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Prepublished online as a Blood First Edition Paper on January 9, 2003; DOI 10.1182/blood-2002-09-2739.
NEOPLASIA
From the Department of Biochemistry, Division of
Hematology/Oncology, Department of Medicine, and Department of
Radiation Oncology, Virginia Commonwealth University/Medical College of
Virginia, Richmond.
Interactions between the protein kinase C (PKC)
activator/down-regulator bryostatin 1 and paclitaxel have been examined
in human myeloid leukemia cells (U937) and in highly
paclitaxel-resistant cells ectopically expressing a Bcl-2
phosphorylation loop-deleted protein ( Apoptosis represents a genetically regulated cell
death program characterized by the engagement of a family of cysteine
proteases termed caspases.1 Currently, two major apoptotic
pathways have been identified: the intrinsic, or mitochondrial pathway,
and the extrinsic, or receptor-related pathway.2 The
former, which is typically initiated by cytotoxic drugs, involves
perturbations in mitochondrial homeostasis, resulting in cytochrome
c release from the inner mitochondrial space. Once in the
cytoplasm, cytochrome c binds to a cytoskeletal protein
referred to as apoptotic protease-activating factor-1 (Apaf-1), which
binds to and activates caspase-9. The resulting "apoptosome"
complex activates the caspase cascade that induces cell
death.3 Release of cytochrome c is regulated by complex interactions between proapoptotic (eg, Bid, Bax, Bak) and
antiapoptotic (eg, Bcl-2, Bcl-xL) members of the Bcl-2
family.4 In contrast, the extrinsic apoptotic pathway is
initiated by binding of ligands such as tumor necrosis factor (TNF) to
members of the tumor necrosis factor receptor (TNFR) family, leading to
recruitment of death domain-containing adaptor proteins such as the
Fas-associated death domain (FADD). Death-effector domains (DEDs) on
FADD interact with procaspase-8 prodomains, resulting in cleavage and
activation by self-processing.5 Once activated, caspase-8
either directly cleaves apoptotic substrates or activates effector
caspases (eg, procaspases-3, -6, and -7). Caspase-8 also cleaves and
activates the proapoptotic protein Bid, a BH3-only domain member of the Bcl-2 family.6 Truncated Bid (tBid) translocates to the
mitochondria, resulting in release of cytochrome c into
cytosol. In this way, activation of the extrinsic pathway can serve to
amplify apoptosis initiated by stimuli (eg, cytoxic drugs) primarily
associated with mitochondrial injury.7 Significantly,
apoptosis stemming from activation of the extrinsic pathway is
relatively insensitive to blockade by Bcl-2 and related
proteins.8
Paclitaxel (Taxol), a member of the taxane family, is a natural product
that exhibits activity against a wide range of human hematopoietic and
nonhematopoietic tumor cell types.9,10 Paclitaxel binds to
microtubules, thereby promoting polymerization of tubulin heterodimers
and inhibiting tubulin depolymerization, leading to Bryostatin 1 is a macrocyclic lactone obtained from the marine bryozoan
Bugula neritina, which has shown significant preclinical antitumor activity16,17 and is currently undergoing phase
1 and 2 clinical evaluation in humans.18 On acute
exposure, bryostatin 1 activates protein kinase C (PKC), whereas on
chronic exposure, it induces down-regulation of PKC
activity.19,20 In leukemic cells, bryostatin 1 has also
been shown to lower the threshold for apoptosis induced by cytotoxic
drugs (eg, ara-C).21-23 In view of evidence that PKC
activation opposes apoptosis24 and that pharmacologic PKC
inhibitors are potent inducers of cell death,25 it is
tempting to relate the proapoptotic actions of bryostatin 1 to
interruption of PKC cytoprotective actions.26-28
In previous studies, we29 and others30 have
reported that bryostatin 1 potentiates the lethal effects of paclitaxel
in a dose-dependent manner. Moreover, bryostatin 1 circumvents, at least in part, the capacity of ectopic expression of Bcl-xL
to block paclitaxel-mediated apoptosis.31 However, the
mechanism by which bryostatin 1 lowers the threshold for
