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Vol. 289, Issue 2, 781-790, May 1999
Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, and Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida (Q.P.D.); and Department of Pharmacology, University of Pittsburgh School of Medicine, and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania (Q.P.D., T.F.M., Y.P., B.A.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The chimeric oncogene bcr-abl is detected in virtually every case of chronic myelogenous leukemia. It has been shown that cells (such as K562) expressing Bcr-Abl/p210, a protein tyrosine kinase, not only undergo cellular transformation but also demonstrate multiple drug resistance. Recent studies also demonstrate that the proteasome is involved in the survival signaling pathway(s). In the current study, we tested the hypothesis that the proteasome might play a role in regulating Bcr-Abl function. We have demonstrated by using a variety of inhibitors that inhibition of the proteasome, but not of the cysteine protease, activity is able to activate the apoptotic cell death program in K562 cells. Proteasome inhibition-induced apoptosis is demonstrated by condensation and fragmentation of nuclei, appearance of an apoptotic population with sub-G1 DNA content, the internucleosomal fragmentation of DNA, and cleavage of poly(ADP-ribose) polymerase, and can be blocked by a specific caspase-3-like tetrapeptide inhibitor. Western blot analysis with specific antibodies to c-Abl and Bcr proteins show that treatment of K562 cells with a proteasome inhibitor results in significant reduction of Bcr-Abl protein expression, which occurs several hours before the onset of apoptotic execution. Levels of c-Abl/p145 and Bcr/p160 proteins, however, remain essentially unaltered at that time. Furthermore, reduced Bcr-Abl expression is reflected in significantly attenuated Bcr-Abl-mediated protein tyrosine phosphorylation. Taken together, these results indicate that proteasome inhibition is sufficient to inactivate Bcr-Abl function and subsequently activate the apoptotic death program in cells that are resistant to apoptosis induced by chemotherapy.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Apoptosis,
or programmed cell death, is a highly regulated, complex cellular
process occurring in two physiological stages, commitment and execution
(Earnshaw, 1995
). The molecular events involved in the apoptotic
commitment are not clearly understood. However, it has been found that
the execution stage of apoptosis is initiated by activation of members
of a specific cysteine protease family, named caspases, which cleave
specific intracellular proteins such as poly(ADP-ribose) polymerase
(PARP) (Martin and Green, 1995). These molecular events lead to
morphological changes that are characteristic of apoptosis, including
plasma membrane blebbing, cytoplasmic and nuclear condensation, nuclear
fragmentation, and cellular fragmentation into apoptotic bodies
(Earnshaw, 1995).
Chronic myelogenous leukemia (CML) is characterized by a translocation
that creates a fusion between the bcr gene on chromosome 22 and the c-abl gene on chromosome 9 to form what is known as the Philadelphia t(9;22) chromosome (Ph1)
(Mes-Masson et al., 1986). This translocation produces a chimeric oncogene, bcr-abl, which encodes a 210-kDa fusion protein
(Bcr-Abl) with unregulated tyrosine kinase activity (Konopka et al.,
1984). The tyrosine kinase activity of Bcr-Abl, which is the
principal driving force behind its oncogenic potential, is responsible
for mediating tyrosine phosphorylation of specific cellular proteins and Bcr-Abl itself (Lugo et al., 1990).
Although there is some evidence that the Bcr-Abl oncoprotein acts as a
proliferative activator (Daley et al., 1990; Elefanty et al., 1990;
Skorski et al., 1996), its major function appears to be to act as an
apoptotic suppressor. This idea is supported by the following evidence.
First, in vivo studies have demonstrated that massive clonal expansion
of myeloid precursor cells in CML patients is due to an increased
survival ability, but not to an increased proliferative rate (Koeffler
and Golde, 1981). Indeed, primary Ph1-positive
leukemic cells expressing Bcr-Abl demonstrate an increased resistance
to apoptosis induced by serum deprivation, irradiation, and
chemotherapeutic agents (Bedi et al., 1994; Nishii et al., 1996).
Second, transfection of the bcr-abl gene into Ba/F3 cells endues these cells with the drug-resistance phenotype (Nishii et al.,
1996) and protects them against the apoptotic effects of growth factor
withdrawal (Bedi et al., 1994). Third, when the Bcr-Abl level in K562
cells was decreased by using its antisense oligonucleotides, cell
growth was reduced due to increased apoptosis, but not due to decreased
DNA synthesis; the treated K562 cells had also lost their
multidrug-resistant phenotype and became sensitized to drug-induced
programmed cell death (McGahon et al., 1994; Rowley et al.,
1996).
