Roles of NF-
B and 26 S Proteasome in Apoptotic Cell Death
Induced by Topoisomerase I and II Poisons in Human Nonsmall Cell Lung
Carcinoma*
Masahiro
Tabata,
Rika
Tabata,
Dale R.
Grabowski,
Ronald M.
Bukowski,
Mahrukh K.
Ganapathi, and
Ram
Ganapathi
From the Experimental Therapeutics Program, Taussig Cancer Center,
Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, October 27, 2000, and in revised form, November 28, 2000
 |
ABSTRACT |
Activation of signaling pathways after DNA damage
induced by topoisomerase (topo) poisons can lead to cell death by
apoptosis. Treatment of human nonsmall cell lung carcinoma (NSCLC-3 or
NSCLC-5) cells with the topo I poison SN-38 or the topo II poison
etoposide (VP-16) leads to activation of NF-
B before
induction of apoptosis. Inhibiting the degradation of I
B
by
pretreatment with the proteasome inhibitor MG-132 significantly
inhibited NF-
B activation and apoptosis but not DNA damage induced
by SN-38 or VP-16. Transfection of NSCLC-3 or NSCLC-5 cells with
dominant negative mutant I
B
(mI
B
) inhibited SN-38 or VP-16
induced transcription and DNA binding activity of NF-
B without
altering drug-induced apoptosis. Regulation of apoptosis by
mitochondrial release of cytochrome c and activation of
pro-caspase 9 followed by cleavage of poly(ADP-ribose) polymerase by
effector caspases 3 and 7 was similar in neo and mI
B
cells
treated with SN-38 or VP-16. In contrast to pretreatment with MG-132,
exposure to MG-132 after SN-38 or VP-16 treatment of neo or mI
B
cells decreased cell cycle arrest in the S/G2 + M fraction
and enhanced apoptosis compared with drug alone. In summary, apoptosis
induced by topoisomerase poisons in NSCLC cells is not mediated by
NF-
B but can be manipulated by proteasome inhibitors.
 |
INTRODUCTION |
Human nonsmall cell lung carcinoma
(NSCLC)1 is clinically
responsive to chemotherapy with topoisomerase (topo) poisons (1). Etoposide (VP-16), which poisons the nuclear enzyme topoisomerase II
(topo II), is currently used in a number of therapeutic protocols. More
recently, the topoisomerase I (topo I) poison, e.g.
irinotecan, has also shown activity in the clinical management of NSCLC
(1). The chemotherapeutic efficacy of topoisomerase I and II poisons is
presumed to be due to stabilization of a topoisomerase-DNA-cleavable complex leading to protein-linked DNA breaks and cell death by apoptosis (2-4). Tumor cell resistance to apoptosis induced by topoisomerase poisons has primarily focused on the membrane efflux pumps affecting cellular drug pharmacokinetics. A major impact of these
studies has been the demonstration that reduced cellular drug levels
due to overexpression of P-glycoprotein encoded by mdr1 gene leads to reduced DNA damage, apoptosis, and
cell death. (5). Both experimental and clinical studies suggest that
overexpression of P-glycoprotein is frequently associated with
resistance to topoisomerase II poisons and, occasionally, with
resistance to topoisomerase I poisons (5). However, unlike the vinca
alkaloids, the magnitude of alterations in cellular drug levels
per se mediated by P-glycoprotein does not correlate with
DNA damage or apoptosis in topoisomerase II poison-treated cells (6).
Alternatively, reduced formation of drug-stabilized
topoisomerase-DNA-cleavable complex can be due to decreased
topoisomerase I/II protein levels or mutations in the enzyme, which can
directly impact on sensitivity to drug-induced apoptosis. (2-4).
Although a cause and effect relationship of DNA damage leading to
apoptosis appears relatively straightforward, determining signaling
events and linking them to DNA damage and apoptosis could potentially
lead to understanding the mechanistic basis governing topoisomerase
poison-induced apoptotic cell death.
NF-
B is an inducible transcription factor involved in the regulation
of genes during inflammatory, acute phase, and immune responses (7, 8).
The inappropriate regulation of NF-
B has been implicated in a
variety of diseases including cancers (9). Specifically, the activation
of NF-
B by tumor necrosis factor and the subsequent induction of
apoptosis would suggest that these events are linked. However, it has
been recently suggested that the activation of NF-
B can indeed be
anti-apoptotic in response to tumor necrosis factor or topoisomerase
poisons (10, 11). In addition to tumor necrosis factor, topoisomerase
poisons also induce activation of NF-
B (11). Since apoptotic cell
death is frequently observed in topoisomerase I/II poison-treated cells (12), establishing a functional link between NF-
B and drug-induced apoptosis has been pursued (11, 13, 14).
