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Experimental Therapeutics, Preclinical Pharmacology |
Division of Medical Oncology [C. G. F., C. T., S. W. S., G. J. P., H. M. P., G. G.] and Department of Pathology [A. J. K.], University Hospital Vrije Universiteit Amsterdam, and Department of Autoimmune Diseases, Centraal Laboratorium Bloedtransfusiedienst, and Laboratory of Experimental and Clinical Immunology, Academic Medical Centre, University of Amsterdam, Amsterdam [T. v. L.], the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytotoxic drugs, irrespective of their intracellular target, have been shown to cause cell death in sensitive cells, at least partly, by inducing apoptosis (2) . Nevertheless, the precise molecular requirements that trigger the apoptotic pathway remain largely unknown (3) and have not been extensively studied in lung cancer cells.
Fas (CD95/APO1) is a Mr 45,000 type I transmembrane glycoprotein that belongs to the nerve growth factor/tumor necrosis factor receptor superfamily (4) . Cross-linking of Fas by either the natural ligand or an agonistic antibody transduces a signal that results in rapid induction of apoptosis in susceptible cells (5) . FasL (CD95-L) is a Mr 40,000 type II transmembrane protein that is a member of the tumor necrosis family of cytokines (6 , 7) . FasL also exists in a soluble form, released from the cell surface after cleavage by metalloproteinases (8 , 9) . After the cross-linking by the ligand, the cytoplasmic region of the Fas receptor, called death domain (10) , recruits proteins present in the cytoplasm, including MORT1/FADD (11 , 12) . Subsequently, caspase-8, or MACH/FLICE (13 , 14) , is activated, and this cysteine protease plays an essential role in the proteolytic cascade that finally leads to apoptosis mediated by Fas and other death receptors (15) . A role for the Fas system as a mediator of the drug-induced apoptosis has been proposed (16, 17, 18, 19, 20, 21, 22, 23) . Chemotherapy would induce an up-regulation of FasL (19 , 23) , leading to an autocrine/paracrine activation of Fas signaling, and this may constitute a potential mechanism in the mediation of anticancer drug-induced apoptosis. Alternatively, other studies have shown that drug-induced apoptosis occurs independently from the Fas signaling (24, 25, 26, 27, 28, 29) . As a consequence, the exact role of the Fas system in apoptosis induced by chemotherapy remains unsettled.
We previously described cellular changes indicative of apoptosis in lung cancer cell lines treated with topotecan and gemcitabine, two active drugs in lung cancer (30) . The present work was designed to study the Fas system in the context of its expression, functional status, and possible relation with chemotherapy-induced apoptosis in lung cancer cell lines. Our results show that the Fas/FasL pathway is not involved in chemotherapy-induced apoptosis in lung cancer cells but indicate that the occurrence of caspase-8 activation induced by chemotherapy does not require Fas/FasL signaling.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
Human NSCLC (NCI-H460 and NCI-H322) and SCLC (NCI-H187 and NCI-N417)
cell lines were kindly provided by Dr. A. Gadzar. Both GLC4 and the
mrp-overexpressing GLC4/ADR (generated by continuous culture in the
presence of doxorubicin) were obtained from Dr. E. De Vries. The Jurkat
human T-cell leukemia cell line was obtained from Dr. T. van Lopik.
Cells were cultured in RPMI 1640 supplemented with 10%
heat-inactivated FCS (Life Technologies, Inc., Breda, the Netherlands),
2 mM L-glutamine, 50 IU/ml penicillin, and 50
µg/ml streptomycin and incubated at 37°C in a humidified atmosphere
with 5% CO2. The cell lines were tested
regularly for the presence of Mycoplasma infection and found
to be negative. Cells from exponentially growing cultures were used for
all of the experiments.
Detection of Fas and Fas Ligand Expression.
