Impairment of the proapoptotic activity of Bax by missense mutations found in 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 were found in this codon, replacing the wild-type threonine by alanine or methionine. To compare the proapoptotic activity of these Bax mutants with wild-type Bax, we established an ecdysone (muristerone A)-inducible system in cultured human embryonal kidney 293 cells. Addition of muristerone A induced a dose-dependent decrease in the viability of cells transfected with wild-type BAX, but this loss of viability was inhibited in cells transfected with BAX mutants. Furthermore, muristerone A induced morphological changes characteristic of apoptosis, including cell shrinkage, rounding, formation of 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 threonine 169 did not affect the binding of Bax to Bax, Bcl-2, or Bcl-X(L). These results demonstrate that missense mutations at codon 169 of BAX are functional because they inhibit its apoptotic activity. This is the first report of the functional significance of missense mutations in BAX, or any other proapoptotic member of the Bcl-2 family, in primary human tumors.
Gil
J
J
The Burnham Institute, La Jolla, California 92037, USA.
Yamamoto
H
H
Zapata
J M
JM
Reed
J C
JC
Perucho
M
M
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000235:genetics*; D001692:Biological Transport; D002461:Cell Line, Transformed; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis; D019281:Dimerization; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D016368:Frameshift Mutation; D005770:Gastrointestinal Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D007668:Kidney / Q000166:cytology; D008715:Methionine / Q000737:chemistry; D018895:Microsatellite Repeats; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology; D011485:Protein Binding; D055503:Protein Multimerization; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000187:drug effects / Q000235:genetics; D011987:Receptors, Steroid / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000187:drug effects; D047488:Retinoid X Receptors; D013912:Threonine / Q000737:chemistry; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics; D014162:Transfection; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D003062:Codon; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C055469:ecdysone receptor; D013912:Threonine; C055348:muristerone A; D004441:Ecdysterone; D008715:Methionine; D000409:Alanine