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NEOPLASIA
From the Departments of Medicine, Microbiology,
Pharmacology, and Biochemistry, Medical College of Virginia, Virginia
Commonwealth University, Richmond, VA.
Cotreatment with a minimally toxic concentration of the protein
kinase C (PKC) activator (and down-regulator) bryostatin 1 (BRY)
induced a marked increase in mitochondrial dysfunction and apoptosis in
U937 monocytic leukemia cells exposed to the proteasome inhibitor
lactacystin (LC). This effect was blocked by cycloheximide, but not by
Mammalian cells, both normal and neoplastic,
undergo apoptosis in response to a variety of stimuli, including DNA
damage, cytokine deprivation, and dysregulation of
oncoproteins.1-3 Survival is governed by a family of
regulatory proteins, whose expression is modified by cellular signals;
together, these determine whether cells undergo differentiation,
proliferation, or apoptosis.4,5 Such cellular signals
exert either cytoprotective or cytotoxic actions, depending on the
specific downstream pathways affected (ie, stress-activated protein
kinase [SAPK] or mitogen-activated protein kinase [MAPK]). There is
accumulating evidence that the dynamic balance between the proapoptotic
JNK (c-Jun N-termina kinase)/p38 (SAPK) pathway and the growth and
differentiation-associated ERK (extracellular receptor kinase; MAPK)
pathway represents an important determinant of cell survival or
death.6 Recently we have shown that in U937 and HL-60
human leukemia cells, the extent of apoptosis depends on the coordinate
regulation of the SAPK and MAPK cascades.7 In view of
their potential importance in regulating neoplastic cell survival,
these signaling pathways are currently the subject of intense interest
as potential targets for chemomodulation.
Bryostatin 1 (BRY), a macrocyclic lactone isolated from the marine
invertebrate Bugula neritina, has shown significant
antitumor activity in preclinical studies8-10 and is
currently undergoing clinical evaluation in humans.11,12
BRY acutely activates protein kinase C (PKC), whereas prolonged
exposure of cells to this compound potently down-regulates enzyme
activity.13,14 The mechanism by which BRY induces
down-regulation of PKC has recently been attributed to ubiquitination
of cPKC The ubiquitin-proteasome system provides a major mechanism by which
intracellular proteins are degraded.21,22 Furthermore, proteasomal inhibitors induce apoptosis in diverse cell
types,23,24 suggesting an important role for the
proteasome in the regulation of cell survival.25 However,
the mechanism by which proteasome inhibitors induce cell death remains
obscure. In a recent communication, Song et al reported that the
proteasome inhibitor MG132 blocked BRY-mediated differentiation and p53
phosphorylation/degradation in the promyelocytic leukemic cell line
NB4, and that these effects were mimicked by the specific MAPK
inhibitor PD98059.26 Such findings raise the possibility
that interactions between BRY and the ubiquitin/proteasome system may
proceed through a MAPK-dependent pathway, at least as far as cellular
maturation is concerned.
The present studies were prompted by a desire to define the role of
bryostatin-induced PKC down-regulation in the cell death process more
rigorously, with particular emphasis on apoptosis induced by disruption
of proteasome function. A second goal was to relate these events to
functional alterations in stress (eg, SAPK) and survival (eg, MAPK)
signaling pathways. To this end, we have used lactacystin (LC), a
Streptomyces product known to inhibit proteasome function in
diverse systems.21,27 Here we report that exposure of U937
monocytic leukemia cells to a minimally toxic concentration of BRY (or,
to a lesser extent, the tumor promoters phorbol 12-myristate 13-acetate
[PMA] or mezerein [MEZ]) results in a dramatic potentiation of
apoptosis in LC-treated cells. Evidence obtained from a cell line
expressing TAM67, a c-Jun transactivation domain-deficient mutant
protein, and studies using the p38 MAP kinase inhibitor SB203580,
suggest that this interaction does not involve signaling through the
c-Jun/AP1 or p38/RK pathways. In marked contrast, enhanced apoptosis in
LC/BRY-treated cells is temporally associated with sustained MAPK
activation and is substantially attenuated by agents that disrupt the
PKC/Raf/MEK/EPK cascade, including bisindolylmaleimide I, geldanamycin,
PD98059, U0126, and SL327. Collectively, these findings suggest a
functional role for activation/dysregulation of the PKC/MEK/EPK module
in the synergistic induction of leukemic cell apoptosis by LC/BRY.
