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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 265-274
Myeloma Cells Selected for Resistance to CD95-Mediated Apoptosis
Are Not Cross-Resistant to Cytotoxic Drugs: Evidence for Independent
Mechanisms of Caspase Activation
By
Terry H. Landowski,
Kenneth H. Shain,
Marc M. Oshiro,
Ibrahim Buyuksal,
Jeffrey S. Painter, and
William S. Dalton
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ABSTRACT |
We have previously shown that selection for resistance to the
anthracenes, doxorubicin or mitoxantrone, results in coselection for
resistance to CD95-mediated apoptosis (Landowski et al: Blood 89:1854, 1997). In the present study, we were interested in determining if the converse is also true; that is, does selection for CD95 resistance coselect for resistance to chemotherapeutic drugs. To
address this question, we used two isogenic models of CD95-resistant versus CD95-sensitive cell lines: 8226/S myeloma cells selected for
resistance to CD95-mediated apoptosis; and K562 cells expressing ectopic CD95. Repeated exposure of the CD95-sensitive human myeloma cell line, 8226/S, to agonistic anti-CD95 antibody resulted in a cell
line devoid of CD95 receptor surface expression and completely resistant to CD95-mediated apoptosis. Multiple clonal populations derived from the CD95-resistant cell line showed no difference in
sensitivity to doxorubicin, mitoxantrone, Ara-C, or etoposide, demonstrating that cross-resistance between Fas-mediated apoptosis and
drug-induced apoptosis occurs only when cytotoxic drugs are used as the
selecting agent. Using the inverse approach, we transfected the
CD95-negative cell line, K562, with a CD95 expression vector. Clones
expressing variable levels of cell-surface CD95 were isolated by
limiting dilution, and analyzed for sensitivity to CD95-mediated apoptosis and response to chemotherapeutic drugs. We show that CD95
surface expression confers sensitivity to CD95-mediated apoptosis; however, it does not alter response to chemotherapeutic drugs. Similarly, doxorubicin-induced activation of caspases 3 and 8 was
identical in the CD95-sensitive and CD95-resistant cell lines in both
isogenic cell systems. In addition, prior treatment with the CD95
receptor-blocking antibody, ZB4, inhibited CD95-activated apoptosis in
8226/S cells, but had no effect on doxorubicin cytotoxicity. These
results show that CD95 and chemotherapeutic drugs use common apoptotic
effectors, but the point of convergence in these two pathways is
downstream of CD95 receptor/ligand interaction.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IN RECENT YEARS, evidence has been
accumulating to support the hypothesis that chemotherapeutic drugs
induce an apoptotic response in target cells.1-3 This
cytotoxic response reportedly uses signal transduction pathways common
to physiological mechanisms of programmed cell death. For example,
treatment of the human leukemia cell line U937 with either etoposide or
agonistic anti-CD95 monoclonal antibody (MoAb) results in the
activation of cysteine proteases and DNA fragmentation.4
The cytotoxic effects of both these agents can be inhibited by the
caspase 3 (CPP32/apopain) specific tetrapeptide inhibitor DEVD,
demonstrating the participation of caspase 3 in both signal
transduction cascades. In a second study, treatment of human glioma
cell lines with the broad specificity caspase inhibitor YVAD was shown
to reduce cell sensitivity to both cisplatin- and
CD95-mediated cell death.5 Although these studies, and
others, indicate that chemotherapeutic drugs and immune effectors share
common mediators, specific enzyme activation events and the point of
convergence in the apoptotic signal transduction pathway remain to be defined.
Conflicting data has been reported regarding the role of CD95/CD95
ligand interactions in drug-induced cell death. Studies using a variety
of cell lines in which the CD95 receptor and CD95 ligand were shown to
be induced by drug treatment have been interpreted as an indication
that drug-induced cell death is mediated by the CD95/CD95 ligand
system.6-9 This activity was reported to be inhibited by
anti-APO-1 F(ab')2 fragments. Subsequent studies by
the same group demonstrated deficient activation of the caspases as a
potential mechanism of cross-resistance between drug- and CD95-mediated
cell death.10,11 In contrast, a study by Villunger et
al12 showed that treatment with the inhibitory anti-CD95 MoAb ZB4 or the anti-CD95 ligand MoAb Nok2 had no effect on the cytotoxicity of cisplatin, doxorubicin, or fludarabine. However, both
were capable of inhibiting apoptosis mediated by the agonistic CH-11
antibody or recombinant human CD95 ligand.12 Villunger et
al provided further support for the hypothesis that cytotoxic drugs and
CD95 cross-linking initiate independent signals to programmed cell
death using CEM T-cell leukemia cells rendered resistant to CD95 by
expression of the cowpox virus protein crmA. This protein has been
shown to inhibit the activity of caspase 8, which is requisite to
CD95-mediated apoptosis.13 However, its expression had no
effect on the cell sensitivity to doxorubicin, cisplatin, or high-dose
fludarabine in the expressing cells. Similarly, Eischen et
al14 found no inhibitory effects of the nonagonist
anti-CD95 antibody ZB4 in the cytotoxic effects of etoposide on Jurkat
T cells; however, this antibody did block apoptosis induced by ligation of the T-cell receptor.
