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
Apoptosis
Necrosis
Purposes of programmed cell
death
II
I Achieve and
maintain normal
physiology:
-EMBRYONIC
DEVELOPMENT
-PROPER
III
MAINTENANCE OF
CERTAIN ORGANS
Checkpoint
mechanisms in
cell division
cycle:
- P53
- RB1
- SURVIVIN
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,1998
Multiple pathways/
Multiple mechanisms
Jaattela, 1999
Therapeutic applications of
regulating apoptosis
Promote apoptosis Prevent apoptosis
in cancer cells:
in certain disorders
and degenerative
Lymphoma/leukemia
diseases:
Oral cancer
AIDS
Brain tumors
Ischemia
Prostate
Alzheimer's/Parkinson's
Colon
etc.
etc.
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
Anti-apoptotic:
• Bcl-2
• Bcl-XL
• Bcl-W
• Mcl-1
• A1 (BFL-1)
• Boo
Pro-apoptotic:
• Bax
• Bad
• Bak
• Bik
• Bid
• Diva
• Bim
• Bcl-Xs
• Hrk
• Blk
Viral Vectors
• Retroviruses (permanent integration).
• Adeno-associated viruses (permanent
integration, small genome, requires
helper virus for growth, nonpathogenic).
• 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 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.