paclitaxel-mediated lethality remains unclear. The goal of the present
studies was to gain insights into the factors responsible for this
phenomenon, with an emphasis on characterizing the relative
contributions of the intrinsic versus extrinsic pathways to bryostatin
1/paclitaxel lethality. In addition, parallel studies have been
conducted in cells ectopically expressing a phosphorylation
loop-deleted mutant to determine what role, if any, Bcl-2
phosphorylation might play in potentiation of lethality. The present
results suggest that bryostatin 1-mediated induction of TNF- Cell lines
Drugs and chemicals
Experimental protocols Cells in logarithmic-phase growth were suspended at 2 × 105/mL and exposed to test agents for various intervals as indicated in a 37°C, 5% CO2 incubator. In the experimental paradigm, paclitaxel was added 15 to 30 minutes before the addition of bryostatin 1. In experiments involving kinase inhibitors and TNF soluble receptors, cells were pretreated with each agent 30 minutes before the addition of paclitaxel with or without bryostatin 1.Assessment of apoptosis Cell morphology and apoptosis was monitored by examining cytocentrifuge preparations stained with the Diff-Quik stain set (Dade Behring, Deerfield, IL) as described previously23 or by annexin V and propidium iodide (PI) positivity. Briefly, following drug treatments, cells were costained with annexin V conjugated to fluorescein isothiocyanate (FITC) and PI per instruction provided by the manufacturer (BD PharMingen, San Diego, CA). The percentage of apoptotic (annexin V-positive and PI-positive) cells was determined by flow cytometric analysis.Cytochrome c and Smac/DIABLO release assay After treatment, cells were harvested by centrifugation at 600g for 10 minutes at 4°C. Cytosolic fractions were obtained by selective plasma membrane permeabilization with digitonin. Briefly, 2 × 106 cells were lysed 1 to 2 minutes in lysis buffer (75 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA [ethylenediaminetetraacetic acid], and 350 µg/mL digitonin). The lysates were centrifuged at 12 000g for 1 minute, and the supernatant was collected and added to an equal volume of 2 × sample buffer. The protein samples were quantified, separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subjected to immunoblot analysis as described in the next paragraph.Western blot analysis Western blot analysis was performed essentially as described previously.23 In brief, for each sample, 25 µg of protein per lane was separated by 4% to 20% SDS-PAGE (Invitrogen, Carlsbad, CA) and electroblotted to nitrocellulose (Schleicher & Schuell, Keene, NH). Subsequently, after incubation in phosphate-buffered saline (PBS)-Tween 20 (0.05%) supplemented with 5% nonfat dry milk for 1 hour at 22°C, the blots were incubated for 2 hours at 22°C in fresh blocking solution with an appropriate dilution of primary antibodies as follows: cytochrome c, caspase-3, and caspase-8, 1:1000 (BD PharMingen); and Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI), 1:500 (BIOMOL Research Laboratories, Plymouth Meeting, PA); blots were washed 3 times for 5 minutes in PBS-Tween 20 and then incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 hour at 22°C. Blots were washed 3 times for 5 minutes in PBS-Tween 20 and then developed by enhanced chemiluminescence (Amersham Biosciences, Braunschweig, Germany).Caspase activity assay Caspase activity was measured per instructions provided by the manufacturer using a colorimetric assay kit (Biovision, Palo Alto, CA). Caspase activity in cytosolic extracts from appropriate treatments was measured by spectrophotometric detection of the chromophore P-nitroanilide (pNA) following its cleavage from the labeled substrate DEVD-pNA for caspase-3 and IETD-pNA for caspase-8.Enzyme-linked immunosorbent assay (ELISA) U937 cells (4 × 106) were exposed to drug treatment at various time intervals. Cell culture supernatants were collected by centrifugation. TNF- protein levels in the supernatants
were monitored by the ELISA OptEIA kit (BD PharMingen) and normalized against untreated controls.
