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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1831-1837
RAPID COMMUNICATION
Cytokine-Based Tumor Cell Vaccine Is Equally Effective Against
Parental and Isogenic Multidrug-Resistant Myeloma Cells: The Role
of Cytotoxic T Lymphocytes
By
Alexander A. Shtil,
Joel G. Turner,
John Durfee,
William S. Dalton, and
Hua Yu
From the Clinical Investigations Program and Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; and the
Department of Internal Medicine and the Department of Medical
Microbiology and Immunology, University of South Florida College of
Medicine, Tampa, FL.
 |
ABSTRACT |
Tumor cells that survive initial courses of chemotherapy may do so
by acquiring a multidrug-resistant phenotype. This particular mechanism
of drug resistance may also confer resistance to physiological effectors of apoptosis that could potentially reduce the efficacy of
immune therapies that use these pathways of cell death. We have
previously demonstrated high efficacy for a cytokine-based tumor cell
vaccine in a murine MPC11 myeloma model. In the present study, the
effects of this vaccination were compared in MPC11 cells and their
isogenic sublines selected for mdr1/P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). Immunization with MPC11 cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) led to long-lasting protection of
mice against subcutaneous (sc) challenge with both
parental cells or their MDR variants. Similarly, immunization with
GM-CSF/IL-12-transfected MDR sublines caused rejection of
transplantation of both parental cells and the MDR sublines. Whereas
MPC11 cells and their MDR variants were resistant to APO-1/CD95/Fas
ligand, the immunization generated potent granzyme B/perforin-secreting
cytotoxic T lymphocytes (CTLs) that were similarly effective against
both parental and isogenic MDR cells. We conclude that MDR mediated by
mdr1/Pgp did not interfere with lysis by pore-forming CTLs.
Immunotherapy based on pore-forming CTLs may be an attractive approach
to the treatment of drug-resistant myeloma.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE RESISTANCE OF tumor cells to various
therapeutic regimens remains a major obstacle for cure of patients with
multiple myeloma. Recurrence of the disease after initial courses of
chemotherapy indicates that a population of myeloma cells emerges due
to the acquisition of multidrug resistance (MDR). This type of
resistance is commonly mediated by P-glycoprotein (Pgp), a
transmembrane pump capable of effluxing many agents out of the
cell.1 The role of Pgp overexpression as a factor of poor
prognosis in patients with multiple myeloma is widely
recognized.2-4 Thus, the cells that acquired MDR in the
course of chemotherapy may constitute a significant component of
minimal residual disease in patients with multiple myeloma. This, in
turn, raises the necessity to develop novel therapeutic approaches
capable of eliminating the drug-resistant population of cells.
In recent years, immunotherapy has been gaining popularity as an
adjunct to cancer chemotherapy. The expression of Igs that contain the
unique antigenic determinants (idiotypes) in myeloma cells provides a
possibility to generate anti-idiotypic immunity. The idiotype-specific
T-cell-mediated immune responses against B-cell lymphomas have been
demonstrated in a number of experimental reports and clinical
trials.5-8 Tumor cell-associated antigens other than
idiotypic Ig may also be recognized by the immune system and processed
to evoke proliferation of specific clones of cytotoxic T lymphocytes
(CTLs). The high therapeutic efficacy of vaccination of mice with
irradiated tumor cells transfected with cytokine genes9-11
demonstrates the potential value of using multiple tumor-associated antigens for generation of antitumor immunity. We have demonstrated effective CTL-mediated antitumor immunity in animals immunized with
poorly immunogenic MPC11 myeloma cells transfected with
granulocyte-macrophage colony-stimulating factor (GM-CSF)
cDNA.11 These data underscore the possibility of using
immunotherapy in combination with conventional therapeutic modalities
in multiple myeloma.
Using immunotherapy as an approach to eliminating minimal residual
disease requires that MDR tumor cells be sensitive to the effector
mechanisms of the immune system. However, the susceptibility of
drug-resistant tumor cells to these mechanisms has been questioned. It
has been shown that several tumor cell lines selected for resistance to
different chemotherapeutic drugs were also resistant to
lymphokine-activated killers (LAK) or natural killer (NK)
cells.12,13 Kimmig et al14 have found that two
of three independently selected MDR sublines of the T-cell leukemia
line, CCRF-CEM, were significantly less susceptible to LAK-mediated
lysis, whereas one subline was as sensitive as the parental cells. In
contrast, Scheper et al15 have demonstrated that the
selection of RPMI-8226 myeloma cells for resistance to doxorubicin
(Dox; Pgp-mediated MDR) or mitoxantrone (Pgp-unrelated resistance) did
not alter the sensitivity to in vitro lysis by LAK or NK. Given that
T-cell response against human myeloma has been
demonstrated16-18 and that CTL-mediated killing of myeloma
cells in mice has been shown to be highly effective,11 we
examined the question of whether the selection for Pgp-mediated MDR
affects susceptibility of myeloma cells to a CTL-based vaccine.
