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Experimental Therapeutics |
Division of Clinical and Translational Research, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, Florida 33136
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m), and an increase in the reactive oxygen species.
These were also associated with the processing of procaspase-8 and
Bid (cytosolic, proapoptotic BH3 domain containing protein).
Transient transfection of Apaf-1 into the Apaf-1-containing mouse
embryogenic fibroblasts (MEFs; Apaf-1+/- MEFs) or Apaf-1-/- MEFs
also induced the processing of procaspase-9 and procaspase-8, Bid
cleavage, and apoptosis. These events were secondary to the activity of
the downstream caspases induced by Apaf-1. This conclusion is supported
by the observation that in HL-60/Apaf-1 cells, ectopic expression of
dominant negative caspase-9, its inhibitory short isoform caspase-9b,
or XIAP or treatment with the caspase inhibitor zVAD (50
µM) inhibited Apaf-1-induced caspase-8 and Bid
cleavage, mitochondrial m, release of cyt c, and
apoptosis. In contrast, a transient transfection of dominant negative
caspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk
inhibited the processing of procaspase-8 and Bid but did not inhibit
the cytosolic accumulation of cyt c in either the
untreated HL-60/Apaf-1 cells or the etoposide-treated HL-60/Apaf-1 and
HL-60/neo cells. These results indicate that Apaf-1 overexpression
lowers the apoptotic threshold by activating caspase-9 and caspase-3.
This triggers the mitochondrial m and cyt c
release into the cytosol through a predominant mechanism other than
cleavage of caspase-8 and/or Bid. This mechanism may involve a
cytosolic mitochondrial permeability transition factor, which may be
processed and activated by the downstream effector caspases, thereby
completing an amplifying feedback loop, which triggers the
mitochondrial events during apoptosis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Rb,
DNA-PK, and so forth, resulting in the morphological features
and DNA fragmentation of apoptosis (1
, 2
, 10)
. Recently, Apaf-1 was identified as the human homologue of the Caenorhabditis elegans CED-4 protein and was shown to participate as an adaptor molecule in the sequential activation of caspase-9 and caspase-3 (11, 12, 13) . The NH2-terminal region of Apaf-1 contains a CARD, which can bind to the corresponding motif in procaspase-9. Apaf-1 also contains a central region homologous to the proapoptotic CED-4 protein, including a conserved P-loop, and a COOH-terminal long WD repeat domain that lacks homology with CED-4. In the presence of dATP and cyt c released from the mitochondria by a number of apoptotic stimuli, the monomeric Apaf-1 is transformed into an oligomeric complex made of at least eight subunits (14, 15, 16) . This Apaf-1-cyt c complex can bind and process procaspase-9, followed by the release of mature caspase-9 (15 , 17) . This, is turn, can process procaspase-3, triggering the caspase cascade of activities that results in apoptosis (15 , 17) . Recent studies have demonstrated that an endogenous alternately spliced isoform of caspase-9 (caspase-9b), a mutation in the active site of caspase-9, or the deletion of caspase-9 from the S-100 fraction of cytosol blocks cyt-c-induced and Apaf-1-mediated ultimate cleavage and activation of caspase-3 (12 , 13 , 18 , 19) . The importance of Apaf-1-induced activity of caspase-9 followed by caspase-3 has been highlighted by reports that demonstrate that Apaf-1 or caspase-9 deficiency results in embryonic lethality due to defective neuronal apoptosis (20, 21, 22, 23) . Furthermore, inactivation of Apaf-1 or caspase-9 has been shown to substitute for p53 loss in promoting oncogenic transformation of the Myc-expressing cells (24) .