Cell lines
Drugs and reagents
Assessment of apoptosis Cell morphology and apoptosis was monitored by examining cytocentrifuge preparations stained with the Diff-Quik stain set (Dade Behring, Deerfield, IL) by light microscopy, or by TUNEL staining and fluorescent microscopy, as previously described.32 For each study, triplicate experiments were performed in which a total of 15 randomly selected fields, encompassing at least 1500 cells, were evaluated for each condition. In some cases the extent of apoptosis was confirmed by size threshold readings on a Coulter Z2 channelyzer (Opa Locka, FL).Clonogenic assays Following drug treatment for 24 hours, cell number was determined by hemacytometer count, and cells were washed 3 times in drug-free medium. Their ability to form colonies in soft agar was determined by a previously described technique.33 Colonies, consisting of groups of 50 cells, were scored at day 10 using an Olympus (Melville) Model CK inverted microscope.Western blot analysis Treated cells were washed once in cold phosphate-buffered saline (PBS) and either lysed in 1 × Laemmli buffer and immunoblotted for procaspase 3, Raf-1 (both 1:1000; Transduction Laboratories, San Diego, CA), PARP (1:1000; Biomol), PKC , PKC I,
PKCII (all 1:3000; Santa Cruz Biotechnology, Santa Cruz,
CA), actin (1:4000; Sigma), tubulin (1:2000; Sigma), or as per the
manufacturer's instructions for phospho-ERK, ERK, and phospho-JNK (all
1:2000; New England Biolabs, Beverly, MA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using either 25 µg cell extracts or 5 × 105
cell equivalents.
Assessment of mitochondrial function/integrity At the indicated intervals, cells were harvested and 2 × 105 cells were incubated with 40 nM 3,3-dihexyloxacarbocyanine (DiOC6; Molecular Probes, Eugene, OR) for 15 minutes at room temperature, as previously described.30 Cells were analyzed on a Becton Dickinson FACScan cytofluorometer and the percentage of cells exhibiting low levels of DiOC6 relative to control cells, reflecting loss of mitochondrial membrane potential, was determined using CyCLOPS 2000 Version 4.0 software.PKC activity The SignaTECT PKC assay system (Promega, Madison, WI) was used as per the manufacturer's instructions. Briefly, drug-treated cells were washed 1 × with cold PBS, lysed on ice in cold extraction buffer (25 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM-mercaptoethanol, 1 µg/mL leupeptin/aprotinin, and 0.5 mM
phenylmethylsulfonyl fluoride [PMSF]) and sonicated. Total
cell lysate was centrifuged at 4°C for 5 minutes at
14 000g and 50 µg supernatant was incubated in
coactivation buffer (0.25 mM EGTA, 0.4 mM CaCl2, 0.1 mg/mL
bovine serum albumin [BSA]), 0.1 mM ATP, 0.5 µCi
 32P] ATP (3000 Ci/mmol), and 100 µM PKC 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) for 5 minutes at 30°C. Reactions
were terminated by the addition of guanidine hydrochloride (2.5 M) and
spotted on SAM2 membrane. Purified PKC was used as a
positive control and myristoylated PKC peptide inhibitor was used to
indicate specificity. Values are expressed as the percentage of PKC
activity relative to those of untreated cell extract (100%).
