1. by Xuefang Cao, Xingming Deng, and W. Stratford May
Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Bax
by Xuefang Cao, Xingming Deng, and W. Stratford May
Blood
Volume 102(7):
October 1, 2003
©2003 by American Society of Hematology

2. Apoptosis-associated Bax cleavage following drug treatment or IL-3 withdrawal and Bax cDNAs constructs.
Apoptosis-associated Bax cleavage following drug treatment or IL-3 withdrawal and Bax cDNAs constructs. (A) HL-60 cells were treated with various doses of camptothecin, a DNA topoisomerase I inhibitor, for 20 hours. (B) H-7 cells were deprived of IL-3 for the indicated times. (Upper panels) Proteins were extracted and subjected to SDS-PAGE and Western blotting with anti-Bax antibody. (Lower panels) DNA fragmentations were determined as described in “Materials and methods” to confirm that cells underwent apoptosis. (C) K-562, U-937, and A-549 cells were treated with 20 μM cisplatin for 36 hours. (D) K-562, U-937, and A-549 cells were treated with 50 μM etoposide for 36 hours. Cell viabilities were measured by trypan blue exclusion method. Proteins were extracted and subjected to SDS-PAGE and Western blotting with anti-Bax antibody. Error bars indicate means ± SD (n = 3). (E) WT Bax cDNA was cloned from H-7 cells with a T7 tag at the N-terminal. Asp33Ala, Glu6Ala, and N-terminal-deleted p18 (Δ1-33) Bax cDNA mutants were constructed as described.
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

3. Ponasterone A (PA)-inducible expression of Bax; the effect of cathepsin inhibitors on Bax expression and viability studies.
Ponasterone A (PA)-inducible expression of Bax; the effect of cathepsin inhibitors on Bax expression and viability studies. Three separate groups of WT, Asp33Ala, p18, Glu6Ala, or empty vector-transfected 293 cells were used for inducible Bax expression and viability studies as described. (A) Representatives of time course-inducible Bax expressions. Following addition of 6 μM PA, proteins were extracted at the indicated times and subjected to SDS-PAGE and Western blotting with mouse-specific anti-Bax antibody and anti-actin antibody for loading control. (B) Cell viabilities were measured by trypan blue exclusion method following PA-inducible expression of Bax. DNA fragmentations were determined to confirm that cells underwent apoptosis. (C) Twenty-four-hour PA induction of Bax was performed in the presence of 20 μM calpeptin (inhibits calpain) or 40 μM ALLM (inhibits both calpain and cathepsin) or 20 μM cathepsin inhibitor I (CI) or 2 μg/mL adriamycin as described in “Materials and methods.” Lane alignment: 0, vector control; 1, WT; 2, Asp33Ala; 3, p18; 4, Glu6Ala. The representatives of 3 independent experiments were shown. (D) Cell viabilities following 24-hour PA induction of Bax in the presence of CI. (E) Cell viabilities following PA induction of Bax in the presence of ALLM. (F) Cell viabilities following 24-hour combination of PA induction and adriamycin treatment. Error bars in B, D-F indicate means ± SD (n = 3).
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

4. Comparison of induced Bax and endogenous Bax.
Comparison of induced Bax and endogenous Bax. (A) Comparison between PA-induced Bax and endogenous Bax in 293 cells. Following induction with 6 μ MPA, proteins were extracted at the indicated times and 100 μg lysate of each time point was subjected to SDS-PAGE and Western blotting with anti-Bax antibody (Cell Signaling Technology; reactive to both the induced mouse Bax and endogenous Bax of human 293 cell); (B) Comparison of endogenous Bax between different cell lines. 100 μg lysate from each cell line was subjected to Western blotting with the anti-Bax antibody described above.
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

5. p18 Bax has a reduced half-life that can be enhanced by ALLM
p18 Bax has a reduced half-life that can be enhanced by ALLM. (A) The half-life of WT and p18 Bax was first determined by [35S]methionine metabolic labeling and pulse-chase as described in “Materials and methods.” (B) The half-life of WT, Asp33Ala, p18, and...
p18 Bax has a reduced half-life that can be enhanced by ALLM. (A) The half-life of WT and p18 Bax was first determined by [35S]methionine metabolic labeling and pulse-chase as described in “Materials and methods.” (B) The half-life of WT, Asp33Ala, p18, and Glu6Ala Bax was determined again using the cyclohexamide method as described in “Materials and methods.” (C) The effect of ALLM on Bax protein half-life was determined by cyclohexamide method. Half-life was determined by densitometry analysis.
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Expression of Asp33Ala or p18 Bax induces increased caspase-9/-3 activation compared with WT or Glu6Ala Bax.
Expression of Asp33Ala or p18 Bax induces increased caspase-9/-3 activation compared with WT or Glu6Ala Bax. Caspase-3 activity assay was performed following inducible Bax expression as described in “Materials and methods.” For caspase-9/-3 cleavage studies, proteins were extracted and subjected to SDS-PAGE and Western blotting with cleaved caspase-9/-3-specific antibodies and full-length procaspase-9/-3-specific antibodies, respectively. (A) Time course caspase-3 activity and caspase-9/-3 cleavage following inducible Bax expression. (B) Caspase-3 activity and caspase-9/-3 cleavage following inducible Bax expression in the presence of 40 μM ALLM. Error bars indicate means ± SD (n = 3).
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

