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B in TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis of Melanoma Cells1
Department of Oncology and Immunology Unit, David Maddison Clinical Sciences Building, Newcastle, New South Wales, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B can
inhibit apoptosis induced by a number of stimuli. It is also known that
TNF-related apoptosis-inducing ligand (TRAIL) can activate NF-B
through the death receptors TRAIL-R1 and TRAIL-R2, and decoy receptor
TRAIL-R4. In view of these findings, we have investigated the extent to
which activation of NF-B may account for the variable responses of
melanoma lines to apoptosis induced by TRAIL and other TNF family
members. Pretreatment of the melanoma lines with the proteasome
inhibitor
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal
(LLnL), which is known to inhibit activation of NF-B, was shown to
markedly increase apoptosis in 10 of 12 melanoma lines with death
receptors for TRAIL. The specificity of results for inhibition of
NF-B activation was supported by an increase of TRAIL-induced
apoptosis in melanoma cells transfected with a degradation-resistant
IB
. Furthermore, studies with NF-B reporter constructs
revealed that the resistance of melanoma lines to TRAIL-induced
apoptosis was correlated to activation of NF-B in response to TRAIL.
TRAIL-resistant sublines that were generated by intermittent exposure
to TRAIL were shown to have high levels of activated NF-B, and
resistance to TRAIL could be reversed by LLnL and by the superrepressor
form of IB. Therefore, these results suggest that activation of
NF-B by TRAIL plays an important role in resistance of melanoma
cells to TRAIL-induced apoptosis and further suggest that inhibitors of
NF-B may be useful adjuncts in clinical use of TRAIL against
melanoma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-related apoptosis-inducing ligand (TRAIL)/apo-2 (4, 5), and apo-3 ligand (apo-3L; Ref. 6). TRAIL
appears to be particularly important, as it appears to be able to
induce apoptosis in a wide range of neoplastic but not normal cells
(4). TRAIL can induce apoptosis by interaction with two
receptors referred to as DR4 (TRAIL-R1) (7, 8) and
DR5/TRAIL-R2 (9, 10, 11). These receptors were found to be
widely expressed on normal tissues, but the latter are believed to be
protected from apoptosis by two additional receptors, TRAIL-R3/DcR1
(12, 13, 14) and TRAIL-R4/DcR2 (15, 16). The
latter are believed to inhibit apoptosis either by competition for
binding of TRAIL (e.g., acting as decoys) or by providing signals that
inhibit apoptosis (3, 17). Activation of the transcription
factor NF-B may be one of the main pathways involved in the latter,
as it is known to be activated by TRAIL-R4 (17) and,
paradoxically, also by death receptors for TRAIL (8).
NF-B is known to regulate the expression of several proteins that
inhibit apoptosis, such as inhibitors of apoptosis (XIAP, IAP1, and
IAP2) that block apoptosis by inhibition of caspase-8 (18)
and caspase-3 and -9 (19) and the protein A1, which is a
Bcl-2 homologue (20).
Evidence for the importance of activation of NF-B in resistance to
apoptosis came from studies on leukemia cells in which inhibition of
NF-B was associated with increased sensitivity to apoptosis induced
by TNF- and activation of CD95 and TRAIL (21).
TRAIL-induced apoptosis of keratinocyte lines was inhibited by IL-1,
which was shown to activate NF-B (22). Some melanoma
cells were shown to become sensitive to TNF- when they were
transfected with a dominant-negative form of IB, which inhibited
NF-B activation (23). Similarly, inhibition of NF-B
was found to sensitize chemoresistant tumors to TNF- and
chemotherapy (24) and to reduce the in vivo survival of
human head and neck squamous cell carcinomas (25).