Statistical analysis The significance of differences between experimental groups was determined using the Student t test for unpaired observations. To assess the interaction between agents, Median Dose Analysis was used34 with a commercially available software program (CalcuSyn; Biosoft, Ferguson, MO). The combination index (CI) was calculated for a 2-drug combination involving a fixed concentration ratio. Using these methods, CI values less than 1.0 indicate a synergistic interaction.
BRY potentiates LC-induced apoptosis in U937 cells Proteasome inhibitors induce apoptosis in proliferating cells,23,24,35 whereas in differentiated neuronal cells or thymocytes they inhibit cell death.36,37 In U937 human leukemic cells, the proteasome inhibitor LC induces caspase 3-dependent cell death.24,38 Furthermore, it has been previously reported that induction or inhibition of cell death by proteasome inhibitors is highly concentration dependent.39 The data in Figure 1A demonstrate that exposure of U937 cells to LC for 24 hours induces apoptosis and reciprocally reduces leukemic self-renewal capacity in a concentration-dependent manner. The LC concentration producing 50% apoptosis was approximately 3 µM. To assess the effects of PKC down-regulation on the response to LC, cells were exposed to a marginally toxic concentration of LC (~15% cell death; 1 µM) for 24 hours in conjunction with a concentration of BRY (10 nM) previously shown to reduce total cellular PKC activity by more than 90% in this cell line.32 Concurrent treatment with BRY (24 hours), which by itself was minimally toxic (8%-10% apoptosis; data not shown), dramatically enhanced LC-mediated lethality, manifested by potentiation of the morphologic features of apoptosis (Figure 1B). In addition, BRY potentiated other aspects of LC-induced cell death, including DNA fragmentation (Figure 2A), loss of mitochondrial membrane potential (  m; Figure 2B), marked degradation/activation
of procaspase-3, and cleavage of the major caspase-3 substrate, PARP
(Figure 2C). Following exposure to LC and BRY, apoptosis was most
apparent after 18 hours of treatment, whereas enhanced loss of
mitochondrial membrane potential was observed as early as 12 hours
after drug exposure. It should be noted that treatment with 1 µM LC alone induced a moderate degree of mitochondrial membrane
discharge (~40%), which was evident at 12 hours but did not increase
further; in contrast, BRY induced less than 15% mitochondrial
discharge at all time intervals examined (not shown). Lastly, median
dose effect analysis was used to characterize the interaction between BRY and LC with respect to induction of apoptosis. Varying
concentrations of LC (0.1-2 µM) and BRY (0.5-10 nM) administered at a
fixed ratio (1:5) resulted in CI values less than 1, indicating a
synergistic interaction (Figure 2D). In separate studies, BRY did not
potentiate apoptosis by other protease inhibitors, including
NH4Cl (2 mM), E64 (1, 10 µM), leupeptin (10 µM),
calpain inhibitor I (1 µM), or calpain inhibitor II (10 µM) (data
not shown), nor did these inhibitors induce apoptosis by themselves,
indicating that inhibition of lysosomal, thiol, and cysteine proteases
is not involved in the initiation of apoptosis. Collectively, these
findings demonstrate that LC and BRY interact synergistically in a
specific manner to induce apoptosis in U937 cells.
LC- and BRY-mediated apoptosis is protein synthesis dependent Cells incubated with proteasome inhibitors display an increase in proteins that are normally degraded through the ubiquitin-proteasome pathway. To determine if LC/BRY-induced apoptosis depended on the accumulation of either new or preexisting cellular proteins, U937 cells were pretreated with varying concentrations of the protein synthesis inhibitor cycloheximide prior to the addition of LC/BRY (Figure 3A). In marked contrast to the results of previous reports, which have shown that cycloheximide renders U937 cells more sensitive to tumor necrosis factor (TNF)-induced apoptosis40 and does not inhibit VP-16-induced cell death in HL-60 cells,41 the enhanced apoptotic response to LC/BRY was completely abolished by cycloheximide. However, inhibition of RNA synthesis with-amanitin or actinomycin D
did not prevent BRY from potentiating LC-induced cell death (Figure
3B,C). These findings suggest that apoptosis induced by LC/BRY
treatment involves a protein synthesis-dependent but
transcription-independent process. Lastly, coadministration of the
reduced glutathione (GSH) precursor L-NAC (5 mM, Figure 3B, or 2.5 mM,
data not shown) effectively blocked both LC- and LC/BRY-induced
apoptosis, raising the possibility that generation of reactive oxygen
species may be involved in the enhanced apoptosis induced by
these agents.
