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NEOPLASIA
From the Department of Haematology, St Bartholomew's
and The Royal London School of Medicine and Dentistry, London, United
Kingdom; and the Department of Microbiology and Immunology, Kimmel
Cancer Institute, Thomas Jefferson University, Philadelphia, PA.
The human leukemia cell lines K562, CEM, CEM/VLB100,
human leukemic blasts, and the bladder cancer J82 cell line have
different sensitivities to UV light-induced apoptosis. It is reported
that resistance to UV light-induced apoptosis occurs at a point in the
apoptotic pathway upstream of caspase-3 but downstream of mitochondrial
cytochrome c release. It is demonstrated that the block is
due to deficiency of Apaf-1, a critical member of the apoptosome.
Sensitivity to apoptosis was independent of caspase-9b or XIAP
(inhibitors of apoptosis proteins) expression or levels of
procaspase-9. Transfection of Apaf-1 conferred sensitivity to apoptosis
in resistant cells. Apaf-1 deficiency may constitute a significant mode
of resistance to apoptosis in human leukemia.
(Blood. 2001;98:414-421) The apoptosome, which includes the protein Apaf-1,
cytochrome c/dATP, and procaspase-9, plays a central role in
the apoptotic process.1-8 Cytochrome c is
released from mitochondria into the cytosol after the induction of
apoptosis by many different stimuli, including CD95, tumor necrosis
factor- Cells from Apaf-1 or procaspase-9 knock-out mice are resistant to
several apoptotic stimuli.8,16-19 Deletions of Apaf-1 or caspase-9 in ras- and myc-transformed
murine cells can replicate the tumorigenic effects of p53
deletion.8 Oncogene (such as E1A)-transformed cells have
increased levels of Apaf-1 and procaspase-9 protein expression and are
sensitized to etoposide-induced apoptosis.5 Inactivation
of Apaf-1 and caspase-9 substantially reduces the number of cells
required to form tumors.8 Transfection of the Apaf-1 gene into leukemic cells increases the sensitivity of
cells to etoposide-induced apoptosis.20
An endogenous, alternatively spliced isoform of caspase-9 (named
caspase-9b), which lacks the central large subunit caspase domain, can
interact with the caspase recruitment domain of Apaf-1 and inhibit the
apoptotic process.21 Even in the presence of functional
Apaf-1 and procaspase-9, inhibitors of apoptosis, such as XIAP
(inhibitors of apoptosis proteins), can prevent the proteolytic processing of procaspase-3 by blocking the cytochrome
c-induced activation of procaspase-9.22
IAPs, in turn, can be regulated by the mitochondrial protein,
Smac.23,24 These data provide strong evidence that
appropriate and functional levels of Apaf-1 or procaspase-9 proteins
are crucial for the inhibition of tumor progression and for maintaining
the sensitivity of tumor cells to apoptosis. However, it is
unknown whether variations in constitutive levels of Apaf-1 have
implications for human leukemia.
Human leukemic cells differ widely in their susceptibility to
apoptosis. This is exemplified by the human leukemic cell lines, K562,
CEM, and CEM/VLB100, which have different sensitivities to
tumor necrosis factor (TNF)- Cell lines, acute myeloid leukemia blasts, and
Apaf-1 gene transfection
Cell lines were cultured in RPMI-1640 medium as described
previously.25 Flag-tagged Apaf-1L (GenBank accession no.