Cells were stained with the anti-Fas (APO-1) mAb CLB-CD95/15, raised in
our group (31)
, or anti-FasL NOK-1 mAb (PharMingen, San
Diego, CA) for 45 min at 4°C. To control for aspecific binding, an
IgG2a antibody (Dako, Santa Barbara, CA) for FasR or an IgG1 for the
ligand (Dako) were used as isotype-matched, nonbinding antibodies. The
equivalent to 1 µg of protein of anti-Fas and FasL mAb antibodies or
the respective isotype antibody was used in each sample. Cells were
then washed twice with cold PBS and incubated with FITC-conjugated
goat-antimouse antibody in the dilution 1:50 at 4°C in the dark for
30 min. Two additional washing steps with cold PBS were performed
before the cells were analyzed by FACScalibur using CELLQuest software
(Becton Dickinson, Mount View, CA). The same instrument settings were
used for all of the experiments, and 5000 events were analyzed. MFI
ratio was defined as: MFI of gated live cells stained with anti-Fas/MFI
of cells stained with isotype-matched antibody.
Growth-Inhibition Assay.
A 100-µl suspension of 20 x 103 cells was
added to each well of flat-bottomed, 96-well plates (Costar, Corning,
NY). After 24 h, various concentrations of the drugs (topotecan,
gemcitabine, cisplatin, and paclitaxel) or the Fas agonistic mAb
CLB-CD95/15 (31)
was added to the cells. After 72 h,
the cells were incubated with a solution of 5 mg/ml of MTT (Sigma
Chemical Co., St. Louis, MO) dye for 4 h. The MTT crystals were
solubilized with 100 µl of DMSO, and absorbance was measured at 540
nm, using Spectra Fluor (Tecan, Salzburg, Austria). Absorbance values
were expressed as a percentage of untreated controls, and
concentrations resulting in cell growth inhibition of 50%
(IC50) and 80% (IC80) were
calculated.
Cell Death Measurement.
Cells were plated at a density of 5 x 106
cells in 75-cm2 tissue culture flasks (Costar,
Cambridge, MA) 24 h before treatment. For determination of
Fas-induced apoptosis, cells were incubated with 5, 10, or 50 µg/ml
of CLB-CD95/15 mAb or 100 ng/ml of recombinant FasL (Alexis
Biochemicals, San Diego, CA) for 4–72 h. Drug-induced apoptosis
was analyzed after incubation of the cells for 4–72 h with
IC50 and IC80
concentrations of topotecan, gemcitabine, cisplatin, or paclitaxel. The
inhibitory anti-Fas mAb CLB-CD95/2 (10 µg/ml; Ref. 31
)
or neutralizing anti-FasL mAb NOK-2 (1 µg/ml; PharMingen, San Diego,
CA) was applied 1 h before drug treatment. The extent of cell
death was determined by PI staining of hypodiploid DNA, and the
measurement of early apoptotic events was performed by annexin V
staining. For the PI staining, 3 x 105
cells were resuspended in Nicoletti buffer as described
(32)
and analyzed by FACScan (Becton Dickinson, Mount
View, CA). The fraction of cells with sub-G1 DNA
content was assessed by the Lysis program (Becton Dickinson). For
annexin V staining (33)
, 3 x
105 treated cells were washed, resuspended in 300
µl of a binding buffer containing 2 mM
Ca2+, and incubated at room temperature for 15
min in the dark with 3 µl of annexin V-phycoerythrin (Nexins
Research, Kattendijk, the Netherlands). Analysis was performed on
FACScalibur using CELLQuest software (Becton Dickinson). The percentage
of specific apoptosis was calculated subtracting the percentage of
spontaneous apoptosis of the relevant controls from the percentage of
total apoptosis.
Western Blot Analysis.
Proteins for Western blot analysis were extracted from whole-cell
pellets and lysed for 30 min at 4°C with 50 µl solution [1%
Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH
7.6), and 5 mM EDTA] per 1 x
106 cells. Protease cocktail inhibitor tablets
(Boehringer-Mannheim, Almere, the Netherlands) were freshly diluted in
PBS before each experiment and added to the lysing solution. Protein
concentration was assayed using the Bio-Rad assay (Bio-Rad
Laboratories, Richmond, CA). Of each sample, 25 µg of protein/lane
were separated on a 10% SDS-PAGE and electroblotted onto
nitrocellulose membranes (Amersham, Braunschweig, Germany). Protein
loading equivalence was assessed by the expression of ß-actin. After
blocking for 30 min in PBS supplemented with 5% BSA (Sigma) and 5%
nonfat dry milk for 1 h at room temperature, immunodetection was
performed using mouse anti-FasR and anti-FasL mAbs (Transduction
Laboratories, Lexington, KY) at a concentration of 1:500 or
anti-caspase-8 mAb (Immunotech, Marseille, France) at a concentration
of 1:1000 at 4°C overnight, followed by horseradish
peroxidase-conjugated goat-antimouse antibody. Enhanced
chemiluminescence (ECL; Amersham, Braunschweig, Germany) was used for
detection, and protein expression was quantified by densitometry of
autoradiographs (model GS-690 Imaging densitometer; Bio-Rad, Richmond,
CA).
