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.