Early studies on CD95-mediated apoptosis showed that forced
overexpression of the cytoplasmic death domain of the CD95 receptor was
sufficient to induce programmed cell death, suggesting apoptotic signal
transduction involved aggregation of this region.15 This observation has been further supported by a recent study showing that
UV-irradiated keratinocytes initiated the caspase signal transduction
pathway via CD95 receptor aggregation without ligation by CD95
ligand.16 UV activation of caspase 3 and subsequent apoptosis of the cells could be inhibited by a dominant negative mutant
of FADD, but was not affected by antibodies directed at external
epitopes of CD95 or CD95 ligand. Taken together, all of these studies
show the complexity of the signal transduction pathway leading to
apoptosis, and suggest multiple mechanisms of caspase activation in
response to various stressful stimuli.
We recently showed that in vitro selection for resistance to the
anthracenes, doxorubicin, or mitoxantrone, results in the coselection
of cell lines resistant to CD95-mediated apoptosis.17 Two
mechanisms of CD95 resistance were identified in drug-resistant cells.
One mechanism is associated with a dose-dependent reduction in the
surface expression of the CD95 receptor in cells chronically exposed to
anthracenes. This reduction of CD95 receptor expression in
drug-resistant cells occurred at the level of mRNA transcription. The
second mechanism of resistance to CD95-mediated apoptosis was likely
related to alterations in the apoptotic signal transduction pathway
that may be common to CD95- and drug-induced apoptosis. In the present
study, we were interested in determining whether cells selected for
resistance to CD95-mediated apoptosis were also resistant to
chemotherapeutic drugs. To investigate this possibility, the human
myeloma cell line, RPMI 8226, was selected for CD95 resistance by
repeatedly exposing cells to the apoptosis-inducing MoAb, CH-11, and
examining these cells for sensitivity to various chemotherapeutic
drugs. If chemotherapeutic agents induce programmed cell death via
interactions of the CD95 receptor and CD95 ligand, as some reports have
suggested, then we would anticipate cross-resistance to cytotoxic drugs
in the CD95-resistant cell lines. Clonal populations derived from the
CD95-resistant 8226 cell line showed no differences in sensitivity to
drug when compared with the CD95-sensitive 8226 parental cell line.
To further define the role of CD95 in sensitivity to chemotherapeutic
drugs, we used an erythroleukemia cell line, K562, which does not
express CD95 and is inherently resistant to CD95-mediated apoptosis.
This cell line also displays a relatively high level of resistance to
most cytotoxic drugs, probably due to the expression of the Bcr-abl
oncogene.18 If CD95 plays a direct role in apoptosis induced by chemotherapeutic drugs, we would anticipate enhanced sensitivity to cytotoxic drugs in cells with enforced expression of
CD95. K562 cells were transfected with CD95 under the control of a
cytomegalovirus (CMV) promoter, and clonal populations
analyzed for sensitivity to CD95-mediated apoptosis and
chemotherapeutic drugs. Although CD95 expression conferred sensitivity
to the agonistic antibody CH-11, clones expressing high levels of CD95
were no more sensitive to doxorubicin, melphalan, or etoposide than
were the untransfected or empty vector control cells. Results of our experiments indicate that (1) treatment with agonistic anti-CD95 MoAb
selects for a cell line that fails to express the CD95 receptor, and is
resistant to CD95-mediated apoptosis; and (2) the presence or absence
of CD95 receptor expression has no effect on sensitivity to
chemotherapeutic agents.
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MATERIALS AND METHODS |
Cell culture.
The human multiple myeloma cell line 8226 was originally obtained from
American Type Culture Collection (ATCC; Rockville MD), and
maintained in RPMI (GIBCO, Grand Island, NY) supplemented with 5%
fetal calf serum (FCS), 100 mmol/L L-glutamine, and 100 U/mL
penicillin/streptomycin (Gemini, Calabasas, CA). Selection for the
CD95-resistant variant, 8226/F4, was done by repeated exposure to the
apoptosis inducing anti-CD95 MoAb CH-11 (MBL, Watertown, MA). Antibody
was added to the tissue culture medium at 200 ng/mL, and the cells
incubated for 72 hours at 37°C. This treatment initially induced
55% to 70% apoptosis in the parental 8226/S cell line. Surviving
cells were isolated on a Ficoll gradient (Pharmacia, Piscataway, NJ),
and expanded in complete media without MoAb for 2 weeks. The process
was repeated a total of four times, and the 8226/F4 cell line was
maintained in culture without further selection until analysis. Clonal
populations of the 8226/F4 cell line were obtained by limiting dilution.