The granzyme B/perforin and APO-1/CD95/Fas ligand (FasL) pathways are
two main mechanisms mediating the lytic effects of
CTLs.19,20 However, it has been shown that myeloma cells
can be resistant to Fas receptor cross-linking before
chemotherapy.21 Moreover, the selection of myeloma cells
for drug resistance may result in coselection for cross-resistance to
Fas-mediated apoptosis.22 Furthermore, Pgp may confer an
additional protection of tumor cells from caspase-dependent apoptotic
signals, including Fas-induced death.23 Consequently, the
functionality of signaling pathways involved in lysis of MDR myeloma
cells by granzyme B/perforin is likely to be an important prerequisite
for the efficacy of CTL-based immunotherapy.
In the present study, we examined the effects of myeloma cell-based
vaccine against isogenic tumor cells selected in vitro for low levels
of Pgp-mediated MDR. In particular, we investigated whether CTLs
generated in response to immunization with cytokine-expressing myeloma
cells were capable of killing isogenic MDR variants. Our results
provide evidence that immunization with GM-CSF/interleukin-12 (IL-12)-transfected myeloma cells caused highly efficient and prolonged cross-protection of mice from challenge with either parental
or isogenic MDR myeloma cells and that this effect was mediated by
granzyme B/perforin-secreting CTLs.
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MATERIALS AND METHODS |
Animals.
Six- to 8-week-old female Balb/c mice (National Cancer Institute,
Bethesda, MD) were used for all experiments. Mice were housed in the
animal facility located at H. Lee Moffitt Cancer Center and Research Institute.
Drugs.
Dox, vincristine, and melphalan (all from Sigma Chemical Co, St Louis,
MO) were reconstituted in distilled water or acidified 70% aqueous
methanol (melphalan) as 100× to 1,000× stock solutions and
kept at  20°C. Aqueous solution of dexamethasone was
purchased from Genisa Pharmaceuticals, Inc (Irvine, CA). Recombinant
soluble human FasL (Alexis Corp, San Diego, CA) was reconstituted in
sterile phosphate-buffered saline (PBS), pH 7.4, and stored at less
than 20°C. Concanamycin A (CMA; follimycin; Calbiochem, San
Diego, CA) was dissolved in dimethyl sulfoxide at 100 µmol/L
immediately before the experiments.
Cell lines and selection for MDR.
Mouse myeloma cell line MPC11 and T-cell leukemia line S49.1 (both
derived from Balb/c mice) were purchased from American Type Culture
Collection (Manassas, VA) and propagated according to the vendor's
instructions. Cells were routinely tested and found to be free of
Mycoplasma. Cells in logarithmic phase of growth were used for
all experiments. For selection of Dox-resistant sublines, MPC11 cells
were cultured in the presence of increasing concentrations of Dox
starting from 20 nmol/L. After 8 to 10 weeks, two independently
selected sublines, MPC11Dox10-1 and MPC11Dox10-2, were established and
propagated in the presence of 100 nmol/L Dox. For cytotoxicity assays,
cells (2 × 104 in 100 µL of culture medium) were
plated into a 96-well plate. Increasing concentrations of cytotoxic
agents were added, and cells were incubated for 72 hours at 37°C,
5% CO2. The percentage of survival was determined in a
colorimetric CellTiter 96 Aqueous Non-Radioactive Cell Proliferation
Assay (Promega Corp, Madison, WI).
Reverse transcription-polymerase chain reaction (RT-PCR) analysis of
the mdr1 gene expression.