LC-BRY-induced apoptosis does not depend on an intact c-Jun/AP-1 signaling pathway Although previous studies have suggested that JNK activation is essential for apoptosis induced by proteasome inhibitors in general, and LC in particular,42 an increase in phospho-JNK expression following either LC or LC/BRY treatment could not be demonstrated (data not shown). In contrast, HSP72 expression was increased following exposure to LC as reported previously in the case of MG132,42 but BRY did not enhance this effect (data not shown). To examine the functional role of this pathway in LC/BRY-mediated cell death, U937 cells stably expressing a dominant-negative c-Jun construct (TAM67)43 were used. This construct contains the DNA binding domain but lacks the AP-1 transactivation domain, thereby blocking an immediate downstream target of the JNK cascade. U937/TAM67 cell lines, and their empty vector controls, were exposed to LC/BRY for 24 hours, after which apoptosis and loss of mitochondrial membrane potential were assessed (Figure 4A,B). In the control and both TAM67 subclones, combined LC/BRY treatment induced apoptosis in about 80% of cells (Figure 4A) and exhibited equivalent loss of mitochondrial membrane potential (Figure 4B). In separate control studies, expression of the TAM67 protein substantially protected cells from sphingomyelinase-induced (100 mU/mL) apoptosis, as previously reported7 (data not shown). One of the TAM67 subclones exhibited slight protection against apoptosis induced by LC alone, a finding analogous to that previously described in the case of human kidney 293 cells transfected with the TAM67 construct.42 Lastly, cells pretreated with curcumin (0.5 µM; 24 hours), an agent that blocks the JNK/AP-1 pathway by interfering with MEKK or other upstream signals,44 also failed to protect cells against LC/BRY-induced apoptosis (data not shown). The failure of a dominant negative c-Jun construct or pharmacologic disruption of the JNK/AP1 pathway to protect against enhanced apoptosis following LC/BRY treatment argues against the involvement of c-Jun/AP1 signaling in this phenomenon.
LC/BRY-induced apoptosis does not require p38/RK signaling Although the p38/RK signaling cascade is activated in response to proteasome inhibitors, this pathway is reportedly not involved in proteasome inhibitor-induced cell death.42 To determine if potentiation of LC-induced apoptosis involved p38/RK, cells were pretreated with the specific p38/RK inhibitor SB203580 before the addition of LC/BRY. Neither 10 nor 20 µM SB203580 inhibited apoptosis induced by LC alone (not shown) or by LC/BRY (Figure 4C), suggesting that p38/RK signaling does not mediate U937 cell death induced by either LC or the combination of LC/BRY.Potentiation of LC-mediated apoptosis by BRY requires PKC activation To determine if potentiation of LC-induced apoptosis could be generalized to include other agents that activate the PKC/MAPK pathway, or is instead restricted to BRY, induction of cell death was monitored in cells treated with LC in conjunction with other well-characterized PKC activators (Figure 5). Of these, PMA and MEZ (but not the negative control 4-PMA) significantly increased apoptosis in LC-treated cells, although the extent of potentiation was
somewhat less than that observed with BRY. In contrast, cotreatment with bacterial phospholipase C (PLC), which acutely activates PKC via
the generation of endogenous diaglycerides, failed to enhance
LC-induced cell death; instead, it marginally protected cells from
LC-induced apoptosis (P = .05 versus LC alone). These studies demonstrate that agents, which, on chronic exposure,
down-regulate PKC (eg, BRY, PMA, MEZ) potentiate LC-induced apoptosis,
whereas an agent known to induce sustained activation of PKC (eg, PLC) fails to do so.