The 26S proteasome, composed of a 20S catalytic core and two associated
700-kDa regulatory proteins, is a large multicatalytic protease
demonstrating at least trypsin-like, chymotrypsin-like, and
peptidylglutamyl peptidase activities (Hochstrasser, 1995). Chains of
polyubiquitin, covalently linked to 
-amino groups of lysine
residues, mark a protein to be targeted to the proteasome for
hydrolysis. Several cell cycle and apoptosis regulatory proteins, including cyclins, cyclin-dependent kinase inhibitors, tumor suppressor p53, and transcription factors E2F and nuclear factor 
B, are regulated through proteolysis via the ubiquitin/proteasome pathway (Hopkin, 1997).
Recently, studies using selective inhibitors of the proteasome
have provided direct evidence that indicates that the proteasome functions both in promoting apoptosis and in protecting cells against
apoptosis. These proteasome inhibitors include lactacystin, a highly
specific inhibitor of the 20S proteasome (Fenteany et al., 1995), and
tripeptide aldehydes, inhibitors of the proteasome chymotrypsin-like
activity (Rock et al., 1994). It has been found that these proteasome
inhibitors block the apoptotic process in thymocytes (Grimm et al.,
1996) and neurons (Sadoul et al., 1996). In contrast, the same
proteasome inhibitors induce apoptosis in human or mouse leukemia
(Imajoh-Ohmi et al., 1995; Shinohara et al., 1996; Drexler, 1997) and
other proliferating cell lines (Lopes et al., 1997). By using novel
dipeptidyl proteasome inhibitors, we also found that inhibition of the
proteasome activity is sufficient to rapidly induce apoptosis in human
Jurkat T cells overexpressing Bcl-2 and also in all human prostate,
breast, tongue, and brain tumor cell lines tested (An et al., 1998).
Furthermore, dipepdidyl proteasome inhibitors selectively accumulate
the cyclin-dependent kinase inhibitor p27 and induce apoptosis in
simian virus 40-transformed fibroblasts, but not in the parental
normal, human fibroblasts (An et al., 1998). In the current
study, we have investigated the ability of proteasome inhibitors,
including tripeptide aldehydes and lactacystin, to induce apoptosis in
K562 human chronic myelogenous leukemic cells, which express Bcr-Abl
and are multidrug-resistant (Martin et al., 1990; McGahon et al., 1994;
Rowley et al., 1996). We report here that these proteasome inhibitors
first decrease the levels and tyrosine kinase activity of the Bcr-Abl
protein and, subsequently, activate the apoptotic death program in
these cells.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials. N-carbobenzoxy-L-leucyl-L-leucyl-norvalinal (LLnV), N-acetyl-L-leucyl-L-leucyl-norleucinal (LLnL), N-carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL), N-acetyl-L-leucyl-L-leucyl-L-methioninal (LLM), etoposide (VP-16), (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester (E-64d), dimethyl sulfoxide (DMSO), antipain, iodoacetamide, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, and phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co. (St. Louis, MO). Lactacystin was obtained from Calbiochem (La Jolla, CA) and acetyl-DEVD-fluoromethyl ketone (DEVD-FMK) was obtained from Kamiya Biomedical Company (Seattle, WA). Stocks of LLnV, LLnL, LLL, LLM, lactacystin, VP-16, and DEVD-FMK were prepared by dissolving in DMSO such that the final concentration of the solvent in the medium did not exceed 0.1%. Purified mouse monoclonal antibody to caspase-3 (CPP32) was obtained from Transduction Laboratories (Lexington, KY); to PARP from Unité de Santé et Environnement (Québec, Canada), and to c-Abl (Ab-3; derived from a fusion protein corresponding to the carboxyl region of the v-abl protein) from Oncogene Research Products (Cambridge, MA). Rabbit polyclonal antibody to Bcr, recognizing the peptide sequence corresponding to amino acids 2 to 21 of human Bcr, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and to ubiquitin from Sigma. Horseradish peroxidase-conjugated anti-phosphotyrosine antibody was from Transduction Laboratories (Lexington, KY).