Antibodies and measurement of apoptosis and cytotoxicity.
Surface expression of the CD95 receptor was determined by flow
cytometry using the nonapoptosis-inducing MoAb UB-2 (MBL). Mouse
anti-human IgG1 (Dako, Carpinteria, CA) serum served as the isotype control. CD95-mediated cell death was assayed by staining with Annexin V-FITC (Clontech, La Jolla, CA) and flow
cytometry analysis.19 Cells were plated at 5 × 105/mL and incubated with CH-11 MoAb at the indicated
concentrations and times. Samples were washed in phosphate-buffered
saline (PBS) and stained with Annexin V-FITC and propidium iodide (PI)
according to the manufacturer's protocol (Clontech). Apoptosis was
measured on a FACScan flow cytometer and analyzed with CellQuest
software (Becton Dickinson, Mountain View, CA). Apoptosis was confirmed by fluorescent microscopic examination of CH-11 and doxorubicin-treated cells using Annexin V-FITC and Dapi counterstain (Vector Laboratories, Burlingame, CA).
Cytotoxicity of doxorubicin, etoposide, Ara-C (all from Sigma, St
Louis, MO) and mitoxantrone (Wyeth-Ayerst, Pearl River, NY) were
determined by MTT assay as previously
described.20 Cells were plated at 8 × 104/mL for the 8226-derived cell lines, or 5 × 104 for K562 cell lines, in 96-well plates and incubated
with serial dilutions of cytotoxic drug. After 96 hours of drug
exposure, 50 µL of MTT dye (2 mg/mL in PBS; Sigma) was added to each
well, and allowed to incubate 4 hours. Plates were centrifuged and the media replaced with 100 µL dimethyl sulfoxide (DMSO; Fisher
Scientific, Pittsburgh, PA) to solubilize the insoluble formazan
complex. Optical density at 540 nm was determined with a Dynex II Elisa plate reader (Dynatech, Chantilly, VA). IC50 was calculated
by linear regression analysis of the log-linear plot for percent survival versus drug concentration. Student's t-test was used to analyze differences in drug response from three independent experiments (.05 significance level). For competition with the inhibitory anti-CD95 MoAb, cells were preincubated for 1 hour at
37°C with 0.5 µg/mL ZB4 (MBL) before addition of the drug or CH-11 agonist antibody. Drug-induced apoptosis was confirmed by labeling fragmented DNA with terminal deoxynucleotidyl transferase (TdT) using the ApopTag kit (Oncor, Gaithersburg, MD) and flow cytometric analysis.
RNA extraction and reverse transcriptase-polymerase chain reaction
(RT-PCR).
Total RNA was extracted from 107 cells in log growth phase
by lysis in guanidine isothiocyanate followed by cesium chloride density centrifugation and ethanol precipitation. Total RNA was digested with RNase-free DNase (Boehringer Mannheim, Indianapolis, IN) for 15 minutes at 37°C and repurified by the
RNeasy kit according to the manufacturer's protocol (Qiagen, La Jolla,
CA). CD95 antigen analysis was performed by 30 cycles of RT-PCR as
previously described.17 For detection of CD95 ligand,
DNase-treated RNA was transcribed to cDNA by extension of dT primers
with 200 U of Superscript II RT (GIBCO) followed by 30 cycles of PCR with primers 5'-TAAAACCGTTTGCTGGGGC-3' and
5'-CTCAGCTCCTTTTTTTCAGGGG-3'.21 The
identity of all products were confirmed by direct sequencing, and a
215-bp fragment of Histone 3.3 was amplified as a control for mRNA
integrity and quantitation.22 The full-length CD95 cDNA was
inserted into pcDNA3.1 expression vector according to the
manufacturer's protocol. K562 cells were transfected with 2 µg of
plasmid DNA with Superfect (Qiagen) and selected with 800 µg/mL G418.
CD95-expressing cells were enriched by FACS sorting, followed by
limiting dilution cloning.
RNase protection assay.
RNase protection was performed using the Pharmingen RiboQuant hAPO-3
kit according to the manufacturer's protocol
(Pharmingen, San Diego, CA). The multi-probe template was prepared by
32P incorporation in an in vitro transcription reaction,
and free nucleotide removed on a G50 column (5 Prime  3 Prime, Inc.,
Boulder, CO). Purified probe (1 to 1.5 × 106 cpm specific activity) was combined with 20 µg of
total RNA, isolated from 5 × 106 cells in 1 mL of
Trizol reagent (GIBCO). Hybridization was allowed to proceed through a
temperature range of 90°C to 56°C over 16 hours before RNase
digestion. After RNase digestion for 45 minutes at 37°C, samples
were separated on a 5% denaturing gel, and protected fragments
quantitated by phosphor imaging using Image Quant software (Molecular
Dynamics, Sunnyvale, CA).