To define the role of PKC activation in this phenomenon further, a
highly selective inhibitor of PKC, bisindolylmaleimide I (GF109203X),
was used to inhibit basal and BRY-related activation of PKC.
Pretreatment of cells with GFX (1 µM; 0.5 hour) prior to the addition
of LC/BRY dramatically inhibited apoptosis (eg, by ~60%; Figure
6A). To establish whether the duration of
PKC activation by BRY represented an important determinant of
LC/BRY-mediated cell death, GFX was added at increasing exposure
intervals following treatment of cells with LC/BRY (Figure 6A). The
enhanced apoptotic response was abrogated when GFX was added at early
intervals following BRY (eg, < 4 hours), whereas at later
time points (eg, > 6 hours) BRY retained its ability to potentiate
cell death in LC-treated cells. These findings suggest that the initial
activation of PKC by BRY plays a critical role in its ability to
augment LC-mediated apoptosis.
Lactacystin (50 µM) prevents BRY-induced down-regulation of PKC in
renal epithelial cells and fibroblasts.15,45 To determine whether the LC/BRY doses and exposure intervals used in the present studies produced a similar response, PKC activity and isoform expression was monitored following 24 hours of exposure of cells to
LC/BRY (Figure 6B,C). U937 cells exposed to BRY for 24 hours displayed
an approximately 70% decrease in PKC activity, whereas LC-treated
cells exhibited only a marginal decrease (< 10%; Figure 6B). In
contrast to previous findings,15,45 LC did not prevent BRY-induced down-regulation of PKC activity inasmuch as combination treatment produced an approximate 80% decrease in enzyme activity. Consistent with these results, PKC isoform expression revealed decreased levels of PKC Raf-1 is essential for LC/BRY-induced apoptosis Studies using dominant-negative and constitutively active PKC mutants have shown that activation of Raf-1 represents a critical component of signaling through the PKC/MAPK pathway.46 In addition, BRY actives Raf-1 in a PKC-dependent manner through direct serine phosphorylation.47 To determine if Raf-1 activation was essential for potentiation of LC-induced cell death by BRY in U937 cells, geldanamycin, a benzoquinone ansamycin that binds to HSP-90 and disrupts the Raf-1/HSP90 complex, resulting in Raf-1 destabilization,48 was used to interrupt this pathway. Cells were either pretreated with 1 µM geldanamycin for 18 hours before the addition of LC/BRY or were treated acutely with LC/BRY. Geldanamycin pretreatment reduced Raf-1 protein expression to undetectable levels (Figure 7, inset). Concomitant with decreased levels of Raf-1 protein, the extent of apoptosis in LC/BRY-treated cells was reduced to levels observed in cells exposed to geldanamycin alone (Figure 7). Moreover, cells treated simultaneously with geldanamycin and LC/BRY (ie, without the 18-hour preincubation interval) exhibited an approximate 40% decrease in apoptosis (P = .05 compared to LC/BRY alone; data not shown). These results suggest that Raf-1 plays a critical role in the enhanced apoptosis observed in U937 cells exposed to LC/BRY.