Cell Culture and Treatment with Proteasome Inhibitors. K562 human CML cells were maintained in RPMI 1640 medium containing 10% fetal calf serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin, and 3 mM glutamine (growth medium) in a 5% CO2 atmosphere at 37°C. K562 cells were treated with a proteasome inhibitor at a concentration for the length of time noted in the legends to Figs. 1 to 8.
Nuclear Staining, Flow Cytometry, and DNA Fragmentation
Assays.
To assay nuclear morphology, K562 cells were washed with
PBS, fixed with 70% ethanol, and stained with Hoechst 33258 (1 mM) for
30 min. The nuclear morphology of cells was visualized by a
fluorescence microscope (Olympus BH2; Olympus Optical Co., LTD, Tokyo,
Japan). DNA content analysis by flow cytometry was performed as
described previously (Nicoletti et al., 1991). Briefly, K562 cells were
fixed with 70% ethanol, stained with propidium iodide (50 µg/ml) for
30 min at room temperature, and analyzed immediately in a flow
cytometer. DNA fragmentation was assayed as described (Grant et al.,
1992). At each time point, K562 cells were washed in PBS and
resuspended in 0.7 ml of a buffer containing 10 mM Tris-HCl, 10 mM
EDTA, 0.5% SDS, and 200 µg/ml protease K. The cell mixtures were
incubated at 55°C for 2 h and then treated with 25 µg/ml RNase
at 37°C for 1 h. After incubation, DNA was precipitated with 1.5 volume of ethanol and resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The prepared DNA samples were analyzed in a 2% agarose gel
containing 0.1% SDS, followed by staining with ethidium bromide.
Whole-Cell Extracts and Western Blot Assay.
Whole-cell
extracts and the enhanced chemiluminescence Western blot assay were
performed as described previously (An and Dou, 1996). Each lane
contains an equal amount of protein (40-70 µg).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Tripeptidyl Aldehyde LLnV Activates Caspase Cascade and Induces
Apoptosis in K562 Cells.
By using different proteasome inhibitors,
several groups have demonstrated that the proteasome is involved in the
survival-signaling pathway(s) (Imajoh-Ohmi et al., 1995; Shinohara et
al., 1996; Drexler, 1997; Lopes et al., 1997; An et al., 1998). Because
the Bcr-Abl oncoprotein functions as a proliferative activator (Daley et al., 1990; Elefanty et al., 1990; Skorski et al., 1996) as well as
an apoptotic suppressor (Bedi et al., 1994; Nishii et al., 1996), we
hypothesized that the proteasome might play a role in the regulation of
Bcr-Abl function and that inhibition of the proteasome activity might
overcome Bcr-Abl-mediated drug-resistance and induce apoptosis in
Bcr-Abl-expressing cells. To test this hypothesis, we used K562 human
CML cells that express Bcr-Abl (Martin et al., 1990). When K562 cells
were treated for 24 to 48 h with 50 µM LLnV, a tripeptidyl
aldehyde that can block the proteasome activity effectively (Rock et
al., 1994), apoptosis indeed occurred, as demonstrated by condensation
and fragmentation of nuclei (Fig. 1A),
appearance of an apoptotic population with sub-G1
DNA content (Fig. 1B, indicated by Ap), and the internucleosomal fragmentation of DNA (Fig. 1C, DNA ladders). Exposure of K562 cells to
LLnV for 24 h also induced the processing of caspase-3/CPP32 (data
not shown), which is required for its activation (Martin and Green,
1995), and complete cleavage of PARP to a p85 fragment (Fig. 1D, lane 6 versus lane 1). None of these LLnV-induced events were observed in K562
cells treated with the solvent DMSO (Fig. 1), demonstrating drug
specificity. Because K562 cells lack functional p53 protein (Shao et
al., 1996), LLnV-induced apoptosis in these cells is p53-independent.
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). When incubated with LLnV-treated
K562 cells, DEVD-FMK effectively prevented, in a
concentrationdependent manner, induction of apoptotic nuclear changes: ~70% inhibition at 30 µM and >90% at 100 µM (Fig.
2A). Consistent with that, inhibition of
PARP cleavage by DEVD-FMK was also concentration-dependent: ~75% at
30 µM and 90 to 100% at 100 or 300 µM (Fig. 2B, lanes 3-5 versus
lane 2). These data demonstrate that LLnV-induced apoptosis in K562
cells requires activation of caspases.
