Impairment of the proapoptotic activity of Bax by missense mutations foundin gastrointestinal cancers

We have reported previously that codon 169 of the proapoptotic gene BAX is a mutational hot spot in gastrointestinal cancer. Two different mutations w ere found in this codon, replacing the wild-type threonine by alanine or me thionine, To compare the proapoptotic activity of these Bar mutants with wi ld-type Bar, we established an ecdysone (muristerone A)-inducible system in cultured human embryonal kidney 293 cells. Addition of muristerone A induc ed a dose-dependent decrease in the viability of cells transfected with wil d-type BAX, but this loss of viability was inhibited in cells transfected w ith BAX mutants, Furthermore, muristerone A induced morphological changes c haracteristic of apoptosis, including cell shrinkage, rounding, formation o f apoptotic bodies, detachment and nuclear condensation and fragmentation, in cells transfected with wild-type BAX, These hallmarks of apoptosis were clearly diminished in cells transfected with BAX mutants. Mutation of threo nine 169 did not affect the binding of Bar to Fax, Bcl-2, or Bcl-X-L, These results demonstrate that missense mutations at codon 169 of BAX are functi onal because they inhibit its apoptotic activity, This is the first report of the functional significance of missense mutations in BAX, or any other p roapoptotic member of the Bcl-2 family, in primary human tumors.