In the present study, using pharmacological inhibitors of proteasome
function and the molecular strategy of transfecting a dominant negative
I
B
to manipulate the activation of NF-
B in topoisomerase I and
II poison-treated human NSCLC cells, we determined the signaling
pathways contributing to apoptotic cell death. Our data in two
independent model systems of human NSCLC suggest that inhibiting the
activation of NF-
B by the proteasome inhibitor or transfection of a
dominant negative I
B
result in markedly different responses to
apoptosis induced by topoisomerase I and II poisons. Although the
dominant negative I
B
and proteasome inhibitor MG-132 do not
affect DNA damage induced by topoisomerase I and II poisons, pre- or
post-treatment with proteasome inhibitor MG-132 inhibits or enhances
apoptosis respectively. In addition, after DNA damage induced by
topoisomerase I or II poisons, neither activation of transcription or
the DNA binding activity of NF-
B affects drug-induced apoptosis,
based on the regulation of mitochondrial release of cytochrome
c and activation of caspase 9 followed by cleavage of
poly(ADP-ribose) polymerase by effector caspases 3 and 7.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The topo I poison SN-38 (active metabolite of
irinotecan) and the topo II poison VP-16 were obtained from Pharmacia & Upjohn and Sigma, respectively. Stock solutions of these drugs were
prepared in dimethyl sulfoxide (Sigma) and stored frozen at
20 °C.
The dominant negative I
B (S32A/S36A) cDNA cloned into pUSEamp(+) expression vector and the empty control pUSEamp(+) expression vector
were obtained from Upstate Biotechnology Inc., Lake Placid, NY. The
PathDetect cis-reporting system pNF-
B-Luc reporter plasmid and
pFC-MEKK positive control plasmid were obtained from Stratagene, La
Jolla, CA. Antibodies to caspase 8 were purchased from Santa Cruz
Biotechnology Inc., Santa Cruz, CA; antibodies to caspase 9 and
cytochrome c were obtained from BD Pharmingen, San Diego, CA; antibodies to I
B
were obtained from Upstate Biotechnology; antibodies to PARP were purchased from Enzyme Systems Inc., Livermore, CA. The fluorogenic substrate leucine-glutamic acid-histidine-aspartic acid (LEHD) coupled to 7-amino-4-trifluoromethylcoumarin (AFC) (LEHD-AFC) for determining caspase 9 activity was obtained from BioVision Inc. Palo Alto, CA. Cell culture medium and fetal bovine serum were obtained from BioWhittaker, Inc., Gaithersburg, MD. All
other chemicals of analytical grade were obtained from commercial sources.
Cell Lines and Transfection--
The human nonsmall cell lung
carcinoma model systems NSCLC-3 and NSCLC-5 were established in culture
in the laboratory using specimens obtained from patients during
surgical resection of the tumor (15). The parental wild-type NSCLC-3/wt
and NSCLC-5/wt cells were cultured in RPMI 1640 supplemented with 10%
fetal bovine serum and 2 mM L-glutamine and
maintained at 37 °C in a humidified 5% CO2 plus 95%
air atmosphere. Doubling time in vitro for the NSCLC-3/wt
and NSCLC-5/wt cells was ~35 h.
Transfection of parental wild-type NSCLC-3 or NSCLC-5 cells with a
dominant negative I
B
(S32A/S36A) in pUSEamp(+) expression vector
under control of the cytomegalovirus promoter or the empty pUSEamp(+)
expression vector was carried out using 4 µg of DNA/2 × 106 cells/1.2 ml containing 6 µl of DMRIE-C (Life
Technologies, Inc.). Stable transfectants were selected by culturing in
1 mg/ml G418.
Measurement of Apoptosis, Drug-stabilized DNA-Topo-cleavable
Complex Formation, and Drug Cytotoxicity--
The cells in all
experiments were either pre- or post-treated for 30 min with the
proteasome inhibitor MG-132 (20 µM). Treatment with the
desired concentration of SN-38 or VP-16 for 60 min either followed or
preceded treatment with the proteasome inhibitor MG-132. After the
required drug treatment, control and treated cells were washed in
drug-free medium and re-incubated in drug-free medium to determine
(a) target protein levels and/or their activity and (b) potential signaling events of apoptosis.
DNA-topoisomerase-cleavable complex formation induced by SN-38 or VP-16
was determined by a modification of the SDS-KCl method (16) using the
NSCLC-3 or NSCLC-5 cells labeled overnight with
[14C]thymidine. Apoptosis in drug-treated cells was
determined using the technique of Muscarella et al. (17).
Briefly, 2 × 105 control or treated cells were
re-suspended in 100 µl of staining solution (70 µg/ml Hoechst 33342 and 100 µg/ml propidium iodide in phosphate-buffered saline) and
incubated at 37 °C for 15 min. The stained cells were viewed in a
fluorescence microscope with the appropriate filters so as to visualize
simultaneously the blue fluorescence from Hoechst 33342 and the red
fluorescence from propidium iodide. Normal viable cells fluoresce blue
within the nucleus, and the apoptotic cells show condensation of
chromatin and formation of small masses of varying sizes. Necrotic
cells stain pink, but these cells are swollen, and the chromatin is not
condensed and fragmented as in apoptotic cells. Flow cytometry for cell
cycle traverse perturbations was carried out after staining with
propidium iodide as described earlier (18). Cytotoxicity induced by
SN-38 or VP-16 was determined by a soft agar colony-forming assay.
Cells were treated with a range of drug concentrations for 60 min at
37 °C in a humidified 5% CO2 plus 95% air atmosphere. After treatment, cells were washed and 3 × 104 cells
plated in triplicate in 35 × 10-mm Petri dishes using RPMI 1640 supplemented with 2 mM L-glutamine and 20%
fetal bovine serum. Colonies were counted after incubation of the Petri
dishes for 10-12 days in a humidified 5% CO2 plus 95%
air atmosphere.