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Inhibition of Activity of the Proteasome, but Not Cysteine
Proteases, Is Responsible for Induction of Apoptosis in K562
Cells.
The tripeptidyl aldehydes that were originally developed as
inhibitors of the cysteine proteases (such as calpain I and cathepsin B) also inhibit the proteasomal activity (Rock et al., 1994). However,
LLnV and LLnL were 50- to 800-fold more potent than LLM in inhibition
of the purified 20S or 26S proteasome activity, whereas the three
aldehydes inhibit calpain and cathepsin with similar potencies (Rock et
al., 1994). To demonstrate that induction of apoptosis in K562 cells is
due to blockade of activity of the proteasome but not of the cysteine
proteases, the following experiments were performed. First, K562 cells
were treated for 18 h with a variety of trypeptidyl aldehydes at a
fixed concentration (50 µM), followed by measurement of
apoptosis-specific PARP cleavage. LLnV and LLnL induced an almost
complete cleavage of PARP, whereas LLL had a partial effect. In
contrast, LLM was completely inactive at inducing PARP cleavage (Fig.
3A, lanes 2-5). The rank of the apoptosis-inducing activity for these inhibitors in K562 cells, therefore, was LLnV = LLnL > LLL 
LLM. Treatment of K562
cells with the DNA-damage agent VP-16 failed to induce the process of PARP cleavage (Fig. 3A, lane 6), confirming that K562 cells are resistant to this drug (Bedi et al., 1994; Nishii et al., 1996).
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).
To provide direct evidence that induction of apoptosis in K562 cells is
due to inhibition of the proteasome but not cysteine proteases, we used
more specific inhibitors of the proteasome and cysteine proteases.
Lactacystin, a microbial metabolite, specifically inhibits the MB1
subunit of the proteasome (Fenteany et al., 1995) and is structurally
distinct from tripeptide aldehyde proteasome inhibitors. Furthermore,
lactacystin has no or little effect in the inhibition of the activity
of a purified calpain (Mellgren, 1997). When K562 cells were treated
with 10 µM lactacystin for 24 to 48 h, apoptosis occurred, as
evidenced by specific morphological changes in the nuclei (Fig.
4A versus the control
in Fig. 1A), an increase in the level of apoptosis-associated
sub-G1 cell population (indicated by Ap, Fig. 4B
versus the control in Fig. 1B), and the internucleosomal fragmentation
of DNA (Fig. 4C). A 24-h treatment also caused cleavage of PARP to the
p85 fragment (Fig. 4D, lane 3). By contrast, the specific cysteine
protease inhibitor E-64d (McGowan et al., 1989), when used at 50 µM
for up to 32 h, did not induce either apoptotic nuclear changes
(Fig. 5A) or PARP cleavage (Fig. 5B).
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Inhibition of the Proteasome Activity in K562 Cells Leads to a
Dramatic Decrease of Bcr-Abl Expression Before Induction of
Apoptosis.
Because the multidrug-resistance phenotype of K562
cells is conferred by the chimeric Bcr-Abl oncoprotein (McGahon et al., 1994; Rowley et al., 1996), we hypothesized that inactivation of
Bcr-Abl function should have occurred before apoptosis induction in
K562 cells after the treatment with proteasome inhibitors. To test this
hypothesis, we measured levels of Bcr-Abl protein in the experiments
described above (Figs. 1-5).
). Expression of this protein was nearly
completely lost after 7 h of LLnV treatment (Fig. 7B, lane 2 versus lane 1). More importantly, after 7 or 8 h of treatment, no
apoptotic changes such as PARP cleavage had been observed (see Fig. 1D,
lane 4). The level of Bcr-Abl protein expression remained low after
12 h of treatment, and further decreased after 24 h (Fig. 7A,
lanes 5 and 6 versus lane 7). Only after 24 h of treatment was the
level of Abl protein expression notably decreased (Fig. 7A, lane 6 versus lane 7). The process of PARP cleavage started at 12 h of
treatment and completed at 24 h of treatment (Fig. 1D, lanes
5-6). These results demonstrate that LLnV treatment of K562
cells leads to a significant reduction of Bcr-Abl protein expression
and subsequent induction of apoptosis. Neither reduction of Bcr-Abl
expression nor induction of apoptosis was seen in K562 cells treated
with DMSO (Figs. 1 and 7A), demonstrating that apoptosis-associated Bcr-Abl reduction is drug-specific.