We have shown previously that TRAIL (26, 27, 28) but not other
members of the TNF family (29, 30) can induce apoptosis in
approximately two-thirds of melanoma cell lines. There was marked
variability in response to TRAIL, which in some lines was related to
loss of TRAIL receptor expression (27) but in other lines,
the basis for resistance to TRAIL was not clear. In the present study,
we have assessed the role of NF-B in susceptibility of melanoma
cells to apoptosis induced by TRAIL and other members of the TNF family
by use of agents which inhibit NF-B activity. We also have generated
TRAIL-resistant melanoma cells by culture in TRAIL and show that
activation of NF-B appears to be a key transcription factor involved
in resistance to TRAIL but not other TNF family members.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanoma cell lines with the prefix Mel were isolated from fresh surgical biopsies from patients attending the Sydney and Newcastle Melanoma Units and established in the laboratory. FH, CV, AT, and GL were from lymph nodes. MC was from skin. RM, JG, and SP were from bowel. The cell lines had been in culture for 2–6 mo at the time of these studies. MM200, Me1007, Me10538, Me4405, and IgR3 were from primary melanoma. The derivation of MM200, Me1007, Me10538, and Me4405, are described elsewhere (27, 30). SK-MEL-110 and SK-MEL-28 were a kind gift from Dr. A. Albino (American Health Foundation, Valhalla, NY) and S. Ralph (Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia), respectively. All melanoma cell lines were positive for tyrosinase and MART-1 mRNA by RT-PCR tests described elsewhere (31), except for Mel-SP, which was positive for Tyrosinase but not MART-1. All cell lines were cultured in DMEM containing 5% FCS (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia).
Plasmid vectors and transfectants
A superrepressor form of human IB protein S32A/36A
resistant to degradation (in the vector PRC/CMV) was kindly provided by
Steve Gerondakis (Walter and Eliza Hall Institute, Melbourne
Victoria, Australia) (32). Stable and transient
transfectants were conducted by electroporation by a Gene Pulser
(Bio-Rad, Hercules, CA) as follows: MM200, 0.36 kV and 960 µF;
Mel-RM, 0.33 kV and 500 µF; Mel-FH, 0.36 kV and 500 µF; SK-MEL-28,
0.32 kV and 500 µF; Mel-CV, 0.36 kV and 500 µF; and Me4405, 0.30 kV
and 500 µF. Twenty-four hours after electroporation, G418 was added
to a final concentration of 400 µg/ml for 21 days or until colonies
appeared on the plate. For the analysis of NF-B activation in
transient transfectants, the cells were cotransfected with 30 µg of
pNF-B-d2EGFP, a reporter vector encoding the green fluorescent
protein (GFP) under the control of the enhancer element (Clontech,
Palo Alto, CA) for 24 h. Fluorescence was measured by flow
cytometry, as mentioned in Materials and Methods. To
determine the efficiency of transfection, the cells were cotransfected
with 30 µg of a vector encoding the LacZ cDNA, and percentage of blue
cells were estimated 24 h after transfection.
In some experiments, the mutant IB-containing vector at 30 µg
DNA/2,000,000 cells in 400 µl was cotransfected with pEG FP-N1GFP
vector (Clontech) at 1.5 µg of DNA. Control cultures were transfected
with the GFP vector at 30 µg of DNA/2,000,000 cells.