AF134397) pCMV2 plasmid4,14 was grown in Escherichia
coli DH5 Determination of DNA damage-alkaline unwinding assay
Western blotting Proteins were subjected to standard SDS-PAGE at 20 to 40 mA/gel and were transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hertfordshire, United Kingdom) for 1 to 2 hours at 100 V after they were probed for various proteins using monoclonal or polyclonal antibodies (as described individually in the figure legends). Bound antibodies were detected using appropriate horseradish peroxidase-conjugated secondary antibodies, followed by detection using SuperSignal enhanced chemiluminescence (ECL; Pierce, Rockford, IL). The density of each band was analyzed using an AlphaImager 2000 Densitometer (Alpha Innotech, San Jose, CA).Measurement of apoptosis by flow cytometry To induce apoptosis in intact cells, leukemic cells (5 × 105/mL) were exposed to UV irradiation (120 mJ/cm2) (model TM-20; Chromato-UV-E Transilluminator) for 2 minutes and then further cultured for up to 12 hours. Irradiation was performed with a -ray source (cesium Cs 137; Gamma Cell 1000, Atomic
Energy of Canada, Ontario) at a dose of 100 Gy. Cells were permeabilized with 70% ethanol, stained with 100 µg/mL propidium iodide (Sigma), and measured by flow cytometry (FACScan, Becton Dickinson).26
Preparation of subcellular fractions Cells were suspended in 1 mL Buffer A (250 mM sucrose, 10 mM HEPES-KOH, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 20 µM cytochalasin B) and incubated for 20 minutes on ice. Cells were then broken with a glass Dounce homogenizer (Jencons, Leighton Buzzard, United Kingdom). Mitochondrial purification was described previously.10 S-100 fraction was obtained by ultracentrifuge cytosol at 100 000g for 1 hour.Measurement of activity of caspase-3 and caspase-9 Fifteen microliters S-100 (50 µg protein) was diluted to 95 µL with Buffer A. The reaction was initiated by the addition of 5 µL 400 µM (final concentration was 20 µM) fluorescent substrates, Ac-Leu-Glu-His-Asp-AFC (Ac-LEHD-AFC; Calbiochem) for caspase-9 or Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC; Calbiochem) for caspase-3. After incubation at 30°C for 15 minutes, the reaction was stopped by the addition of 50 µL 1% sodium acetate trihydrate in 175 mM acetic acid. Fluorescence at 400/505 nm for caspase-9 or at 380/460 nm for caspase-3 was measured with a TD-700 fluorometer. Measurements were calibrated against a standard linear regression curve of AFC or AMC. Caspase activity was defined as µM AFC or AMC release per milligram protein per hour (µM/h per milligram protein).Coupled in vitro transcription/translation [35S] Methionine-labeled caspase-9 was in vitro transcribed and translated using the T7 polymerase TNT kit (Promega, Southampton, United Kingdom). One microgram pRSC-LacZ-caspase-9 plasmid4 was used in a 50 µL transcription/translation reaction mixture containing 1 µL [35S] methionine. Translation was completed at 30°C for 2 hours.Immunodepletion of Apaf-1 One microliter monoclonal anti-Apaf-1 antibody was added to 100 µL S-100 (300 µg protein) and incubated for 2 hours at 4°C on a rotor. Thirty microliters protein G Sepharose (Pharmacia Biotech, Amersham, United Kingdom) was then added into S-100 to bind Apaf-1 and antibody complex, and the incubation was continued for 2 hours. After spinning down, the supernatant was used for the cell-free reactions.Cell-free reactions Cell-free reactions were set up in 20 µL reaction volumes. For cytochrome c/dATP-induced activation of caspases, 50 µg S-100 in Buffer A was incubated with or without bovine heart cytochrome c (50 ng)/dATP (10 nm) at 30°C up to 1 hour. To detect cytochrome c/dATP-induced activation of exogenous procaspase-9, 0.5 µL [35S]-labeled procaspase-9 was added to the reaction system before cytochrome c/dATP. The protein was subjected to 10% SDS-PAGE. The gel was dried and exposed to x-ray film. For granzyme B-induced caspase-3 and caspase-9 activation, 2 U granzyme B (Calbiochem) was added to each reaction and incubated at 30°C for 1 hour. Activities of caspase-3 and -9 were measured by fluorogenic assay as described above. Cleavage of the caspase proteins was confirmed by Western blot analysis. Monoclonal anticaspase-9 antibody5 was used at a 1:1000 dilution for the detection of the procaspase-9 cleaved into 37-kd and 35-kd fragments. Monoclonal anti-caspase-3 antibody (Transduction Laboratories, Lexington, KY) was used in 1:1000 dilution to detect the cleavage of procaspase-3. A reduction in density or disappearance of procaspase-3 bands defined the cleavage of caspase-3. Monoclonal anti- -actin antibody (Sigma) (1:10 000) was used as a
control protein.
