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NEOPLASIA
From Equipe Propre de l'Institut National de la
Santé et de la Recherche Médicale (INSERM) E9910, Institut
Claudius Régaud, Toulouse, France.
Granzyme B (GrB) and perforin (PFN) are the major components of
cytoplasmic granules contained in immune cellular effectors. The
granule secretory pathway is one of the mechanisms by which these cells
exert their cellular cytotoxicity. Recently, it has been reported that
GrB and PFN are also present in circulating hemopoietic
CD34+ progenitor cells mobilized by chemotherapy and
granulocyte-colony stimulating factor, whereas these proteins are
undetected in steady-state peripheral CD34+ cells. In this
study, we hypothesized that anticancer agents may increase GrB and PFN
expression in immature myeloid leukemic cells and that these treated
leukemic cells become cellular effectors through a granule-dependent
mechanism. Our results show that KG1a, HEL, and TF-1 CD34+
acute myeloblastic leukemia cells expressed both GrB and PFN. Moreover,
ionizing radiation, aracytine, and etoposide not only increase GrB and
PFN expression but also conferred potent cellular cytotoxicity to these
cells toward various cellular targets. Cellular cytotoxicity required
cell-cell contact, was not influenced by anti-tumor necrosis factor The granule secretory pathway is one of the
mechanisms by which cytotoxic lymphocytes (CTLs) and natural killer
(NK) cells exert their cytotoxicity against virus-infected,
alloreactive, or transformed cells. The contact between effector cells
and aberrant target cells induces exocytosis from the CTL or the NK
cells, of granules that contain the potential cytolytic effector
molecules. The most prominent components of cytotoxic granules are
perforin (PFN) and a family of proteases, most importantly granzyme A
(GrA) and granzyme B (GrB) in humans.1 Although it has
been documented that PFN alone may induce, at least in some
circumstances, cell lysis,2 it is generally believed that
the main function of PFN is to create pores in the target cell
membrane, which may disrupt osmoregulation and facilitate transfer of
the granzymes.3,4 Although exogenous granzymes can also
enter target cells by themselves, via a classical endocytic pathway,
they are not cytotoxic unless liberated from endosomes into the cytosol
by PFN or another endosomolytic agent, such as
adenovirus.5 Previous studies have established that the
GrB/PFN system plays a central role in cellular immunity. Indeed,
targeted mutation of the PFN gene in mice results in profound immunosuppression and altered skin and tumor allograft
rejections,6 whereas GrB homozygous null mutant mice have
a severely depressed ability to cause target cell lysis.7
Moreover, it has been previously shown that interleukin-2-induced
NK/lymphokine activated killer (LAK) cell cytotoxic activity measured
in a 51Cr-release short-term assay can be mainly attributed
to PFN.8
More recently, GrB was detected in normal and tumor nonlymphoid cells,
such as keratinocytes,9 Kupffer cells,10 and
Reed-Sternberg cells.11 Albeit
controversial,12 this protein was also found in some
myeloid cell lines, including FDCP-Mix, a murine growth factor-dependent stem cell line,13 as well as KG1a, a
human acute myeloid leukemia (AML) cell line derived from an early
stage of hemopoiesis as attested by its CD34+
CD38 In this study, we showed that genotoxic stress increased the expression
of both GrB and PFN and influenced their cellular distribution in KG1a,
HEL, and TF-1 CD34+ AML cells. Moreover, we have found that
when treated with ionizing radiation (IR), etoposide (VP-16), and
aracytine (Ara-C), these cells became capable of inducing rapid cell
lysis of both lymphoid and myeloid cellular targets through a
GrB-dependent mechanism.
