mechanisms of apoptosis/review ribozyme inhibition of bcl-2 expression/summary sandra a. gibson...
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![Page 1: Mechanisms of Apoptosis/Review Ribozyme Inhibition of Bcl-2 Expression/Summary Sandra A. Gibson Hudson Ph.D](https://reader035.vdocuments.us/reader035/viewer/2022062516/56649d575503460f94a36ce7/html5/thumbnails/1.jpg)
Mechanisms of Apoptosis/Review
Ribozyme Inhibition of Bcl-2 Expression/Summary
Sandra A. Gibson Hudson Ph.D.
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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.
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Morphology
ApoptosisApoptosis NecrosisNecrosis
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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
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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
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Multiple pathways/Multiple mechanisms
Hetts,1998Hetts,1998
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Multiple pathways/Multiple mechanisms
Jaattela, 1999Jaattela, 1999
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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.
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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.
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Potential applications of ribozymes in gene
therapy
•Inhibit viral replication•Inhibit gene expression or
protein expression•Repair defective RNAs
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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.
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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.
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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
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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).
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The anti-bcl-2 ribozyme and its target
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Growth curves of ribozyme-infected cells
TU183 cellsTU183 cells
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Immunoblot of Bcl-2 protein expression in ribozyme-infected cells (24 hours)
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Apoptosis after 24 hour infection with Av1Rz279 or Av1LacZ4
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Apoptosis after 24 hour infection with Av1Rz279 or Av1LacZ4
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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.
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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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