RT-PCR detection for caspase-9b/procasapse-9 Total RNA was extracted with the RNeasy kit (QIAGEN). Two micrograms RNA was used for reverse transcription, which was performed using Retroscript first-strand synthesis kit for RT-PCR (Ambion, TX). Reverse-transcribed products were amplified by PCR using 2 caspase-9-specific primers, Mch6-ATG (ATGGACGAAGCGGATCGG) and Mch6-TAA (CCCTGGCCTTATGATGTT). Samples were subjected to an initial 5-minute denaturation at 95°C, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 1.5 minutes. Additional extension was at 72°C for 10 minutes. Twenty-five cycles were previously determined to be on the linear part of the curve for caspase-9b cDNA amplification under these conditions (data not shown); 18S served as the housekeeper gene. Mch6 (procaspase-9)-pRSC-LacZ and Mch6b (caspase-9b)-pcDNA3 plasmids served as positive controls for procaspase-9 and caspase-9b.21
Sensitivity of leukemic cells to UV light-induced apoptosis is not correlated with DNA damage or cytochrome c release UV light-induced DNA damage after 1 hour was assessed by the alkaline unwinding assay.28 There was no difference in the sensitivity of these cell lines to UV light-induced double-strand breaks (Figure 1A). Leukemic cells started to undergo apoptosis after 4 hours of exposure to UV light. As shown in Figure 1B, leukemic cells had different susceptibilities to UV light-induced apoptosis. The K562 cell line was remarkably resistant to UV light-induced apoptosis. However, the CEM/VLB100 cell line showed greater sensitivity to UV light-induced apoptosis than either K562 or its parental CEM cells. The sensitivity of leukemic cells to UV light-induced apoptosis did not correlate with the degree of DNA double-strand breakage (correlation, P > .5).
Cytochrome c translocation from mitochondria to cytosol was convincingly demonstrated in all cell lines after UV light stimulation for 3 hours, as detected by Western blot analysis (Figure 1C) and confirmed by fluorescence microscopy (10 and data not shown). Again, however, we observed that the amount of released cytochrome c in each cell line did not correlate with the extent of apoptosis in response to UV light. These results suggest that the resistance to UV light-induced apoptosis does not occur at the point of release of cytochrome c. Block conferring resistance to UV light-induced apoptosis is upstream of caspase-3 activation Differential sensitivity of leukemic cells to UV light-induced caspase-3 activation was shown by fluorescent AMC release (Figure 2A). The CEM/VLB100 cell line showed significantly increased susceptibility to UV light-induced caspase-3 activation compared with the parental CEM cell line. The K562 cell line was remarkably resistant to UV light-induced caspase-3 activation. The degree of UV light-induced apoptosis was proportional to the activity of caspase-3 (P < .001), suggesting that UV light-induced apoptosis in these cell lines was associated with caspase-3 activity. This result also suggests that the block conferring the resistance is upstream of caspase-3 activation.
