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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 265-274
By
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.
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.
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).
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 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).
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
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 10
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.
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 × 10
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.
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
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).
7 mol/L doxorubicin.
), 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).