Northern Blot Analysis.
Total RNA was extracted using RNAzol B (CINNA/Biotecx Laboratory, Inc.,
Houston, TX). RNA was denatured at 55°C for 15 min in 50% formamide,
electrophoresed through a 1% agarose gel containing formaldehyde, and
blotted onto Qiabrane nylon membranes (Qiagen, Hilden, Germany). The
membranes were hybridized with the full-length cDNA for Fas antigen and
FasL (4
, 7)
, kindly provided by Dr. Shigekazu Nagata,
Osaka University Medical School, Osaka, Japan. The membranes
were washed and autoradiographed for 48 h at -80°C. A cDNA
probe for glyceraldehyde-3-phosphate dehydrogenase was used as a
control for RNA loading.
p53 Gene Sequencing.
The p53 gene of the six lung cancer cell lines was sequenced
from exon 5 to exon 9 according to methods described previously
(34, 35, 36)
. In brief, DNA was isolated, and a PCR reaction
using primers for exons 5–9 was performed. The product was submitted
to a new PCR round using specific 
-33P
end-labeled primers for each of the five exons to be sequenced.
Subsequently, the product was separated on a 6% PAGE gel, fixed, and
autoradiographed at room temperature overnight.
Caspase-8 Proteolytic Activity.
Caspase-8 activity was assessed using ApoAlert Caspase-8 Fluorescent
Assay kit (Clontech Laboratories, Inc., Palo Alto, CA). Whole-cell
pellets obtained from 2 x 106 cells were
resuspended in 50 µl of chilled lysis buffer and incubated on ice for
10 min. The cell lysates were centrifuged at 12,000 rpm at 4°C for 3
min, and the supernatants were collected. Subsequently, 50 µl of a
reaction buffer containing DTT at a concentration of 10 mM
and 5 µl of 1 mM IETD-AFC-conjugated substrate were added
to the supernatants. The supernatants were then incubated at 37°C for
1 h in a water bath. Fluorescence was detected using a fluorometer
equipped with a 400-nm excitation and a 505-nm emission filter.
Fold-increase in the protease activity was determined by comparing the
levels of the treated cells with the untreated controls.
Statistics.
Quantitative experiments were analyzed by use of Student’s
t test. All Ps resulted from the use of two-sided
tests and were considered significant when <0.05. Correlative data
were analyzed using the Pearson correlation coefficient.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, all
cell lines revealed Fas and FasL protein expression. In four cell lines
(H460, H322, GLC4, and GLC4/ADR), expression was quantitatively similar
to that of Jurkat T-leukemia cells, used as a positive control, but
H187 and N417 cells showed lower levels of both Fas and FasL (Fig. 1
and Table 1
). Western blot analysis,
using mAb directed against different epitopes (see "Materials and
Methods") and Northern blotting confirmed the FACS results for both
Fas and FasL (data not shown).
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. To investigate whether
this growth inhibition could be translated into apoptosis, cells were
again exposed to different concentrations of the Fas agonistic mAb, and
apo-ptosis was analyzed at different time points (4–72 h) by
PI-stained hypodiploid DNA. As shown in Fig. 2B
, Fas-induced
apoptosis was observed in four of six lung cancer cell lines. Compared
with Jurkat cells, apoptosis in lung cancer cell lines occurred at a
lower extent (not exceeding 25%) and started at later time points
(24–48 h; Fig. 2B)
. The complete resistance to the Fas
agonistic mAb found in the growth inhibition assay in H187 and N417
lines was also detected when Fas-induced apoptosis was analyzed in
these cells (Fig. 2)
. No clear difference in Fas sensitivity between
NSCLC and SCLC cell lines was found, but a correlation
(R2 = 0.95; P = 0.06)
between Fas expression and sensitivity to Fas-induced cytotoxicity was
observed among the cell lines (Table 1)
. Fas-induced apoptosis was also
assessed by annexin V staining and forward/side scatter analysis,
leading to similar results (data not shown). Because the functionality
of the Fas pathway is a prerequisite to investigate its role in
drug-induced apoptosis, the four Fas-sensitive lung cancer lines (H460,
H322, GLC4, and GLC4/ADR) were selected for the following experiments.