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) is less potent than LLnV (IC50 = 21 nM;
Rock et al., 1994) in inhibiting the activity of a purified proteasome,
although lactacystin is more specific for the inhibition of the
proteasome activity. Their different potencies in inhibiting the
proteasome activity may be responsible for their different abilities in
reducing Bcr-Abl expression and killing K562 cells (Figs. 1, 3, 4, and
7). Regardless of that, our data strongly suggest that inhibition of
the proteasome activity in K562 cells first leads to reduction of
Bcr-Abl protein expression and, subsequently, induction of apoptosis.
To establish that the decreased Bcr-Abl expression leads to attenuation
of Bcr-Abl-mediated protein tyrosine phosphorylation, the lysates,
which were prepared from K562 cells treated with 50 µM LLnV for
7 h (identical with those in Fig. 7B), were also analyzed by
immunoblotting with an anti-phosphotyrosine antibody. As shown in Fig.
8, several heavily
tyrosine-phosphorylated proteins were observed in control K562 cell
lysates (lane 1), which is typical of Bcr-Abl-expressing cells (Okuda
et al., 1996). A band of ~210 kDa is Bcr-Abl itself (indicated in
lane 1) because Bcr-Abl undergoes autophosphorylation on tyrosine
residues (Lugo et al., 1990) and because this band was not detected in
an HL-60 cell lysate (data not shown). After 7 h of LLnV
treatment, the levels of tyrosine phosphorylation for nearly all of the
major phosphotyrosine-containing proteins, as well as Bcr-Abl itself,
were significantly reduced (Fig. 8, lane 2 versus lane 1). This
demonstrates that LLnV-mediated reduction of Bcr-Abl protein expression
in K562 cells leads to inactivation of Bcr-Abl tyrosine kinase
function, which is responsible for the decrease in the level of
Bcr-Abl-mediated protein tyrosine phosphorylation before the initiation
of apoptotic execution.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The chimeric oncoprotein Bcr-Abl is responsible for conferring
both transformation properties (Daley et al., 1990; Elefanty et al.,
1990; Skorski et al., 1996) and the multiple drug-resistance phenotype
(Bedi et al., 1994; Nishii et al., 1996) to human CML cells. In the
current study, we have shown that the death-promoting activity of
proteasome inhibitors is able to overcome the antiapoptotic function of
Bcr-Abl in K562 cells (Figs. 1, 3, and 4) and that proteasome
inhibitor-induced apoptosis in these cells is p53-independent and
requires activation of the caspases (Fig. 2). In addition, we have
found that before induction of apoptosis, proteasome inhibition decreases the levels and tyrosine kinase activity of the Bcr-Abl protein (Figs. 7 and 8), suggesting a novel mechanism for inactivating the Bcr-Abl function.
The following evidence supports the hypothesis that inhibition of the
proteasome, but not of the cysteine protease, activity is responsible
for induction of apoptosis in K562 cells. First, LLnV, previously shown
to be a potent proteasome inhibitor in vitro and in vivo (Rock et al.,
1994), effectively induces apoptosis in K562 cells (Figs. 1 and 3).
Other structurally similar peptide aldehydes (LLnL and LLL), which also
inhibit the proteasome in vitro and in vivo (Rock et al., 1994), also
induce apoptosis-associated PARP cleavage in K562 cells (Fig. 3A). In
contrast, LLM, an inhibitor of the cysteine proteases but only a weak
inhibitor of the proteasome (Rock et al., 1994), does not activate the
caspase cascade at even a very high concentration (Fig. 3C). The
relative potencies of LLnV to LLM for inducing apoptosis correspond to
the abilities of these two compounds to inhibit the activity of the
isolated proteasome but not calpain and cathepsin B (Rock et al.,
1994). Second, the microbial metabolite lactacystin, a specific
proteasome inhibitor which is structurally distinct from tripeptide
aldehydes (Fenteany et al., 1995), is also able to induce programmed
cell death in K562 cells (Fig. 4), whereas the specific cysteine
protease inhibitor E-64d (Mellgren, 1997) cannot induce apoptosis in
K562 cells even when used at a higher concentration for a longer period of time (Fig. 5). Third, LLnV induces a rapid accumulation of high
molecular weight polyubiquitinated proteins to maximum levels within
1 h of treatment (Fig. 6). These results, together with the fact
that proteasome inhibition induces apoptosis in other cell lines
(Imajoh-Ohmi et al., 1995; Shinohara et al., 1996; Drexler, 1997; Lopes
et al., 1997; An et al., 1998), strongly suggest that inhibition of the
proteasome activity in K562 cells leads to activation of the apoptotic
death program.