Analysis of caspase activity.
For analysis of caspase 3 and caspase 8 activation, doxorubicin or
anti-CD95 treated cells (2 to 4 × 106) were washed
with PBS and resuspended in lysis buffer (30 mmol/L HEPES, 10 mmol/L
NaCl, 5 mmol/L MgCl2, 25 mmol/L NaF, 1 mmol/L EGTA, 1 mmol/L EGTA, 1% Triton X-100, 10% glycerol, 2 mmol/L
Na-orthovanadate, 25 µg/mL leupeptin, 10 µg/mL aprotinin, 2 mmol/L
phenylmethyl sulfonyl fluoride [PMSF], and 10 µg/mL soybean trypsin
inhibitor) on ice for 30 minutes. Lysates were centrifuged at 14,000 rpm for 15 minutes at 4°C. Total protein determination was done
using Bio-Rad Bradford Reagent (Bio-Rad, Hercules, CA), and 100 µg of protein separated on 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane. Immune detection of caspase 3 was done using a rabbit polyclonal antibody which recognizes both p32 procaspase and the p20/p17 activated
subunits (generously provided by Dr H-D. Wang, H. Lee Moffitt Cancer
Center). Caspase 8 was detected using a goat polyconal antibody (Santa
Cruz Biotech, Santa Cruz, CA). Secondary antibodies were horseradish
peroxidase (HRP)-conjugated (Dako), and blots were developed with the
ECL detection system (NEN, Boston, MA).
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RESULTS |
Selection of a CD95-resistant cell line.
Chemotherapeutic agents commonly used in the treatment of malignant
disease have been shown to activate common apoptotic pathways in target
cells.1-3 Although the intracellular activity of many of
these compounds has been extensively studied, it is still unclear how
the cellular damage incurred is translated into a signal for programmed
cell death. We have previously reported that in vitro selection for
resistance to the anthracenes, doxorubicin, or mitoxantrone results in
a coselection for cells that are resistant to CD95-mediated apoptosis.17 To determine if selection for resistance to
CD95-mediated apoptosis also selected for resistance to cytotoxic
drugs, we subjected the human myeloma cell line 8226/S to repeated
exposure of the agonistic anti-CD95 MoAb CH-11. Treatment of 8226/S
with 200 ng/mL was found to evoke the maximal response in the 8226/S cell line, resulting in 55% to 70% apoptosis of the unselected population.17,23,24 Surviving cells were rescued on a
Ficoll-Hypaque gradient and expanded in culture for four consecutive
selections. After repeated exposure to CH-11, the anti-CD95-selected
cell line was maintained in culture without further selection pressure for 4 months before analysis. This CD95-resistant cell line, designated 8226/F4, did not respond to cross-linking with agonistic anti-CD95 MoAb
(Fig 1) or with soluble recombinant CD95
ligand (data not shown). To ensure the resistance to CD95-mediated
apoptosis was not simply caused by clonal variation, we examined seven
subclones of the 8226/F4 cell line derived by limiting dilution. Clonal populations of the 8226/F4 cell line were uniformly resistant to
CD95-mediated apoptosis in response to CH-11 or soluble CD95 ligand.
This is in contrast to 62% cell death in the parental 8226/S cell
line, and 12% death in the multidrug-resistant cell line 8226/Dox40,
which is maintained under continuous selection with 4 × 10 7 mol/L doxorubicin.25 Extended
exposure of the cells for 48 hours with 200 or 1,000 ng/mL CH-11 MoAb
did not increase the CD95-mediated cell death in any of the cell lines
examined. Both 8226/F4 and 8226/Dox40 were highly resistant to
cross-linking by the agonistic CD95 antibody at all time points and
concentrations examined (Fig 1).
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| Fig 1.
CD95-mediated apoptosis in 8226 myeloma cell lines. Cells
were treated with 200 ng/mL or 1,000 ng/mL anti-CD95 antibody CH-11 and
incubated for the indicated times. Apoptosis was determined by staining
with Annexin V FITC and flow cytometry analysis.
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To identify the mechanism of CD95 resistance, we first examined the
8226/F4 cell line for surface expression of the CD95 receptor. These
cells were stained with the nonapoptosis-inducing anti-CD95 MoAb UB2
and analyzed by flow cytometry. In contrast to the parental 8226/S cell
line, which displays relatively high expression of the CD95 receptor,
cell-surface expression of the CD95 receptor was entirely negative in
the 8226/F4 cell line (Fig 2A). Using primers derived from the cytoplasmic region of the CD95 receptor, mRNA
expression was examined by RT-PCR. Only very minimal levels of CD95
mRNA could be detected with 30 cycles of amplification (Fig 2B).
Examination of seven clones derived from the 8226/F4 cell line showed
similar results, with negligible CD95 expression and function in all
cases (data not shown).
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| Fig 2.
