Role of BAX in the Apoptotic Response to Anticancer Agents

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Role of BAX in the Apoptotic Response to
Anticancer Agents

• Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B.
  2000. Role of BAX in the apoptotic response to
  anticancer agents. Science. 290(5493)989.
• Presented By Trina Geiss

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Purpose

• The role of BAX in drug-induced apoptosis in
  human colorectal cancer cells.
• Generate cells that lack functional BAX genes.
• Find implications for cancer chemoprevention
  strategies.

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Terms

• ApoptosisProgrammed Cell Death
• BAX Pro-Apoptotic Gene
• P53 Regulator of BAX, tumor suppressor gene.
• Bcl-2 Apoptosis Inhibiting Protein, silences
  effect of p53.
• MMR (mismatch repair)Excise incorrect
  nucleotides.
• Chemotherapeutic agents 5-flourouracil(5-FU)
  sulindac

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Background

• BAX is expressed in normal tissue
• Becomes inactive in the presence of certain
  cancers.
• This causes proliferation of cancer cells.
• Lower BAX activity means poor response to
  chemotherapy, fast tumor progression, and shorter
  survival.

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Role of p53

• Drugs may induce BAX expression through
  p53-dependent transcription.
• BAX plays no role in murine p53 gene.
• Murine BAX has no p53-binding site in its
  promotor region.
• BAX deficiency in mice may promote apoptosis.

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Role of Bcl-2

• Intimately involved in apoptosis.
• Role is confusing in drug-induced death.
• Interaction between BAX and Bcl-2 may cause cell
  death.
• Bcl-2, BAX, and p53 analysis could be good
  prognostic marker for patients who have already
  had surgery.

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Chemotherapeutic Agents

• 5-FU is the mainstay of treatment for colorectal
  cancer.
• Antimetabolite that induces cell death in a
  p53-dependent manner.
• Sulindac is the usual chemopreventive agent for
  those with colorectal cancer predisposition.
• It is a NSAID and it binds to and inhibits
  cellular proteins necessary for cell cycling.

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Overview

• Target of chemotherapeutic agentshuman
  epithelial cells
• Clarify role of BAX
• Create colorectal cancer cells that differ only
  in presence of BAX gene.

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Obtaining the Cells

• Exploit MMR-deficient cells to mutate
  mononucleotide tracts.
• BAX has unstable tract often mutated in
  MMR-deficient tumors.
• Created cells with two BAX alleles (/), one BAX
  allele (/-), and two mutant alleles (-/-).
• Confirm BAX (-/-) with Western Blot.

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Methods

• Culture cells with 5-FU and Sulindac for 48
  hours.
• Cell proteins separated by SDS-polyacrylamide gel
  electrophoresis.
• Immunoblotting with polyclonal antibody to BAX.
• Apoptosis assessed through microscopic
  visualization.

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Apoptosis
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Results
BAX genotype
/ /- -/-
5-FU Slight induction of BAX protein by agents that activate p53. Equivalent amounts of apoptosis Slight induction of BAX protein by agents that activate p53. Equivalent amounts of apoptosis No p53 deficiency. Apoptosis activity is reduced.
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Confirm the Results

• Make sure that it is BAX deficiency that caused
  the effect.
• Inactivate wild-type allele in BAX /-.
• Transfect to colorectal cancer cells.
• Treat cells with 5-FU and sulindac.
• Cells behaved just like BAX -/- cells.

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Applying the Results

• Parental cell populations treated with NSAIDs
  would be enriched in cells with mutations of BAX.
  
• Recovery of clones growing after NSAID treatment.
  
• The clones with BAX mutations were resistant to
  NSAIDs.

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NSAID Mechanisms

• NSAIDS cause BAX-dependent apoptosis by
• 1. Alterations in the ratio between
  proapoptotic and antiapoptotic members of the
  Bcl-2 family, can determine apoptotic
  sensitivity.
• 2. Expression of antiapoptotic Bcl-XL reduced by
  NSAIDs.

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Conclusions

• BAX and Bcl-XL proteins plays a major role in
  sensitivity to NSAIDs.
• Change in BAX/Bcl-XL should cause apoptosis
  through a mitochondrial pathway.
• BAX disruption will lead to differences in NSAID
  sensitivity.

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Importance of Experiment

• Use of NSAIDs on tumor cells has shown growth
  arrest, apoptosis, and necrosis.
• Used 3 different approaches for generating cells
  with disrupted BAX genes.
• Chemoprevention is best hope for nonsurgical
  management of hereditary colorectal cancer.

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References

• Fearon, ER. 2000. Human cancer syndromes clues
  to the origin and nature of cancer. Science
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• Russell PJ. 2000. Fundamentals of Genetics (2nd
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  Francisco.
• Terdiman JP, Gum JR, Conrad PG, Miller GA,
  Weinberg V, Crawley SC, Levin TR, Reeves C,
  Schmitt A, Hepburn M, Sleisenger MH, Kim YS.
  2001. Efficient detection of hereditary
  nonpolyposis colorectal cancer gene carriers by
  screening for tumor microsatellite instability
  before germline genetic testing.
  Gastroenterology. 12021-30.