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. The results
were analyzed by Western blotting and Northern blotting at different
time points (4–72 h). In none of the cell lines, regardless of the
drug or concentration used, was FasL expression significantly increased
when compared with the expression levels observed for the untreated
controls (Fig. 3A)
. Fas
expression was assessed during the same experiments described above; a
2–3-fold increase in the Fas expression at protein and RNA levels was
found in H460 but not in H322 cells (Fig. 3B)
. GLC4 and
GLC4/ADR cell lines showed similar results to H322 cells (data not
shown). To have a better insight into the influence of p53 status on
the up-regulation of the Fas levels upon drug exposure, the
p53 gene was submitted to direct sequencing. H460 was found
to be the only wt-p53 cell line (Table 3)
, whereas the other cell lines were
shown to harbor p53 mutations. Similar results of FasL and Fas
expression upon exposure to anticancer drugs were obtained using FACS
analysis (data not shown).
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. Furthermore, the
doxorubicin-resistant cell line GLC4/ADR, also cross-resistant to
topotecan and paclitaxel (Table 2)
, was five and three times more
sensitive than the parental cell line GLC4 to Fas-induced growth
inhibition and apoptosis, respectively (Table 2)
. A discrepancy between
drug and Fas-induced cytotoxicity was also observed between the NSCLC
cell lines H460 and H322 (Table 2)
. The correlation coefficient
(R2) between the sensitivity of the
cells to IC80 concentration of cisplatin and to
50 µg/ml of the CLB/CD95–15 analyzed at 24–72 h was <0.05. Similar
values were obtained when the other drugs (topotecan, gemcitabine, and
paclitaxel) were used.
Drug-induced Apoptosis after Blockage of the Fas Signaling.
To further assess the role of the Fas pathway in drug-induced apoptosis
in lung cancer cell lines, we also tested the ability of the anticancer
drugs to induce apoptosis in the absence of a functionally active Fas
signaling. Cells were preincubated either with the FasR-blocking
specific mAb CLB-CD95/2 or the FasL-neutralizing mAb NOK-2 1 h
preceding drug treatment. These two antibodies blocked almost
completely apoptosis induced, respectively, by Fas agonistic mAb and
recombinant FasL in Jurkat and lung cancer cell lines (Table 4)
. Nevertheless, the addition of these
antibodies failed to protect from apoptosis induced by cisplatin,
topotecan, gemcitabine, and paclitaxel in all of the lung cancer cell
lines tested, regardless of the drug used. Similar results of the
impact of Fas signaling blockade were obtained when different time
points (4, 24, and 48 h) and lower drug concentrations
(IC30) were analyzed or if another
FasL-neutralizing mAb (NOK-1) was used (data not shown). To further
validate our results, we tested Jurkat T-cells (a prototype of a
Fas-sensitive line) in similar experiments; the application of the
blocking antibodies did not protect Jurkat T cells from drug-induced
apoptosis (Table 4)
.
