1. Mechanisms of Apoptosis/Review Ribozyme Inhibition of Bcl-2 Expression/Summary Sandra A. Gibson Hudson Ph.D.

2. APOPTOSIS “a distinct and important type of cell death” (Kerr 1971). A regulated decision triggered by the appearance or loss of an external signal. Cell shrinkage and apoptotic body production.

3. Morphology Apoptosis Necrosis

4. Purposes of programmed cell death I Achieve and maintain normal physiology: -EMBRYONIC DEVELOPMENT -PROPER MAINTENANCE OF CERTAIN ORGANS II Checkpoint mechanisms in cell division cycle: - P53 - RB1 - SURVIVIN IIISurvival of the organism: -RESPONSE TO EXTERNAL STIMULI

5. Apoptotic Pathway Cell death triggers: Cell death receptors: Cell death effector domains: Cell death enforcers: Cell death substrates: external or physiologic factors TNFR-1; FAS/APO-1/CD95 TRADD; RAIDD; FADD; RIP; FLICE caspases; endonucleases PARP; actin; DNA

6. Multiple pathways/ Multiple mechanismsHetts,1998

7. Jaattela, 1999

8. Therapeutic applications of regulating apoptosis Promote apoptosis in cancer cells: Lymphoma/leukemia Oral cancer Brain tumors Prostate Colon etc. Prevent apoptosis in certain disorders and degenerative diseases: AIDS Ischemia Alzheimer's/Parkinson's etc.

9. Ribozyme Inhibition of Bcl-2 Expression Apoptosis removes damaged cells from the body. The bcl-2 gene prevents this. The role of bcl-2 in oral cancer and glioblastoma is unexplored. We constructed a hammerhead ribozyme that would digest the bcl-2 mRNA message and delivered it to oral cancer and glioblastoma cells with an adenovirus vector.

10. Potential applications of ribozymes in gene therapy Inhibit viral replication Inhibit gene expression or protein expression Repair defective RNAs

11. B-cell Leukemia/Lymphoma 2 (Bcl-2) Bcl-2 is an integral membrane protein with many possible functions. Bcl-2 represses apoptotic cell death. Bcl-2 becomes deregulated in tumor cells. Overexpression of Bcl-2 protein allows for further genetic changes.

12. Bcl-2 family of proteins Bcl-2 family members participate in cell death regulation. Heterodimerization verses homodimerization of various bcl-2 family members can help determine the fate of a cell. Inhibiting Bcl-2 protein production in cells where it is overexpressed will eliminate a survival advantage and allow apoptosis to occur.

13. Bcl-2 family members Pro-apoptotic: Bax Bad Bak Bik Bid Diva Bim Bcl-Xs Hrk Blk Anti-apoptotic: Bcl-2 Bcl-X L Bcl-W Mcl-1 A1 (BFL-1) Boo

14. Viral Vectors Retroviruses (permanent integration). Adeno-associated viruses (permanent integration, small genome, requires helper virus for growth, non- pathogenic). Adenoviruses (transient expression, infect non-replicating cells, large genome). Herpes simplex virus (transient expression, infect non-replicating cells, large genome).

15. The anti-bcl-2 ribozyme and its target

16. Growth curves of ribozyme-infected cells TU183 cells

17. Immunoblot of Bcl-2 protein expression in ribozyme-infected cells (24 hours)

18. Apoptosis after 24 hour infection with Av1Rz279 or Av1LacZ4

20. Summary A hammerhead ribozyme was designed that would cleave bcl-2 mRNA. The ribozyme was expressed from an adenovirus vector. When oral cancer cells were infected with the vector they underwent apoptosis. Anti-bcl-2 ribozymes offer a new approach to treatment of tumors that express Bcl-2 such as, oral cancer, lymphomas and glioblastomas.

21. Future Directions Use a promoter that is stronger than the MMTV promoter. Design ribozymes with shorter and longer binding arms. Test the ribozyme in multiple cancer cell lines. Test the ribozyme in an animal model. Test the ribozyme with chemotherapeutic agents.