(A) Surface expression of the CD95 antigen in cells
selected for resistance to CD95-mediated apoptosis. Cells were stained
with the non-apoptosis-inducing antibody UB2 and analyzed by flow
cytometry. In the 8226/F4 cell line, the UB2-specific staining
completely overlaps the isotype control. (B) RT-PCR of CD95 antigen
mRNA expression. Total RNA was extracted from 8226/S and 8226/F4 cells,
and the CD95 antigen expression determined by RT-PCR as previously
described. Histone 3.3 was amplified as a control for RNA integrity and
equal gel loading.
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To examine the possibility that selection for CD95 resistance altered
other components of the CD95 apoptotic pathway, we used an RNase
protection assay with a multiprobe template for the expression of known
effectors (Fig 3). This template includes
the constitutive genes GAPDH and L32 for relative quantitation of
protected RNA hybrids. Analysis of CD95 receptor mRNA by RNase
protection correlated with RT-PCR and flow cytometry (see Fig 2),
showing high expression in the parental 8226/S cell line, very low
expression in the doxorubicin-resistant 8226/Dox40, and no CD95
receptor mRNA detected in the CD95-resistant 8226/F4. Comparison of the
downstream mediators of CD95-mediated apoptosis demonstrates 30% and
37% reduction in the level of caspase 8 (FLICE/MACH/Mch5), in 8226/F4,
and 8226/Dox40, respectively, compared with the parental 8226/S.
Additionally, expression of the CD95-associated phosphatase FAP-1 is
reduced in the cell lines with reduced CD95 expression. FAP-1 is
reported to be constitutively associated with the cytoplasmic region of
the CD95 receptor.26 The CD95-deficient cell lines 8226/F4
and 8226/Dox40 fail to express FAP-1, implying a potential coregulation
of FAP with the CD95 receptor. However, this possibility remains to be
investigated. No significant differences were found in the expression
levels of the adapter proteins FADD and TRADD. Other death receptors, including TNFRI and DR3, are equally expressed in all three cell lines.
Although the multiprobe template used in this study contains a CD95
ligand fragment, we were not able to detect CD95 ligand mRNA by RNase
protection assay. Therefore, we examined CD95 ligand expression by
RT-PCR using previously published conditions.21 All cell
lines expressed low constitutive levels of CD95 ligand, which were
likely below the limits of detection of the RNase protection assay. No
significant differences in CD95 ligand expression were seen in any of
the cell lines (data not shown).
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| Fig 3.
RNase protection assay. Total RNA was isolated from
8226/S, 8226/F4, and 8226/Dox40 cell lines and hybridized with a
multitemplate probe before digestion by RNase. Protected fragments were
separated on a denaturing acrylamide gel and analyzed by PhosphorImage
analysis. Fragment assignment was determined by migration distance
relative to unprotected standards.
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Cytotoxicity analysis.
If cytotoxic drugs initiate cell death via CD95 receptor/ligand
interactions, then we would predict that the CD95 receptor negative
cell line, 8226/F4, would display resistance to cytotoxic agents as
compared with the parental cell line, 8226/S. Using the MTT dye
reduction assay, we examined the cytotoxicity of doxorubicin, mitoxantrone, Ara-C, vincristine, and VP-16 in 8226/F4 versus 8226/S.
The P-glycoprotein-expressing cell line 8226/Dox40 was included as a
control for multidrug resistance.25 Drug sensitivity profiles for the 8226/F4 cell line were virtually identical to the
parental cell line 8226/S for all agents tested
(Fig 4).
Additionally, individual clones of the 8226/F4 cell line were equally
sensitive, despite the absence of CD95 receptor expression in all cell
lines examined. This response was highly reproducible over a wide range of drug concentrations. In contrast, 8226/Dox40, an MDR1-positive cell
line, is cross-resistant to all agents except Ara-C, as previously described.17,25 Because the MTT dye reduction assay does
not directly demonstrate apoptotic cell death, 8226/S and 8226/F4 cells
were treated with 105 mol/L VP-16 for 24 hours, and
flow cytometry used to analyze for TdT labeling. Again, there was no
difference in the degree of DNA fragmentation in the CD95-positive cell
ine, 8226/S, compared with the CD95-negative cell line, 8226/F4 (data
not shown).
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| Fig 4.
Dose-response profiles of 8226 myeloma cells selected for
resistance to CD95-mediated apoptosis. Cells were plated in 96-well
plates at 5 × 104/mL with the indicated concentrations of
chemotherapeutic drugs. Percent survival was determined by MTT dye
reduction after 96 hours of drug exposure, and the IC50
calculated by linear regression analysis. Each drug was examined by
three independent assays with eight replicates per assay. (A) VP-16;
(B) ara-C; (C) vincristine; (D) mitoxantrone; (E) doxorubicin; (F)
doxorubicin ± ZB4. ( ), 8226/S; ( ), 8226/Dox40; ( ), 8226/F4.