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) U937 cells (4 × 106) were exposed to paclitaxel with or without bryostatin 1 for various time points, and total RNA was isolated using the Qiagen RNeasy Mini Kit; 10 ng RNA was used to conduct Taqman One-step RT-PCR (Applied Biosystems, Foster City, CA). Each tube contained both the TNF- gene probe/primer and
the human actin control probe/primer. Briefly, oligonucleotide probes
were labeled at the 5' end with FAM 6-carboxyfluorescein and at the 3'
end with the quencher dye N,N',N'-tetramethyl-6 carboxyrhodamine. The
TNF- primers were (F) 5'-CCCCAGGACCTCTCTCAATC-3'; (R)
5'-CATGGGCTACAGGCTTGTCA-3', and the probe sequence was
5'-CCCAGGCAGTCAGATCATCTTCTCGAA-3'. The results for the experimental
gene were normalized to actin levels as specified by the manufacturer.
PKC activity A minor modification of a previously described method33 was employed to assay membrane, cytosolic, and total PKC activity in U937 cells exposed to bryostatin 1 with or without paclitaxel. Briefly, cell pellets were homogenized in 20 mM Tris (tris(hydroxymethyl)aminomethane), 0.5 mM EDTA, 0.5 mM EGTA (ethylene glycol tetraacetic acid), pH 7.5, containing 25 µg/mL protease inhibitors. The homogenate was incubated on ice for 30 minutes and centrifuged for 30 minutes at 100 000g to yield a cytosolic supernatant and a membrane pellet, which was homogenized in the same buffer containing 0.5% Triton X-100. After incubation on ice for 30 minutes followed by centrifugation for 5 minutes at 12 000g, the supernatant, representing the membrane fraction, was isolated. PKC activity in each fraction was monitored using a commercially available kit (Signa TECT PKC assay system; Promega, Madison, WI). For both fractions, normalized quantities of protein were added to coactivation buffer (2.5 × 10 5 Ci/mL [9.3 × 103
MBq/mL] [ -32P]ATP, 2 × 105 M
adenosine triphosphate [ATP], and 5 × 105 M
biotinylated peptide substrate) in the presence or absence of
activation buffer (0.32 mg/mL phosphatidylserine, 0.032 mg/mL diacylglycerol, 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2). After
a 5-minute incubation at 30°C, reactions were terminated by the addition of guanidine hydrochoride (2.5 M) and spotted on
SAM2 membrane (Promega). The membranes were
washed thoroughly, and radioactivity was measured by
liquid-scintillation counting. Purified PKC was used as a positive
control, and myristoylated PKC peptide inhibitor was used to indicate
specificity. The enzyme activity of PKC was determined by subtracting
the activity of the enzyme in the absence of phospholipids (control
buffer) from that of the enzyme in the presence of phospholipids
(activation buffer). Kinase activity was calculated as picomoles of
[-32P]ATP incorporated per minute per microgram of
protein. Values are expressed as the percentage of PKC activity
relative to those of untreated cell extracts (100%).
Cell synchronization To assess further the effects of bryostatin 1-mediated signaling pathway on cell cycle traverse, aphidicolin block was employed to synchronize cells in the S phase. In brief, U937 cells were exposed to 100 nM aphidicolin for 24 hours and washed 3 times in drug-free medium. At various times after washout of aphidicolin, cells were treated with rhTNF (0.025 ng/mL). Cell death was determined by annexin V and PI positivity as described in "Assessment of apoptosis."MPM-2/propidium iodide bivariate flow cytometry Following treatment of U937 cells with paclitaxel with or without bryostatin 1 for 18 hours, cells were harvested and fixed with 1% paraformaldehyde and 70% ethanol. After washing with PBS containing 0.05% Tween 20 and 1% fetal bovine serum, cells were labeled with MPM-2 antibody (final concentration of 6 µg MPM-2 antibody per milliliter; Upstate Biotechnology, Lake Placid, NY) for 1 hour at 4°C. Cells were washed twice with PBS and incubated with goat antimouse-FITC (Boehringer Mannheim, Mannheim, Germany) for 1 hour at room temperature in the dark. Cells were washed twice with PBS and resuspended in 5 µg/mL PI containing 50 µg/mL RNase A. Samples were analyzed on a Becton Dickinson FACScan, and data were analyzed using CellQuest software.Statistical analysis The significance of differences between experimental groups was determined using the Student t test for unpaired observations.