Electromobility Shift Assays--
Nuclear extracts from control
and treated cells were prepared as described by Dignam et
al. (19). Electromobility shift assays (EMSA) were carried out
using nuclear extracts containing equivalent amounts of protein (10 µg) that were incubated with 32P-labeled oligonucleotide
containing the consensus sequence 5'-GGGACTTTCC-3', corresponding to
the
-light chain enhancer motif (20). Assays for supershift were
carried out by incubation of nuclear extracts with antibodies to the
p65 subunit and the labeled oligonucleotide before analysis by EMSA.
Cells transfected with PathDetect pNF-
B-Luc plasmid cis-reporting
system were used to test the effect of drug treatment on
transcriptional activation of NF-
B. Transfection with the pFC-MEKK
plasmid was used as the positive control.
Cell Lysis and Western Blotting--
Cell lysates prepared in 50 mM Tris, pH 7.4, 1% Nonidet P-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitors (1 µg/ml each aprotinin, leupeptin, pepstatin), and phosphatase
inhibitors (1 mM Na2VO4, 1 mM NaF) were used for detection of I
B
protein levels
in Western blots. Cell lysates (50 µg protein) were resolved by 10%
SDS-polyacrylamide gel electrophoresis, electroblotted onto
nitrocellulose (0.45 µm), and blocked by incubation in 3% nonfat dry
milk in phosphate-buffered saline for 3 h at room temperature. The
membranes were probed with antibody to I
B
(1 µg/ml) overnight
at 4 °C followed by incubation with secondary antibody for 1 h
at room temperature for signal detection by chemiluminescence.
Cytosolic extracts were prepared in extraction buffer (21) containing
220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, and protease inhibitors to determine
cytochrome c protein levels. Control or treated cells were
incubated on ice in extraction buffer for 30 min followed by disruption
with a "B" pestle in a glass Dounce homogenizer. After
centrifugation of the cell homogenates at 14,000 × g,
the supernatant containing 50 µg of cytosolic protein was resolved on
a 15% SDS-polyacrylamide gel. After electrophoresis, the gels were
electroblotted onto polyvinylidene difluoride membrane (0.2 µm) and
blocked by incubation in 3% bovine serum albumin, 3% nonfat milk,
0.1% Tween 20 in phosphate-buffered saline for 3 h at room
temperature. The polyvinylidene difluoride membrane was probed by
incubating with antibodies to cytochrome c (dilution 1: 500)
overnight at 4 °C followed by horseradish-coupled secondary antibodies for 1 h at room temperature for signal detection by chemiluminescence. Lysates prepared from aliquots of control and treated cells were tested for caspase 9 activity using the fluorogenic substrate LEHD-AFC.
Cell lysates prepared in 62.5 mM Tris, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.003% bromphenol blue,
5%
-mercaptoethanol were used for detection of caspase 9 and PARP
cleavage. The lysate samples from control and treated cells were
resolved on 10% SDS-polyacrylamide gel, electroblotted onto
nitrocellulose, blocked in 5% nonfat dry powdered milk in
phosphate-buffered saline, and probed with antibody to caspase 9 or
antibody C2.10 for PARP followed by horseradish-coupled secondary
antibody for detection by chemiluminescence. The antibody to caspase 9 detects both pro- and cleaved active forms of caspase 9, and C2.10
antibody detects the 116-kDa monomeric PARP and cleaved 89-kDa
PARP.
 |
RESULTS |
Differential Effect on DNA Binding Activity of NF-
B in Cells
Treated with SN-38, VP-16, Cis-platinum, or Paclitaxel--
Results in
Fig. 1A demonstrate that after
treatment of NSCLC-3/wt cells with the topo I poison SN-38 or the topo
II poison VP-16, there is a significant increase in the DNA binding
activity of NF-
B compared with the untreated control. The activation
of NF-
B by SN-38 and VP-16 is dose-dependent and
specific for topoisomerase poisons, since treatment with the
microtubule poison paclitaxel (PCT) or the DNA-damaging
agent cis-platinum (CDDP) produced no measurable increase in
the DNA binding activity of NF-
B. In the next series of experiments
we determined the time course governing the enhanced DNA binding of
NF-
B in topo poison-treated cells, and the results in Fig.
1B demonstrate that maximal enhancement of DNA binding
activity of NF-
B in topo poison-treated cells occurs at 2-3 h. The
results in Fig. 1C indicate that with the various drugs at
concentrations tested for activation of NF-
B there is significant
apoptosis in the NSCLC-3/wt cells by 24-48 h, and the topo poisons
SN-38 and VP-16 induce apoptosis much more rapidly than cis-platinum or
paclitaxel. As shown in Fig. 2, treatment
of NSCLC-5/wt cells with SN-38 or VP-16 but not cis-platinum or
paclitaxel significantly enhanced the DNA binding activity of NF-
B.
The data in Figs. 1 and 2 suggest that in NSCLC-3/wt or NSCLC-5/wt
cells treated with a topo I or topo II poison, activation of NF-
B
precedes the subsequent induction of apoptosis.

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 1.