Cell cultures
Drugs and reagents
Flow cytometry analysis of GrB and PFN expression Cells were fixed with 4% (w/v) paraformaldehyde for 10 minutes at room temperature (RT), then permeabilized by 0.3% saponin-PBS (10 minutes at RT), and resuspended in 20% FCS-PBS for 20 minutes at RT. Cells were then incubated for 25 minutes at RT with anti-human GrB monoclonal antibody (MoAb; clone GrB-7; Euromedex, Souffelweyersheim, France). This MoAb is an immunoglobulin G2a (IgG2a) that reacts specifically with human serine protease GrB and does not cross-react with human GrA.11,15-17 Alternatively, cells were incubated with an anti-PFN MoAb (clone KM585; Kamiya, Seattle, WA). This MoAb is a rat IgG2a that recognizes murine PFN but cross-reacts with human PFN.18 Nonimmune mouse or rat IgG2a was used as control (Beckman-Coulters, Roissy, France). Each antibody was used at 1:20 in PBS containing 1% FCS. After two washes in PBS, cells were incubated for 30 minutes at RT with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (GrB) or anti-rat (PFN) IgG (Beckman-Coulters, Roissy, France) diluted at 1:50 in 1% FCS-PBS. Before analysis on a flow cytometer (FACScan Becton Dickinson Co, San Jose, CA), cells were resuspended in 0.5% formaldehyde-PBS. rh-IL-2 treated peripheral blood lymphocytes (PBL-IL2) were used as positive controls for GrB and PFN expression.Confocal laser scanning microscopy (CLSM) analysis Aliquots of 1-2 × 105 cells were cytocentrifuged and fixed with 4% (w/v) paraformaldehyde (10 minutes at RT), washed twice with PBS, and permeabilized using 100% methanol (2 minutes at RT). Coverslips were washed with PBS, saturated for 20 minutes at RT with 20% FCS-PBS and incubated with anti-human GrB MoAb or mouse isotype-matched control, both at a 1:20 dilution in 1% FCS-PBS, followed by FITC-conjugated goat anti-mouse IgG (Beckman-Coulters) diluted at 1:100 in 1% FCS-PBS for 25 minutes at RT. After washing, the coverslips were sealed and examined with confocal imaging system that was a Zeiss (Oberkochen, Germany) scanning assembly incorporating argon and helium/neon lasers coupled to a Zeiss Axiovert 100 fluorescence microscope.Western blot analysis Ten million cells were washed twice in serum-free medium and lysed by resuspension in lysis buffer containing 10 mmol/L Hepes (pH 7.8), 100 mmol/L EDTA, 100 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 2 µmol/L pepstatin A, 0.6 µmol/L leupeptin, and 1 µg/mL aprotinin on ice for 15 minutes. Nonidet P-40 was then added at 0.6% final, and the nuclear pellet was recovered after centrifugation at 1200g for 30 minutes at 4°C. Cytoplasmic lysate (postnuclear supernatant) was collected and stored at 20°C. The nuclear pellet was resuspended in 20 mmol/L Hepes (pH 7.8), 400 mmol/L NaCl, 1 mmol/L EDTA, and 1 mmol/L EGTA. Aliquots were then incubated at 4°C for 30 minutes, centrifuged at 21 000g for 30 minutes at 4°C, and supernatants containing nuclear protein were collected. Nuclear and cytoplasmic fractions were boiled in sample buffer containing 0.4% -mercaptoethanol, separated on 12.5% (w/v)
SDS-PAGE, and transferred electrophoretically onto nylon membranes
(Hybond-C extra; Amersham Life Science, Cergy-Pontoise, France).
Nonspecific binding sites were blocked in 10 mmol/L Tris buffered
saline (TBS) containing 0.1% Tween-20, and 10% nonfat milk. Membranes
were incubated overnight at 4°C with mouse anti-GrB or rat anti-PFN MoAb both diluted at 1:200 in 10 mmol/L TBS containing 0.1% Tween-20 and 1% nonfat milk. Membranes were then washed five times at RT, and
bound Ig was detected with anti-mouse IgG or anti-rat IgG coupled to
horseradish peroxidase (Beckman-Coulters). The signal was visualized by
enhanced chemiluminescence (Amersham, Buckinghamshire, UK) and autoradiography.
Cytotoxicity assays The CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) was used to evaluate stress-induced cytotoxicity.19,20 This assay is a colorimetric alternative to 51Cr-release cytotoxicity assay based on the measurement of lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released on cell lysis, in much the same way as 51Cr is released in radioactive assay and that allows discrimination between effectors and targets LDH release.21 Variations on this technology have been reported for measuring natural cytotoxicity and have been demonstrated to be identical (within experimental error) to values determined in parallel 51Cr-release assays and was performed according to manufacturer recommendations.Briefly, effector cells (KG1a, HEL, TF-1, PBL-IL2, and NKL:
8 × 106 or 4 × 106 cells/mL) were
pretreated with cytotoxic agents, washed, then resuspended in RPMI-1640
supplemented with 5% FCS, and mixed with target cells (U937, Jurkat,
and K562: 1 × 105 cells/mL) in U-bottom 96-microwell
plates (Nunc, Roskilde, Denmark) at various effector-to-target (E:T)
ratio in triplicates. Microplates were spun for 3 minutes at
200g and incubated for 4 hours at 37°C, 5%
CO2. Supernatant (50 µL) was collected from each well and
added to 50 µL reconstituted Substrate Mix for 30 minutes in the dark at RT. Enzymatic reaction was stopped by adding Stop Solution. Counting
was realized by recording absorbance at 490 nm. Maximum release (TM)
was determined by lysing target cells with 20 µL of Lysis Solution.