Bryostatin 1 potentiates paclitaxel-induced apoptosis in human leukemia cells To define the effects of bryostatin 1 on paclitaxel-induced apoptosis, U937 cells were exposed to 5 nM paclitaxel in combination with increasing concentration of bryostatin 1 (10 to 100 nM). As shown in Figure 1A, bryostatin 1 treatment alone exerted minimal toxicity (6% to 10% apoptosis) but dramatically enhanced paclitaxel-induced apoptosis. An increase in paclitaxel-induced apoptosis by a minimally toxic concentration of bryostatin 1 (10 nM) was also observed at higher paclitaxel concentrations (eg, 10 to 20 nM) although potentiatiation was most marked at paclitaxel concentrations of about 5 nM (Figure 1B). Bryostatin 1 also increased paclitaxel lethality in HL-60 and THP-1 leukemia cells (Figure 1C-D) as well as in primary blast cells obtained from the peripheral blood of a patient with acute myeloid leukemia (AML) (data not shown). In each of the cell lines examined, the effects of combined treatment with bryostatin 1 and paclitaxel was significantly greater than the sum of the effects of each agent administered alone (P < .05 in each case).
Addition of bryostatin 1 circumvents leukemic cell resistance to paclitaxel conferred by ectopic expression of full-length as well as an N-terminal phosphorylation loop-deleted Bcl-2 protein To investigate the role of Bcl-2 and its phosphorylation in the response of leukemic cells to the bryostatin 1/paclitaxel regimen, U937/neo, U937/Bcl-2, and U937/ Bcl-2 cells were exposed to
paclitaxel (10 nM) with or without bryostatin 1 (10 nM) for 24 hours.
As we have previously reported, ectopic expression of full-length Bcl-2
significantly protected cells from paclitaxel lethality, and deletion
of loop region, which contains the major phosphorylation
sites,14 conferred a very high degree of protection (Figure 2A). In marked contrast, ectopic
expression of either full-length Bcl-2 or the loop-deleted Bcl-2 failed
to protect cells from apoptosis induced by the combination of
bryostatin 1 and paclitaxel. In parallel studies, bryostatin 1 also
significantly reduced the clonogenic survival of paclitaxel-treated
U937/Bcl-2 and U937/Bcl-2 cells (eg, from 5.82% and 7.66% to
0.42% and 0.41% respectively; Figure 2B). Similar results were
obtained when cells were exposed to 5 nM paclitaxel (data not shown).
These findings indicate that administration of bryostatin 1 can
circumvent blockade of paclitaxel-mediated apoptosis conferred by the
full-length and loop-deleted Bcl-2 protein and argue against a role for
modulation of Bcl-2 phosphorylation in this process. They also indicate
that the ability of these agents to inhibit the clonogenic growth of leukemia cells may far exceed their capacity to induce apoptosis, at
least at early exposure intervals, and raise the possibility that other
mechanisms of reproductive cell death may be involved in the
paclitaxel/bryostatin 1 interaction.
Addition of bryostatin 1 overcomes the ability of full-length or loop-deleted Bcl-2 to protect leukemic cells from paclitaxel-induced mitochondrial dysfunction As shown in Figure 3, ectopic expression of full-length Bcl-2 significantly attenuated the capacity of paclitaxel (10 nM) to induce release of cytochrome c and the inhibitor of apoptosis antagonist Smac/DIABLO34,35 into the cytosolic S-100 fraction, and the loop-deleted protein was even more effective in this regard. However, when cells were exposed to bryostatin 1, which exerted minimal effects by itself, paclitaxel-induced redistribution of cytochrome c and Smac/DIABLO in cells ectopically expressing full-length or loop-deleted Bcl-2 was restored to levels equivalent to those observed in empty-vector controls. Similar results were obtained when cells were exposed to 5 nM paclitaxel (data not shown). These findings suggest that bryostatin 1 acts by interfering with the ability of Bcl-2 to attenuate paclitaxel-mediated release of proapoptotic mitochondrial proteins into the cytosol and that this phenomenon does not depend upon Bcl-2 phosphorylation.