A, DNA binding activity of
NF- B determined by EMSA in NSCLC-3/wt cells treated with etoposide
(VP-16), active metabolite of irinotecan (SN-38),
paclitaxel (PCT), or cis-platinum (CDDP) for
1 h. B, time course of DNA binding activity of NF- B
detected in NSCLC-3/wt cells. Cells were treated for 1 h with 100 µM VP-16, washed, re-incubated in drug-free medium and
nuclear extracts from aliquots of control (C), and treated cells
retrieved at 0-4 h were analyzed for NF- B DNA binding activity by
EMSA. C, apoptosis induced by VP-16, SN-38, paclitaxel, or
cis-platinum (CDDP) in NSCLC-3/wt cells treated for 1 h. After treatment, cells were re-incubated in drug-free medium, and
apoptosis was analyzed by fluorescent microscopy at 4, 24, and 48 h.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
DNA binding activity of
NF- B determined by EMSA in NSCLC-5/wt cells
treated with etoposide (VP-16), active metabolite of
irinotecan (SN-38), paclitaxel (PCT),
or cis-platinum (CDDP) for 1 h.
|
|
The Proteasome Inhibitor MG-132 Inhibits Topo Poison-induced
NF-
B DNA Binding Activity and Apoptosis--
It is well recognized
that degradation of phosphorylated I
B by the 26 S proteasome
regulates the DNA binding of NF-
B subunits (22). Thus, using MG-132
as a pharmacological inhibitor of proteasome activity, we determined
the effect of pretreatment for 30 min of NSCLC-3/wt cells with MG-132
on the DNA binding activity of NF-
B in cells treated with SN-38 or
VP-16. The results in Fig. 3A
indicate that pretreatment with 20 µM MG-132 for 30 min
significantly inhibits the DNA binding activity of NF-
B in SN-38- or
VP-16-treated cells. The attenuated activation of NF-
B in cells
pretreated with MG-132 is consistent with the data in Fig.
3B, demonstrating diminished degradation of I
B
protein. In subsequent experiments, we analyzed drug-induced apoptosis
in an attempt to correlate the apoptotic response with the DNA binding
activity of NF-
B in cells pretreated with MG-132. The results in
Table I indicate that pretreatment with
MG-132, which resulted in the inhibition of NF-
B DNA binding
activity, also inhibited SN-38- or VP-16-induced apoptosis.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
A, effect of pretreatment for 30 min
with 20 µM MG-132 followed by 100 µM VP-16
or 0.1 µM SN-38 for 60 min on DNA binding activity of
NF- B detected by EMSA in nuclear extracts from NSCLC-3/wt cells.
B, effect of pretreatment for 30 min with 20 µM MG-132 followed by 100 µM VP-16 or 0.1 µM SN-38 for 60 min on degradation of I B protein in
NSCLC-3/wt cells.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Effect of pre-treatment with 20 µM MG-132 for 30 min on
apoptosis induced in NSCLC-3/wt cells treated for 60 min with 100 µM VP-16 or 0.1 µM SN-38
|
|
Differential Activation of NF-
B and Degradation of I
B
in
Topo Poison-treated neo- or mI
B-transfected Cells--
The data
with MG-132 pretreatment (Fig. 3) suggest that in NSCLC-3/wt cells
treated with SN-38 or VP16 reduced activation of NF-
B, and apoptosis
is correlated. To establish a functional link between NF-
B
activation- and apoptotic-signaling pathways in SN-38- or VP-16-treated
cells, experiments were carried out in NSCLC-3/wt or NSCLC-5/wt cells
stably transfected with vector control (NSCLC-3/neo, NSCLC-5/neo) or
mutant I
B
(NSCLC-3/mI
B
or NSCLC-5/mI
B
). The results
in Fig. 4A demonstrate that
after treatment with SN-38 or VP-16, the significant increase in the DNA binding activity of NF-
B observed in the parental or vector control (neo) cells is absent in cells transfected with dominant negative mutant I
B
(mI
B
). Consistent with this differential response to the DNA binding activity of NF-
B in neo
versus mI
B
cells treated with SN-38 or VP-16, the
transcriptional activation of NF-
B is also inhibited in the
mI
B
but not in neo cells treated with VP-16 (Fig. 4B).
Treatment with VP-16 results in the rapid degradation of I
B
protein in the neo but not mI
B
cells (Fig. 4C).
Similar to the data obtained in NSCLC-3 cells, a differential response
to SN-38- or VP-16-stimulated DNA binding activity of NF-
B,
transcriptional activity of NF-
B, and degradation of I
B
protein is also observed between the neo- and mI
B-transfected NSCLC-5 cells (Fig. 5,
A-C).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
A, effect of treatment with VP-16 or
SN-38 for 60 min on DNA binding activity of NF- B detected by EMSA in
nuclear extracts from neo (NSCLC-3/neo)- and mI B -transfected
NSCLC-3/wt cells. Lanes 1, 4, 7,
10, 12, and 14, control; lanes
2, 5, and 8, 40 µM VP-16;
lanes 3, 6, and 9, 100 µM VP-16; lanes 11, 13, and
15, 0.1 µM SN-38. The NF- B signal and the
nonspecific (N.S.) binding are identified by
arrows. B, transcriptional activation of NF- B
in NSCLC-3/neo and NSCLC-3/mI B cells transfected with the
pNF- B luciferase plasmid cis-reporting system and treated with VP-16
for 60 min. C, degradation of I B protein in
NSCLC-3/neo and NSCLC-3/mI B cells treated with VP-16 for 60 min.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
A, effect of treatment with VP-16 (100 µM) or SN-38 (0.1 µM) for 60 min on DNA
binding activity of NF- B detected by EMSA in nuclear extracts from
neo (NSCLC-5/neo)- and mI B (NSCLC-5/mI B )-transfected
NSCLC-5 cells. B, transcriptional activation of NF- B in
NSCLC-5/neo and NSCLC-5/mI B cells transfected with pNF- B
luciferase plasmid cis-reporting system and treated with VP-16 for 60 min. C, degradation of I B protein in NSCLC-5/neo and
NSCLC-5/mI B treated with VP-16 for 60 min.
