Molecular mechanisms of apoptosis in lung cancer: a role for retinoblastoma binding protein 6 (RBBP6) and its protein products
Date
2010-08-24
Authors
Motadi, Lesetja Raymond
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Abstract
RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts
with both p53 and pRb and has been implicated in mRNA processing. It has also been
identified as an E3 ubiquitin ligase due to the presence of a RING finger domain and also
assumed to have a regulatory role of p53 due to the presence p53BD through MdM2,
although no substrate has been identified up to now. RBBP6 gene mutants are reported to be
resistant to apoptosis inducers, which led to a belief that mutation of this gene might result in
the development of lung cancer. Earlier localization and expression studies have shown that
RBBP6 expression and apoptosis levels are indirectly proportional. The purpose of this study
is to establish the expressional pattern of the RBBP6 gene in lung cancer at both mRNA and
protein levels. The objective is also to characterize the role of this gene and apoptosis in
diverse lung diseases. An understanding of the role of RBBP6 in the development of lung
diseases may lead to insights into developing new therapeutic measures for those lung
diseases in which apoptosis plays a prominent part. This thesis elucidate the possible role of RBBP6 in lung cancer and apoptosis, to establish
tissue distribution and expression levels of RBBP6 at protein and mRNA levels in lung
cancer by immunocytochemistry (ICC), in situ hybridization (ISH) and confirm findings by
quantitative RT-PCR. RBBP6 mRNA transcripts were expressed in the cytoplasm of normal
and tumour lung epithelium. In general, expression was highest in the cytoplasm of welldifferentiated
carcinoma and invasive carcinoma that showed signs of cells undergoing
mitosis. Immunolabelling results further showed high level of expression in all lung cancer
types except in Small and large cell carcinomas. The immunolabeling were confirmed by ISH
experiments and RT-PCR. In relation to p53, RBBP6 mRNA expression was higher in lung cancer cell lines that had
p53 silenced and apoptosis induced by TRAIL and camptothecin. There was no notable
change in the levels of p53 expression following RBBP6 silencing and apoptosis induction.
However, there was a little correlation between RBBP6 expression and apoptosis levels in
both lung cancer tissues by TUNEL and lung cancer cell line following apoptosis induction
by TRAIL. The ratio of Bax/Bcl-2 was seen to be upregulated following p53 and RBBP6
silencing after apoptosis induction. The most common mutation notable after RBBP6 DNA
sequencing was point mutations where only single nucleotide was mutated and mostly they
were observed in lung cancer tissues.
This was the first demonstration that RBBP6 is expressed in lung cancers. Because of the
ubiquitin-like nature of the protein and its localized up-regulation and corresponding proapoptotic
activity in lung cancer cells, it is possible that further characterization of this gene could lead to its manipulation as a diagnostic marker and a potential therapeutic target for
cancer treatment.
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Keywords
RBBP6, Apoptosis, Bax, lung cancer, p53 protein