Spontaneous release (TS) was determined by incubation of target
cells in medium in the absence of effector cells. Effector spontaneous
release (ES) was done with effector cells alone at the same E:T
ratio. Results are expressed as a percentage of cytotoxicity, using
the following formula: % cytotoxicity = [(experimental Blocking experiments were performed with DCIC (50 µmol/L), a broadly reactive serine esterase inhibitor, that has been shown to neutralize granzyme enzymatic activity,22,23 MgCl2/EGTA (1 mmol/L) that blocks degranulation and prevents PFN polymerization,24 or phosphorothioate oligodeoxynucleotides (2 µmol/L) antisense or sense directed against GrB. Statistics Quantitative experiments were analyzed, using the Student t test. All P values resulted from the use of 2-sided tests.
GrB expression in leukemic cell lines The steady-state levels and cellular distribution of GrB in leukemic cells were investigated respectively by flow cytometry, confocal microscopy, and Western blot analysis. As shown in Figure 1A, GrB was detected in CD34+ KG1a, HEL, and TF-1 cells, whereas CD34 U937 cells and
Jurkat cells lacked significant GrB expression. However,
CD34+ AML cell GrB expression was lower than that measured
in PBL-IL2. Remarkably, confocal microscopy showed that GrB was mainly
localized in the nucleus of KG1a cells (Figure 1B), whereas very low
level, if any, was detected in the cytoplasm. Similar results were
obtained in HEL and TF-1 cells (data not shown). Fractionation studies showed that GrB was almost exclusively contained in the nuclear pellet
as shown for KG1a in Figure 1C. These results established that, in KG1a
cells, GrB is predominantly located into the nucleus. In contrast, in
PBL-IL2, GrB was exclusively found in the postnuclear fraction,
suggesting that it is predominantly located in the cytoplasm (Figure 1C).
Effects of IR on GrB expression We next investigated the effects of IR on expression and cellular distribution of GrB in CD34+ leukemic cells. In repeated experiments (n = 5), cytometric analysis showed that -irradiation induced a modest but reproducible increase in GrB
expression in KG1a, HEL, and TF-1 cells (Figure
2A). Indeed, -radiation induced a
dose- and time-dependent increase of GrB expression (data not shown)
with a maximum amplification at 72 hours in cells treated with 4 Gy
exposure. In these conditions, CD34+ cells retained
viability as assessed by trypan blue exclusion (data not shown).
However, -irradiation, used at subtoxic doses, induced no GrB
expression in U937, Jurkat, and all epithelial cell lines tested,
including MCF-7, HeLa, and NIHOVCARIII (Figure 2A). Confocal analysis
showed that nuclear GrB-associated fluorescence was increased in
-irradiated KG1a cells, compared with untreated cells. However, the
most prominent finding was that GrB became easily detectable in the
cytoplasm with a punctate distribution (Figure 2B). Western blot
analysis performed on whole extracts of KG1a cells showed a significant
increase in GrB expression, thus confirming the above findings.
Moreover, fractionation studies revealed that IR-induced GrB expression
increase was still more significant in the cytoplasm compared with the
nuclear fraction (Figure 2C).
Effects of IR on PFN expression Flow cytometry and Western blot analysis showed that KG1a cells expressed a modest level of PFN expression that was significantly increased by-irradiation (Figure 3A).
Moreover, fractionation experiments revealed that KG1a cell nuclei were
devoid of PFN and that IR increased cytoplasmic PFN expression
(Figure 3B).
Effects of genotoxic drugs on GrB and PFN expression The effect of other anticancer agents on GrB and PFN expression was also investigated in KG1a cells. As depicted in Figure 4, treatment with clinically relevant doses of Ara-C (40 µmol/L) or VP-16 (20 µmol/L), but not DNR (1 µmol/L), increased both GrB and PFN expression at 24 hours, compared with untreated cells (Figure 4). Similar results were obtained in drug-treated TF-1 and HEL cell lines (data not shown). At these doses, cell viability remained intact.