|
|
Signaling Events and Apoptosis Induced by SN-38 or VP-16 Are
Similar in neo- or mI
B-transfected Cells--
Since drug-induced
NF-
B activation in the neo- and mI
B
-transfected cells was
different, the temporal regulation of events that lead to the induction
of apoptosis in NSCLC-3/neo or NSCLC-3/mI
B
and NSCLC-5/neo or
NSCLC-5/mI
B
cells treated with SN-38 or VP-16 was determined.
Preliminary studies revealed that SN-38- or VP-16-induced apoptosis in
the neo or mI
B
transfectants was not mediated by Fas or FasL, and
the parental and transfected cells were caspase 8-deficient (data not shown).
Immunoblotting results in Fig.
6A indicate that after
treatment with either SN-38 or VP-16, a detectable increase in
cytosolic cytochrome c was apparent at 4 h. This was
followed by conversion of caspase 9 from the pro- to the active form at
6 h, with maximal levels being detectable at 24 h (Fig.
6A). Consistent with the immunoblot results on activation of
caspase 9, experiments on caspase 9 activity using the specific
substrate LEHD-AFC also revealed detectable activity at 6 h (Fig.
6A). Cleavage of PARP by the effector caspases 3 and 7 after
activation of caspase 9 was detected as early as 6 h, and maximal
levels of cleaved PARP product occurred at 24 h (Fig.
6A). The morphological determination of apoptosis by
fluorescence microscopy indicated that drug-induced apoptosis was
dose-dependent and similar between the neo- and mI
B
-transfected NSCLC-3 (Fig. 6B). Confirming the data
on topo poison-induced apoptosis (Fig. 6B), the results in
Table II indicated that the clonogenic
cell survival of VP-16- or SN-38-treated NSCLC-3/neo and
NSCLC-3/mI
B
is similar in soft agar colony-forming assay. The
data in Fig. 7 show results on
mitochondrial release of cytochrome c and activation of
caspases, which lead to SN-38- or VP-16-induced apoptosis in
NSCLC-5/neo and NSCLC-5/mI
B
cells. Overall, after treatment with
VP-16 or SN-38, results on mitochondrial release of cytochrome
c, the conversion of unprocessed pro-caspase 9 (46-48 kDa)
to active caspase 9 (35 kDa, 37 kDa), caspase 9 activity, and the
cleavage of PARP by effector caspases 3 and 7 suggest no apparent
differences in the temporal regulation of apoptotic pathways between
the neo- and mI
B
-transfected NSCLC-3 or NSCLC-5 cells.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 6.
A, effect of treatment with VP-16 or
SN-38 for 60 min on levels of cytochrome c, caspase 9 levels/activity, and cleavage of PARP in NSCLC-3/neo and NSCLC-3/mI B
cells. B, dose-dependent effects of VP-16 and
SN-38 on induction of apoptosis at 4 and 24 h in NSCLC-3/neo and
NSCLC-3/mI B cells.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of treatment with VP-16 or SN-38 for
60 min on levels of cytochrome c, caspase 9 levels/activity, and cleavage of PARP in NSCLC-5/neo and
NSCLC-5/mI B
cells. Apoptosis at 48 h in NSCLC-5/neo or
NSCLC-5/mI B treated with 100 µM VP-16 and 0.1 µM SN-38 was ~50 and 27%, respectively.
|
|
Post-treatment with the Proteasome Inhibitor MG-132 Potentiates
Topo Poison-induced Apoptosis and Is Independent of NF-
B
Activity--
Earlier experiments (Fig. 3) on pretreatment with the 26 S proteasome inhibitor MG-132 indicated that the DNA binding activity of NF-
B, degradation of I
B
, and apoptosis induced by SN-38 or
VP-16 were inhibited. However, comparative studies with the neo and
mI
B transfectants of NSCLC-3 (Fig. 6) or NSCLC-5 (Fig. 7) cells
demonstrated that SN-38- or VP-16-induced activation of NF-
B may not
be required for apoptosis. To further evaluate the mechanisms
contributing to these discrepant results, we determined the effect of
pre- or post-treatment with MG-132 on SN-38- and VP-16-induced cell
cycle traverse perturbations and apoptosis in NSCLC-3/wt or the neo and
mI
B transfectants of NSCLC-3 or NSCLC-5 cells. Results in Fig.