Isolation of total RNA and RT were performed as
described.24 PCR was performed with amounts of cDNA
corresponding to 50 ng of total RNA, 1.5 mmol/L MgCl2, 200 µmol/L each of deoxynucleotide triphosphate, 1 U Taq polymerase
(Boehringer Mannheim Co, Indianapolis, IN), and 10 ng/µL primers
specific for murine mdr125 and  -actin (internal
standard; R&D Systems, Minneapolis, MN) cDNAs. The reaction conditions
were 94°C for 45 seconds, 55°C for 1 minute, and 72°C for 1 minute. Each cDNA was amplified in separate tubes in duplicate in a
Peltier Thermal Cycler (Watertown, MA). In preliminary experiments we
found that 29 cycles of PCR for mdr1 and 31 cycles for
-actin cDNAs, respectively, yielded clearly detectable mdr1-
(395 bp) and -actin (528 bp)-specific products within the
exponential range. PCR products were resolved in a 1.5% agarose gel
electrophoresis, stained with ethidium bromide, and photographed.
Pgp-mediated transport.
The intracellular accumulation of calcein was used to analyze
Pgp-mediated transport.26 MPC11 cells or Dox-selected
sublines (106 cells in 1 mL of PBS) were loaded with 50 nmol/L calcein/acetoxymethyl ester (AM; Molecular Probes, Eugene, OR)
for 20 minutes at 37°C in the presence or absence of 40 µmol/L
verapamil (Sigma). Cells were preincubated with verapamil for 10 minutes before the addition of calcein/AM. Cell-associated green
fluorescence was detected on a FACScan (Becton Dickinson, San Jose, CA;
excitation 488 nm, emission 524 nm). Ten thousand events were acquired
for each treatment.
Topoisomerase II activity.
A kinetoplast DNA decatenation assay was performed using Topoisomerase
II assay kit (TopoGEN, Inc, Columbus, OH) as recommended by the
manufacturer. Nuclear extracts were prepared as
described.27
Transfection, immunization, and tumor cell challenge.
The vectors expressing GM-CSF or IL-12 cDNA and transfection with the
Accell gene gun (Agracetus/PowderJect, Middleton, WI) have been
described previously.28 The MPC11, MPC11Dox10-1, or MPC11Dox10-2 cells were  -irradiated (40 Gy), transfected with the
vector expressing GM-CSF cDNA28 or mock-transfected (also referred to as irradiated cells), and injected (1.5 × 106 cells) subcutaneously (sc) into the abdominal area of
mice. Seven days later, an equal number of irradiated, IL-12
cDNA-transfected (or mock-transfected) cells were injected sc into the
adjacent area. In all experiments, the amounts of GM-CSF and IL-12 in
the cell supernatants 24 hours posttransfection were 40 to 60 ng and 200 to 250 ng per 106 cells, respectively (as determined by
enzyme-linked immunosorbent assay11). At day 14, 106 nonirradiated cells were inoculated sc into the right
flank. In the cross-immunization experiments, mice were immunized
either with irradiated, GM-CSF/IL-12-transfected parental MPC11 cells followed by engraftment of Dox-resistant cells or with irradiated, GM-CSF/IL-12-transfected Dox-resistant cells and then challenged with
parental MPC11 cells. For challenge, 106 cells/animal, the
fivefold TD100 (the number of tumor cells that induce
tumors in 100% of injected mice), was used. Tumor growth was monitored
3 times/week for 5 weeks. In preliminary experiments, we determined
that tumorigenicity and tumor growth rates in parental cells and MDR
sublines were indistinguishable (data not shown).
CTL activity assay.
Mice were immunized as described above. Single-cell suspensions of
splenocytes pooled from 3 mice were obtained 21 days after the
injection of IL-12-transfected cells. The splenocytes were stimulated
in vitro with irradiated cells used for immunization (ie, MPC11 or
their Dox-resistant counterparts) for 5 days at 37°C, 5%
CO2. Fresh target cells (MPC11 or MDR variants) were labeled with 100 µCi sodium chromate-51 (1 mCi/mL; New England Nuclear Life Science Products, Boston, MA) for 1 hour at 37°C, followed by three washings. The activity of CTLs was tested in a 5-hour
51Cr release assay.29 In some experiments, the
cocultures of splenocytes and stimulator cells after 5 days of
incubation were pretreated with 250 nmol/L CMA for 2 hours before the
addition of target cells.30
| |
RESULTS |
Mechanisms of MDR in MPC11-derived sublines selected in vitro for Dox
resistance.