Monoclonal Abs, recombinant proteins, and peptides
Recombinant human (hu) TRAIL (lot 6321-19) prepared as described
elsewhere (4) and huCD40L (lot 5753-56) were supplied by
Immunex (Seattle, WA). Each preparation was supplied as a leucine
zipper fusion protein, which required no further cross-linking for
maximal activity. Recombinant huFasL, produced from isolated cDNA
(GenBank accession no. U08137) in vector pDC409 and transfected into
COS cells, was kindly supplied as sterile supernatants by Immunex. It
produced 50% lysis of Jurkat T cells at dilutions of 2–4
(30). rTNF- cytokines and control mAb
antitrinitrophenyl (anti-TNP; IgG1) were purchased from BD
PharMingen (Bioclone, Marrickville, Australia). Control mAb 1D4.5
(Ig2a) was against an Ag on Salmonellae (kindly supplied by
L. Ashman, Hansen Cancer Research Centre, Adelaide, Australia).
The MAbs against TRAIL-R1 (IgG2a huTR1-M271; lot: 7136-07), TRAIL-R2
(IgG1 huTRAIL-R2-M413; lot: 5274-96), TRAIL-R3 (IgG1 huTR3-M430; lot:
7313-217), and TRAIL-R4 (IgG1 huTR4-M444; lot: 7136-15) were supplied
by Immunex and are described elsewhere (33). The rabbit
Abs against p65 were raised against the C-terminal end peptide
SGDEDFSSIADMDFSALLSQIS and anti-p75 against the C-terminal end
peptide MMTTSSDSMGETDNPRLLSM, and were purchased from Stressgen
(Victoria, British Columbia, Canada). The rabbit Ab against p50 was
purchased from Fitzgerald (Concord, MA). The control purified rabbit
IgG (prod. no. 15006) was purchased from Sigma (St. Louis, MO). The
proteasome inhibitor (calpain inhibitor 1)
N-acetyl-Leu-Leu-norleucinal (LLnL) (34) was
purchased from Sigma (cat. no. Ab2185). Lactacystin (35)
was purchased from ICN Biochemicals (Biomedicals Australasia, Seven
Hills, New South Wales, Australia).
Flow cytometry
Analysis was conducted with a Facscan flow cytometer (Becton Dickinson, Mountain View, CA) . In studies on relocalization of TRAIL receptors, melanoma cells were grown overnight in 24-well plates and exposed to TRAIL 100 ng/ml for 30 min. Adherent cells were removed by trypsinization in 0.25% trypsin at 37°C for 5 min, washed twice in cold DMEM and once in PBS at 4°C, and fixed in 4% paraformaldehyde. Appropriate concentrations of mAbs were added to the cells in 100 µl of PBS containing 20% human A serum and incubated for 7 min at room temperature. Cells were washed either twice with PBS and analyzed if directly labeled, or if indirectly labeled, cells the were incubated with F(ab')2 affinity-isolated FITC-conjugated sheep anti-mouse Ig (Silenus, cat. no. 985051020; Amrad Biotech, Boronia, Victoria, Australia) plus 20 µl of 100% human serum to block Fc receptors for 7 min at room temperature. A minimum of 5000 cells was analyzed. Studies on permeabilized cells were similar to the methods of Jung et al. (36). The cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% saponin in permeabilization buffer and the Ab added for 30 min at 4°C. The cells were washed and then stained by FITC-labeled F(ab)2 fraction of affinity-isolated sheep anti-mouse Ig (Silenus; Amrad Biotech) at 1/100 dilution for 30 min at 4°C. After washing, the cells were analyzed by flow cytometry. The TRAIL-R-negative Me10538 cell line (27) was included as a negative control in studies on permeabilized cells. The percentage of cells expressing the receptors was calculated as the difference in positive area between the positive and negative control histograms. The positive area was that to the right of the intersection of the two curves (37).
For the detection of nuclear localization of NF-B proteins P50, P65,
and P75, the melanoma cells were stained by Cycletest PLUS DNA reagent
kit (Becton Dickinson, Lane Cove, Australia) as described elsewhere
(38). The cells were resuspended in 5 ml of citrate buffer
and centrifuged at 300 x g for 5 min. The supernatant
was discarded and the cells resuspended in 1.5 ml of citrate buffer and
centrifuged for a further 5 min at 300 x g. The
supernatant was carefully discarded and the cells blotted dry. The
pellet was gently resuspended in a mixture of 250 µl of solution A
(trypsin in a spermine tetrahydrochloride detergent buffer) and 200
µl of solution B (trypsin inhibitor and RNase A in citrate
stabilizing buffer with spermine tetrahydrochloride) for 10 min at room
temperature. Abs against P65, P50, or P75 then were added for 10 min at
room temperature, followed by incubation for 10 min with 2.5 µl of
FITC-conjugated anti-mouse mAb (Amrad Biotech). Percentage of
expression of the proteins was calculated as above for receptor
expression.