Similar to the enforced overexpression of FADD and CRADD, Apaf-1
overexpression was shown to induce apoptosis, through the cleavage and
activation of caspase-9 and caspase-3 (25)
. Higher levels
of Apaf-1 were demonstrated to enhance apoptosis induced by
chemotherapeutic agents, e.g., etoposide and Taxol, by
increasing the sequential cleavage and activities of caspase-9 and
caspase-3 (25)
. However, in these studies, the role of
mitochondrial m and cyt c release in Apaf-1-mediated
sensitization of apoptosis was not determined. In the present studies,
we investigated whether the lowering of the threshold to trigger
apoptosis through Apaf-1 overexpression involves procaspse-8 and Bid
cleavage and activity, resulting in the mitochondrial m and the
release and cytosolic accumulation of cyt c. Our findings
demonstrate that Apaf-1 overexpression mediates mitochondrial m,
cytosolic accumulation of cyt c, and apoptosis predominantly
through a mechanism that involves the activity of caspase-9 and
caspase-3 but not caspase-8 and Bid.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and Transfection of the Genes.
Human myeloid leukemia HL-60 cells were cultured as described
previously (26)
. Viable HL-60 cells (2–3 x 106) were transiently transfected with
0.5–1.0 µg each of the indicated plasmid DNA using LipofectAMINE
PLUS reagent (Life Technologies, Inc., Gaithersburg, MD). Stable
HL-60 cell lines overexpressing the human Apaf-1 protein were developed
as described previously (25)
. Lysates from the selected
clones were evaluated for Apaf-1 expression by immunoblot analyses (see
below). The clones expressing high levels of the specific proteins were
further subcloned by limiting dilution. Representative subclones of
each of the HL-60 transfectants were passaged twice per week and used
for the studies. Data presented are representative of those derived
from at least two independent clonal transfectants of HL-60/Apaf-1
cells. Apaf-1-deficient MEFs (a gift from Dr. Tak W. Mak; The Amgen
Institute, Toronto, Ontario, Canada) were cultured in DMEM (Life
Technologies, Inc., Grand Island, NY) supplemented with 10% fetal
bovine serum (20)
. Apaf-1+/- and Apaf-/- MEFs were
transfected with 0.5–1.0 µg of the appropriate plasmid DNA using the
LipofectAMINE PLUS reagent. Transfection efficiency was 
40–60%,
based on cotransfection of pCMV-GFP-1.
Western Analyses of Proteins.
Western analyses of Apaf-1, Bcl-2, Bcl-xL,
caspase-9, caspase-3, caspase-8, Bid, DFF, PARP, and ß-actin were
performed using specific antisera or monoclonal antibodies according to
previously reported protocols (25
, 27
, 28)
. Horizontal
scanning densitometry was performed on Western blots by using
acquisition into Adobe Photo Shop (Apple, Inc., Cupertino, CA) and
analysis by the NIH Image Program (NIH, Bethesda, MD). The expression
of ß-actin was used as a control.
Preparation of S-100 Fraction for the Analysis of Cytosolic
Accumulation of Cyt c.
Untreated and drug-treated cells were harvested by centrifugation, and
the cell homogenates were centrifuged at 100,000 x g for 30 min at 4°C to obtain the S-100 fraction, as
described previously. The supernatants were collected, and the protein
concentrations of S-100 were determined by using the Bradford method
(Bio-Rad, Hercules, CA). Samples were then analyzed for the release of
cyt c from the mitochondria into the cytosol by Western
blot, as described previously (29)
.
Apoptosis Assessment by Morphology and Annexin V Staining.
After drug treatment or the indicated transfections, 5 x 105 to 1 x 106 cells were washed in PBS, and cytospin
preparations of the cell suspension were fixed and stained with Wright
stain. Morphological evaluation of apoptosis was performed as described
previously (26)
. Cells were also resuspended in 100 µl
of staining solution (containing annexin V fluorescein and PI in a
HEPES buffer; annexin V-FLUOS staining kit; Boehringer Mannheim,
Indianapolis, IN). After incubation at room temperature for 15 min,
cells were analyzed by flow cytometry (25)
. Annexin V
binds to those cells that express phosphotidylserine on the outer layer
of the cell membrane, and PI stains the cellular DNA of those cells
with a compromised cell membrane. This allows for the discrimination of
live cells (unstained with either fluorochrome) from apoptotic
cells (stained only with annexin V) and necrotic cells (stained with
both annexin V and PI; Ref. 30
).