7. Expression of Asp33Ala or p18 Bax induces more cytochrome c release than WT or Glu6Ala Bax.
Expression of Asp33Ala or p18 Bax induces more cytochrome c release than WT or Glu6Ala Bax. Following inducible expression, cytosolic and mitochondrial fractions were generated using a digitonin-based subcellular fractionation technique. Equal amounts (50 μg) of mitochondrial and cytosolic fractions were subjected to SDS-PAGE and Western blotting with anticytochrome c antibody and, for negative control, antiprohibitin antibody. (A) Time course cytochrome c release following PA-inducible Bax expression. (B) Cytochrome c release at 24 hours following PA-inducible Bax expression in the absence or presence of 40 μM ALLM.
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

8. Mitochondrial targeting and membrane insertion of Bax are not affected by N-terminal cleavage or Asp33Ala mutation.
Mitochondrial targeting and membrane insertion of Bax are not affected by N-terminal cleavage or Asp33Ala mutation. Inducible Bax expression was performed in the presence of 40 μM ALLM for the indicated times. The heavy membrane was isolated, and alkali extraction was performed to determine the percentage of the alkali-resistant (integral, nonextractable) Bax contained in the mitochondrial membrane as described in “Materials and methods.” Intermembrane cytochrome c was used as extractable positive control, and inner membrane prohibitin was used as nonextractable negative control.
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

9. N-terminal cleavage or Asp33Ala mutation appears to enhance homooligomerization of Bax in mitochondrial membranes.
N-terminal cleavage or Asp33Ala mutation appears to enhance homooligomerization of Bax in mitochondrial membranes. Inducible Bax expression was performed in the presence of 40 μM ALLM for 24 hours. The heavy membrane was collected, and cross-linking was performed as described in “Materials and methods.” Then, membranes were lysed and 40μg lysate was analyzed by SDS-PAGE and Western blots with anti-T7 tag antibody.
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

10. Cathepsin inhibitor I (CI) amplifies drug-induced apoptosis in HL-60 cells.
Cathepsin inhibitor I (CI) amplifies drug-induced apoptosis in HL-60 cells. (A) HL-60 cells were treated with 2 μM camptothecin only or in the presence of 20 μM calpeptin (calpain inhibitor), or 20 μM cathepsin inhibitor I (CI) or 20 μM ALLM (inhibits calpain and cathepsin) for the indicated times. (B) HL-60 cells were treated with 20 μM cisplatin, or 50 μM etoposide or 0.5 μg/mL adriamycin alone for 20 hours or in the presence of inhibitor. Control cells were left untreated or treated with either the same dose of inhibitor only or a corresponding volume of dimethyl sulfoxide (DMSO). Proteins were extracted and subjected to SDS-PAGE and Western blotting with human-specific anti-Bax antibody, anti-Bcl-2 antibody, and anti-actin antibody for loading control. Cell viabilities were measured by trypan blue exclusion method. Error bars indicate means ± SD (n = 3).
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

11. Calpain inhibitors block while cathepsin inhibitor I (CI) amplifies drug-induced apoptosis in A-549, K-562, and U-937 cells.
Calpain inhibitors block while cathepsin inhibitor I (CI) amplifies drug-induced apoptosis in A-549, K-562, and U-937 cells. K-562, U-937, and A-549 cells were treated with 20 μM cisplatin alone or in the presence of 20 μM calpeptin (calpain inhibitor) or 40 μM CI or 20 μM ALLM for 36 hours. Control cells were left untreated or treated with either the same dose of inhibitor only or a corresponding volume of DMSO. (A) Proteins were extracted and subjected to SDS-PAGE and Western blotting with human-specific anti-Bax, anti-Bcl-2, and anti-Bid antibodies. (B) Cell viabilities were measured by the trypan blue exclusion method. Caspase-3 activity was measured as described in “Materials and methods” at 24 hours following the treatments to confirm the viability results. Error bars indicate means ± SD (n = 3).
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology

12. Proposed model: calpain-mediated cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like cysteine protease may rapidly degrade p18 Bax.
Proposed model: calpain-mediated cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like cysteine protease may rapidly degrade p18 Bax. Although full-length p21 Bax can initiate apoptosis through the mitochondrial pathway, p18 Bax is more potent than WT p21 Bax in disrupting mitochondrial integrity and inducing apoptosis. Therefore, calpain-mediated cleavage of Bax to p18 Bax functions as an amplification step that accelerates the apoptotic process following cytotoxic stress. On the other hand, removal of the N-terminal domain of Bax facilitates the rapid degradation of p18 Bax by a cathepsin-like cysteine protease. This specific proteolytic degradation of p18 Bax may be equivalent to a silencing mechanism for this potent form of Bax to delay cell death or promote survival. Thus, in addition to the executionary consequences of caspase activation, noncaspase cysteine proteases such as calpain and the cathepsin-like protease may be involved in regulating apoptosis through sequential proteolytic processing of Bax and p18 Bax.
Xuefang Cao et al. Blood 2003;102:
©2003 by American Society of Hematology