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.
• Cell shrinkage and apoptotic body production.
Morphology
ApoptosisApoptosis NecrosisNecrosis
Purposes of programmed cell death
I Achieve and maintain normal physiology:
-EMBRYONIC DEVELOPMENT -PROPER MAINTENANCE OF CERTAIN ORGANS
II Checkpoint mechanisms in
cell divisioncycle:
- P53
- RB1- SURVIVIN
III Survival of the organism:
-RESPONSE TO EXTERNAL STIMULI
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
Multiple pathways/Multiple mechanisms
Hetts,1998Hetts,1998
Multiple pathways/Multiple mechanisms
Jaattela, 1999Jaattela, 1999
Therapeutic applications of regulating apoptosis
Promote apoptosis in cancer cells:
Lymphoma/leukemiaOral cancerBrain tumorsProstateColon etc.
Prevent apoptosis in certain disorders and degenerative diseases:AIDSIschemia
Alzheimer's/Parkinson'setc.
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.
Potential applications of ribozymes in gene
therapy
•Inhibit viral replication•Inhibit gene expression or
protein expression•Repair defective RNAs
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.
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.
Bcl-2 family members
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
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).
The anti-bcl-2 ribozyme and its target
Growth curves of ribozyme-infected cells
TU183 cellsTU183 cells
Immunoblot of Bcl-2 protein expression in ribozyme-infected cells (24 hours)
Apoptosis after 24 hour infection with Av1Rz279 or Av1LacZ4
Apoptosis after 24 hour infection with Av1Rz279 or Av1LacZ4
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.
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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