Measurement of Mitochondrial Potential and ROS.
To assess the changes in mitochondrial potential and ROS, 1 x 10 6 cells were incubated for 15 min at
37°C with 40 nM 3,3'-dihexyloxacarbocyanine iodide
(DiOC6; Ref.
3
) and 5 µM
dichlorodihydrofluorescein diacetate, respectively, and analyzed by
fluorescence-activated cell sorting as described previously (31
, 32)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m and
Cytosolic Accumulation of Cyt c.
demonstrates data from a representative clone of each cell
type. As compared with HL-60/neo cells, HL-60/Apaf-1 cells showed
2.5-fold higher levels of Apaf-1 and demonstrated the processing and
activation of procaspase-9 and procaspase-3 (Fig. 1, A and B)
. This was associated with the cleavage of PARP into its
Mr 85,000 fragment and the
cleavage of DFF45 (or ICAD) into its
Mr 30,000 fragment. DFF45 and PARP are
known substrates for caspase-3 (33
, 34)
. As compared with
HL-60/neo, Apaf-1 overexpression in HL-60/Apaf-1 cells was associated
with a slightly increased expression of Bcl-2 (Fig. 1B)
but
not of Bcl-xL or Bax (data not shown). Whereas
oligomerization of Apaf-1 is known to recruit, bind, and activate
caspase-9 and caspase-3, in HL-60/Apaf-1 cells, this was unexpectedly
associated with a 5-fold increase in the cytosolic cyt c
(Fig. 1C)
, as well as an increase in the percentage of cells
with low MMP and increased ROS (Fig. 1D)
. These preapoptotic
mitochondrial events and caspase activations were also observed in
HL-60 cells after transient transfection and overexpression of Apaf-1
or the transfection of its longer, alternately spliced isoform
(Apaf-1L), which possesses an additional WD repeat (data not shown).
Therefore, we used Apaf-1 transfectants in the following experiments
and refer to them henceforth as HL-60/Apaf-1 cells.
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and Fig. 2B
, Apaf-1 overexpression resulted in the
processing of procaspase-8 and Bid, and, as noted previously, Bid
processing caused cytosolic accumulation of cyt c and
apoptosis. Fig. 2A
also shows that an exposure to Taxol (10
nM for 24 h) or etoposide (1.0
µM for 24 h) induced significantly more
apoptosis in HL-60/Apaf-1 cells than in HL-60/neo cells. Cotreatment
with 50 µM zVAD, which exerts more potent
inhibitory effects against the activities of caspase-9 and caspase-3
than against the activities of apical caspase-8 and caspase-10,
also inhibited the processing of caspase-8 and Bid. Consequently, zVAD
inhibited the cytosolic accumulation of cyt c and apoptosis
induced by either Apaf-1 overexpression or cotreatment with etoposide
or Taxol (Fig. 2A
and Fig. 2B
). Fig. 3
demonstrates that the mitochondrial release and cytosolic accumulation
of cyt c caused by Apaf-1 overexpression in HL-60/Apaf-1
cells or caused by cotreatment with etoposide or Taxol in HL-60/neo
cells is also associated with reduced mitochondrial MMP (m) and
increased ROS. These mitochondrial perturbations in HL-60/Apaf-1
cells, as in HL-60/neo cells, were also reversed by cotreatment with
zVAD, indicating that these were secondary to the activities of the
effector caspases.
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, Fas
ligation, and TRAIL. Fig. 4A
demonstrates that Apaf-1+/- MEFs, which express Apaf-1
(Fig. 4B)
, were susceptible to apoptosis induced by
etoposide, doxorubicin, and staurosporine, but were minimally
susceptible to apoptosis induced by Taxol (1
µM for 24 h; Fig. 4A
).