Bryostatin 1 enhances caspase-8 activation and Bid cleavage in parental and Bcl-2-overexpressing U937 cells To assess the effects of bryostatin 1 on paclitaxel-mediated activation of the extrinsic apoptotic pathway, procaspase-8 degradation and Bid cleavage were monitored in empty-vector control cells as well as those ectopically expressing full-length and phosphorylation loop-deleted Bcl-2. As shown in Figure 4, cotreatment with paclitaxel and bryostatin 1 induced equivalent degrees of activation of caspase-8 and Bid cleavage in U937/neo, U937/Bcl-2, and U937/Bcl-2 cells. Such
findings indicate that ectopic expression of these antiapoptotic proteins is ineffective in blocking activation of the extrinsic apoptotic pathway by the bryostatin 1/paclitaxel regimen.
Caspase-8 activation is central to the potentiation of paclitaxel lethality by bryostatin 1 To define further the functional role of the extrinsic cell death pathway in bryostatin 1/paclitaxel lethality, parallel studies were conducted in cells ectopically expressing dominant-negative caspase-8 (U937/casp8-DN). As shown in Figure 5A, ectopic expression of U937/casp8-DN minimally attenuated apoptosis in cells exposed to paclitaxel alone (5 nM), suggesting that the extrinsic pathway plays only a minor role in the lethal effects of this agent. However, in marked contrast, ectopic expression of DN caspase-8 completely abrogated the ability of bryostatin 1 to potentiate paclitaxel-induced cell death. Similar results were obtained when cells were exposed to 10 nM paclitaxel (data not shown). Thus, these data indicate that caspase-8 plays a central role in potentiation of paclitaxel lethality by bryostatin 1 but not in cell death induced by paclitaxel alone.
To confirm further that potentiation of paclitaxel-induced apoptosis by bryostatin 1 was mediated through the death receptor pathway, cells ectopically expressing the serpin CrmA, a potent inhibitor of caspase-8,3 were exposed to paclitaxel (5 nM) with or without bryostatin 1 (10 nM) for 24 hours, after which apoptosis was monitored. As shown in Figure 5B, ectopic expression of CrmA did not modify the apoptotic response to paclitaxel alone but essentially abrogated potentiation of paclitaxel-induced cell death by bryostatin 1. Ectopic expression of CrmA, like DN caspase-8, also substantially blocked apoptosis triggered by the combination of TNF and cycloheximide (CHX). Taken together, these findings argue strongly that potentiation of paclitaxel-induced apoptosis by bryostatin 1 is mediated by the extrinsic cell death pathway. Coadministration of paclitaxel and bryostatin 1 results in
potentiation of TNF- , in hematopoietic cells,36
RT-PCR was employed to assess the impact of bryostatin 1 and paclitaxel exposure on TNF- mRNA production in U937 cells. As shown in Figure 6A, paclitaxel (5 nM) by itself exerted
minimal effects on TNF- mRNA levels, while bryostatin 1 by itself
induced a 10-fold increase in TNF- mRNA levels at 3 hours, which
declined to baseline by 6 hours (Figure 6B). Cotreatment of cells with
paclitaxel and bryostatin 1 did not increase TNF- mRNA levels at 3 hours relative to cells exposed to bryostatin 1 alone. However,
cells treated with both bryostatin 1 with paclitaxel continued to
exhibit a 4-fold increase in TNF- mRNA levels at 6 hours (Figure
6B). Assessment of TNF- protein levels by ELISA revealed
equivalent increases in bryostatin 1- and bryostatin 1 plus
paclitaxel-treated cells at 3 hours (Figure 6C). However,
whereas TNF- protein levels declined to baseline by 24 hours in
cells exposed to bryostatin 1 alone, a persistent increase in protein
levels was noted in bryostatin 1/paclitaxel-treated cells at 24 hours.
Essentially identical results were noted in HL-60 cells (Figure 6D).
These findings indicate that coexposure of U937 cells to the
combination of paclitaxel and bryostatin 1 results in sustained
increases in TNF- mRNA and protein levels.