Measurement of apoptosis
Apoptotic cells were determined by the propidium iodide method
(39). In brief, melanoma cells were adhered overnight in a
24-well plate (Falcon 3047; Becton Dickinson, Lane Cove, Australia) at
a concentration of 1 x 105/well in 10%
FCS. Cells in suspension were added on the day of the assay. Medium was
removed and cells treated for 20 h with the reagents TRAIL,
huFasL, TNF-, or huCD40L. Cells were incubated for a further 24
h at 37°C, the medium removed, and adherent and suspended cells
washed 1 x with PBS. The medium and PBS were placed in 12 x
75 mm Falcon polystyrene tube and centrifuged at 200 x
g. A total of 1 ml of hypotonic buffer (propidium iodide, 50
µg/ml, in 0.1% sodium citrate plus 0.1% Triton X-100; Sigma) was
added directly to the cell pellet of cells grown in suspension or to
adhered cells in the 24-well plate and gently pipetted off, then added
to the appropriate cell pellet in the Falcon tube. The tubes were
placed at 4°C in the dark overnight before flow cytometric analyses.
The propidium iodide fluorescence of individual nuclei was measured in
the red fluorescence with a FACScan flow cytometer (Becton Dickinson,
Mountain View, CA) and the data registered in a logarithmic scale. At
least 104 cells of each sample were analyzed.
Apoptotic nuclei appeared as a broad hypodiploid DNA peak, which was
easily distinguished from the narrow hyperdiploid peak of nuclei in the
melanoma cells.
In cotransfection experiments with the GFP vector, apoptosis was
assessed in cells gated for green fluorescence by annexin V conjugated
with PE (BD PharMingen). TRAIL 100 ng/ml was added 
48 h after
transfection and apoptosis measured 3 h later.
EMSA
For the preparation of whole-cell lysates, melanoma cells
(2 x 106) were lysed in 80 µl of 600 mM
KCl, 20 mM HEPES, pH 7.05, 200 µM EDTA, 1 mM PMSF, 10 µg/ml soybean
trypsin inhibitor, and 2 µg/ml aprotinin for 45 min on ice (all
obtained from Sigma) and centrifuged at 12,000 x g for
5 min at 4°C. For the preparation of cytoplasmic and nuclear extracts
from melanomas, we followed the methods described by Dignam et al.
(40). Cells (5 x 106) were
washed twice with ice-cold 1x PBS and spun down at 500 x
g at 4°C for 5 min. The cell pellet was subsequently
homogenized in 200 µl of Dignam buffer A containing protease
inhibitors as described above. The homogenate was spun down, and the
nuclear pellet was washed twice with buffer A. The nuclear pellet then
was resuspended in 30 µl of Dignam buffer C. A total of 10 µgrams
of supernatant was incubated with 4 µl of 5x gel shift binding
buffer (Promega, Madison, WI) for 30 min at room temperature to
double-stranded oligonucleotides encoding the consensus binding sites
of NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') that had been labeled
with [32P]ATP (GeneWorks, Adelaide, Australia)
by polynucleotide kinase (Promega). For supershift assays, Abs against
P50 and P65 were incubated at room temperature for 30 min with the
melanoma lysates in the presence of the radiolabeled oligo.
Protein-oligo complexes were separated on a nondenaturing 6% PAGE in
0.5 x Tris-borate-EDTA buffer. Gels were dried and exposed
overnight to an x-ray film. Included in each assay was a positive
control of extracts from the Jurkat T cell line exposed to TRAIL and a
negative control of unlabeled NF-B and random AP2 oligonucleotide
included in the Promega NF-B gel shift assay system (cat. no.