Apoptosis of Apaf-1+/- cells was also observed after treatment with
TNF-, Fas ligation, and TRAIL. In contrast, Apaf-1-/- MEFs lacking
Apaf-1 (Fig. 4B)
were resistant to etoposide, doxorubicin,
and staurosporine-induced apoptosis (Fig. 4A)
. Whereas Fas
ligation and TNF- were equally effective in inducing apoptosis in
Apaf-1+/- and Apaf-1-/- cells, TRAIL-induced apoptosis was
partially inhibited in Apaf-1-/- cells (Fig. 4A)
.
Etoposide-induced processing of procaspase-9, procaspase-8, and Bid was
compared between Apaf-1+/- and Apaf-1-/- cells. Fig. 4B
demonstrates that etoposide-induced apoptosis of Apaf-1+/- cells is
associated with the processing of procaspase-9, procaspase-8, and Bid,
whereas these effects of etoposide are absent in Apaf-1-/- cells.
Transient transfection and overexpression of Apaf-1 cDNA increased the
percentage of Apaf-1+/- MEFs demonstrating apoptosis. Apaf-1
transfection also sensitized Apaf-1+/- cells to etoposide-induced
apoptosis (Fig. 5A)
. Transient transfection of Apaf-1 also induced apoptosis
of Apaf-1-/- cells; Apaf-1 also sensitized these cells to
etoposide-induced apoptosis. Fig. 5B
shows that the
transient transfection of Apaf-1 with or without etoposide resulted in
the processing of procaspase-9, procaspase-8, and Bid.
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and Fig. 6B
). In contrast, when procaspase-8 and Bid
processing due to etoposide in HL-60/neo cells or Apaf-1 overexpression
in HL-60/Apaf-1 cells was directly inhibited by transient transfection
of the cDNA of DN caspase-8 (37)
, there was minimal
inhibition of the cytosolic accumulation of cyt c and
apoptosis (Fig. 7A
and Fig. 7B
). In contrast, the processing of
procaspase-8 (p55) and Bid was inhibited (Fig. 7B)
.
Procaspase-3 processing was not inhibited by DN caspase-8 in HL-60/neo
or HL-60/Apaf-1 cells. Similarly, a transient transfection of the cDNA
of CrmA (a cowpox virus encoded serpin-like protease inhibitor), which
specifically inhibits caspase-8 activity (5)
, also did not
inhibit cytosolic accumulation of cyt c or apoptosis (data
not shown). The dominant role of the activities of caspase-9 and
capase-3 over those of caspase-8 followed by Bid in mediating the
feedback effects on the mitochondria is further supported by the data
presented in Fig. 8A
and Fig. 8B
. As shown, the effects of cotreatment
with a relatively specific inhibitor of the activity of either
caspase-9 (i.e., zLEHD-fmk) or caspase-8 (i.e.,
z-IETD-fmk) were compared (38)
. Again, inhibition of
caspase-9 by z-LEHD-fmk inhibited the processing of procaspase-3,
procaspase-8, and Bid and markedly inhibited the cytosolic accumulation
of cyt c and apoptosis induced by etoposide in HL-60/neo
cells or by the ectopic overexpression of Apaf-1 in HL-60/Apaf-1 cells.
Inhibition of caspase-8 activity by 50 µM
z-IETD-fmk, in contrast, had a minimal effect on the levels cytosolic
cyt c and apoptosis in both cell types. The
difference in the pattern of the cleavage fragments of procaspase-3 in
Fig. 8B
versus Fig. 7B
may be due to
the use of tetrapeptide inhibitors in the studies represented in Fig. 8B
but not in those represented in Fig. 7B
. z-LEHD-fmk, but
not z-IETD-fmk, also reversed the loss of mitochondrial m and the
increase in ROS induced by the ectopic overexpression of Apaf-1 in
HL-60/neo cells (data not shown). The differential effect of treatment
with z-LEHD versus z-IETD does not rule out the possibility
that the observed mitochondrial events in HL-60/Apaf-1 cells, including
the release of cyt c, might also be due to the activation of
other effector caspases, in addition to caspase-3, by caspase-9.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or Fas
ligation by an agonist antibody (20)
. In contrast,
apoptosis induced by TRAIL was only partially inhibited in the
Apaf-1-/- cells. This may be because, as compared to Fas ligand or
TNF-, TRAIL-induced processing of procaspase-8 in Apaf-1-/- MEFs
may be modest and delayed due to the presence of DcR1 and/or DcR2
(39
, 40)
, allowing Bid processing and the resultant
mitochondrial release of cyt c. The presence of Bid
in the cytosol clearly would not facilitate caspase-9 and
capase-3 activations in Apaf-1-/- cells (20
, 21)
.