Cellular cytotoxicity of irradiated KG1a cells On the basis of the observation that, in the immune system, CTL and NK/LAK cells can kill their cellular targets through the GrB/PFN system, we hypothesized that stressed CD34+ AML cells could acquire cytotoxic potential because of GrB/PFN overexpression and redistribution.-Irradiated or untreated KG1a cells were co-cultured
with various cellular targets at an E:T ratio between 2.5:1 and 80:1.
Untreated KG1a cells displayed no cytotoxicity toward target cells
(Figure 5). When irradiated at a dose of
4 Gy and then incubated for 72 hours, KG1a cells exhibited significant
cytotoxicity toward U937 cells (Figure 5A), K562 cells (Figure 5B), and
Jurkat cells (Figure 5C). Interestingly, irradiated KG1a cells exerted
a cytotoxic effect comparable to that observed with PBL-IL2 toward
Jurkat target cells. In addition, in the 4-hour assay, we were unable
to detect any apoptotic features in target cells as evaluated by DAPI
staining and DNA fragmentation measurement (data not shown). Moreover,
K562, U937, and Jurkat cells displayed no cytotoxicity toward KG1a
cells (data not shown).
Cellular cytotoxicity of drug-treated KG1a cells We also investigated the influence of drugs on KG1a cellular cytotoxicity. We found that when treated with Ara-C (40 µmol/L) or with VP-16 (20 µmol/L) for 24 hours, but not with DNR, KG1a, HEL, and TF-1 cells displayed significant cytotoxicity toward K562 or Jurkat cells (Table 1).
Effects of GrB and PFN inhibitors To investigate the role of the GrB/PFN system in the mechanism by which irradiated KG1a cells exerted their lytic ability, we used pharmacological agents known to modulate GrB/PFN cytotoxic function. These experiments were performed with K562 and Jurkat cells as targets at various E:T ratios. Therefore, irradiated KG1a cells were pre-incubated with DCIC, a GrB inhibitor, washed, and allowed to react with target cells. As depicted in Table 2, at an E:T ratio of 40:1, pretreatment with DCIC resulted in total abrogation of irradiated-KG1a cell cytotoxicity. Similar results were obtained at the other E:T ratios (data not shown). As expected, in the same conditions, DCIC also significantly reduced PBL-IL2 cytotoxicity. Moreover, when treated KG1a cells or PBL-IL2 were co-incubated with MgCl2/EDTA, a PFN inhibitor, cytotoxicity was no longer observed. To investigate other mechanisms implicated in cellular-mediated cytotoxicity, irradiated KG1a cells and target cells were co-cultured in the presence of anti-tumor necrosis factor (TNF-) or anti-CD95 (Fas/APO-1)
blocking antibodies. As shown in Table 2, these antibodies had no
influence on irradiated-KG1a cellular cytotoxicity.
Effects of GrB antisense oligonucleotides To ascertain the involvement of the lytic protein GrB in cell-mediated cytotoxicity, we first exposed KG1a cells to the action of antisense oligonucleotides directed against GrB. Cells were then irradiated at a dose of 4 Gy, incubated for 72 hours, and then allowed to react with K562 used as target cells at various E:T ratios for 4 hours. In fact, GrB antisense, but not sense, oligonucleotides strongly diminished irradiated-KG1a cell cytotoxicity as depicted in Figure 6a for an E:T ratio of 40:1. In parallel, exposure to antisense oligonucleotide resulted in a decrease of GrB expression, more significantly in the cytoplasm than in the nucleus, whereas exposition to control oligonucleotide did not affect significantly GrB expression (Figure 6B).