Leukemic cells displayed differential sensitivity to exogenous cytochrome c but not granzyme B We were, therefore, interested in whether the cytosol from the different cell lines had a similar response to exogenous cytochrome c because they expressed similar levels of caspase-3 protein (data not shown). Caspase-3 activation in cytosolic extraction from K562, CEM, and CEM/VLB100 cells in response to exogenous cytochrome c was almost identical to that induced by UV light stimulation (P < .0001) (Figure 2A,B). However, caspase-3 activation in the 3 cell lines showed similar sensitivity to granzyme B-induced activation (Figure 2C), and there was no correlation with cytochrome c-induced caspase-3 activation (P > .5). The results strongly suggest that the susceptibility of leukemic cells to apoptosis is cytochrome c-dependent and is delayed or blocked downstream of cytochrome c release and upstream of caspase-3 in these apoptosis-resistant cell lines.Resistance to cytochrome c-induced caspase-3 activation is due to reduced Apaf-1 protein expression in resistant leukemic cells The K562 and CEM cell lines expressed less Apaf-1 than the CEM/VLB100 cell line (Figure 3A) as analyzed by densitometry. CEM and CEM/VLB100 cells expressed similar levels of procaspase-9; however, the K562 cell line, which is most resistant to cytochrome c, expressed more procaspase-9 than the CEM and CEM/VLB100 cell lines. There was no difference in XIAP expression among the 3 cell lines. Caspase-9b mRNA expression was assessed by semiquantitative RT-PCR (Figure 3B). Although the K562 cell line expressed more caspase-9b than the CEM and CEM/VLB100 cell lines, the CEM cell line expressed less caspase-9b than the CEM/VLB100 cell line. This suggests that overexpression of the potential inhibitory caspase-9b was not associated with sensitivity to cytochrome c-induced apoptosis.
In vitro translated procaspase-9 showed different sensitivity to
cytochrome c-induced cleavage in the presence of S-100
extracted from the 3 cell lines (Figure
4A). Cytochrome c-induced
cleavage of procaspase-9, in the presence of cytosol from the
CEM/VLB100 cell line, was observed after 5 minutes but was
less evident in the CEM and K562 cell lines. At 1 hour, complete
degradation of exogenous procaspase-9 was observed in the
CEM/VLB100 cells. This is further confirmation that the
sensitivity of these cell lines to cytochrome c was not
associated with endogenous procaspase-9 expression.
After immunodepletion of Apaf-1 protein from S-100, the effect of cytochrome c-induced exogenous procaspase-9 cleavage completely vanished (Figure 4B). In contrast, the addition of purified Apaf-1 protein14 to K562 and CEM cytosol restored sensitivity to cytochrome c-mediated procaspase-9 activation (Figure 4C). These results demonstrate that the resistance of K562 and CEM cell lines to cytochrome c-dependent activation of procaspases was due to insufficient Apaf-1 protein levels in their cytosol. Apaf-1 gene transfection increased the sensitivity of leukemic cells to cytochrome c-mediated activation of procaspases and apoptosis We next transfected the Apaf-1 gene into leukemic cells. Increased Apaf-1 protein expression was detected after transfection for 24 hours in all 3 cell lines (Figure 5A); however, the differential expression of Apaf-1 still existed after transfection (Figure 5B). S-100 prepared from all Apaf-1-transfected cells significantly increased cytochrome c-mediated activation of procaspase-9 and procaspase-3 in a cell-free system (Figure 6A,B). The enhanced cleavage of procaspase-9 and -3 proteins in Apaf-1-transfected cells was observed by Western blot as a disappearance or reduction in procaspase bands in response to cytochrome c stimulation. As with the response to cytochrome c, Apaf-1-transfected cells were sensitized to the activation of caspase-9 (Figure 7A) and caspase-3 (Figure 7B) with UV light irradiation. In addition, the percentage of apoptotic Apaf-1-transfected cells was significantly greater than wild-type cell lines after exposure to UV light (Figure 7C). The sensitivity of cells to UV light or cytochrome c-induced activation of caspases and apoptosis was dependent on the expression of Apaf-1 in both the wild-type and Apaf-1-transfected cells (P < .001). Mock transfection with vector alone or DMRIE-C, Lipofectamine Plus (Gibco BRL) did not affect Apaf-1 expression or caspase activation (data not shown). Taken together, these data suggest that the restoration of Apaf-1 protein levels can sensitize leukemic cells to cytochrome c-dependent apoptosis.