8A demonstrate that in SN-38-
or VP-16-treated NSCLC-3 cells (a) pretreatment with MG132
significantly inhibits apoptosis, and (b) post-treatment for
30 min with MG-132 results in a significant (>3-fold) increase in
apoptosis. As shown in Fig. 8B, an analysis of cell cycle
traverse perturbations demonstrated that treatment with VP-16 alone or
the pretreatment with MG-132 followed by VP-16 resulted in the
accumulation of cells in the S + G2/M boundary at 24 h
and a measurable increase in the apoptotic (sub-G1) cell
population. In contrast, post-treatment for 30 min with MG-132 after
VP-16 exposure resulted in no remarkable accumulation of cells at the
S-G2 + M boundary but produced a marked increase in the
apoptotic (sub-G1) population, suggesting either apoptosis in this phase or transit through cycle and mitotic catastrophe. The
differential effect of pre- or post-treatment with MG-132 on SN-38- or
VP-16-induced apoptosis is not unique to NSCLC-3/wt cells or dependent
on NF-
B activation, since post-treatment with MG-132 also enhanced
apoptosis in the neo- or mI
B-transfected NSCLC-3 or NSCLC-5 cells
(Fig. 9 and Fig.
10).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 8.
A, effect of pre- or post-treatment for
30 min with 20 µM MG-132 on VP-16- or SN-38-induced
apoptosis at 24 h in NSCLC-3/wt cells. Apoptosis in NSCLC-3/wt
cells treated with VP-16 followed by MG-132 was significantly higher
p < 0.007 than treatment with VP-16 alone; apoptosis
in NSCLC-3/wt cells treated with MG-132 followed by SN-38 was
significantly lower p < 0.03 than treatment with SN-38
alone; apoptosis in NSCLC-3/wt cells treated with SN-38 followed by
MG-132 was significantly higher p < 0.03 than
treatment with SN-38 alone. B, effect of pre- or
post-treatment with MG-132 on VP-16-induced cell cycle traverse
perturbations and apoptosis (% cells in sub G1 fraction)
in NSCLC-3/wt cells.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
A, effect of pre- or post-treatment for
30 min with 20 µM MG-132 on VP-16 (100 µM)
induced apoptosis at 4 and 24 h in NSCLC-3/neo and
NSCLC-3/mI B cells. Apoptosis at 4 and 24 h in NSCLC-3/neo or
NSCLC-3/mI B cells treated with MG-132 followed by VP-16 was
significantly lower (p < 0.03) than treatment with
VP-16 alone; apoptosis at 4 h in NSCLC-3/neo or NSCLC-3/mI B
cells treated with VP-16 followed by MG-132 was significantly lower
(p < 0.01) than treatment with VP-16 alone; apoptosis
at 24 h in NSCLC-3/neo or NSCLC-3/mI B cells treated with
VP-16 followed by MG-132 was significantly higher (p < 0.003) than treatment with VP-16 alone. B, effect of pre- or
post-treatment for 30 min with 20 µM MG-132 on SN-38 (0.1 µM) induced apoptosis at 4 and 24 h in NSCLC-3/neo
and NSCLC-3/mI B cells. Apoptosis at 4 and 24 h in
NSCLC-3/neo or NSCLC-3/mI B cells treated with MG-132 followed by
SN-38 was significantly lower (p < 0.04) than
treatment with SN-38 alone; apoptosis at 4 h in NSCLC-3/neo or
NSCLC-3/mI B cells treated with SN-38 followed by MG-132 was
significantly lower (p < 0.01) than treatment with
SN-38 alone; apoptosis at 24 h in NSCLC-3/neo or NSCLC-3/mI B
cells treated with SN-38 followed by MG-132 was significantly higher
(p < 0.003) than treatment with SN-38 alone.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 10.
A, effect of pre- or post-treatment
with 20 µM MG-132 on VP-16 (100 µM) induced
apoptosis at 48 h in NSCLC-5/neo and NSCLC-5/mI B cells.
Apoptosis in NSCLC-5/neo or NSCLC-5/mI B cells treated with MG-132
followed by VP-16 was significantly lower (p < 0.008)
than treatment with VP-16 alone; apoptosis in NSCLC-5/neo or
NSCLC-5/mI B cells treated with VP-16 followed by MG-132 was
significantly higher (p < 0.03) than treatment with
VP-16 alone. B, effect of pre- or post-treatment with MG-132
on SN-38 (0.1 µM) induced apoptosis at 48 h in
NSCLC-5/neo and NSCLC-5/mI B cells. Apoptosis in NSCLC-5/neo or
NSCLC-5/mI B cells treated with MG-132 followed by SN-38 was
significantly lower (p < 0.001) than treatment with
SN-38 alone; apoptosis in NSCLC-5/neo or NSCLC-5/mI B cells
treated with SN-38 followed by MG-132 was significantly higher
(p < 0.002) than treatment with SN-38 alone.
|
|
 |
DISCUSSION |
Drugs that poison the enzymes topo I or topo II stabilize
topo-DNA-cleavable complex formation, which leads to protein-linked DNA
strand breaks and cell death (2-4). Although it is generally accepted
that topoisomerase I or topoisomerase II poisons produce DNA damage and
induce cell death by apoptosis, it remains to be addressed whether the
signaling pathways that regulate the initiation of apoptosis induced by
topo poisons are dependent on the DNA damage. Since it is well
established that the activation of NF-
B is an inducible stress
response and topo poisons are effective in stimulating this pathway
(20), we examined the functional role for NF-
B activation in
apoptosis induced by SN-38 or VP-16. Our results indicate that NF-
B
(based on an increase in DNA binding activity) is indeed activated
after treatment with the topo I poison SN-38 or the topo II poison
VP-16 but not with cis-platinum or taxol, although all of these agents
are potent inducers of apoptosis. The anti-apoptotic role of NF-
B
has been suggested as a mechanism of resistance to chemotherapy, since
attenuation of NF-
B activity in topoisomerase poison-treated cells
leads to stimulation of an apoptotic response (11). The present data demonstrating reduced apoptosis after inhibition of NF-
B activity by
pretreatment with the proteasome inhibitor MG-132 suggests that
activation of NF-
B mediates apoptosis induced by SN-38 or VP-16.
