De-regulation of the RBBP6 isoform 3/DWNN in human cancers Zukile Mbita • Mervin Meyer • Amanda Skepu • Margot Hosie • Jasper Rees • Zodwa Dlamini Received: 7 June 2011 / Accepted: 3 November 2011 / Published online: 3 December 2011 Ó Springer Science+Business Media, LLC. 2011 Abstract Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apop- tosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis. Keywords RBBP6 isoform 3 Á Cell cycle Á Apoptosis Á Human cancers and FISH Introduction In humans, the retinoblastoma binding protein 6 (RBBP6) gene localized on chromosome 16p12.2 codes for three protein products [1–3]. RBBP6 isoform 1 has been shown to bind two tumour suppressor proteins, retinoblastoma protein (pRb) and p53 [4, 5]. The RBBP6 protein iso- forms, 1, 2 and 3 are derived from two mRNA transcripts, a 1.1 and 6.1 kb transcript. Isoform 1 is encoded by the 6.1 kb transcript, whilst alternative splicing of the 6.1 kb transcript results in isoform 2. Isoform 3 encoded by a 1.1 kb transcript is also known as a domain with no name (DWNN) [6]. The coding region of this gene is composed of 18 exons; isoform 1 is comprised of all 18 exons, whilst isoform 2 has 17 exons (exon 16 is absent, due to alternative splicing) and isoform 3 consists of only the first 3 exons. These three exons that encode DWNN are also found in transcripts 1 and 2. Consequently, all the three RBBP6 isoforms share a common N-terminal domain, DWNN. DWNN was first identified through genetic screening aimed at identifying novel components of the antigen processing and presentation pathway via major histocompatibility class I (MHC class I) mol- ecules [7]. Z. Mbita Á Z. Dlamini (&) College of Agriculture and Environmental Science, University of South Africa, Florida Campus, Corner Christian de Vet Ave and Pioneer, Johannesburg 1710, South Africa e-mail: dlamizl@unisa.ac.za M. Meyer Biotechnology Department, University of the Western Cape, Bellville, Cape Town 7535, South Africa A. Skepu Mintek, National Innovation Centre (NIC) Biolabels, Johannesburg 2125, South Africa M. Hosie School of Anatomical Sciences, University of the Witwatersrand, Parktown 2193, South Africa J. Rees Division of Biotechnology, Agricultural Research Council of South Africa, Hatfield, Pretoria 0001, South Africa 123 Mol Cell Biochem (2012) 362:249–262 DOI 10.1007/s11010-011-1150-5