E3300).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B by LLnL
The proteasome inhibitor LLnL was shown in previous studies to
inhibit the degradation of IB and thereby to prevent activation of
NF-B (21, 34). To determine whether LLnL inhibited
activation of NF-B in melanoma cells, we used EMSA to measure
NF-B activation in the melanoma lines before and after activation by
TRAIL and the effect of LLnL on this activation. As shown in Fig. 1
, enhanced NF-B binding activity
induced by TRAIL was shown in seven of the melanoma lines selected for
study. Constitutive NF-B activation was found in five cell lines,
SK-MEL-28, Mel-CV, Mel-FH, MM200, and Mel-RM, before addition of TRAIL
(Fig. 1). LLnL (5 µM) added 2 h before TRAIL (and throughout
culture with TRAIL) reduced the binding to barely detectable levels,
except in extracts from Me4405 that still had relatively high levels of
activated NF-B after pretreatment with LLnL. The level of NF-B
activation shown in the gel shift assays were consistent with the
levels shown by NF-B reporter assays shown below. As shown in Fig. 2A, NF-B activation
occurred within 5 min and was maximal from 15 to 60 min after exposure
to TRAIL. NF-B activation was maximal at a concentration of 10 ng/ml
(Fig. 2B). This is less than the optimal concentration of
100 ng/ml shown for TRAIL-induced apoptosis (26). Similar
results were found in studies on the Mel-CV line.
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B
can reverse resistance to TRAIL-induced apoptosis
We then examined whether LLnL would inhibit TRAIL-induced
apoptosis of melanoma cells in studies on a large number of melanoma
lines. The dose titration studies shown in Table I indicate that two of the lines, Me1007
and Mel-AT, showed marked sensitivity to LLnL alone, and there was only
a small increase in apoptosis induced by TRAIL in the presence of LLnL.
Three melanoma lines that were partially sensitive to TRAIL-induced
apoptosis had more marked increases in sensitivity to TRAIL in the
presence of LLnL that approached maximum levels in the assay. It also
was noted that treatment of melanocytes, fibroblasts, and resting PBL
with even high concentrations of LLnL did not make them sensitive to
TRAIL-induced apoptosis. However, there was a small increase in
sensitivity of PBL activated by anti-CD3 to TRAIL-induced
apoptosis.
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. Again, there was wide variation in
toxicity of LLnL between lines but in most lines there was a marked
increase in TRAIL-induced apoptosis in the presence of LLnL, the
exceptions being the control cell lines Me10538 and Mel-SP, which did
not have death receptors for TRAIL (27). The
TRAIL-sensitive line Mel-JG was maximally killed by TRAIL alone, and no
further increase was induced by LLnL. Studies with a second proteasome
inhibitor, lactacystin (35), at 10 µM on the cell lines
Me1007, Mel-CV, Mel-FH, and MM200 gave a similar increase in
TRAIL-induced apoptosis (data not shown).
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B with LLnL has minimal effects on the level of
apoptosis induced by other TNF family members
In view of the increased sensitivity of melanoma cells to
TRAIL-induced apoptosis in melanoma cells exposed to LLnL, we also
examined whether activation of NF-B may be involved in resistance of
melanoma cells to TNF-, FasL, and CD40L. Six cell lines that showed
a wide range of sensitivity to TRAIL-induced apoptosis were pretreated
with LLnL 3 h before addition of TRAIL, FasL, TNF-, and CD40L
in optimum concentrations established in previous studies (26, 29, 30). The results shown in Table III indicate that LLnL did not sensitize
the melanoma cells to apoptosis induced by TNF- or CD40L but did
potentiate low levels of apoptosis induced by FasL in the cell line
Mel-CV. In contrast, LLnL increased apoptosis induced by TRAIL in all
but the control TRAIL-R-negative line Me10538. It was of interest that
FasL induced low levels of apoptosis in the latter and this was not
increased by LLnL. All of the cell lines expressed receptors for the
other members of the TNF family except the TRAIL-sensitive line Mel-JG.
The results shown are representative of two experiments.
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B was activated by other members of the TNF
family, NF-B activation was measured in gel-shift assays on two of
the cell lines (SK-MEL-28 and Mel-FH) that did not undergo apoptosis
after exposure to the TNF family members. As shown in Fig. 3, NF-B was activated by FasL and
TRAIL in both melanoma lines and by CD40L in the SK-MEL-28 line, but
NF-B activation was not associated with apoptosis. However, the
results show that the receptors were expressed at sufficient levels to
be functionally active.