We also tested whether differential Apaf-1 levels could determine
the sensitivity of cell lines to
Apaf-1 protein deficiency found in human AML blasts and human solid tumor cells Finally, we were interested in whether Apaf-1 deficiency was associated with resistance to apoptosis in primary leukemic cells and other solid tumor cells. We tested the association of Apaf-1 expression and the sensitivity to UV light-induced apoptosis in some human AML blasts and compared them with the normal human CD34+ hemapoietic cells. One of 3 human AML blast populations showed reduced levels of Apaf-1 protein and was resistant to UV light-induced apoptosis (Figure 9A,B; Table 1). Ten hours after UV light exposure, the percentage of apoptosis in this resistant AML blast population was 6.6% compared with 55.2% in nonleukemic CD34+ cells and with 60.2% and 58% in other 2 AML blast populations with higher Apaf-1 protein levels. Even after 24 hours, apoptosis in the AML blast population with reduced Apaf-1 level was less than 10%. This suggests that AML-1 blast samples showed lower levels of Apaf-1 protein and more resistance to UV light-induced apoptosis. Using a cell-free system, we also confirmed that AML-1 blasts were more resistant to cytochrome c-induced activation of caspases than AML-2 and AML-3 samples. There was a significant correlation (P < .05) between Apaf-1 levels and the sensitivity to cytochrome c-dependent activation of caspases/apoptosis (Table 1).
The bladder cancer J82 cell line expressed less Apaf-1 protein than the
CEM/VLB100 cell line, and the sensitivity of J82 cells to
UV light-induced apoptosis was considerably lower than in
CEM/VLB100 cells (Figure
10A,B). The percentage of apoptosis and
the activation of caspases-3 and -9 were significantly increased after
transfection with the Apaf-1 gene (data not shown). In
addition, decreased colony formation was correlated with increased
levels of Apaf-1 expression as determined by clonogenic assay in
0.3% soft agar (data not shown). Moreover, Apaf-1 transfection
suppressed colony formation in J82 cells after UV light stimulation.
Previous studies have suggested that Apaf-1 is required for mitochondria-dependent apoptosis5,8,18,19 and that overexpression of Apaf-1 can increase sensitivity to apoptosis induced by chemotherapeutic drugs.20 We suggest that we have provided, for the first time, direct evidence that Apaf-1 protein levels vary constitutively in human leukemic cells. In addition, we have shown that a relative deficiency of Apaf-1 can result in resistance to apoptosis in human leukemic cells and tumor cells. Restoration of Apaf-1 protein levels after gene transfection is able to resensitize previously resistant cells to UV light-induced killing. Previous studies on the mechanism of leukemic cell resistance to
apoptosis have focused on mitochondrial components, such as Bcl-2 or
Bcl-XL overexpression,9,29,30 the ratio of Bax to Bcl-2 protein at the mitochondrial membrane,10
functional deficiency of the mitochondrial electron transport
chain,25,31 or factors upstream of the mitochondrial
level, such as NF- We have previously shown that the leukemic cell lines K562, CEM,
and CEM/VLB100, have differential sensitivities to
TNF- UV light induces apoptosis through cytochrome c release and activation of caspase-3.9,19 In our cells, UV light-induced activation of caspase-3 was proportional to the degree of apoptosis. Using a cell-free system, we showed that exogenous cytochrome c was able to activate procaspase-3 to the same extent as UV light. Procaspase-3 itself was not defective because granzyme B, which directly processes caspase-3,15 induced similar levels of caspase-3 activity in all 3 cell lines. Thus, we suggest that the block conferring resistance is between cytochrome c and caspase-3. Tumors in Apaf-1 or caspase-9 knock-out mice are resistant to apoptosis
induced by c-myc.8 T cells from Apaf-1
