1. 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.

2. 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

3. 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.

4. 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.

5. 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.

6. Results Colony PCR

7. Plasmid DNA extraction

8. Restriction Analysis

9. 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

10. 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

11. 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

12. 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

13. 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.