Recombinant human TNF- on paclitaxel-induced
lethality, U937 cells were exposed to paclitaxel (5 nM) in combination with recombinant human TNF- (rhTNF-) (0.025 to 1.0 ng/mL) for 24 hours. As shown in Figure 7A,
paclitaxel-induced cell death was markedly enhanced by rhTNF-.
Coadministration of paclitaxel with rhTNF- also circumvented the
blockade of paclitaxel-mediated apoptosis conferred by ectopic
expression of Bcl-2 protein (Figure 7B), analogous to results obtained
with bryostatin 1. Consistent with these findings, coadministration of
rhTNF- restored the ability of paclitaxel to promote
procaspase-8 degradation and cytochrome c release in
U937/Bcl-2 cells (Figure 7C). Equivalent results were obtained when
cells were exposed to 10 nM paclitaxel (data not shown). These findings
indicate that the capacity of bryostatin 1 to overcome paclitaxel
resistance in leukemia cells ectopically expressing the phosphorylation
loop-deleted Bcl-2 protein is closely mimicked by rhTNF-.
TNF soluble receptor blocks bryostatin 1-mediated potentiation of paclitaxel-induced apoptosis To investigate the functional significance of induction of TNF-
in bryostatin 1-related potentiation of paclitaxel-induced cell death,
U937 cells were exposed to TNF soluble receptor (sTNFR, 100 ng/mL) 30 minutes prior to the addition of paclitaxel and bryostatin 1. As shown
in Figure 8A, sTNFR abrogated the
potentiation of paclitaxel-induced cell death by bryostatin 1, analogous to results obtained when exogenous TNF- was added (data
not shown). The sTNFR also blocked potentiation of paclitaxel lethality
by bryostatin 1 in HL-60 cells (Figure 8B). Consistent with these findings, sTNFR blocked the increase in caspase-8 and caspase-3 activation in U937 cells exposed to the combination of paclitaxel and
bryostatin 1 but exerted minimal effects in cells exposed to paclitaxel
alone (Figure 8C-D). Together, these findings support the notion
that induction of TNF- by bryostatin 1 plays a major role in
potentiating the lethal actions of paclitaxel in U937 and HL-60 cells.
Initial PKC activation is required for potentiation of
paclitaxel-induced TNF-
To determine the relationship between PKC activation and TNF- To evaluate further the possibility that PKC activation might be involved in bryostatin1-related potentiation of paclitaxel-induced cell death, U937 cells were pretreated with GFX (1 µM, 0.5 hours) prior to the addition of paclitaxel/bryostatin1. As shown in Figure 9C, pretreatment of cells with GFX essentially abrogated potentiation of paclitaxel actions by bryostatin 1. To determine whether the duration of PKC activation by bryostatin 1 represents an important determinant of potentiation of paclitaxel-mediated apoptosis, GFX was added at varying exposure intervals before or after treatment of cells with paclitaxel/bryostatin 1. Potentiation of paclitaxel actions by bryostatin 1 was essentially abrogated when GFX was added at time 0 or 30 minutes prior to bryostatin1/paclitaxel. In contrast, addition of GFX at later time points (eg, 3 or 6 hours after bryostatin 1/paclitaxel administration) was associated with the persistence of the ability of bryostatin 1 to potentiate paclitaxel-induced apoptosis. In separate studies, pretreatment of cells with 10 nM bryostatin 1 for 6 hours, an interval when PKC activity was significantly down-regulated (Figure 9A), failed to potentiate paclitaxel-induced apoptosis (data not shown). Collectively, these findings suggest that initial PKC activation, rather than PKC down-regulation or inhibition, is essential for promotion of paclitaxel lethality by bryostatin 1. Bryostatin 1 increases paclitaxel-induced apoptosis in cells that have undergone mitotic arrest As shown in Figure 10, exposure of U937/neo cells to 10 nM paclitaxel in combination with bryostatin 1 for 18 hours resulted in a clear increase in the subdiploid (apoptotic) population and a reciprocal decline in the MPM-2+ (mitotic) fraction compared with cells exposed to paclitaxel alone (P < .02 in each case). In U937/Bcl-2 cells exposed to paclitaxel alone, the subdiploid population was minimal, whereas more
than 50% of cells were MPM-2+, consistent with the notion
that cells protected from apoptosis underwent extensive mitotic arrest.