Because expression of Bcr-Abl oncoprotein in K562 cells confers their
drug-resistance phenotype (Bedi et al., 1994; Nishii et al., 1996; Fig.
3A, lane 6), we hypothesized that proteasome inhibition must have
caused inactivation of the Bcr-Abl oncoprotein before apoptosis
induction in K562 cells. The following evidence supports this
hypothesis. First, treatment of K562 cells with the tripeptidyl
proteasome inhibitor LLnV caused a significant reduction of the Bcr-Abl
protein expression between 4 and 8 h of treatment (Fig. 7A), which
was followed by induction of apoptosis between 12 and 24 h (Fig.
1D). Therefore, the level of the Bcr-Abl oncoprotein was decreased at
least 4 h before apoptosis was detected. The Bcr-Abl reduction can
be detected by specific antibodies to either c-Abl or Bcr protein (Fig.
7, A and B). Because no notable changes in the levels of c-Abl/p145 and
Bcr/p160 were detected at up to 8 h of treatment (Fig. 7, A and
B), the reduction of Bcr-Abl by proteasome inhibition seems selective.
Second, we have found that LLnL and LLL also caused a decrease in the
levels of Bcr-Abl protein (data not shown) and induction of apoptosis
(Fig. 3A). By contrast, LLM at up to 100 µM neither decreased the
level of Bcr-Abl expression (Fig. 7C) nor induced apoptosis (Fig. 3C). However, LLM at 300 µM was able to induce both events (Figs. 7C and
3C). Furthermore, the relative potencies of LLnV to LLM for reducing
the level of Bcr-Abl oncoprotein match exactly those of these two
compounds for inducing apoptosis (Figs. 7C and 3C), which also
correspond to their inhibitory activities toward the isolated
proteasome but not calpain and cathepsin B (Rock et al., 1994). Third,
treatment of K562 with the specific proteasome inhibitor lactacystin
reduced the level of Bcr-Abl protein at 8 h (Fig. 7D) and induced
apoptosis at 24 h (Fig. 4D). In contrast, the specific cysteine
protease inhibitor E-64 days did not induce either of these two events
even when used at a high concentration for a long period of time (Figs.
7E and 5). Fourth, the decreased level of Bcr-Abl oncoprotein is
directly associated with the decreased levels of tyrosine
phosphorylation for nearly all of the major phosphotyrosine-containing
proteins including Bcr-Abl itself (Figs. 7, 8), indicating inactivation
of Bcr-Abl tyrosine kinase activity. Fifth, LLnV induces a rapid
accumulation of high molecular weight polyubiquitinated proteins in
K562 cells to maximum levels within 1 h of treatment (Fig. 6),
which occurred before reduction of Bcr-Abl expression (between 4 and
8 h; Fig. 7A). Therefore, it appears that inhibition of the
proteasome first promotes the removal of the antiapoptotic activity of
Bcr-Abl and subsequently induces programmed cell death in K562 cells.
The following arguments suggest that inactivation of Bcr-Abl function
by proteasome inhibition is essential for induction of apoptosis in
K562 cells. First, overexpression of the Bcr-Abl oncoprotein inhibits
apoptosis induced by multiple stimuli (Bedi et al., 1994; Nishii et
al., 1996). Second, treatment of K562 cells with antisense
oligonucleotides targeting Bcr-Abl decreases Bcr-Abl protein expression
and sensitizes the cells to drug-induced programmed cell death (McGahon
et al., 1994; Rowley et al., 1996). Third, our results from
kinetics experiments (Figs. 1, 4, 7, and 8) demonstrate that the levels
and tyrosine kinase activity of Bcr-Abl are decreased before K562
apoptotic cell death induced by proteasome inhibitors.
