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Mechanisms of Apoptosis/Review Ribozyme Inhibition of Bcl-2 Expression/Summary

Mechanisms of Apoptosis/Review Ribozyme Inhibition of Bcl-2 Expression/Summary. Sandra A. Gibson Hudson Ph.D. APOPTOSIS. “a distinct and important type of cell death” (Kerr 1971). A regulated decision triggered by the appearance or loss of an external signal.

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Mechanisms of Apoptosis/Review Ribozyme Inhibition of Bcl-2 Expression/Summary

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  1. Mechanisms of Apoptosis/ReviewRibozyme 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. IAchieve and maintain normal physiology: -EMBRYONIC DEVELOPMENT -PROPER MAINTENANCE OF CERTAIN ORGANS II Checkpoint mechanisms in cell division cycle: - P53 - RB1 - SURVIVIN III Survival of the organism: -RESPONSE TO EXTERNAL STIMULI Purposes of programmed cell death

  5. 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 Apoptotic Pathway

  6. Multiple pathways/Multiple mechanisms Hetts,1998

  7. Multiple pathways/Multiple mechanisms Jaattela, 1999

  8. 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. Therapeutic applications of regulating apoptosis

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

  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

  19. 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.

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