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B
To further substantiate the gel shift assays of NF-B activation
we examined the site of the activated NF-B proteins in the melanoma
cells by studies on extracts from the nucleus and cytoplasm of the
cells that were prepared as described elsewhere (40). As
shown in Fig. 4A, TRAIL
induced a typical increase in NF-B activation in extracts from the
nuclei of Mel-RM cells and there were only relatively small amounts in
the cytoplasm. The specificity of the probe for the NF-B proteins
also was also shown by the supershift electromobility assays with Abs
to p50 and p65 NF-B proteins, illustrated in Fig. 4B.
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B measured in
reporter assays are higher in TRAIL-resistant compared with
TRAIL-sensitive cell lines
In view of the variable levels of NF-B activation in the
melanoma lines before and after exposure to TRAIL shown in Fig. 1
, we
used more quantitative NF-B reporter assays to examine the relation
between NF-B activation and TRAIL-induced apoptosis. Transient
transfection with the NF-B reporter vector pNF-B-d2EGFP was
possible in eight of nine melanoma lines but was not possible in the
Me4405 line. The results shown in Table IV have been arranged in order of
the sensitivity of the cell lines to TRAIL and %GFP levels have been
adjusted to take into account the variable transfection efficiency. The
relatively resistant cell lines Mel-FH and Me1007 had low basal NF-B
levels, but after treatment with TRAIL, the levels of NF-B increased
by 73 and 166% respectively (Table IV). IgR3, Mel-RM, and SK-MEL-28
had moderate sensitivity to TRAIL and moderately high basal levels of
NF-B, which was increased further by TRAIL. In contrast, the
TRAIL-sensitive lines, Mel-JG and MM200, had high basal levels of
NF-B that were relatively unchanged after treatment with TRAIL.
Mel-CV also had high basal levels and relatively low levels of
TRAIL-induced activation of NF-B but remained relatively resistant
to TRAIL-induced apoptosis. Essentially similar results were found in a
repeat of the study, with the exception of Mel-CV. These results
suggest a reciprocal relation between the TRAIL-induced activation of
NF-B and TRAIL-induced apoptosis of melanoma. Regression analysis of
percentage of apoptosis vs percentage of increase in GFP expression,
including all the results in Table IV, showed a significant inverse
relationship (y = 54.0 - 0.39 x
r2 = 0.55; p =
0.03.).
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B increases TRAIL-induced apoptosis
in melanoma cells
The findings above that TRAIL-induced apoptosis was increased by
treatment with LLnL did not prove that the increase was attributable to
inhibition of NF-B activation, as proteasome inhibitors also inhibit
the breakdown of other proteins that may be involved in apoptosis, such
as p53 (41). In view of this, we transfected a mutated
degradation-resistant form of IB into the melanoma cells that
specifically inhibits activation of NF-B by binding the proteins in
the cytoplasm. Transient transfection of the degradation-resistant
IB was conducted in two cell lines, Mel-FH and Mel-RM. The levels
of IB in the transfectants were confirmed by FACS with
anti-IB (data not shown). As shown in Table V, TRAIL-induced apoptosis was increased
in each of the melanoma lines to levels comparable to that seen with
the proteasome inhibitor LLnL. This was accompanied by decreased
binding of p50, p65, and p75 to the nuclei of Mel-FH and Mel-RM, shown
by flow cytometry on isolated nuclei. Inhibition of NF-B activation
also was shown in NF-B GFP reporter assays on Mel-RM and Mel-FH in
that there was a decrease in %GFP expression after exposure to TRAIL
in the IB-transfected melanoma cells but not in the control cells
transfected with the vector alone.
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B we cotransfected a GFP-expressing vector at a
twentieth of the concentration of the IB vector and apoptosis was
measured in cells gated for green fluorescence by annexin V conjugated
with PE. Given the relatively low concentration of the GFP vector, it
was safe to assume the GFP-containing cells also were transfected with
the mutant IB vector. The results of three studies on the cell
lines Mel-RM and Mel-FH shown in Fig. 5
indicate that transfection of the mutant IB was associated with a
marked increase in apoptosis induced by TRAIL relative to cells
transfected with the vector alone and relative to cells not exposed to
TRAIL. The higher degree of apoptosis associated with transfection of
IB in the absence of TRAIL presumably reflects the greater
sensitivity of the annexin V assay.