knock-out mice exhibited resistance to apoptosis induced by
DNA-damaging agents, such as etoposide or UV light, but were sensitive
to Fas-mediated killing.19 Apaf-1 mRNA is expressed
ubiquitously in human tissue, and normal leukocytes express high levels
of Apaf-1.3 In our study, the leukemic cell lines K562 and
CEM, some human AML blasts, and the tumor cell line J82 The K562 cell line expressed more inhibitory caspase-9b than the CEM/ VLB100 cell line, which can compete with procaspase-9 for binding to Apaf-1.21 However, CEM cells expressed less caspase-9b than CEM/ VLB100 cells. The expression of caspase-9b does not explain the differential sensitivity of the cell lines to cytochrome c-mediated procaspase activation. XIAP inhibitory protein levels were similar among the 3 cell lines. In addition, the ability of exogenous cytochrome c to cleave exogenous procaspase-9 in the presence of cytosol from each of the 3 cell lines studied was identical to the cleavage of endogenous procaspase-3, suggesting that the differential sensitivity was not due to differences in endogenous procaspase-9 levels. In summary, we suggest that Apaf-1 deficiency may be an important mechanism of resistance to apoptosis in human leukemic cells. Further studies are required to assess the overall importance of the mechanism in human leukemia and to understand the mechanism by which Apaf-1 expression is regulated.
We thank Dr Y. A. Lazebnik (Cold Spring Harbor Laboratory, New York) for the gifts of monoclonal anti-Apaf-1 and anti-caspase-9 antibodies, and we thank Dr Michael Neat and Ms Claire Wiggins for clinical samples.
Supported by the Leukemia Research Fund (S.M.K., L.J.) and by Elimination of Leukemia Training and Traveling Fellowship, the Pathology Society of Great Britain and Ireland Traveling Fellowship, and the Cancer Research Committee of the St Bartholomew's Hospital of London (L.J.).
Submitted September 27, 2000; accepted March 20, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Li Jia, Dept of Haematology, St Bartholomew's and The Royal London School of Medicine and Dentistry, Turner Street, London E1 2AD, United Kingdom; e-mail: l.jia{at}mds.qmw.ac.uk.
1. Liu X, Kim N, Yang J, Jemmerson R, Wang X. Induction of apoptosis program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147-157[CrossRef][Medline] [Order article via Infotrieve]. 2. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997;91:479-489[CrossRef][Medline] [Order article via Infotrieve]. 3. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1 a human protein homologous to C. elegans CED-4 participates in cytochrome c-dependent activation of caspase-3. Cell. 1997;90:405-413[CrossRef][Medline] [Order article via Infotrieve]. 4. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell. 1998;1:949-957[CrossRef][Medline] [Order article via Infotrieve].
5.
Fearnhead HO, Rodriguez J, Govek E-E, et al.
Oncogene-dependent apoptosis is mediated by caspase-9.
Proc Natl Acad Sci U S A.
1998;95:13664-13669 6. Hu Y, Benedict MA, Ding L, Núñez G. Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. EMBO J. 1999;18:3586-3595[CrossRef][Medline] [Order article via Infotrieve]. 7. Qin H, Srinivasula SM, Wu G, Fernandes-Alnemri T, Alnemri ES, Shi Y. Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature. 1999;399:549-557[CrossRef][Medline] [Order article via Infotrieve].
8.
Soengas MS, Alarcon RM, Yoshida H, et al.
Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition.
Science.
1999;284:156-159
9.
Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD.
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
Science.
1997;275:1132-1136
10.
Jia L, Macey MG, Yin Y, Newland AC, Kelsey SM.
Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis.
Blood.
1999;93:2353-2359
11.
Cain K, Brown DG, Langlais C, Cohen GM.
Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex.
J Biol Chem.
1999;274:22686-22692
12.