For plots A, B, C, D, and E, open symbols represent individual clones
of 8226/F4. In plot F, open symbols show the effects of 1 hour of
preincubation with 500 µg/mL ZB4 on doxorubicin cytotoxicity or
CH-11-mediated apoptosis (inset).
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Previous reports have shown that antibodies which block CD95
receptor/ligand interactions inhibit drug cytotoxicity.8,9 Therefore, we preincubated the parental 8226/S and four subclones of
the 8226/F4 cell lines with the CD95 receptor-blocking antibody ZB4 for
1 hour before the addition of doxorubicin or the CD95-agonist MoAb,
CH-11, in the MTT assay. Inactivation of the CD95 receptor by 0.5 µg/mL ZB4 did not inhibit the cytotoxicity of doxorubicin in either
the 8226/S or the 8226/F4 cell lines. In contrast, apoptosis mediated
by the CD95-agonist MoAb CH-11 is almost completely inhibited by this
concentration of ZB4 (Fig 4). The inability of ZB4 to inhibit
drug-induced cell death further supports the conclusions that CD95/CD95
ligand interactions do not participate in the apoptotic pathway
initiated by cytotoxic drugs.
CD95 expression does not enhance chemosensitivity in K562 cells.
Because the K562 cell line expresses no CD95, and displays a relatively
high level of intrinsic resistance to chemotherapeutic drugs, we were
interested in determining if CD95 expression would enhance the efficacy
of cytotoxic drugs. Using RT-PCR, we isolated full-length CD95 mRNA
from normal peripheral blood lymphocytes (PBLs), inserted
it into the pcDNA3.1 expression vector, and transfected K562 cells.
After selection with G418, transfectants were cloned by limiting
dilution and analyzed for CD95 expression and function (Fig 5). Data are shown for K562/fasH2,
which expresses high levels of CD95; and K562/fasB7, which expresses
minimally detectable levels of CD95 on the cell surface. Two additional
clones with high CD95 expression and two clones with low or negative
CD95 expression were analyzed, and found to correlate as well. Exposure to 500 ng/mL CD95 agonistic antibody, CH-11, induced no significant cell death in the parental K562 cell line, or cells transfected with
the empty vector, pcDNA3.1. In contrast, clone K562/fasH2, which
expresses high levels of CD95, showed 47% apoptosis after 24 hours of
exposure to CH-11. Clone K562/fasB7, which expresses minimal levels of
CD95, demonstrated apotosis equivalent to background cell death. In all
clones examined, the degree of CH-11-induced cell death correlated
directly with the level of CD95 surface expression.
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| Fig 5.
Expression and function of CD95 in K562-transfected cell
lines. K562 cells were transfected with pcDNAfas, selected with G418,
and cloned by limiting dilution. (A) Surface expression of CD95
detection by staining with UB2-FITC or IgG1-FITC isotype control, and
flow cytometry analysis. Filled peaks represent isotype control. (B)
Annexin V-FITC analysis of CH-11-mediated apoptosis. Cells were
treated with 500 ng/mL CH-11 anti-CD95 antibody for 24 hours followed
by Annexin-V staining and flow cytometric analysis of programmed cell
death. Horizontal axis is Annexin V-FITC, y-axis is propidium iodide
staining. Background levels of apoptosis with mouse IgM was determined
in an identical manner, and found to range from 2% to 12% in all cell
lines.
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Using both MTT and Annexin V analyses, the CD95-expressing K562 clones
were further examined for differential sensitivity to doxorubicin,
melphalan, or etoposide. Linear regression analysis of 96-hour MTT
dose-response curves showed no significant differences in the
IC50 values for all drugs tested comparing the
CD95-expressing clones to untransfected or empty vector control cell
lines (Table 1). Cell death after exposure
to VP-16 was significantly delayed in all of the K562-derived cell
lines compared with the 8226 myeloma-derived cell line. However, again,
no significant differences were seen in the level of VP-16-induced
apoptosis as measured by Annexin V staining in CD95 transfected cell
lines as compared with the K562 parental cell line, or K562/pcDNA3.1
(data not shown).
Analysis of caspase activity.
To establish that cytotoxic drugs used the caspase signal transduction
cascade in these cells, 8226/S and 8226/F4 cells were exposed to 5 × 106 mol/L doxorubicin and assayed for
activation of caspase 3 and caspase 8. Western blot analysis showed
equivalent activation in the 8226/S and 8226/F4 cell lines with the
appearance of catalytic fragments after 4 hours of doxorubicin exposure
(Fig 6). As previously reported, caspase
activation in the K562-derived cell lines was significantly delayed
compared with 8226 myeloma cells.27 However, no differences
were identified in the CD95-expressing cells as compared with the
parental cell line or empty vector transfectants. These data are
compatible with studies in many other cell lines showing that cytotoxic
drugs use apoptotic pathways common to physiological stimuli. However,
the lack of cross-resistance to chemotherapeutic drugs in cells that do
not express CD95 indicates distinct pathways of caspase activation by
CD95 cross-linking compared with activation by cytotoxic drugs.
