Oncogene (2012) 31, 3177–3189 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1’s inhibitory effect on Bak C Wang and RJ Youle Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA The intrinsic mitochondrial apoptotic pathway acts apoptosis induced by certain specific stimuli, at least in through two core pro-apoptotic proteins Bax (Bcl2- human cells. For example, Bak or Bax single knock- associated X protein) and Bak (Bcl2-antagonist/killer down renders HeLa cells resistant to Ngo or cisplatin- 1). Although Bax and Bak seem to have redundant roles in induced apoptosis (Kepp et al., 2007). Bax-deficient or apoptosis, accumulating evidence also suggests that they Bak-knockdown human glioblastoma multiforme tumor might not be interchangeable under certain conditions, at cells are also refractory to UV, staurosporine (STS) and least in some human cell lines. Here we report the doxorubicin (Cartron et al., 2003). The BH3-only generation of Bak knockout as well as BaxBak double protein natural born killer (NBK)/Bcl-2-interacting knockout HCT116 human colon carcinoma cells. We killer (Bik) has been shown to act entirely through Bax show that Bak is dispensable for apoptosis induced by a in human cells (Gillissen et al., 2003), but is solely variety of stimuli including ABT-737 but not for dependent on Bak in mouse cells (Shimazu et al., 2007). fluorouracil-induced apoptosis. In addition, Bax deficiency Unlike Bax-deficient MEF cells, BaxÀ/À HCT116 cells only provides partial protection against camptothecin and are extremely resistant to non-steroidal anti-inflamma- cisplatin-induced apoptosis and no protection against tory drugs (NSAIDs) (Zhang et al., 2000) and tumor killing by Puma or ABT-737 plus Noxa overexpression. necrosis factor-related apoptosis-inducing ligand Moreover, Bak is activated normally in response to many (TRAIL) (Deng et al., 2002; von Haefen et al., 2004). chemotherapeutic drugs in the presence of Bax, but As Bak is expressed at similar levels in BaxÀ/À as in the remains kept in check by Mcl-1 in the absence of Bax. wild type (WT) HCT116 cells and is functional Our data suggest that Bax and Bak are functionally (Theodorakis et al., 2002), it suggests that Bax is redundant, but they are counteracted by distinct anti- predominantly required for a large variety of apoptotic apoptotic Bcl-2 family proteins in different species. stimuli in certain cell lineages. Another possibility is that Oncogene (2012) 31, 3177–3189; doi:10.1038/onc.2011.497; Bak is completely suppressed by some inhibitors such as published online 7 November 2011 Bcl-xL and Mcl-1 (Willis et al., 2005). Although Bak activation is absent in BaxÀ/À HCT116 cells upon Keywords: HCT116; Bax; Bak; DKO; BH3-only; apoptosis cisplatin exposure (Kepp et al., 2007) or NBK over- expression (Gillissen et al., 2007), it has never been tested whether or not Bak activation occurs in WT HCT116 cells in response to those particular stimuli. Introduction Hence, it is not clear whether Bax is required for Bak activation (directly or indirectly) or certain apoptotic The essential roles of Bax and Bak in apoptosis were stimuli only specifically activate Bax but not Bak. The demonstrated by the complete resistance of BaxBak roles of Bax and Bak can only be explicitly dissected by double knockout (DKO) mouse embryonic fibroblast establishing BakÀ/À and BaxÀ/ÀBakÀ/À DKO HCT116 (MEF) cells to a variety of apoptotic stimuli (Wei et al., cell lines. 2001) and the finding that pro-apoptotic BH3-only Here we report the generation of Bak single and proteins such as Bim and Puma act through Bax/Bak BaxBak DKO HCT116 cell lines. We show that BaxÀ/À (Zong et al., 2001). Bax and Bak appear functionally BakÀ/À DKO cells are more resistant to various redundant, as loss of Bax or Bak alone does not provide apoptotic agents than BaxÀ/À cells, indicating that Bak significant protection against apoptosis (Lindsten et al., is still involved in many apoptotic responses. In cases 2000, Wei et al., 2001). However, growing evidence where BaxÀ/À cells are as resistant as BaxÀ/ÀBakÀ/À DKO suggests that Bax and Bak have non-redundant roles in cells, for instance, to ABT-737 treatment, Bak is activated in the WT cells but becomes restrained by / Correspondence: Dr RJ Youle, Biochemistry Section, Surgical Mcl-1 in BaxÀ À cells, highlighting the important role of Neurology Branch, National Institute of Neurological Disorders and Bax in removing Mcl-1’s grip on Bak. Our data also Stroke, National Institutes of Health, SNB/NINDS, Building 35, suggest that Bax is not restrained