[CANCER RESEARCH 59, 2034 –2037, May 1, 1999] Advances in Brief Impairment of the Proapoptotic Activity of Bax by Missense Mutations Found in Gastrointestinal Cancers 1 Joan Gil, Hiroyuki Yamamoto, Juan M. Zapata, John C. Reed, and Manuel Perucho 2 The Burnham Institute, La Jolla, California 92037 Abstract 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. Ad- dition 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, muris- terone A induced morphological changes characteristic of apoptosis, in- cluding cell shrinkage, rounding, formation of apoptotic bodies, detach- ment 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 demon- strate 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. Introduction Homeostasis of tissues is regulated by a balance between cell proliferation and apoptosis. Therefore, dysregulation of apoptosis may be involved in the initiation and progression of human cancer (1). The Bcl-2 family of proteins plays a pivotal role in the regulation of apoptosis (2). Some member of this family, such as Bcl-2 and Bcl-X L , function as cell death suppressors, whereas others such as Bax induce apoptosis. The Bcl-2 oncogene was identified at t(14:18) chromo- somal translocation breakpoints in B-cell neoplasms (3), representing the first genetic alteration in human cancer of a gene encoding for a protein involved in the control of apoptosis. Several lines of evidence indicate that the proapoptotic protein Bax (4) plays a tumor suppressor role. Mice deficient in bax grow nor- mally but eventually develop lymphoid hyperplasia (5). The tumor suppressor p53 is a direct transcriptional activator of BAX (6). Abla- tion of BAX in mice reportedly decreased apoptosis induced by a transgene expressing a truncated T antigen that selectively binds Rb and induces p53-dependent apoptosis (7). In addition, chemothera- peutic agents induce apoptosis in embryonic fibroblasts in a p53- dependent manner, and elimination of BAX prevents approximately half of those chemotherapy-induced deaths (8). Reduced Bax expres- sion is associated with poor responses to chemotherapy and shorter survival in women with breast adenocarcinoma (9). Overexpression of BAX in breast cancer cells restores sensitivity to a variety of apoptotic stimuli and reduces tumor growth in nude mice (10). The finding of somatic frameshift mutations in the BAX gene in colon cancers of the MMP 3 provided genetic evidence for the role of BAX inactivation in human tumor progression (11). Later studies have found frameshift mutations of BAX in other MMP+ tumors (12, 13). BAX mutations have also been found in cell lines derived from hematopoietic malignancies (14, 15). The existence of other types of nonframeshift somatic mutations in BAX added further support to the concept that this gene functions as a tumor suppressor (12–14, 16, 17). Interestingly, some of these mutations introduce single amino acid substitutions (missense muta- tions). Analysis of these spontaneous mutations thus could contribute to an improved understanding of the mechanisms by which the Bax protein induces apoptosis. We found a mutational hot spot at codon 169 in MMP+ gastroin- testinal cancers (12, 13). Two different mutations occurred in this codon, replacing the wild-type threonine by alanine or methionine. This residue lies just proximal of the COOH-terminal hydrophobic region implicated in insertion of Bax into membranes (18 –20). Here we demonstrate that mutation of threonine 169 either to alanine or methionine decreases the proapoptotic activity of Bax. Materials and Methods Plasmid Constructions. A 0.6 Kb human BAX-cDNA subcloned into the EcoRI site of pcDNA3 (Invitrogen, Carlsbad, CA) was liberated by EcoRI digestion, and the fragment was subcloned into the EcoRI site of pIND (Invitrogen), creating pIND-BAX. Mutant BAX plasmids were created using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and pIND-BAX as the template with the following mutagenic primers: pIND- BAXT169A, 5'-CTTTGGGACGCCCGCGTGGCAGACCG-3' and 5'-CG- GTCTGCCACGCGGGCGTCCCAAAG-3' and pIND-BAX T169M 5'-CAC- GGTCTGCCACATGGGCGTCCCAAAG-3' and 5'-CACGGTCTGCCACA- TGGGCGTCCCAAAG-3. Clones with the wild or mutant BAX cDNA inserted in sense orientation were identified by restriction digestion and confirmed by DNA sequencing using the ABI PRISM dye terminator cycle sequencing kit (Perkin-Elmer, Branchburg, NJ). Generation of Stable Transfectants with Inducible Bax Expression. To generate stable transfectants with inducible Bax expression, we used human transformed primary embryonal kidney cells (HEK-293) transfected with pVgRXR (293VgRXR; Invitrogen). pVgRXR encodes for a heterodimer of the ecdysone receptor and the retinoid X receptor, which binds to the ecdysone response element (encoded by pIND) in the presence of muristerone A (21). 293VgRXR cells were transfected with pIND-BAX, pIND-BAX-T169A, and pIND-BAX-T169M by a calcium phosphate precipitation method, and stable transfectants were selected with 1 mg/ml Geneticin (Life Technologies, Inc., Gaithersburg, MD). After 3 weeks, antibiotic-resistant cells were maintained in DMEM supplemented with 10% FCS, 2 mML-glutamine, 100 units/ml penicillin, Received 12/29/98; accepted 3/18/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA63585 and CA38579 (to M. P.). J. G. was supported by a fellowship from Ministerio de Educacaion y Cultura (Spain). H. Y. was supported by a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science. J. M. Z. was supported by the Breast Cancer Research Program of the University of California. 2 To whom requests for reprints should be addressed, at The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (619) 646-3112; Fax: (619) 646-3190; E-mail: mperucho@ljcrf.edu. 3 The abbreviations used are: MMP, microsatellite mutator phenotype; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4',6-diamidino-2- phenylindole. 2034 Research. on December 1, 2015. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from