We have previously shown that immunization of mice with irradiated
MPC11 cells transfected with GM-CSF expression vector resulted in the
rejection of subsequent engraftment of intact MPC11
cells.11 We sought to test whether this vaccination was
also effective in protecting mice from MPC11 cells selected in vitro
for MDR. The stepwise selection of parental MPC11 cells with increasing concentrations of Dox resulted in the establishment of MPC11Dox10-1 subline. We found that the MPC11Dox10-1 cells were resistant to Dox
(IC50, 460 nmol/L v 50 nmol/L in MPC11 cells;
9.2-fold resistance) and cross-resistant to vincristine
(IC50, 60 nmol/L v 8 nmol/L in MPC11
cells; 7.5-fold resistance). No significant resistance to melphalan
(IC50, 10 µmol/L in MPC11Dox10-1 cells v 9 µmol/L in MPC11 cells) or dexamethasone (0.3 µmol/L v 0.3 µmol/L, respectively) was observed in MPC11Dox10-1 cells, suggesting
typical Pgp-mediated MDR. Indeed, the increased levels of mdr1
mRNA were detected in MPC11Dox10-1 subline by RT-PCR
(Fig 1A). In addition, accumulation of
calcein, a measure of Pgp-mediated transport,26 was
decreased in the selectants, indicating that MPC11Dox10-1 cells
expressed functional Pgp (Fig 1B; compare profiles 2 and 5). We also
examined the expression of the mrp and lrp genes as
well as the activity of topoisomerase II, mechanisms known to be
involved in the resistance to Dox,31-34 in
MPC11 cells and in Dox-selected variants. No changes in the
steady-state levels of the mrp or lrp mRNAs (by RT-PCR) or topoisomerase II activity (by decatenation assay) were found in
MPC11Dox10-1 cells as compared with their parental counterparts (data
not shown). Finally, blocking of Pgp transport with verapamil completely abrogated the resistance of MPC11Dox10-1 cells to Dox (data
not shown). Together, these results indicate that the overexpression of
mdr1/Pgp is the main mechanism of acquired MDR in MPC11Dox10-1 cells.
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| Fig 1.
MDR in MPC11Dox10-1 cells is conferred by
mdr1/Pgp. (A) Detection of increased mdr1 mRNA in
MPC11Dox10-1 cells by RT-PCR. Total RNA was isolated from MPC11 and
MPC11Dox10-1 cells, reverse transcribed, and amplified with primers
specific for the mdr1 and -actin genes as described in
Materials and Methods. The experiments were repeated twice with the
same results. (B) Calcein accumulation is decreased in MPC11Dox10-1
cells. MPC11 (upper panel, profiles 1 through 3) and MPC11Dox10-1
(lower panel, profiles 4 through 6) cells were left untreated (1 and 4)
or treated with 50 nmol/L calcein/AM in the absence (2 and 5) or
presence (3 and 6) of 40 µmol/L verapamil. After washing, cellular
fluorescence was measured on a FACScan (excitation 488 nm, emission 524 nm) on FL1. The experiments were performed twice with comparable
results.
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Provided that the selection procedure may upregulate different
mechanisms of MDR and also change the malignant potential of selectants,35,36 we performed another independent selection of MPC11 cells for resistance to Dox. This selection resulted in the
establishment of MPC11Dox10-2 subline propagated in the presence of 100 nmol/L Dox. The levels of cross-resistance to chemotherapeutic drugs in
this subline were essentially the same as in MPC11Dox10-1 variant.
Also, the overexpression of the mdr1 gene and the decreased
calcein accumulation were detected in MPC11Dox10-2 cells (data not
shown) indicating typical Pgp-mediated MDR. Each Dox-selected subline
was tested separately in the following in vivo experiments (see below).
Cross-protection from challenge with MPC11 cells and MDR derivatives
in mice vaccinated with irradiated, GM-CSF/IL-12-expressing myeloma
cells.
Despite the fact that GM-CSF cDNA-based vaccination resulted in potent
protection against MPC11 cells, under more stringent conditions in
which tumor challenge was performed 1 week after immunization, only
approximately 60% of mice were protected.11 An additional
immunization with IL-12 cDNA-transfected MPC11 cells at least 1 day
before tumor cell challenge improved the antitumor protection to
greater than 90% (manuscript in preparation). Based on these data, we
used the combination of GM-CSF- and IL-12-transfected tumor cells
(MPC11 or their MDR variants) for vaccination experiments. Figure 2 shows the kinetics of tumor take
in control animals (naïve or immunized with irradiated cells)
and mice immunized with GM-CSF/IL-12-transfected cells (both MPC11 and
MPC11Dox10-1). Typically, in control cohorts tumors were registered by
days 8 to 12 after the engraftment of tumor cells. In contrast, mice
immunized with GM-CSF/IL-12-transfected cells remained tumor-free for
at least 35 days postchallenge.
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| Fig 2.