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. Comparable kinetic
cleavage was obtained with all lung cancer cell lines and also
different drugs. To further confirm that the processed caspase-8 was
active, its proteolytic activity was assessed upon drug exposure. A
concentration- and time-dependent increase in the caspase-8 proteolytic
activity up to 9-fold was observed, compared with the untreated
controls, corroborating the results of the Western blot (Fig. 4B)
. The increase in the caspase-8 proteolytic activity was
similar, regardless of the drug used. Regarding the other cell lines,
GLC4 and GLC4/ADR showed similar results to H460, but in H322 cells, a
less intense increase in the proteolytic activity was observed (data
not shown). Similar results in both Western blot and proteolytic
activity assay were observed when lung cancer cells were incubated with
Fas-antagonistic mAb CLB-CD95/2 prior to drug exposure in all lung
cancer cell lines.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition, the Fas system was shown to be functional in four of the cell lines analyzed (H460, H322, GLC4, and GLC4/ADR), because apoptosis was induced in these cells after exposure to Fas agonistic antibody. Despite showing similar levels of Fas expression as compared with Jurkat cells, the lung cancer cells (H460, H322, GLC4, and GLC4/ADR) showed less sensitivity to Fas-induced apoptosis. A possible explanation for that is the expression of natural inhibitors of apoptosis in these lung cancer cell lines, such as decoy receptor 3 and FLICE-inhibitory protein (40 , 41) . These inhibitors block the Fas pathway acting, respectively, at the level of FasL (40) or FADD (41) and have been shown to be highly expressed in lung cancer tumors (40 , 41) .
Despite the presence of a functional Fas pathway, we observed that drug-induced apoptosis in lung cancer cells occurred independently from Fas/FasL signaling. This conclusion was substantiated by different results: (a) drug-induced apoptosis was not accompanied by a significant FasL up-regulation compared with untreated controls, irrespective of the compound, cell line, or drug concentration; (b) lack of correlation between sensitivity to chemotherapy and Fas-induced growth inhibition and apoptosis; (c) blockade of the Fas signaling system, either by a Fas-antagonistic mAb or a FasL-neutralizing mAb failed to inhibit chemotherapy-induced apoptosis.
The lack of FasL induction above the level observed for the untreated
controls suggests no activation of the Fas/FasL signaling upon drug
exposure. The absence of FasL up-regulation after drug exposure also
found at the RNA level rules out a possible posttranscriptional or
posttranslational mechanism that could mask an increase in FasL levels
after drug exposure. This finding is in contrast with results reported
in different cell lines (19, 20, 21, 22, 23
, 29)
but is in accordance
with other reports (28
, 39)
. This discrepancy may be
attributable to different cell types or distinct experimental
conditions. The results showing up-regulation of FasL protein and RNA
levels in both treated and untreated cells (Fig. 3A)
suggest
that the levels of this molecule may vary physiologically in these
cells or be induced by changes in cell culture conditions and highlight
the importance of one control for each time point analyzed. We observed
drug-induced Fas up-regulation in the wt-p53 H460 cells; however,
because it occurred in the absence of FasL induction, we can speculate
that it was probably not sufficient to activate the proposed
autocrine/paracrine death loop (19)
in this cell line. In
fact, this Fas up-regulation might constitute an epiphenomenon, being a
result of p53 up-regulation, because we described p53 induction in this
cell line upon drug exposure (30)
. The absence of Fas
induction in the mt-p53 cell lines (H322, GLC4, and GLC4/ADR) is in
line with previous reports (21
, 42)
and suggests
that the Fas gene is under the transcription control of a
functional p53. The discrepancy between drug- and Fas-induced
cytotoxicity was evidenced by the fact that the most Fas-sensitive lung
cancer cell lines were not necessarily the most drug-sensitive ones. In
particular, there was a lack of correlation between drug- and
Fas-induced cytotoxicity when GLC4 and the mrp-overexpressing GLC4/ADR
were compared. GLC4/ADR has been shown to be at least 10 times more
resistant to doxorubicin than the parental line (43)
, and
in our hands, it was also found to be cross-resistant to topotecan and
paclitaxel (Table 2)
. However, GLC4/ADR was three times more sensitive
to Fas-induced apoptosis than the parental cell line GLC4. Moreover,
because the lack of correlation between Fas and chemotherapy-induced
cytotoxicity was irrespective of the drug used, the possibility of a
misleading analysis attributable to a drug-specific mechanism of
resistance is unlikely. These findings contrast with previous reports
that showed cross-resistance between Fas and drug-induced apoptosis, as
well as reduced Fas antigen expression in the resistant cell lines in
comparison with the parental ones (20
, 44)
. Our findings
suggest that chemotherapy and Fas-induced apoptosis do not share the
same pathway. This, however, does not rule out that both pathways may
converge at some downstream point, as suggested recently
(24)
. No protection from drug-induced apoptosis was
provided by the blockage of the Fas signaling system, either by
Fas-antagonistic mAb or a FasL-neutralizing mAb, in any of the cell
lines tested. The same results were obtained irrespective of the drug
used, excluding the possibility of the results being attributable to
cell line, drug concentration, or anticancer agent-specific effect.