When paclitaxel-resistant U937/Bcl-2 cells were treated with the
combination of paclitaxel and bryostatin 1, there was a very
substantial increase in the subdiploid fraction and a large decline in
the MPM-2+ population. Taken together, these findings
suggest that paclitaxel-treated U937 cells that have undergone mitotic
arrest are particularly susceptible to potentiation of apoptosis by
bryostatin 1.
Cells synchronized in G2M display enhanced
susceptibility to TNF- -induced lethality. To this end, U937/neo and U937Bcl-2
cells were synchronized with aphidicolin (100 nM, 24 hours), washed
free of drug, and after 6 hours exposed to 0.025 ng/mL rhTNF for 24 hours. As shown in Figure 11A, about 75% of cells were in
G2M at the time of TNF- administration. The results
shown in Figure 11B indicate that
empty-vector and Bcl-2 cells synchronized in G2M display
a marked increase in apoptosis following TNF- exposure compared with
their unsynchronized counterparts (P < .01). Together,
these findings raise the possibility that induction of TNF- by
bryostatin 1 in cells arrested in G2M by paclitaxel
contributes to the marked potentiation of apoptosis accompanying
combined drug exposure.
While previous studies have demonstrated that bryostatin 1 potentiates the lethal actions of paclitaxel in both hematopoietic29 as well as nonhematopoietic cells,30 the mechanism underlying this phenomenon remains unclear. One possibility is that bryostatin 1 promotes those events responsible for paclitaxel-mediated mitochondrial injury and apoptosis. In this regard, the lethal actions of paclitaxel have been linked to phosphorylation of Bcl-2 and interference with the capacity of this protein to block mitochondrial injury and cell death.38 Consistent with this model, bryostatin 1-mediated circumvention of paclitaxel resistance in leukemic cells ectopically expressing Bcl-2 has been shown to be associated with Bcl-2 phosphorylation.23 An alternative possibility is that chronic exposure to bryostatin 1, which induces PKC down-regulation,20,39 generically sensitizes cells to a variety of noxious stimuli, including cytotoxic drugs.25,40 The observations that PKC inhibitors promote drug-induced apoptosis33 and that activation of PKC opposes hematopoietic cell death,41,42 including that induced by paclitaxel,43 support this notion. However, results of the present studies argue against either of these
mechanisms; instead, they strongly suggest that potentiation of
paclitaxel-mediated lethality in human myeloid leukemia cells stems
from bryostatin 1-mediated stimulation of TNF- Analogously, the present results are also difficult to reconcile with
the notion that potentiation of paclitaxel-mediated apoptosis by
bryostatin 1 stems solely from interruption of the PKC cytoprotective
pathway. While bryostatin 1 initially activates PKC,47 on
chronic administration it down-regulates the enzyme48 through a process that involves proteasomal degradation.49
Thus, chronic exposure to bryostatin 1 may mimic the actions of
pharmacologic PKC inhibitors (eg, staurosporine), many of which have
been shown to be potent inducers of apoptosis.23 In this
regard, the capacity of bryostatin 1 to promote apoptosis induced by
the antimetabolite ara-C in human leukemia cells has also been
correlated with PKC down-regulation.25 However, the
observation that GFX, a highly specific PKC inhibitor,50
blocked potentiation of paclitaxel lethality by bryostatin 1 argues
strongly against the possibility that interruption of the PKC pathway
is solely responsible for synergism between these agents. Moreover,
preexposure of cells to bryostatin 1 for 6 hours failed to promote
paclitaxel lethality despite down-regulating PKC activity (Figure 9A).
Instead, these findings, as well as the observation that PKC activation
is required for bryostatin 1-mediated potentiation of TNF- The notion that bryostatin 1 potentiates paclitaxel-mediated apoptosis
by increasing TNF release is supported by several lines of evidence.