Time course of initiation of tumor growth in
mock-immunized mice and mice vaccinated with irradiated,
GM-CSF/IL-12-transfected MPC11 cells. Mice were vaccinated with
irradiated MPC11 cells with or without cytokine gene transfection. Both
MPC11 and MPC11Dox10-1 cells were used for tumor challenge. Tumor
growth was monitored for up to 35 days. Data shown represent cumulative
results of three independent experiments (n = 15).
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The results presented in Table 1 show that
both the parental MPC11 and the MDR variants were capable of producing
tumors in nonimmunized (naïve) animals. Only 1 mouse (of 15)
vaccinated with irradiated MPC11 cells rejected subsequent tumor cell
challenge, whereas all animals vaccinated with cytokine-expressing
cells were tumor-free. Importantly, 90% of mice immunized with
GM-CSF/IL-12-expressing MPC11 cells were protected from the
engraftment of MDR variants. Furthermore, all animals vaccinated with
cytokine-expressing MPC11Dox10 sublines rejected the challenge with
parental cells (Table 1).
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Table 1.
Cross-Protection of Mice Immunized With
GM-CSF/IL-12-Transfected Tumor Cells From Challenge With MPC11 Cells
or MDR Variants
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The cytotoxic effect of vaccination is mediated by granzyme
B/perforin-secreting CTLs.
Previous studies have demonstrated that immunization with
cytokine-based tumor cell-based vaccines generated T-cell-mediated antitumor immunity.9-11 We investigated whether CTLs
generated by vaccination with cytokine gene-based MPC11 cells were
capable of lysing MPC11Dox10 sublines. After in vitro stimulation with respective myeloma cells for 5 days, the splenocytes from mice immunized with GM-CSF/IL-12-transfected MPC11 cells lysed both MPC11
cells and isogenic MDR cell variants (Fig
3A). Also, CTLs from mice vaccinated with GM-CSF/IL-12-transfected
MPC11Dox10 cells effectively lysed both parental cells and MDR variants
(Fig 3B). The results indicate similar sensitivity to CTL-mediated killing of both parental cells and their isogenic sublines with acquired MDR.
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| Fig 3.
CTL activity in mice immunized with irradiated cells and
GM-CSF/IL-12-expressing vaccine. Mice were immunized with irradiated
only or irradiated, GM-CSF/IL-12-transfected cells (MPC11 [A] or
MPC11Dox10-1 [B]). At day 21 after the initial vaccine injection,
animals were killed, and CTL activity was tested as described in
Materials and Methods. CTL activity is expressed as the percentage of
specific lysis of 51Cr-labeled target cells. Each value
represents mean of duplicate samples. The error between duplicates was
less than 10%. The experiments were repeated twice with similar
results.
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To specify which mechanism(s) was involved in rejection of engraftment
of MPC11 and MPC11Dox10 cells, two sets of experiments were performed.
We first tested whether inhibition of perforin secretion would
attenuate CTL activity. It has been previously shown that CMA at
submicromolar concentrations caused degradation of perforin and
abrogated perforin-mediated lytic activity of CTLs.30,37
Figures 4A and B show that pretreatment of
cocultures of splenocytes and stimulator cells with 250 nmol/L CMA for
2 hours significantly decreased the ability of splenocytes from mice
vaccinated with GM-CSF/IL-12-expressing cells (parental or MDR
sublines) to lyse both MPC11 cells and MDR variants. Furthermore, treatment with recombinant FasL showed that MPC11 and MPC11Dox10-1 cells were resistant to cross-linking of Fas receptor (Fig 4C). The
MPC11Dox10-2 subline was also resistant to FasL (data not shown).
Together, the results suggest that immunization with cytokine-based tumor cell vaccine generates CTLs that are capable of killing both
MPC11 cells and their MDR variants via a granzyme B/perforin-sensitive and Fas-independent mechanism.
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| Fig 4.
CTLs from mice immunized with GM-CSF/IL-12-expressing
vaccines lyse MPC11 cells and their MDR variants via granzyme
B/perforin- but not Fas-dependent mechanism. Mice were immunized with
GM-CSF/IL-12-expressing MPC11 cells (A) or MPC11Dox10-1 cells (B) as
described. Splenocytes isolated at day 21 after the initial injection
of the vaccine were stimulated with irradiated MPC11 (A) or irradiated
MPC11Dox10-1 (B) cells for 5 days, and CTL activity in the absence
( ) or presence ( ) of 250 nmol/L CMA was determined. (C) Cells
were incubated with recombinant FasL for 17 hours. The cytotoxicity was
determined in a colorimetric test (see Materials and Methods). The
experiments were repeated twice with similar results. The S49.1 cell
line was included as a positive control for FasL-mediated apoptosis.