Moreover, the observation of similar findings when Jurkat T cells (a
prototype of a Fas-sensitivity line) were used exclude that the results
obtained in lung cancer cell lines were attributable to a relatively
low Fas sensitivity of these cell lines.
Taken together, our results indicate independence of drug-induced apoptosis from the Fas/FasL signaling pathway in lung cancer cells. Our findings are consistent with recent reports showing that chemotherapy-induced apoptosis occurs in the absence of Fas/FasL interaction (24, 25, 26, 27, 28, 29 , 45) . According to these reports, different approaches to inhibit the Fas pathway, such as antagonistic antibodies (26 , 28 , 29) , overexpression of natural inhibitors like FLICE-inhibitory protein (27) , or the use of FADD-null cells (45) , do not protect from chemotherapy-induced apoptosis. Nevertheless, our results are in contrast with previous data proposing that the Fas/FasL signaling pathway mediates drug-induced programmed cell death (16, 17, 18, 19, 20, 21, 22, 23) . The differences among these reports, especially those in solid tumor lines (21 , 22) and ours, might be attributable to differences in the cell type or to difference in the drugs used in the studies, because it has been suggested that not all of the drugs would depend on the Fas pathway to induce apoptosis (46) . Nonetheless, the use of a panel of lung cancer cells, composed of both NSCLC and SCLC lines, the confirmation of our findings in lung cancer cells when additional experiments using Jurkat T-cells were performed, in addition to observation of similar results with the use of four anticancer drugs with different pharmacological mechanisms of action, give consistence to our results. Differences in the reagents used, in particular the specificity of the Fas-antagonistic mAbs, should also not be neglected as a possible explanation for the discrepancy in the results.
Because caspase-8 is an essential caspase in the Fas pathway, its status was also analyzed. Although drug-induced apoptosis did not require Fas/FasL signaling, caspase-8 was activated in a drug-, time-, and concentration-dependent fashion in all of the cell lines. This suggests that caspase-8 can also be activated by chemotherapy in a Fas-independent way. To the best of our knowledge, this is the first report of Fas-independent caspase-8 activation induced by conventional chemotherapeutic agents in solid tumor cells, being in line with recent reports obtained in leukemia cells (25 , 47, 48, 49, 50) . Because caspases form a redundant system, the finding of chemotherapy-induced caspase-8 activation in lung cancer cells certainly requires further investigation to define whether this is relevant for drug-induced apoptosis in solid tumor cells or whether it constitutes only a secondary event. Another issue that requires additional investigation is the mechanism responsible for caspase-8 processing and activation independently of Fas. Activation downstream of caspase-9, as recently described in cell-free extracts (47) , or an interaction with apoptotic protease activating factor-1, reported previously (51) , may constitute possible scenarios for drug-induced caspase-8 activation in lung cancer cells. Nevertheless, a role for another death receptor, such as tumor necrosis factor-related apoptosis-inducing ligand or tumor necrosis factor, in the processing of caspase-8 cannot be ruled out.
The results presented here may have clinical relevance, not only because we show that lung cancer cells do not depend upon Fas/FasL signaling to undergo drug-induced apoptosis but also because we identify a functional Fas pathway in most of the lung cancer cell lines. The possibility of inducing Fas-mediated apoptosis in vivo has been proposed as a potential approach for anticancer therapy (5 , 52) . Some reports have suggested a synergistic effect in vitro when chemotherapy and FasL or Fas-agonistic mAbs are combined (28 , 53) . Because drug and Fas-induced apoptosis involve two different and not completely overlapping pathways in lung cancer cells, the possibility to combine chemotherapy and immunotherapy can be envisaged as a new strategy for anticancer treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from Fundação
Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES-Brasil; to C. G. F.). 
2 Present address: Department of Medical Oncology,
Ioannina University Hospital, 45332 Greece.