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| Fig 6.
Western blot analysis of caspase 3 and caspase 8 activation after doxorubicin treatment in 8226 and K562 cell lines.
Cells were exposed to 5 × 106 mol/L doxorubicin for
the indicated times, and cell lysates analyzed for caspase 3 and
caspase 8 active subunits. (A) 8226/S and 8226/F4; (B) K562
untransfected, K562/pcDNA3.1 empty vector, and the CD95 high-expressing
K562/fasH2. Although procaspase 8 can be detected in K562 cells, the
active subunits are below the limits of detection at the time points
examined. Data shown are representative of three independent
experiments.
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DISCUSSION |
Signal transduction pathways for cytotoxic drugs and physiological
mediators of apoptosis have been shown to converge into a common final
pathway.28 Thus, alterations in shared effectors could
potentially result in cross-resistance between chemotherapeutic drugs
and CD95-mediated apoptosis. We have previously shown that selection
for resistance to chemotherapeutic drugs results in a coselection for
resistance to CD95-induced apoptosis.17 In this report, we
show that selection for resistance to CD95-mediated apoptosis does not
coselect for resistance to chemotherapeutic drugs. Using the human
myeloma cell line 8226, which has long served as a useful model for
multidrug resistance, we selected a cell line resistant to
CD95-mediated apoptosis, 8226/F4. Analysis of this cell line, and
multiple subclonal populations derived from this cell line, showed no
resistance to several cytotoxic agents as compared with the parental,
CD95-sensitive cell line. Furthermore, no differences in the activation
of distal effectors of apoptosis were observed between the
CD95-sensitive and CD95-resistant cell lines when exposed to
chemotherapeutic drugs. Thus, while selection for resistance to
chemotherapeutic agents coselects for resistance to CD95-mediated
apoptosis, we find that in the human myeloma cell line 8226, selection
for resistance to CD95-mediated apoptosis does not select for
resistance to chemotherapeutic drugs. In addition, constitutive
expression of CD95 in the K562 cell line resulted in CD95-induced
apoptosis, but no changes in the sensitivity to chemotherapeutic drugs.
These data have important implications for current understanding of
mechanisms contributing to cellular response to chemotherapeutic
agents, and the drug-resistant phenotype.
Our data agree with that published by Eischen et al,14 who
selected for CD95 resistance in Jurkat T-cell leukemia cells after
pretreatment with mutagenic substances. Using immunoblotting and an
affinity labeling technique to identify specific caspase activity,
these investigators showed that the cytotoxic activity of etoposide,
doxorubicin, topotecan, and cisplatin in CD95-resistant Jurkat cells is
indistinguishable from that of the parental cell line. In their study,
one CD95-resistant clone was reported to be negative for surface
expression of the CD95 receptor, while four additional clones selected
for CD95 resistance were reported to express normal levels of the CD95
receptor. Thus, it is likely that their protocol selected for
alterations in signal transduction effector molecules that participate
in CD95-mediated, but not drug-mediated, cell death.
Our selection protocol, which included no mutagenic pressure, resulted
in a cell line that did not express the CD95 receptor. In addition,
multiple subclones of the CD95-selected cell line were also negative
for CD95 receptor. These results indicate that the mechanism of
resistance to CD95-mediated apoptosis in the selected cells is directly
related to the absence of receptor expression. To gain insight into
mediators of apoptosis that may be differentially affected by selection
for CD95 resistance versus drug resistance, we examined the expression
of several known mediators of the apoptotic signal transduction cascade
using an RNase protection assay. This assay showeded a slight reduction
in caspase 8 mRNA expression in both CD95- and drug-resistant cell
lines. Expression of the death-inducing signaling complex (DISC)
components FADD and caspase 8 are not significantly changed in the
8226/F4 cell line, and the absence of CD95 receptor does not affect the
activation of the proteolytic cascade in response to cytotoxic drugs.
These data emphasize the existence of at least two distinct routes for caspase activation, and demonstrate that the point of convergence in
the signaling pathways of drug- and CD95-mediated apoptosis is before
the activation of caspase 3, but downstream of CD95 receptor/ligand interaction.