However, these results contradict data obtained with the neo- and
mI
B
-transfected NSCLC-3 or NSCLC-5 cells, wherein the differential activation of NF-
B did not alter apoptosis or
clonogenic cell survival (NSCLC-3/neo and NSCLC-3/mI
B
) in a soft
agar colony assay after treatment with SN-38 or VP-16.
A proposed sequence of events regulating chemically induced apoptosis
involves release of cytochrome c followed by activation of
initiator caspases 8 and/or 9 and the effector caspases 3 and 7 (21).
In the present study we were able to detect changes in the protein
levels and activity of initiators and effectors of apoptosis in a
temporal manner after treatment of NSCLC-3 or NSCLC-5 cells with SN-38
and VP-16. Measurable increases in the mitochondrial release of
cytochrome c, an initiator of chemically induced apoptosis,
were observed 2-4 h after treatment with SN-38 and VP-16, and maximal
levels were apparent between 24 and 48 h after drug treatment. In
these caspase 8-deficient NSCLC-3 and NSCLC-5 cells, the mitochondrial
release of cytochrome c was followed by the detection of
unprocessed inactive pro-form and the active proteolysed forms of
caspase 9 (in Western blots) as well as changes in caspase 9 activity.
The subsequent cleavage of PARP by effector caspases 3 and 7 was
maximal at 24-48 h. Signaling pathways regulating SN-38- or
VP-16-induced apoptosis in neo and mI
B transfectants of NSCLC-3 and
NSCLC-5 cell were also similar, since no differences in the levels or
activity of proteins initiating or effecting apoptosis were apparent on
a temporal basis. Thus, these results suggest that in human nonsmall
cell lung carcinoma cells, NF-
B may not be functionally involved in
affecting the initiation or execution of topoisomerase I/II
poison-induced apoptosis.
The differential apoptotic response with the proteasome inhibitor
MG-132, whether the treatment precedes or follows exposure to SN-38 or
VP-16, is unique and indeed suggests that the downstream apoptotic
response can be manipulated without any apparent change in the
magnitude of the DNA damage induced by topoisomerase poisons. The
apoptotic response, which is decreased with pretreatment and increased
by post-treatment with MG-132 is not due to altered degradation of
either topo I (23) or topo II in the SN-38- and VP-16-treated cells,
respectively (data not presented). The data on reduction in apoptosis
with MG-132 pretreatment suggest that either a delay in mitochondrial
release of cytochrome c or the activation of the
pro-caspases may be involved. However, the increased apoptosis at
24 h with MG-132 post-treatment suggests that an alternate
mechanism based on cell cycle traverse perturbations may exist. Data on
cell cycle traverse in VP-16-treated cells indicate that pretreatment
with MG-132 does not affect cell cycle arrest in the S + G2/M fraction. However, post-treatment with the proteasome
inhibitor, which does not result in sustained arrest in the S + G2/M fraction, leads to enhanced apoptosis, possibly due to
continued cell cycle transit. Although a precise mechanism for this
response is not readily apparent, the data strongly support the
possibility that interference with proteasome function after DNA damage
induced by topo poisons can affect cell cycle arrest in the late S + G2/M fraction. In both pre- or post-treatment with MG-132,
activation of NF-
B induced by SN-38 or VP-16 is inhibited. However,
the role of NF-
B mediating this enhanced apoptotic response is
countered by data demonstrating that post-treatment with the proteasome
inhibitor (which inhibits NF-
B activity in neo cells) results in
enhancement of SN-38- or VP-16-induced apoptosis in the neo- or
mI
B
-transfected NSCLC-3 or NSCLC-5 cells.
In summary, the present results demonstrate that in human NSCLC cells
treated with a topo I poison, e.g. SN-38 or a topo II poison, e.g. VP-16, the apoptosis downstream of drug-induced
DNA damage is initiated by the mitochondrial release of cytochrome c, followed by the processing of caspase 9, and the
subsequent cleavage of PARP by the effector caspases 3 and 7. These
apoptotic pathways are not regulated by activation of NF-
B induced
by topoisomerase poisons. In contrast, although inhibitors of the 26 S
proteasome do not affect topoisomerase poison-induced DNA damage, the
use of a proteasome inhibitor after treatment with SN-38 or VP-16 remarkably affects the course of drug-induced cell cycle traverse perturbations and significantly enhances the apoptotic response independent of NF-
B activation. Future studies on the role of proteasome function in cellular response to chemically induced DNA
damage could provide additional information on the signaling pathways
of apoptosis that may be useful in improving the therapeutic benefit of
topoisomerase poisons in cancer chemotherapy.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Dr. Ian Hickson and
Dr. Chris Norbury, Institute of Molecular Medicine, Imperial
Cancer Research Fund, University of Oxford, UK for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by United States Public Health
Service Grants RO1 CA35531 and RO1 CA74939 and an educational/research grant from Rodney A. Beason, Medical Sciences Liaison Oncology, Medical
Operations and Scientific Affairs, Pharmacia & Upjohn.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Taussig Cancer Center,
R40, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH
44195. Tel.: 216-444-2085; Fax: 216-444-7115; E-mail: ganapar@ cc.ccf.org.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M009831200
 |
ABBREVIATIONS |
The abbreviations used are:
NSCLC, nonsmall cell
lung carcinoma;
topo, topoisomerase;
mI
B
, mutant I
B
;
PARP, poly(ADP-ribose) polymerase;
LEHD-AFC, L-leucine-glutamic
acid-histidine-aspartic acid coupled to
7-amino-4-trifluoromethylcoumarin;
wt, wild type;
EMSA, electrophoretic
mobility shift assay;
PIPES, 1,4-piperazinediethanesulfonic acid.