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To identify factors associated with resistance to TRAIL more
clearly we generated TRAIL-resistant lines by exposure to TRAIL in
vitro. Three melanoma cell sublines, Mel-FHSel, MM200Sel, and
Mel-RMSel, were grown in the presence of TRAIL. Briefly, the parental
cell lines were seeded in 25 cm2 flasks and TRAIL
(100 ng/ml) was added. The surviving cells were fed every three
days for 3–6 wk with tissue culture medium containing TRAIL until they
reached confluence and were resistant to TRAIL-induced apoptosis. The
levels of NF-B measured by the NF-B GFP reporter construct in the
Mel-RM and MM200 TRAIL-selected cells shown in Table VI were higher than the parental cell
lines in the study shown in Table IV. Similar results were shown in a
repeat of this study.
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B the in vitro generated TRAIL-resistant
melanoma lines Mel-FHSel, MM200Sel, and Mel-RMSel were pretreated with
LLnL (2–5 µM) and their sensitivity to TRAIL examined. As shown in
Table VII, there was a marked increase
in sensitivity of the TRAIL-resistant lines MM200Sel, Mel-RMSel, and
Mel-FHSel to TRAIL in the presence of LLnL that exceeded the level of
apoptosis in the parental lines. The results shown are representative
of two experiments. Evidence for the specificity of these results for
inhibition of NF-B was obtained by transient transfection of the
TRAIL-resistant MM200Sel and Mel-RMSel lines with the
degradation-resistant IB (66 and 48% transfection efficiency,
respectively). As shown in Table VII, the cells transfected with
the latter had increased sensitivity to TRAIL-induced apoptosis that
was similar to that seen in melanoma cells treated with LLnL. When the
transfection efficiency is allowed for, the percentage of apoptosis
levels induced by TRAIL approximates that in the lines pretreated with
LLnL. The results were similar in a repeat of the study and confirm
that activation of NF-B was responsible in part for the resistance
of the cell lines to TRAIL-induced apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (8, 17). This was confirmed in the present studies by gel shift
assays, GFP reporter constructs for NF-B, and nuclear staining with
Abs specific for p50, p65, and p75. NF-B is known to activate a
number of genes coding for proteins that inhibit apoptosis by the
transmembrane signaling pathway (18, 43) and mitochondrial
stress pathway (20, 44). To investigate the possible role
of NF-B in resistance to TRAIL-induced apoptosis, we used an
inhibitor, LLnL, that blocks the ability of proteasomes to degrade
IB. Therefore, IB remains bound to NF-B in the cytoplasm and
prevents its relocation to the nucleus. A mutant form of IB that
was not degraded by proteasomes also was used to confirm the
specificity of the effects for inhibition of the activation of
NF-B.
The results indicated that LLnL reversed the resistance to
TRAIL-induced apoptosis in 10 of 12 melanoma lines with death receptors
for TRAIL. Two lines did not show an increase in sensitivity to TRAIL
in the presence of LLnL. One (Me10538) did not express TRAIL-R
(27) and another line, Me1007, was difficult to interpret
because it underwent cell death in the presence of LLnL alone. The
latter result was similar to studies on Hodgkin’s lymphoma cells that
found that proliferation and viability was dependent on activation of
NF-B in the cells (45). Another line, Mel-JG, was very
sensitive to TRAIL-induced apoptosis alone, and LLnL could not further
increase this sensitivity. These results suggest that NF-B
activation is a significant factor in protection against TRAIL-induced
apoptosis of melanoma, as reported by others in studies on lymphoid
cells (21) and other cancers (24, 25). In
contrast to the increased sensitivity to TRAIL induced by LLnL, the
latter did not increase the sensitivity of the melanoma lines to
apoptosis induced by CD40L, TNF-, or FasL, the exception being small
increases in sensitivity of one line to FasL.
However, proteasome inhibitors are not specific for NF-B and are
responsible for degradation of proteins involved in cell cycle
regulation (41), which might have indirect effects on
induction of apoptosis. Therefore, we transfected melanoma cells with a
degradation-resistant form of IB that binds NF-B proteins in
the cytoplasm and thereby specifically inhibits activation of NF-B.