Several investigators have shown induced expression of the CD95
receptor and ligand by chemotherapeutic drugs.6,7,12,21 This observation has led to the suggestion that the cytotoxicity of
these agents is at least partially due to engagement of the CD95
receptor and initiation of the apoptotic cascade. However, attempts to
provide direct evidence of receptor/ligand interactions in these
studies have yielded conflicting reports. Kasibhatla et
al21 showed a reduction of etoposide cytotoxicity in Jurkat T cells with the use of a recombinant CD95-Fc chimeric protein, which
inhibits CD95 receptor/ligand interactions. Likewise, Fulda et
al8 and Friesen et al6 reported that
preincubation with Fab fragments of the anti-CD95 antibody APO-1
inhibited the cytotoxicity of doxorubicin, etoposide, and cisplatin. In
contrast, Eischen et al14 and Villunger et al12
showed no inhibitory effects of the non-cross-linking antibody ZB4, as
we also found in this study. This discrepancy may be due to the
particular reagents used for competition. The ZB4 and APO-1 antibodies
are likely directed to different epitopes of the CD95 receptor, and one
potential explanation for the disparate results reported in the
literature may be related to the different MoAbs used in these studies.
A second possibility for the differential effects of CD95 inhibitory
reagents in reducing drug cytotoxicity may be related to the particular
cell lines analyzed. For example, Scaffidi et al29 recently
proposed two classifications of tissues based on DISC formation and
kinetics of caspase activation. In their study, the T-cell leukemias
CEM and Jurkat were both classified as type II, based on their delayed
induction of caspases 8 and 3 in response to cross-linking by anti-CD95
MoAb. In these cell lines, caspase activation and subsequent apoptosis
were inhibited by forced overexpression of the anti-apoptotic proteins
Bcl-2 or Bcl-XL, indicating that caspase 8 and caspase 3 activation occurs downstream of mitochondrial perturbation. In
contrast, a variety of other cell types, including the B-cell line SKW6 and the T-lymphoma cell line H9 were classified as type I. Type I cells
showed rapid DISC formation and direct activation of caspases 8 and 3 by CD95 agonist antibody. This activity could not be blocked by Bcl
family members, indicating caspase 8 activity and commitment to
apoptosis occurred independent of mitochondrial perturbation. These
observations suggest two independent routes to caspase activation, one
which is initiated by caspase 8 at the level of DISC formation before
mitochondrial perturbation, and one which occurs downstream of the
mitochondrial permeability transition and cytochrome c release. Our
data suggest that cytotoxic drugs may use a DISC-independent route to
caspase activation, similar to that identified in the type II cells.
Further definition of these alternative pathways and identification of
factors involved may be relevant to defining strategies to overcome the
drug-resistant phenotype.
In our previous study, we showed that selection for resistance to the
anthracenes doxorubicin or mitoxantrone coselects for resistance to
CD95-mediated apoptosis. Although the primary mechanism of
cross-resistance was found to be a reduction in the cell-surface expression of the CD95 receptor, some cell lines exhibited a reduced sensitivity to CD95 mediated without reduced receptor levels, indicating a potential alteration in downstream effectors. Similarly, a
study published by Friesen et al11 demonstrated reduced
expression of the CD95 receptor in CEM and SHEP neuroblastoma cells
selected for resistance to doxorubicin. However, in contrast to their
study, we did not find any cross-resistance to cytotoxic drugs in
myeloma cell lines selected for resistance to CD95-mediated apoptosis, nor did we find chemosensitization in the cell lines engineered to
express high levels of CD95. One potential explanation for the
differences seen in these studies may be related to the particular cells used in each study and the type of stress response they display.
For example, the K562 cell line expresses bcr-abl, which has been shown
to confer resistance to apoptotic death in some cell
types.18 In our hands, enforced expression of CD95 was sufficient to overcome the inhibitory effects of the Abl kinase and
confer sensitivity to CD95-mediated apoptosis, but CD95 expression did
not alter the response of the K562 cell lines to chemotherapeutic drugs. Therefore, using two isogenic cell lines with variable levels of
CD95 expression, we have shown that the CD95 receptor is not required
for drug cytotoxicity.
Because apoptosis is vital to the process of development and the
maintenance of homeostasis, it is not surprising that multiple pathways
exist to achieve a common end. For example, daunorubicin and
CD95-mediated apoptosis have both been reported to induce apoptosis
through a ceramide-activated ras signal transduction pathway.30,31 Likewise, Jun kinase (JNK), stress-activated kinase (SAPK), and extracellular signal-regulated kinases (ERK1 and 2)
have also been implicated in signal transduction cascades leading to
programmed cell death induced by drugs and physiological stimuli.32-35 Although many of these apoptotic signal
transduction pathways appear to converge, the specific point of
convergence and commonalities are not yet fully defined. Further
definition of these mechanisms and their common effectors may
contribute to the future development of strategies to enhance
cytotoxicity of anticancer agents.
| |
FOOTNOTES |
Submitted August 12, 1998; accepted March 1, 1999.
Supported in part by a grant from the National Cancer Institute, No.
CA77859 (W.S.D.). T.H.L. is a Cure for Lymphoma Foundation Fellow.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to William S. Dalton, MD, PhD, Clinical
Investigations Program, H. Lee Moffitt Cancer Center and Research
Institute, University of South Florida, 12902 Magnolia Dr, Tampa FL
33612.
| |
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TOP
ABSTRACT
INTRODUCTION