 |
REFERENCES |
1.
|
Hoffman, P. C.,
Mauer, A. M.,
and Vokes, E. E.
(2000)
Lancet
355,
479-485[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Schneider, E.,
Hsiang, Y-W.,
and Liu, L. F.
(1990)
Adv. Pharmacol.
21,
149-183[Medline]
[Order article via Infotrieve]
|
3.
|
Chen, A. Y.,
and Liu, L. F.
(1994)
Annu. Rev. Pharmacol. Toxicol.
36,
191-218
|
4.
|
Pommier, Y.,
Leteurtre, F.,
Fesen, M. R.,
Fujimori, A.,
Bertrand, R.,
Solary, E.,
Kohlhagen, G.,
and Kohn, K. W.
(1994)
Cancer Invest.
12,
530-542[Medline]
[Order article via Infotrieve]
|
5.
|
Chen, K-V.,
Pasta, I.,
and Gottesman, M. M.
(1993)
Adv. Cancer Res.
60,
157-180[Medline]
[Order article via Infotrieve]
|
6.
|
Ganapathi, R.,
Kuo, T.,
Teeter, L.,
Grabowski, D.,
and Ford, J.
(1991)
Mol. Pharmacol.
39,
1-8[Abstract]
|
7.
|
Lenardo, M. J.,
and Baltimore, D.
(1989)
Cell
58,
227-229[Medline]
[Order article via Infotrieve].
|
8.
|
Barnes, P. J.,
and Karin, M.
(1997)
N. Engl. J. Med.
336,
1066-1071[Free Full Text]
|
9.
|
Rayet, B.,
and Gelinas, C.
(1999)
Oncogene
18,
6938-6947[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Beg, A. A.,
and Baltimore, D.
(1996)
Science
274,
782-784[Abstract/Free Full Text]
|
11.
|
Cusack, J. C.,
Liu, R.,
and Baldwin, A. S.
(1999)
Drug Resistance Updates
2,
271-273[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Schmitt, S. E.,
Sane, A-T.,
and Bertrand, R.
(1999)
Drug Resistance Updates
2,
21-29[Medline]
[Order article via Infotrieve]
|
13.
|
Bentires-Alj, M.,
Hellin, A-C.,
Ameyar, M.,
Chouaib, S.,
Merville, M-P.,
and Bours, V.
(1999)
Cancer Res.
59,
811-815[Abstract/Free Full Text]
|
14.
|
Pajonk, F.,
Pajonk, K.,
and McBride, W. H.
(1999)
J. Natl. Cancer Inst.
91,
1956-1960[Abstract/Free Full Text]
|
15.
|
Ganapathi, M. K.,
Weizer, A. K.,
Borsellino, S.,
Bukowski, R. M.,
Ganapathi, R.,
Rice, T.,
Casey, G.,
and Kawamura, K-I.
(1996)
Cell Growth Differ.
7,
923-929[Abstract]
|
16.
|
Grabowski, D.,
Holmes, K. A.,
Aoyama, M.,
Ye, Y.,
Rybicki, L. A.,
Bukowski, R. M.,
Ganapathi, M. K.,
Hickson, I. D.,
and Ganapathi, R.
(1999)
Mol. Pharmacol.
56,
1340-1345[Abstract/Free Full Text]
|
17.
|
Muscarella, D. E.,
Rachlinski, M. K.,
Sotiriadis, J.,
and Bloom, S. E.
(1998)
Exp. Cell Res.
238,
155-167[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Kawamura, K-I,
Grabowski, D.,
Krivacic, K.,
Hidaka, H.,
and Ganapathi, R.
(1996)
Biochem. Pharmacol.
52,
1903-1909[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Dignam, J. D.,
Lebovitz, R. M.,
and Roeder, R. G.
(1983)
Nucleic Acids Res.
11,
1475-1489[Abstract]
|
20.
|
Boland, M. P.,
Foster, S. J.,
and O'Neill, L. A. J.
(1997)
J. Biol. Chem.
272,
12952-12960[Abstract/Free Full Text]
|
21.
|
Sun, X-M,
MacFarlane, M.,
Zhuang, J.,
Wolf, B. B.,
Green, D. R.,
and Cohen, G. M.
(1999)
J. Biol. Chem.
274,
50553-5060
|
22.
|
Palombella, V. J.,
Rando, O. J.,
Goldberg, A. L.,
and Maniatis, T.
(1994)
Cell
78,
773-785[Medline]
[Order article via Infotrieve]
|
23.
|
Desai, S. D.,
Liu, L. F.,
Vazquez-Abad, D.,
and D'Arpa, P.
(1997)
J. Biol. Chem.
272,
24159-24164[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.