Our data show that Apaf-1 overexpression in HL-60 cells also resulted
in the processing of procaspase-8 and Bid. This is most likely due to
the activity of caspase-9 and caspase-3, which can result in the
processing of procaspase-8 (10
, 22)
. Neither caspase-6 nor
caspase-7 has been shown to cleave Bid (41)
. In addition
to the processing of procaspase-8 and Bid, ectopic overexpression of
Apaf-1 was also associated with the mitochondrial m and the
release of cyt c into the cytosol. As shown in Fig. 9
, these preapoptotic mitochondrial events could be due directly to
either the activities of caspase-9 followed by caspase-3 or the
intervening processing of procaspase-8 and Bid. It should be
noted that the inhibition of the activities of caspase-9 and caspase-3
by transient ectopic expression of caspase-9b, DN caspase-9, or XIAP
(Fig. 6B)
or by cotreatment with the caspase-9-specific
inhibitor zLEHD-fmk (Fig. 8B)
markedly inhibited not only
the processing of procaspase-8 and Bid but also the preapoptotic
mitochondrial release of cyt c in HL-60/Apaf-1 cells. In
contrast, cotreatment with the caspase-8-specific inhibitor zIETD-fmk
(Fig. 8B)
or transient overexpression of DN caspase-8 (Fig. 7B)
or CrmA (data not shown) did not inhibit the
mitochondrial release of cyt c. This indicates that the
procaspase-8 and Bid processing mediated by ectopic overexpression of
Apaf-1 in HL-60 cells does not play a dominant role in causing the
feedback effects on the mitochondria. Therefore, the following question
arises: how do caspase-9 and/or caspase-3 (or another executioner
caspase) activities triggered by ectopic Apaf-1 mediate mitochondrial
m and cyt c release, if not through Bid activity? A
recent report by Bossy-Wetzel and Green (41)
suggested
that the effector caspases such as caspase-3, caspase-6, and caspase-7
that do not act directly on the mitochondria to release cyt
c might cleave and activate another cytosolic substrate
(other than Bid) that could then promote the mitochondrial m and
the release of cyt c. Our data also support the existence of
such a putative cytosolic MPTF. As shown in Fig. 9
, MPTF may be the
direct target for processing by caspase-9 and/or caspase-3 or
caspase-7. Although Bid cleavage and activity triggered by death
receptor signaling has clearly been shown to exert an amplifying role
during apoptosis by causing mitochondrial m and cyt
c release, our findings suggest that this may not be the
sole mechanism or may be part of the feedback mechanism by which
mitochondria are recruited for their role during apoptosis.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Division of Clinical and Translational Research,
Sylvester Comprehensive Cancer Center, University of Miami School of
Medicine, 1550 NW 10th Avenue (M710), Miami, FL 33136.
Phone: (305) 243-5907; Fax: (305) 243-5885; E-mail: kbhalla{at}med.miami.edu 
2 The abbreviations used are: CARD, caspase
recruitment domain; cyt c, cytochrome c;
ROS, reactive oxygen species; MEF, mouse embryonic fibroblast; DN,
dominant negative; MPTF, mitochondrial permeability transition factor;
PARP, poly(ADP-ribose) polymerase; DFF, DNA fragmentation factor;
TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TNF,
tumor necrosis factor; MMP, mitochondrial membrane potential; PI,
propidium iodide.
