The role of RbBP6 gene in apoptosis and the pathogenesis of lung cancer. Progress report Lesetja Motadi Supervisors: Dr Z Dlamini and Prof KD Bhoola School of Molecular and Cell Biology, University of the Witwatersrand.

Introduction Lung cancer (LC) is the most common- in SA L.C- cause 12% of deaths worldwide Causes-Smoking, abestosis and irradiation p53 mutation common genetic lesion in L.C

RbBP-6  RbBP6, both animal and plant kingdom.  Located at 16p11.2- p12  Homologues- PACT and P2PR  Human- 13kD and 200kD  200kD- Zinc, Ring finger, Rb and p53 binding domains.  Ring, DWNN and p53 domains-p53 dependent apoptosis.

Aims and Objectives Characterize, RbBP6 gene and apoptosis in L.C. Correlation between p53, caspases and Bcl2. Knockdown RbBP6 using RNAi. Mutation analysis if any.

Methodology Tissue culture- RNA extraction Probe preparation, clone by Zukile Cloning into P GEM T Easy- plasmid DNA extraction- restriction analysis- pst1 and apa1. Fluorescent In situ hybridization (FISH)- mRNA expression TUNEL- for apoptosis detection. Immunohistochemistry (ICC)- detection of protein expression.

Results Colony PCR

Plasmid DNA extraction

Restriction Analysis

ICC Results Figure4: 5’ 13 DWNN protein expression in the cytoplasm of normal alveolar (A) and in bronchial mucosa bronchial mucosa of the adeno-squamous. A B

Figure 5: 5’ 13 DWNN protein expression in the cytoplasm of the glandular tumour cell of the adenocarcinoma (C) and smooth muscle of the bronchial mucosa of the adeno-squamous carcinoma (D) C D

FISH Results Figure 6: 5’13 mRNA localization in the cytoplasm of the normal alveolar cells (A) and that of squamous cell carcinoma of the bronchial mucosa (B). A B

Figure 7: 5’ 13 mRNA highly localized in the cytoplasm of pulmonary adenocarcinoma (C) and well differentiated adenocarcinoma ( D) as compared to the normal tissue. D C

Future studies and analysis of results Results- suggest involvement of DWNN in apoptosis. Future studies include- Microarray analysis, Western and Northern Blot, RNAi and DWNN mutation analysis in L.C. Targeting RbBP6/DWNN as potential marker for inducing apoptosis in cancer.