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DISCUSSION |
The major findings of this study are (1) vaccination with myeloma cells
expressing transfected GM-CSF/IL-12 confers cross-protection of mice
from challenge with poorly immunogenic MPC11 cells as well as from
their MDR counterparts and (2) granzyme B/perforin-secreting CTLs can
efficiently eliminate myeloma cells that are resistant to
Pgp-transported chemotherapeutic drugs and FasL.
In accordance with the data from in vivo experiments, CTLs from immune
mice showed high in vitro toxicity against MPC11 cells or MDR sublines
regardless of which cell line was used for immunization. CTLs exert
cytolytic effect on tumor cells by two mechanisms, namely, via soluble
or CTL membrane-bound FasL, and/or by exocytosis of 70-kD protein
perforin that forms pores in the plasma membrane of tumor
cells.38 The latter event allows granzyme B, a serine protease produced by CTLs, to enter the target cell and initiate apoptotic cascade.38 It has been shown that perforin is
stored in acidic compartments of the cell38 and that the
increase of pH in these organelles by blocking vacuolar type
H+-ATPase by CMA leads to degradation of
perforin.37 In our model, the FasL-mediated lytic mechanism
was eliminated because neither MPC11 cells nor MPC11Dox10 variants were
sensitive to FasL. In contrast, pretreating of splenocytes derived from
immunized mice with CMA significantly attenuated the CTL lytic activity
against MPC11 cells and their MDR variants. These results demonstrate that immunization with GM-CSF/IL-12-secreting myeloma cells can generate potent CTLs that are capable of killing both parental and
isogenic MDR myeloma cells via perforin/granzyme B-mediated mechanism.
The fact that the lysis of myeloma cells by CTLs was observed in a
short-term (5-hour) 51Cr release assay suggests
perforin-induced necrosis a mechanism of CTL cytotoxicity. However, we
cannot rule out the possibility that granzyme B acted as an
apoptosis-initiating agent in MPC11 and MPC11Dox10 cells. Several
proenzyme caspases, including caspase 3, are the substrates for
granzyme B.39 Moreover, caspase 3 may be directly cleaved
by granzyme B in vivo.40 These data imply that apoptosis
triggered by granzyme B requires functional activity of signaling
mechanisms downstream of caspase 3. It remains to be determined whether
these mechanisms execute granzyme B-initiated programmed death in MPC11
cells and whether the selection for MDR impairs these pathways. If that
is the case, perforin and granzyme B may act in concert to ensure
killing of parental and MDR myeloma cells. Taking into consideration
that the selection for resistance to chemotherapeutic drugs may render
tumor cells less sensitive to several caspase-dependent apoptotic
stimuli, including Fas cross-linking,22,23 the lysis via
perforin should be sufficient to effect antitumor immunity in MDR myeloma.
Our data indicate that myeloma cells that acquire MDR in the course of
selection with Dox are still sensitive to lysis by CTLs. This implies
potential therapeutic value for CTL-based immunotherapy in patients
with minimal residual disease, given that proliferative T-cell
responses are not compromised by preceding treatment.18 Collectively, we provide experimental evidence that immunization with
cytokine-based tumor cell vaccine is an efficient adjunct to
conventional therapeutic protocols in MDR myeloma.
| |
ACKNOWLEDGMENT |
The authors are grateful to Agracetus/PowderJect (Middleton, WI) for
providing us with the gene gun, to M. Oshiro for selecting the
MPC11Dox10-1 subline, to Dr T. Landowski for critical discussion of the
manuscript, and to J. Kroeger (Flow Cytometry Core, H. Lee Moffitt
Cancer Center and Research Institute) and J. Szucs (Multimedia
Educational Resource Center, H. Lee Moffitt Cancer Center and Research
Institute) for computer graphic assistance.
| |
FOOTNOTES |
Submitted November 16, 1998; accepted December 16, 1998.
A.A.S. and J.G.T. contributed equally to this work.
Supported by grants from Kathy Guisti Myeloma Research Foundation and
National Institutes of Health (CA 75243 and CA 77859) and by the Dr.
Tsai-Fan Yu Cancer Research Endowment.
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 Hua Yu, PhD, Immunology Program, H. Lee
Moffitt Cancer Center and Research Institute, 12902 Magnolia Dr, Tampa,
FL 33612-9497.
| |
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