ERK activation is essential for potentiation of LC-mediated apoptosis by BRY MEK (MAPK/ERK kinase), a physiologic substrate of Raf-1, phosphorylates and activates ERK1/2,49 which in turn phosphorylates multiple downstream targets. Proteasome inhibitors have been shown to enhance MAPK activation through prolonged translocation of MAPK to the nucleus.50 To determine whether cells exposed to LC exhibit altered MAPK activation in response to BRY treatment, levels of phosphorylated (ie, activated) ERK1 and ERK2 were compared in extracts from BRY- and LC/BRY-treated cells (Figure 8A). BRY induced maximal ERK phosphorylation within 1 hour of treatment, after which expression rapidly declined to basal levels by 4 hours. In marked contrast, cells exposed to BRY in conjunction with LC exhibited pronounced expression of phosphorylated ERK throughout the 8-hour treatment interval, and continued to display elevated phospho-ERK expression for as long as 12 hours (data not shown). Total ERK expression was unaltered by treatment with BRY or the LC/BRY combination (Figure 8A); moreover, 1 µM LC alone did not induce ERK phosphorylation nor did it alter ERK expression (data not shown).
To investigate the functional significance of MAPK activation in
potentiation of LC-mediated apoptosis by BRY, the specific MEK
inhibitors, PD98059,51 U0126,52 and
SL32753 were used. The data in Figure 8B demonstrate that
MEK inhibitors partially blocked loss of
LC/BRY-mediated apoptosis does not involve the MAPK downstream target p21CIP1 Finally, we have previously reported that dysregulation of the cyclin-dependent kinase inhibitor p21CIP1, a downstream target of MAPK,54 modifies the susceptibility of U937 cells to apoptosis induced by the cytotoxic agent 1--D-arabinofuranosylcytosine (ara-C).55 To
determine whether p21CIP1 might be involved in potentiation
of LC-mediated apoptosis by BRY, stable p21CIP1
antisense-expressing cells (p21AS) and their vector control
counterparts (pREP4) were exposed to BRY and LC alone and in
combination (Figure 10). In marked
contrast to results obtained with ara-C,55 dysregulation of p21CIP1 did not lead to a statistically significant
increase in LC/BRY-mediated apoptosis, nor in apoptosis induced by LC
alone. This finding indicates that apoptosis induced by LC/BRY
treatment, in contrast to that induced by the antimetabolite ara-C,
proceeds through a pathway that is independent of the MAPK downstream
target, p21CIP1.
The results described herein suggest the mechanism by which the
PKC modulator BRY 1 enhances cell death in U937 human leukemia cells
exposed to the proteasome inhibitor LC involves perturbations in the
MAPK signaling cascade. Potentiation of cell death was documented by an
increase in morphologic characteristics of apoptosis (ie, cell
shrinkage, nuclear condensation, formation of apoptotic bodies),
augmented DNA fragmentation (manifested by TUNEL positivity), and an
increase in the percentage of cells exhibiting low levels of
DiOC6, reflecting loss of mitochondrial membrane potential ( The ubiquitin/proteasome pathway represents the primary mechanism by
which the bulk (eg, 80%-90%) of cellular proteins in proliferating cells are degraded. Numerous proteins are targeted for
degradation by this pathway including (1) cell cycle proteins (eg,
mitotic and G1 cyclins, cyclin-dependent kinase inhibitors), (2)
oncogenic transcription factors (eg, c-Myc, c-Myb, p53), and (3) early
response transcription factors (eg, c-Jun, c-Fos, I Currently, the mechanism by which proteasome inhibitors induce apoptosis is poorly understood, and relatively little information is available concerning the relationship between their actions and activation of signal transduction pathways, particularly the PKC/Raf/MEK/EPK cascade. However, evidence for cross-talk between this pathway and LC-related actions have recently emerged from studies involving the macrocyclic lactone, BRY.26 The most characteristic function of BRY lies in its ability to modulate PKC; that is, acute exposure induces PKC activation, whereas chronic exposure results in down-regulation.32,62,63 In this context, it is noteworthy that 2 other agents known to trigger initial activation and, on chronic exposure, down-regulation of PKC (eg, PMA and