In some experiments, cotransfection of a GFP expression vector was used
to identify the transfected cells. These studies showed that the
transfected melanoma cells had increased sensitivity to TRAIL-induced
apoptosis and decreased binding of NF-B proteins in the nucleus.
NF-B reporter assays indicated that some melanoma had high
constitutive levels of NF-B activation but this was not necessarily
related to resistance to TRAIL-induced apoptosis. Instead, it appeared
from the NF-B reporter assays that resistance to apoptosis
correlated more with the increase in the level of NF-B activation
after exposure to TRAIL. This was shown particularly in studies on
Mel-FH and Me1007 and in studies on melanoma lines that had been
selected for resistance to TRAIL, such as Mel-RMSel.
Melanoma cells with high constitutive levels of NF-B activation
(Mel-JG and MM200) underwent a small increase in NF-B activation
after exposure to TRAIL and were relatively sensitive to TRAIL-induced
apoptosis. High constitutive levels of activated NF-B in melanoma
were reported previously to be associated with the level of oxidative
radicals in the cells (46) or autocrine production of
IL-1, which is known to activate NF-B (47). A third
possible mechanism was accelerated decay of IB in melanoma cells
allowing NF-B proteins to enter the nucleus (48).
Reasons for the sensitivity of melanoma cells to TRAIL-induced
apoptosis despite the presence of high constitutive levels of activated
NF-B remain the subject of ongoing studies.
Studies on melanoma lines made resistant to TRAIL by prolonged culture
in TRAIL indicated that NF-B appeared to be activated to a greater
extent in the TRAIL-selected resistant lines compared with the parental
line. This was consistent with the marked increases in sensitivity to
TRAIL-induced apoptosis in the TRAIL-resistant sublines after treatment
with LLnL to inhibit NF-B. Transfection of the degradation-resistant
IB into the cells also resulted in an increase in TRAIL-induced
apoptosis but not to the same level as seen with the proteasome
inhibitor. The latter results may reflect the transfection efficiency
or indicate that factors other than those involved in activation of
NF-B are involved in resistance of the cells to TRAIL-induced
apoptosis.
Some insights into how activation of NF-B may inhibit apoptosis
resulting from signals triggered from the same receptor was derived
from kinetic studies, which suggested that NF-B was activated much
earlier than the caspase pathway and at lower concentrations of TRAIL;
e.g., activation of NF-B was evident by 5 min and at concentrations
of 1 ng/ml. This compares with reported activation of caspase-8 30 min
after exposure to TRAIL (42) and peak activation of the
main effector, caspase-3, by 2–4 h (49) and at
concentrations of 100 ng (26). These possible
differences in kinetics of the two pathways needs to be confirmed by
studies on the induction of NF-B proteins involved in inhibition of
apoptosis such as the Bcl-2 homologue A1 and members of the IAP family.
If confirmed, these findings might suggest that TRAIL may only induce
apoptosis when present at high concentrations and that low
concentrations may act to increase resistance to apoptosis.
In summary, these studies on melanoma lines and TRAIL-selected lines
support the view that mechanisms dependent on activation of NF-B
play a key role in resistance of melanoma cells to TRAIL-induced
apoptosis and suggest that inhibitors of NF-B may be a valuable
adjunct to treatment of patients with TRAIL. Further studies on fresh
melanoma tissue and studies in vivo are needed to support these
conclusions.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter Hersey, Department of Oncology and Immunology, Room 443, David Maddison Building, Corner of King and Watt Streets, Newcastle, NSW 2300, Australia.
3 Abbreviations used in this paper: FasL, Fas ligand; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, receptors for TRAIL; hu, human; DcR, decoy receptor for TRAIL; DR, death receptor for TRAIL; GFP, green fluorescent protein; LLnL, N-acetyl-Leu-Leu-norleucinal; IAP, inhibitors of apoptosis.
Received for publication April 12, 2000. Accepted for publication February 16, 2001.
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