First, coexposure of cells to paclitaxel and bryostatin 1 resulted in a
marked increase in caspase-8 activation and Bid cleavage, events that
are generally associated with activation of the extrinsic apoptotic
pathway. Second, coadministration of these agents resulted in sustained
elevations in TNF- Induction of TNF- A model that might account for the marked potentiation of
paclitaxel-induced apoptosis by bryostatin 1 is shown in Figure 12. In this model, acute exposure to
bryostatin 1 leads to the PKC-dependent induction of TNF-
Although taxanes such as paclitaxel have been employed in the treatment
of leukemia,57 activity to date has been limited. In this
regard, intrinsic resistance of primary leukemia cells to
paclitaxel-mediated lethality has been postulated.58
However, the ability of agents such as bryostatin 1 to potentiate
paclitaxel-induced apoptosis in human leukemia cells raises the
possibility that combined treatment with these agents might exhibit
increased activity in acute leukemia. The observation that bryostatin 1 potentiates paclitaxel-induced apoptosis in at least some primary AML
specimens (data not shown) supports this notion. In this context,
encouraging activity for a regimen combining paclitaxel and bryostatin
1 in patients with esophageal cancer has been reported.59
Whether this phenomenon can be generalized to additional leukemia cell specimens and, if so, whether it stems from enhanced TNF-
Submitted September 16, 2002; accepted December 15, 2002.
Prepublished online as Blood First Edition Paper, January 9, 2003; DOI 10.1182/blood-2002-09-2739.
Supported by awards CA 63785 and CA 63753 from the National Institutes of Health.
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: Steven Grant, Division of Hematology/Oncology, Virginia Commonwealth University/Medical College of Virginia, MCV Station Box 230, Richmond, VA 23298; e-mail: stgrant{at}hsc.vcu.edu.
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  W.-C. Chou, C. Jie, A. A. Kenedy, R. J. Jones, M. A. Trush, and C. V. Dang Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells PNAS, March 30, 2004; 101(13): 4578 - 4583. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
microtubular stabilization and the formation of nonfunctional microtubular bundles.
32-80) to
protect cells from paclitaxel-induced mitochondrial injury and
apoptosis supports a functional role for Bcl-2 phosphorylation in the
lethal response of cells to such agents.
), U937/Bcl-2 (
), and U937/
) cells
were treated with paclitaxel (Tax; 10 nM) with or without bryostatin 1 (Bry; 10 nM) for 24 hours. Apoptotic cells were quantified by annexin V
and PI positivity as described in "Materials and methods." (B)
Cells were treated as in panel A, after which they were washed
free of drug and plated in soft agar, after which colonies consisting
of at least 50 cells were scored after 12 days. Values were expressed
as a percentage relative to untreated controls (Con). Values for panels
A and B represent the means ± SE for 3 separate experiments
involving different U937/Bcl-2 and U937/
) or in
combination with paclitaxel (5 nM, 
). Total cellular PKC activity
was assessed as described in "Materials and methods." (B) U937
cells were exposed to paclitaxel/bryostatin 1 in the presence or
absence of GFX (1 µM) for 3 hours. Total RNA was isolated, and
TNF-
30 or 0 minutes) or
after (3 or 6 hours) administration of paclitaxel (5 nM) and bryostatin
1 (10 nM). Cell death was assessed after 24 hours by annexin and PI
staining as described in "Materials and methods." Results for
panels A and C correspond to 3 separate experiments (means ± SE).
that is, as a consequence of caspase-8 cleavage by caspase-3,
which was in turn activated by the caspase-9/cytochrome
c/Apaf-1 complex. In contrast, paclitaxel lethality was
unperturbed by interventions that blocked the extrinsic pathway,
indicating that cell death induced by this agent, at least when
administered alone, proceeds largely through the intrinsic pathway. On
the other hand, the ability of bryostatin 1 to potentiate this process
by engaging the extrinsic cascade shares similarities with the capacity
of agents such as Trail (TNF receptor apoptosis-inducing ligand) to
enhance the lethality of cytotoxic drugs.
is differentially down-regulated by bryostatin 1 and tetradecanoyl phorbol 13-acetate in SH-SY5Y human neuroblastoma cells.
Biochem Biophys Res Commun.
1993;191:472-478