The apoptotic response in HCT116^{<it>BAX-/- </it>}cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

• Main Authors: Semaan Sheila J, Nickells Robert W
• Format: Article
• Language: English
• Published: BMC 2010-10-01
• Series: BMC Cancer
• Online Access: http://www.biomedcentral.com/1471-2407/10/554

<p>Abstract</p> <p>Background</p> <p>Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the <it>BCL2 </it>gene family. Chemoresistance is often associated with abnormalities in concentrations of <it>BCL2 </it>family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood.</p> <p>Methods</p> <p>Exogenous GFP-murine <it>Bax </it>fusion constructs were transfected into <it>BAX</it>-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116^{<it>BAX-/- </it>}cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei.</p> <p>Results</p> <p>HCT116^{<it>BAX-/- </it>}cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations.</p> <p>Conclusion</p> <p>Within the limitations of this experimental system, <it>BAX</it>-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the translocation step of BAX activation may be impaired.</p>