It remains unclear how the decrease in the levels and tyrosine kinase
activity of Bcr-Abl by proteasome inhibition triggers K562 cell
apoptosis. We propose that reduction of the Bcr-Abl protein expression
and consequent inactivation of the Bcr-Abl function could be involved
in committing a K562 cell to undergoing apoptotic death. Because
specific targeting of Bcr-Abl protein is not sufficient for induction
of apoptosis (McGahon et al., 1994; Rowley et al., 1996), a proteasome
inhibitor, a single agent, therefore, should have dual functions:
inactivation of Bcr-Abl and induction of apoptosis. It is possible that
these two functions of the proteasome inhibitor work together to
trigger K562 cellular apoptosis. This hypothesis is supported by the
following arguments. It has been reported that Bcr-Abl-mediated
protection is regulated by Bcl-2 (Sanchez-Garcia and Grutz, 1995) or
Bcl-XL (Amarante-Mendes et al., 1998a), two
apoptosis inhibitory proteins, and can be overcome by overexpression of
Bax (Kobayashi et al., 1998), another Bcl-2 family protein and an
apoptosis inducer. Furthermore, the Bcl-2 family proteins are involved
in regulating the proapoptotic mitochondrial release of cytochrome
c into cytosol, which activates the caspase-3 pathway
involved in the execution of apoptosis (Green and Reed, 1998). Indeed,
overexpression of Bcr-Abl blocks release of cytochrome c
from mitochondria to the cytosol (Martins et al., 1997; Amarante-Mendes
et al., 1998b). We also found that when K562 cells were treated with a
proteasome inhibitor, cytochrome c was released into cytosol
immediately after the reduction of the Bcr-Abl protein expression (B. Li, K. Morrow, and Q.P.D., unpublished data). We are currently
investigating the detailed molecular mechanisms for the link between
reduction of Bcr-Abl protein expression and induction of apoptosis by
proteasome inhibition.
The molecular mechanisms responsible for the proteasome
inhibition-induced decrease in the levels of Bcr-Abl oncoprotein remain unknown. We have found that it cannot be inhibited by a variety of the
tested protease inhibitors (data not shown), which suggests that the
Bcr-Abl level reduction, if due to proteolytic degradation, must be
mediated by an unique protease. Alternatively, a different mechanism
may be responsible. One possibility is that inhibition of the
proteasome could lead to inhibition of Bcr-Abl transcription. This
remains to be determined by future studies. Regardless of that, the
results presented herein suggest that reduction of the Bcr-Abl
oncoprotein is a novel mechanism by which proteasome inhibitors overcome multidrug-resistance that Bcr-Abl confers and induce p53-independent apoptosis. Because loss of functional p53 protein may
play a role in the transition from the chronic phase of CML to the
blast crisis in the latter stages of the disease (Skorski et al.,
1996), proteasome inhibitors might have potential as chemotherapeutic agents for treating CML and related diseases.
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Footnotes |
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Accepted for publication December 8, 1998.
Received for publication August 5, 1998.
1 This work was supported in part by research funds from the Department of Pharmacology, University of Pittsburgh School of Medicine (to Q.P.D.), H. Lee Moffitt Cancer Center and Research Institute (to Q.P.D.), and the University of Pittsburgh Cancer Institute (to T.F.M.).
Send reprint requests to: Q. Ping Dou, Ph.D., Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12902 Magnolia Dr., Tampa, FL 33612-9497. E-mail: douqp{at}moffitt.usf.edu
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Abbreviations |
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PARP, poly(ADP-ribose) polymerase; CML, chronic myelogenous leukemia; LLnV, N-carbobenzoxy-L-leucyl-L-leucyl-norvalinal; LLnL, N-acetyl-L-leucyl-L-leucyl-norleucinal; LLL, N-carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; LLM, N-acetyl-L-leucyl-L-leucyl-L-methioninal; VP-16, etoposide; E-64d, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester; DMSO, dimethyl sulfoxide; DEVD-FMK, acetyl-DEVD-fluoromethyl ketone.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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-converting enzyme-like protease as candidate.
Cancer Res
56:
438-442-D-arabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: Association with enhanced fragmentation of mature DNA.
Cancer Res
52:
6270-6278
New concepts.
N Engl J Med
304:
1269-1274[Medline].This article has been cited by other articles:
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