Adrain C, Slee EA, Harte MT, Martin SJ.
Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region.
J Biol Chem.
1999;274:20855-20860
13.
Hu Y, Ding L, Spencer DM, Núñez G.
WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation.
J Biol Chem.
1998;273:33489-33494
14.
Saleh A, Srinivasula SM, Acharya S, Fishel R, Alnemri ES.
Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation.
J Biol Chem.
1999;274:17941-17945
15.
Yang X, Stennicke HR, Wang B, et al.
Granzyme B mimics apical caspases: description of a unified pathway for trans-activation of executioner caspase-3 and -7.
J Biol Chem.
1998;273:34278-34283 16. Hakem R, Hakem A, Duncan GS, et al. Differential requirement for caspase-9 in apoptotic pathways in vivo. Cell. 1998;94:339-352[CrossRef][Medline] [Order article via Infotrieve]. 17. Kuida K, Haydar TF, Kuan CY, et al. Reduced apoptosis and cytochrome c-mediated caspase-activation in mice lacking caspase-9. Cell. 1998;94:325-337[CrossRef][Medline] [Order article via Infotrieve]. 18. Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA, Gruss P. Apaf-1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell. 1998;94:727-737[CrossRef][Medline] [Order article via Infotrieve]. 19. Yoshida H, Kong YY, Yoshida R, et al. Apaf-1 is required for mitochondrial pathways of apoptosis and brain development. Cell. 1998;94:739-750[CrossRef][Medline] [Order article via Infotrieve].
20.
Perkins C, Kim CN, Fang G, Bhalla KN.
Overexpression Apaf-1 promotes apoptosis of untreated and paclitaxel- or etoposide-treated HL-60 cells.
Cancer Res.
1998;58:4561-4566
21.
Srinivasula SM, Ahmad M, Guo Y, et al.
Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis.
Cancer Res.
1999;59:999-1002 22. Deveraux QL, Roy N, Stennicke HR, et al. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 1998;17:2215-2223[CrossRef][Medline] [Order article via Infotrieve]. 23. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell. 2000;102:33-42[CrossRef][Medline] [Order article via Infotrieve].
24.
Srinivasula SM, Datta P, Fan X-J, Fernandes-Alnemri T, Huang Z, Alnemri ES.
Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway.
J Biol Chem.
2000;275:36152-36157
25.
Jia L, Kelsey SM, Grahn MF, Newland AC.
Increased activity and sensitivity of mitochondrial respiratory enzymes to TNF-
26.
Jia L, Doumashkin RR, Allen PD, Gray AB, Newland AC, Kelsey SM.
Inhibition of autophagy abrogates TNF-
27.
Jia L, Dourmashkin RR, Newland AC, Kelsey SM.
Mitochondrial ultracondensation, but not swelling, is involved in TNF- 28. Kanter PM, Schwartz HS. A fluorescence enhancement assay for cellular DNA damage. Mol Pharmacol. 1982;22:145-151[Abstract].
29.
Yang J, Liu X, Bhalla K, et al.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science.
1997;275:1129-1132 30. Vander-Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cel |
Top
Abstract
Introduction
, UV irradiation, and chemotherapeutic and DNA-damaging
agents.
), and Apaf-1-transfected cells are
indicated as Apaf-1 (+). (A) Cytochrome c-mediated
procaspase-9 activation. The increased activity of caspase-9 in
Apaf-1-transfected cells was compared with their wild type
(*P < .0001). Processing of procaspase-9 was confirmed by
immunoblotting. Caspase-9 precursors are indicated by an asterisk, and
the processed caspase-9 are indicated by an arrowhead. (B) Cytochrome
c-mediated procaspase-3 activation.
*P < .0001; **P < .01. Processing of
procaspase-3 protein was indicated by immunoblotting. Caspase-9
precursors are indicated by an asterisk, and 
B, IAPs,
all of which
are relatively resistant to UV light