3 To whom requests for reprints should be
addressed, at Department of Medical Oncology, Vrije Universiteit
Hospital, De Boelelaan 1117, 1081HV Amsterdam, the Netherlands. Phone:
31-20-44-3300; Fax: 31-20-444-3844; E-mail: g.giaccone{at}azvu.nl
4 The abbreviations used are: NSCLC, non-small
cell lung cancer; SCLC, small cell lung cancer; FasR, Fas receptor;
FasL, Fas ligand; FACS, fluorescence activated cell sorter; mAb,
monoclonal antibody; wt, wild type; mt, mutant; MFI, mean fluorescence
intensity; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; PI, propidium iodide.
Received 8/11/99; revised 10/18/99; accepted 10/20/99.
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 M. S. Park, M. De Leon, and P. Devarajan Cisplatin Induces Apoptosis in LLC-PK1 Cells via Activation of Mitochondrial Pathways J. Am. Soc. Nephrol., April 1, 2002; 13(4): 858 - 865. [Abstract] [Full Text] [PDF]
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M. P. Piechocki, F. Lonardo, J. F. Ensley, T. Nguyen, H. Kim, and G. H. Yoo Anticancer Activity of Docetaxel in Murine Salivary Gland Carcinoma Clin. Cancer Res., March 1, 2002; 8(3): 870 - 877. [Abstract] [Full Text] [PDF]
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 D. R. Jones, R. M. Broad, L. D. Comeau, S. J. Parsons, and M. W. Mayo Inhibition of nuclear factor {kappa}B chemosensitizes non-small cell lung cancer through cytochrome c release and caspase activation J. Thorac. Cardiovasc. Surg., February 1, 2002; 123(2): 310 - 317. [Abstract] [Full Text] [PDF]
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C. Huisman, C. G. Ferreira, L. E. Broker, J. A. Rodriguez, E. F. Smit, P. E. Postmus, F. A. E. Kruyt, and G. Giaccone Paclitaxel Triggers Cell Death Primarily via Caspase-independent Routes in the Non-Small Cell Lung Cancer Cell Line NCI-H460 Clin. Cancer Res., February 1, 2002; 8(2): 596 - 606. [Abstract] [Full Text] [PDF]
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 D. T. Jones, K. Ganeshaguru, A. E. Virchis, N. I. Folarin, M. W. Lowdell, A. B. Mehta, H. G. Prentice, A. V. Hoffbrand, and R. G. Wickremasinghe Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation Blood, November 1, 2001; 98(9): 2800 - 2807. [Abstract] [Full Text] [PDF]
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 J. Pan, G. Xu, and S.-C. J. Yeung Cytochrome c Release Is Upstream to Activation of Caspase-9, Caspase-8, and Caspase-3 in the Enhanced Apoptosis of Anaplastic Thyroid Cancer Cells Induced by Manumycin and Paclitaxel J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4731 - 4740. [Abstract] [Full Text] [PDF]
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S. Liekens, J. Neyts, E. De Clercq, E. Verbeken, D. Ribatti, and M. Presta Inhibition of Fibroblast Growth Factor-2-induced Vascular Tumor Formation by the Acyclic Nucleoside Phosphonate Cidofovir Cancer Res., July 1, 2001; 61(13): 5057 - 5064. [Abstract] [Full Text] [PDF]
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C. G. Ferreira, S. W. Span, G. J. Peters, F. A. E. Kruyt, and G. Giaccone Chemotherapy Triggers Apoptosis in a Caspase-8-dependent and Mitochondria-controlled Manner in the Non-Small Cell Lung Cancer Cell Line NCI-H460 Cancer Res., December 1, 2000; 60(24): 7133 - 7141. [Abstract] [Full Text]
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Y. Chen and M.-Z. Lai c-Jun NH2-terminal Kinase Activation Leads to a FADD-dependent but Fas Ligand-independent Cell Death in Jurkat T Cells J. Biol. Chem., March 9, 2001; 276(11): 8350 - 8357. [Abstract] [Full Text] [PDF]
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10%. B, the percentage of specific Fas-induced
apoptosis was determined by FACS analysis of PI-stained nuclei (see
"Materials and Methods") after exposure to the anti-Fas mAb
CLB-CD95/15, used at the concentration of 50 µg/ml at different time
points (4–72 h). Results depicted represent a mean of three
independent experiments in which the SD was 