MEZ64,65) also enhanced apoptosis in LC-treated cells, although not to the same extent as BRY. It is conceivable that such differences reflect disparate effects on PKC isoform expression,14 or possibly the extent of PKC down-regulation. In marked contrast, PLC, a physiologic PKC modifier that activates but does not down-regulate PKC,66 did not potentiate cell death, but instead protected the cells from LC-induced apoptosis. This observation is consistent with evidence of an antiapoptotic role for chronic PKC activation.67 Collectively, these findings are consistent with the concept that down-regulation of PKC contributes to the enhanced cell death observed in cells exposed to LC/BRY. Although PKC down-regulation (eg, by PMA or BRY) has been implicated in the induction of apoptosis,67,68 it remained possible that the initial activation of PKC by BRY (or other PKC activators) contributed to promotion of LC-mediated cell death. The ability of the specific PKC inhibitor bisindoylmaleimide I (GF109203X) to attenuate the lethal actions of LC/BRY strongly suggests that this is the case. In addition, the finding that increasing the interval prior to GFX administration led to a progressive decline in cytoprotection indicates that early events (eg, those occurring within the initial 4-6 hours) following PKC activation are responsible for the enhanced lethality of the LC/BRY combination. It is important to note that Lee and coworkers have shown that the ability of BRY to down-regulate PKC in renal epithelial cells and nonimmortalized human fibroblasts stemmed from PKC dephosphorylation, ubiquitination, and targeting for proteasomal degradation.15,42,63 However, in contrast to the results of these studies, in which considerably higher LC concentrations were used (eg, > 50 µM), LC, when administered to U937 cells at low concentration (eg, 1 µM), failed to block BRY-induced PKC down-regulation at 24 hours. Although such a discrepancy may reflect dose- or cell type-specific differences, the present findings demonstrate that chronic activation of PKC by BRY is not required for enhanced lethality of the LC/BRY combination. In addition, the possibilities that LC interferes with degradation of alternative, as yet to be identified proapoptotic proteins in BRY-treated cells, or that LC-related cell death involves actions other than proteasome inhibition, cannot be excluded. In this regard, the relatively early onset of enhanced cell death in LC/BRY-treated cells, and our failure to detect changes in levels of expression or alterations in gel mobility of apoptotic regulatory proteins over this interval (eg, Bcl-2 and Bax; J.A.V. et al, unpublished observations) would argue against a primary role for such events in mediating lethality. However, the possibility that these agents induce subtle qualitative changes in Bcl-2 phosphorylation status that might influence the susceptibility of cells to apoptosis69 cannot presently be excluded. Bryostatin has been shown to activate Raf through PKC-dependent phosphorylation of S497 and S619 (single-letter amino acid codes) residues.47 Consistent with a report demonstrating that depletion of Raf (eg, by geldanamycin) attenuates paclitaxel-mediated apoptosis,70 the presence of Raf was found to be required for enhanced apoptosis in LC/BRY-treated cells. In addition, BRY, which activates MAPK through the PKC/Raf/MEK pathway, transiently induced phosphorylation of ERK1/2, whereas this effect was markedly prolonged in cells coexposed to LC. Significantly, each of the 3 specific MEK inhibitors examined (eg, PD98059, U0126, and SL327) inhibited both ERK phosphorylation and LC/BRY-induced apoptosis in a dose-dependent manner. MAPK activation was also essential for induction of the mitochondrial membrane permeability transition observed in LC/B-treated cells. Although activation of the MAPK cascade has generally been associated with antiapoptotic action,6,71 it has recently been shown that MAPK activation may, under some circumstances, promote cell death.72 Taken in conjunction with the preceding results, such findings strongly suggest that dysregulation of the MAPK signaling pathway, either upstream at the level of PKC or Raf, or further downstream at the level of MEK, plays a key functional