by Mcl-1 in human Room 2C917, 35 Lincoln Drive, Bethesda, MD 20892, USA. E-mail: [email protected] cells and the differential expression levels of Mcl-1 in Received 22 April 2011; revised and accepted 1 September 2011; MEFs and HCT116 cells might underlie the distinct published online 7 November 2011 phenotypes of Bax deficiency in these two species. BaxBak DKO in HCT116 cells C Wang and RJ Youle 3178 Results essential for mitochondrial localization of Bak and the BH3 domain is central to its apoptotic activity, either of Generation of BakÀ/À and BaxÀ/ÀBakÀ/À DKO cells these truncated proteins would be inactive. A promoter- Bax seems to be the key pro-apoptotic factor in human less gene-targeting vector was employed (Topaloglu cells as BaxÀ/À HCT116 cells are extremely resistant to et al., 2005). In combination with the effective rAAV apoptosis (Zhang et al., 2000) and Bak appears to be transduction system (Kohli et al., 2004), the promoter- dispensable for apoptosis based on gene-knockdown less gene-targeting vector exhibits high recombination studies (Cartron et al., 2003; Chandra et al., 2005; efficiency. Overall, 12 out of 36 clones were Bak þ /À and Gillissen et al., 2007, 2010). In order to explicitly dissect 5 out of 24 clones of BaxÀ/À cells were Bak þ /À, about 30 the role of Bak in apoptosis, we sought to knock out and 20% targeting efficiency, respectively. A similar Bak by homologous recombination in HCT116 cells. We targeting frequency was achieved in the second round targeted exon 5 of the Bak gene. Exon 5 encodes the targeting. Western blotting confirmed the successful BH1 domain and a part of the BH2 domain, whereas the abolishment of Bak expression in clones #29, 36, 44 BH3 domain is located on exon 4 and the transmem- and 49 yielding BakÀ/À and clones #2 and 19 yielding brane domain resides on exon 6 (Figure 1a). Targeting BaxÀ/ÀBakÀ/À DKO (Figure 1b). The premature stop exon 5 would result in a C-terminal-truncated protein codon introduced by the targeting vector (Figure 1d) lacking the transmembrane domain (due to the pre- likely triggers mRNA decay and aberrant splicing does mature stop codon introduced by the targeting vector, not occur, as no truncated Bak is produced in BakÀ/À Figure 1d) or an N-terminal truncated protein lacking and BaxÀ/ÀBakÀ/À DKO cells (Figure 1c), indicating that the BH3 domain. As the transmembrane domain is our BakÀ/À cells are truly null. NC 74 90 117 136 170 183 191 211 BH3 BH1 BH2 TM Bak protein structure 1234 5 6 Bak genomic locus IRES-Neo Targeting vector 123455IRES-Neo 6 After targeting 1234655 After Neo removal HCT116 MEF Bax+/+ Bax-/- KDa WT Bak-/-Bax-/-DKO WT Bak-/-Bax-/-DKO 28 +/- +/- Bak #29 #36 #44 #49 #2 #19 KDa #14 17 28 Bak 14 6 17 Bax 3 48 Actin 38 Figure 1 Generation of Bak KO and BaxBak DKO HCT116 cells. (a) Schematic diagram of gene targeting of Bak locus. The top panel shows BH3, BH1, BH2 and the transmembrane (TM) domains of Bak protein in orange, green, pink and blue. The exons encoding the corresponding domains are labeled in the same color in the Bak genomic locus and are shown as rectangles. Shaded regions of rectangles represent coding exons. Triangles indicate LoxP sites. (b) Confirmation of Bak knockout by western blot. #29, 36, 44 and 49 are homozygous Bak KO and #2 and #19 are homozgyous BaxBak DKO clones. Asterisk indicates a non-specific band detected by anti-Bax antibody. (c). No truncated or aberrant Bak was produced in Bak KO and BaxBak DKO HCT116 cells. Bak KO and BaxBak DKO MEFs were used as a control. Anti-Bak NT recognizes the N-terminal region of Bak. (d) Final Bak ORF sequence after gene targeting. Sequence underlined is introduced by the targeting vector. Lox P site is shown in red. The two premature stop codons are shown as bold italic. Alternating coding exons are shown in black or blue color. Note that exon 1 is a non-coding exon. Oncogene BaxBak DKO in HCT116 cells C Wang and RJ Youle 3179 HCT116 Bak-/- *** Bax-/- Bax Bak DKO *** n.s 50% *** HCT116 Bak KO Bax KO BaxBakKDa DKO 40% *** n.s PARP Camptothecin 48h 97 30% cleaved 97 PARP Indomethacin 24h 20% cleaved 28 Cell Viability (%) 10% Bak 17 Bax 0% indomethecin sulindac acid HCT116 Bak-/- Bax-/- *** HCT116 Bax Bak DKO Bak KO 100% *** *** *** Bax KO µ BaxBak DKO 20 M ABT-737 80% 80000 ** 70000 60% 60000 50000 40% ** n.s n.s n.s 40000 ** 30000 Cyt.c release (%) 20% 20000 10000 0% 0 Caspase-3/7 activity (a.u.) 0h 24h 48h indo 48h camp 24h camp 48h ABT-73724h ABT-73748h Control Sulindac acid 3 dayss 1 2 1 2 9 HCT116 ABT-737 8 Bak KO 3 4 3 4 7 Bax KO 6 BaxBak DKO 5 1.