Received 11/12/99. Accepted 1/19/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Y. Furukawa, N. Nishimura, Y. Furukawa, M. Satoh, H. Endo, S. Iwase, H. Yamada, M. Matsuda, Y. Kano, and M. Nakamura Apaf-1 Is a Mediator of E2F-1-induced Apoptosis J. Biol. Chem., October 11, 2002; 277(42): 39760 - 39768. [Abstract] [Full Text] [PDF]
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M. Huigsloot, I. B. Tijdens, G. J. Mulder, and B. van de Water Differential Regulation of Doxorubicin-induced Mitochondrial Dysfunction and Apoptosis by Bcl-2 in Mammary Adenocarcinoma (MTLn3) Cells J. Biol. Chem., September 20, 2002; 277(39): 35869 - 35879. [Abstract] [Full Text] [PDF]
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 T. J. Page, S. O'Brien, C. R. Jefcoate, and C. J. Czuprynski 7,12-Dimethylbenz[a]anthracene Induces Apoptosis in Murine Pre-B Cells through a Caspase-8-Dependent Pathway Mol. Pharmacol., August 1, 2002; 62(2): 313 - 319. [Abstract] [Full Text] [PDF]
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 M. Kondo, T. Shibata, T. Kumagai, T. Osawa, N. Shibata, M. Kobayashi, S. Sasaki, M. Iwata, N. Noguchi, and K. Uchida 15-Deoxy-Delta 12,14-prostaglandin J2: The endogenous electrophile that induces neuronal apoptosis PNAS, May 28, 2002; 99(11): 7367 - 7372. [Abstract] [Full Text] [PDF]
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F. Guo, R. Nimmanapalli, S. Paranawithana, S. Wittman, D. Griffin, P. Bali, E. O'Bryan, C. Fumero, H. G. Wang, and K. Bhalla Ectopic overexpression of second mitochondria-derived activator of caspases (Smac/DIABLO) or cotreatment with N-terminus of Smac/DIABLO peptide potentiates epothilone B derivative-(BMS 247550) and Apo-2L/TRAIL-induced apoptosis Blood, May 1, 2002; 99(9): 3419 - 3426. [Abstract] [Full Text] [PDF]
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 P. Matarrese, L. Di Biase, L. Santodonato, E. Straface, M. Mecchia, B. Ascione, G. Parmiani, F. Belardelli, M. Ferrantini, and W. Malorni Type I Interferon Gene Transfer Sensitizes Melanoma Cells to Apoptosis via a Target Activity on Mitochondrial Function Am. J. Pathol., April 1, 2002; 160(4): 1507 - 1520. [Abstract] [Full Text] [PDF]
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J. F. Kidd, M. F. Pilkington, M. J. Schell, K. E. Fogarty, J. N. Skepper, C. W. Taylor, and P. Thorn Paclitaxel Affects Cytosolic Calcium Signals by Opening the Mitochondrial Permeability Transition Pore J. Biol. Chem., February 15, 2002; 277(8): 6504 - 6510. [Abstract] [Full Text] [PDF]
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X. W. Meng, M. P. Heldebrant, and S. H. Kaufmann Phorbol 12-myristate 13-Acetate Inhibits Death Receptor-mediated Apoptosis in Jurkat Cells by Disrupting Recruitment of Fas-associated Polypeptide with Death Domain J. Biol. Chem., January 25, 2002; 277(5): 3776 - 3783. [Abstract] [Full Text] [PDF]
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H. Yamaguchi, S. R. Paranawithana, M. W. Lee, Z. Huang, K. N. Bhalla, and H.-G. Wang Epothilone B Analogue (BMS-247550)-mediated Cytotoxicity through Induction of Bax Conformational Change in Human Breast Cancer Cells Cancer Res., January 1, 2002; 62(2): 466 - 471. [Abstract] [Full Text] [PDF]
