeeb. Adebodun, Bankole, Mervin Meyer, Faghri February,
and David Jasper Gilbert Rees
Retinoblastoma-binding protein 6 (RBBP6) is one of the few proteins that interacts with both tumour suppressor proteins p53 and Rb and by implication plays an important role in cell regulation. Previous studies carried out to monitor the role of RBBP6 targeted mouse spliced variants (P2P-R and PACT) of the gene. These studies showed that over-expression of the P2P-R protein promote camptothecin-induced apoptosis, whilst knocking out the PACT domain using homologous recombination promoted apoptosis. In this study, the role of RBBP6 in camptothecin induced apoptosis was investigated by knocking down the whole gene by targeting the 5’ DWNN domain of RBBP6 using RNA interference (RNAi). Two distinct small interference RNA (siRNA) oligonucleotides designated DWNN-A and DWNN-B targeting different regions of the gene were designed and two stable siRNA-expressing cell lines were established, namely RU6A and GU6B expressing DWNN-A and DWNN-B respectively. Qualitative fluorescent microscopy and quantitative Real-Time polymerase chain reaction (qRT-PCR) analysis were used to evaluate the silencing effect on RBBP6 expression in the NIH 3T3 parent cell line as compared to the stable cell lines RU6A and GU6B. The DWNN-A oligonucleotide effect appeared to be more potent than that exerted by DWNN-B. The stable cell lines proved to be significantly more resistant to apoptosis induced by camptothecin compared to the parental NIH 3T3 cell line using several apoptosis assays. Camptothecin induced apoptosis was restored to that observed in the NIH 3T3 cell line after the re-introduction of the full-length RBBP6 cDNA (DWNN-200). Furthermore, the results suggest that Apoptosis mediated through RBBP6 is dependent on p53 expression and possibly follows an intrinsic pathway as shown by the Bax/Bcl-2 ratio. Bcl-2 was higher than Bax in RU6A as compared to NIH 3T3 where Bax was higher compared to Bcl-2. Growth of the siRNA expressing cell lines RU6A and GU6B were also shown to be restricted in the G1 phase of the cell cycle implicating the RBBP6 gene in cell cycle progression. In conclusion the role of RBBP6 was established in camptothecin induced apoptosis and the cell cycle although the exact mechanisms have not been fully elucidated.
Keywords: Apoptosis, Cell cycle, RBBP6, RNA interference Camptothecin, p53, bax, bcl-2