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. 2012 Jun 28;31(26):3177-89.
doi: 10.1038/onc.2011.497. Epub 2011 Nov 7.

VSports手机版 - Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak

C Wang  1 , R J Youle
Affiliations

"VSports在线直播" Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak

C Wang et al. Oncogene. .

"VSports app下载" Abstract

The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax (Bcl2-associated X protein) and Bak (Bcl2-antagonist/killer 1). Although Bax and Bak seem to have redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for fluorouracil-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax, but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant, but they are counteracted by distinct anti-apoptotic Bcl-2 family proteins in different species VSports手机版. .

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Figures

Figure 1
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. LoxP 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.
Figure 2
Figure 2
Bak deficiency provides no protection against many apoptotic stimuli. (A) Cell viability assay in wild type, Bak KO, Bax KO and BaxBak DKO cells with 500 μM indomethacin and 120 μM sulindac acid treatment for 48h. Cell viability was measured with Promega’s CellTiter-Glo luminescent cell viability assay and normalized to non-treatment controls. The experiment was repeated three times and representative data were shown as average±SD. Since non-treated cells proliferate at a greater rate than the treated cells, this assay underestimates the survival rate of treated cells. (B) PARP cleavage in indicated cell lines with 1 μM camptothecin and 500 μM indomethacin treatment. (C) Cyt. c release in indicated cell lines with 1 μM camptothecin (camp), 500 μM indomethacin (indo) and 20 μM ABT-737. The experiment was repeated three times and representative data were shown as average±SD. (D) Caspase-3/7 activity in indicated cell lines with 20 μM ABT-737 treatment. The experiment was repeated twice and representative data were shown as average±SD. (E) Caspase-3/7 activity in indicated cell lines with different concentrations of ABT-737 for 24h. Representative data were shown as average±SD. (F) Clonogenic assay of tested cell lines with 120 μM sulindac acid, 0.2 μM staurosporine and 20 μM ABT-737 treatment. The experiment was repeated three times and a representative image was shown. The p-value was obtained using the Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 3
Figure 3
Bak deficiency leads to protection against 5-FU. (A) Cyt. c release in tested cell lines with 100 μM cisplatin or 375 μM 5-FU treatment. The experiment was repeated twice and representative data were shown as average±SD. (B) Caspase-3/7 activity in tested cell lines with 375 μM 5-FU or 10 ng/ml TRAIL plus 50 uM 5-FU treatment. The experiment was repeated twice and representative data were shown as average±SD. (C) Re-introduction of Bak in Bak KO cells restores the sensitivity to 5-FU (375 μM for 48h) indicated by Cyt.c release. The experiment was repeated twice and representative data were shown as average±SD. The p-value was obtained using the Student’s t-test.*, p<0.05; **, p<0.01; ***, p<0.001.
Figure 4
Figure 4
Bak is kept in check by Mcl-1 in Bax KO cells. (A) BH3-only proteins display different killing profiles to Bax KO cells shown by caspase-3/7 activity normalized to GFP-C1 vector control. BH3-only proteins were transiently expressed in the indicated cell lines and caspase-3/7 activity was measured 18 h after transfection. The experiment was repeated three times and representative data were shown as average±SD. (B) Noxa and NBK sensitize Bax KO cells to ABT-737. Puma, NBK or Noxa was transiently transfected and 20 μMABT-737 was added to the cells during transfection. Caspase-3/7 activity was measured 24 h after transfection and normalized to GFP-C1 vector control. The experiment was repeated twice and representative data were shown as average±SD. (C) Bcl-xS sensitizes cells to ABT-737 and Noxa sensitizes Bax KO cells to NBK. Untagged Bcl-xS, GFP-NBK and GFP-Noxa were transiently transfected and 20 μM ABT-737 was added during transfection. The experiment was repeated twice and representative data were shown as average±SD. (D-E) Protein levels of transiently expressed pro-and anti-apoptotic genes detected by Western blotting in HCT116 WT cells (E) and BaxBak DKO HCT116 cells (E). The p-value was obtained using the Student’s t-test.*, p<0.05; **, p<0.01; ***, p<0.001.
Figure 5
Figure 5
Interdependence of Bak and Bax activation. (A) A Bak mutant (m2, I85A/N86A) that fails to be bound by Bcl-xL and Mcl-1 sensitizes BaxBak DKO cells to camptothecin. GFP-Bak or GFP-Bak m2 was stably expressed in BaxBak DKO cells. Cells were treated with 1 μM camptothecin and Cyt.c release was examined. The experiment was repeated twice and representative data were shown as average±SD. (B) Bak expression levels in transiently expressed or stably expressed cells shown by Western blotting. Asteries indicate degraded Bak bands. (C) Caspase-3/7 acitivity was measured in indicated cell lines transiently transfected with GFP-Bax, GFP-Bak or in combination with GFP-Mcl-1 or GFP-Bcl-xL (1:3 ratio of DNA amount). Fold change was normalized with cells transfected with GFP-C1 vector control. The experiment was repeated twice and representative data were shown as average±SD. (D) Bak activation occurs normally in wild type cells in response to camptothecin and ABT-737 treatment detected by immunoprecipitation with anti-Bak (ab-1) antibody. Asteria indicates the light chain of Ig. (E) Bak activation indicated by Bak foci. GFP-Bak was stably expressed in Bak KO and BaxBak DKO cells. Cells were treated with 20 μM ABT-737, 1 μM camptothecin or 50 ng/ml TRAIL plus 375 μM 5-FU in the presence of 10 μM Q-VD for 24 h. (F) Bak activation in GFP-Bak stably expressing cells. Cells were treated and immnuoprecipitated as in (D). (G) Bcl-xL, VDAC2 and Mcl-1 express normally in Bax KO and Bak KO cells. The p-value was obtained using the Student’s t-test.*, p<0.05; **, p<0.01; ***, p<0.001.
Figure 6
Figure 6
Mcl-1 determines the resistance of Bax KO cells. (A) Knockdown level of Mcl-1 siRNA and Bcl-xL siRNA shown by Western blotting. Caspase-3/7 activity in control and Mcl-1 siRNA cells treated with 0.2 μM staurosporine (B) and 20 μM ABT-737 (C) for 24h was measured and normalized to untreated controls. Data were shown as average±SD. (D-E) Cyt.c release in control, Mcl-1 siRNA and Bcl-xL siRNA cells treated with 20 μM ABT-737 (D) and 1 μM camptothecin (E) for 24h. Data were shown as average±SD. The p-value was obtained using the Student’s t-test.*, p<0.05; **, p<0.01; ***, p<0.001.
Figure 7
Figure 7
Comparison of Bax/Bak-mediated cell death in HCT116 and MEF cells. (A-B) Caspase-3/7 activity in indicated MEF (A) or HCT116 (B) cell lines treated with 0.2 μM staurosporine. Fold change of caspase activity was normalized to untreated controls. The experiment was repeated twice and representative data were shown as average±SD. (C-D) Caspase-3/7 activity (C) or Cyt.c release (D) in indicated MEF cell lines treated with ABT-737. The experiment was repeated twice and representative data were shown as average±SD. (E-F) Cyt.c release in HCT116 (E) or MEF (F) cells transfected with GFP-tagged BH-3 only proteins and GFP-Bcl-xS for 24 hrs. GFP-C1 vector was used as a control. The experiment was repeated twice and representative data were shown as average±SD. The p-value was obtained using the Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001.
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References

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