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A. R. Jazirehi, C.-P. Ng, X.-H. Gan, G. Schiller, and B. Bonavida Adriamycin Sensitizes the Adriamycin-resistant 8226/Dox40 Human Multiple Myeloma Cells to Apo2L/Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated (TRAIL) Apoptosis Clin. Cancer Res., December 1, 2001; 7(12): 3874 - 3883. [Abstract] [Full Text] [PDF]
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C. N. O'Donovan, D. Tobin, and T. G. Cotter Prion Protein Fragment PrP-(106-126) Induces Apoptosis via Mitochondrial Disruption in Human Neuronal SH-SY5Y Cells J. Biol. Chem., November 16, 2001; 276(47): 43516 - 43523. [Abstract] [Full Text] [PDF]
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 L. Qiao, E. Studer, K. Leach, R. McKinstry, S. Gupta, R. Decker, R. Kukreja, K. Valerie, P. Nagarkatti, W. E. Deiry, et al. Deoxycholic Acid (DCA) Causes Ligand-independent Activation of Epidermal Growth Factor Receptor (EGFR) and FAS Receptor in Primary Hepatocytes: Inhibition of EGFR/Mitogen-activated Protein Kinase-Signaling Module Enhances DCA-induced Apoptosis Mol. Biol. Cell, September 1, 2001; 12(9): 2629 - 2645. [Abstract] [Full Text] [PDF]
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V. M. Dirsch, H. Stuppner, and A. M. Vollmar Helenalin Triggers a CD95 Death Receptor-independent Apoptosis That Is Not Affected by Overexpression of Bcl-xL or Bcl-2 Cancer Res., August 1, 2001; 61(15): 5817 - 5823. [Abstract] [Full Text] [PDF]
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R. Nimmanapalli, E. O’Bryan, and K. Bhalla Geldanamycin and Its Analogue 17-Allylamino-17-demethoxygeldanamycin Lowers Bcr-Abl Levels and Induces Apoptosis and Differentiation of Bcr-Abl-positive Human Leukemic Blasts Cancer Res., March 1, 2001; 61(5): 1799 - 1804. [Abstract] [Full Text]
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R. Nimmanapalli, M. Porosnicu, D. Nguyen, E. Worthington, E. O’Bryan, C. Perkins, and K. Bhalla Cotreatment with STI-571 Enhances Tumor Necrosis Factor {{alpha}}-related Apoptosis-inducing Ligand (TRAIL or Apo-2L)- induced Apoptosis of Bcr-Abl-positive Human Acute Leukemia Cells Clin. Cancer Res., February 1, 2001; 7(2): 350 - 357. [Abstract] [Full Text]
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W. F. Symmans, M. D. Volm, R. L. Shapiro, A. B. Perkins, A. Y. Kim, S. Demaria, H. T. Yee, H. McMullen, R. Oratz, P. Klein, et al. Paclitaxel-induced Apoptosis and Mitotic Arrest Assessed by Serial Fine-Needle Aspiration: Implications for Early Prediction of Breast Cancer Response to Neoadjuvant Treatment Clin. Cancer Res., December 1, 2000; 6(12): 4610 - 4617. [Abstract] [Full Text]
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J. Wen, N. Ramadevi, D. Nguyen, C. Perkins, E. Worthington, and K. Bhalla Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells Blood, December 1, 2000; 96(12): 3900 - 3906. [Abstract] [Full Text] [PDF]
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G. Fang, C. N. Kim, C. L. Perkins, N. Ramadevi, E. Winton, S. Wittmann, and K. N. Bhalla CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs Blood, September 15, 2000; 96(6): 2246 - 2253. [Abstract] [Full Text] [PDF]
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)/necrotic (
) cells detected by annexin V/PI staining.
Error bars, SE for the mean percentage of apoptotic
cells under the various treatment conditions. B, Western
blot analyses of procaspase-8 (p55), Bid (p22), and its p15 cleaved
product as well as the cytosolic levels of cyt c.
ß-Actin was used as a control for equal protein loading.