role in the enhanced apoptosis accompanying LC/BRY treatment of U937 cells. Activation of JNK/SAPK has been shown to be critical for apoptosis induced by numerous environmental stresses, including proteasomal inhibitors such as MG132.42,73-75 However, in contrast to evidence suggesting a requirement for JNK activation in MG 132-induced apoptosis in U937 and 293 human kidney cells,42 a functional role for the SAPK pathway in the LC/BRY-mediated apoptosis of U937 cells could not be demonstrated. More specifically, our results indicated that (1) JNK activation was not enhanced following LC/BRY treatment; (2) LC/BRY-induced apoptosis was not inhibited by a dominant-interfering form of c-JUN (TAM67) or by pretreatment with curcumin, a pharmacologic inhibitor of the AP-1 pathway; and (3) LC/BRY-induced apoptosis was not inhibited by the specific p38 inhibitor, SB203580. Exposure to LC did induce HSP72 as reported previously in the case of MG13242; however, coexposure to BRY did not alter HSP72 expression. Thus, in the U937 cell system, c-Jun/AP-1 or p38/RK signaling does not appear to be involved in LC/BRY-induced cell death. In marked contrast, ceramide and TNF-mediated apoptosis is critically dependent on an intact c-Jun/AP-1 axis in these cells,75 indicating that LC/BRY-mediated apoptosis proceeds through a ceramide-independent pathway. The recent observation that the proteasome inhibitor MG132 potentiates
sodium butyrate-induced apoptosis and caspase activation in Y79 human
retinoblastoma cells76 has potential relevance for the
present study. In the Y79 system, dysregulation of the transcription
factors p53, N-myc, and I In summary, the present findings demonstrate that the combination of a
proteasome inhibitor (LC) with BRY or other agents that perturb a
putatively cytoprotective signaling pathway (eg, PKC/Raf/EPK) potently
induce apoptosis in U937 human leukemia cells. More specifically, they
suggest that initial activation and subsequent down-regulation of PKC,
as well as sustained activation of MAPK, contribute to the increased
susceptibility of these cells to LC-mediated lethality. Although the
downstream targets of MAPK responsible for this phenomenon remain to be
identified, the results of this study, particularly those shown in
Figure 10, argue against the involvement of p21CIP1. An
alternative MAPK target is NF
The assistance of Dr Paul Dent, Department of Radiation Oncology, Medical College of Virginia, who made U0126 and SL327 available for these studies, is gratefully acknowledged. We also thank Lora Kramer and Aida Saunders for their technical assistance, Dr M. Birrer for the U937/TAM67 cells, and Dr Z. Wang for the U937/p21AS cells.
Submitted September 11, 2000; accepted November 21, 2000.
Supported by awards CA63753, CA72955, and CA77141 from the National Institutes |
Top
Abstract
Introduction
) or their self renewal capacity by their ability
to form colonies in soft agar (
). (B) U937 cells
(2 × 105 cells/ml) were exposed to 1µM LC (
]) for 1 hour before the addition of LC/BRY.
Following a 24-hour exposure, the cells were either (B) assayed for
loss of mitochondrial membrane permeability by determining the
percentage of cells exhibiting low levels of DiOC6; values
are expressed as the percentage inhibition of LC/BRY-mediated
mitochondrial damage compared to cells that were not exposed to MEK
inhibitors or (C) the percentage of apoptotic cells was determined by
morphologic analysis as above. In each case, values represent the means
for triplicate experiments (± SEM).
m). Enhanced cleavage of the proform of
caspase-3 was also noted in LC/BRY-treated cell extracts, as well
as degradation of one of its principal downstream substrates, PARP.
This response was cycloheximide sensitive indicating that up-regulation
of short-lived proteins may be critical for LC/B-induced cell death.
Taken together, these findings raise the possibility that coexposure of
U937 cells to LC and BRY stimulates the synthesis of an as yet to be
identified protein(s) that triggers the apoptotic cascade. Lastly, the
ability of L-NAC to block LC/BRY-induced lethality is similar to
results recently described by Mansat-de Mas et al, who found that 10 mM NAC opposed daunorubicin-induced apoptosis in U937
cells.
B, NF