In this report, we show that KG1a, TF-1, and HEL cells
express both GrB and PFN as assessed by flow cytometry, confocal
microscopy, and Western blot analysis. For GrB detection, we have used
GrB-7 MoAb that has been characterized elsewhere.15
Briefly, GrB-7 has been raised against recombinant GrB and reacts with
isolated GrB from activated cytotoxic lymphocytes, but it does not
recognize GrA.15 In fact, this antibody has been used in a
large number of immunohistochemical studies.11,15-17,25,26
Moreover, we have used in parallel B18.1 MoAb that also reacts
specifically against human GrB.9,14,27 B18.1 MoAb provided
similar results with GrB-7 in flow cytometry and confocal microscopy
studies (data not shown) but, at least in our hands, it was found to be
less reliable than GrB-7 for Western blot analysis. For this reason, the study was conducted with GrB-7 MoAb. For PFN detection, we have
used KM585 MoAb,18 a rat MoAb raised against mouse
recombinant PFN that cross-reacts with human PFN.18 This
antibody was also used in immunohistochemical studies and Western blot
analysis.18,28-31 Moreover, we have used in parallel The fact that KG1a cells express both GrB and PFN has been previously reported.14 However, intracellular distribution of these lytic proteins have not been documented. In our study, cellular fractionation followed by Western blot analysis showed that PFN was exclusively distributed in the cytoplasm of KG1a cells. In contrast, we found that GrB was mainly contained in the nucleus. This result contrasts with that generally observed in activated T or NK/LAK cells in which GrB and PFN are internalized into cytoplasmic granules.35 Although nuclear localization of GrB has been previously described,36 the fact that KG1a cells express cytoplasmic PFN and nuclear GrB while retaining viability is somewhat intriguing. Indeed, recent work37 supports the idea that translocation of GrB to the nucleus plays a central role in effecting the nuclear changes associated with cell death. One can hypothesize that GrB is complexed to other molecule(s) in the nucleus and stored in an inactive form as it has been previously proposed.36 Our study shows that IR and some chemotherapeutic compounds
increase PFN and GrB expression in KG1a, HEL, and TF-1 cells. The
reason why DNR did not induce GrB and PFN expression is still unclear
but could be due to vesicular drug sequestration as we have previously
described.38 Fractionation studies combined with Western
blot analysis showed that PFN remained localized in the cytoplasm,
whereas GrB expression increased in both nuclear and cytoplasmic
compartments. This finding suggests that GrB is not translocated from
the nucleus to the cytoplasm and that GrB redistribution is due to de
novo synthesis activated by genotoxic stress. Indeed, we observed that
actinomycin D as well as cycloheximide treatments dramatically
decreased stress-induced GrB expression (data not shown). The mechanism
by which anticancer agents enhanced GrB and PFN expression remains to
be determined. However, it should be noted that, on one hand, GrB
promoter contains AP-1 binding sites, and that, on the other hand,
In this study, we show for the first time that leukemic cells treated with genotoxic agents acquire major histocompatibility complex (MHC)-unrestricted potent lytic ability toward targets from various origins. This finding may have important implications in AML therapy. Indeed, it is conceivable that Ara-C, which is used as part of front-line therapy, renders blast cells cytotoxic for nonleukemic bone marrow cells, including residual normal myeloid progenitors and immune cellular effectors. Moreover, blast lytic activity should be facilitated by a high E:T ratio in the overt phase of the disease. The mechanism by which stress agents render leukemic cells
cytotoxic has been examined. Irradiated or drug-treated KG1a cells displayed no lytic ability when separated from target cells by nylon
membrane (data not shown). The fact that cell-cell contact is required
for cellular cytotoxicity precludes the role of soluble cytotoxic
molecules, including those which have been found to be produced under
stress conditions, such as TNF- On the basis of these findings and considerations, we hypothesized that GrB and PFN could be involved in the cellular cytotoxicity of stressed leukemic cells. Several lines of evidence support this hypothesis: (1) the requirement of cell-cell contact for target lysis, (2) the blocking effects of serine esterase inhibitor and MgCl2/EGTA, and (3) the blocking effect of antisense oligonucleotides directed against GrB. To conclude, we propose a model in which some leukemic cells constitutively express cytoplasmic PFN and nuclear GrB. Under normal conditions, these proteins are sequestered in distinct intracellular compartments and confer no lytic activity to leukemia cells. However, under genotoxic stress, both PFN and GrB are newly synthesized, stored in cytoplasmic cytotoxic granules, and confer potent MHC-unrestricted cellular cytotoxicity to leukemia cells.
Submitted March 27, 2000; accepted May 2, 2000.
Supported by the Association pour la Recherche contre le Cancer (ARC) (grant 9296) and la Faculté de Médecine Toulouse-Rangueil. A.P.B. is the recipient of a grant from the Ministère de l'Education Nationale, de l'Enseignement Supérieur, et de la Recherche (MENESR).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Anne Quillet-Mary, INSERM E9910, Institut Claudius Régaud, 20 rue du Pont St Pierre, 31052 Toulouse, France; e-mail: quillet_mary{at}icr.fnclcc.fr.
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Abstract
Introduction
ES 
), irradiated KG1a (
), NKL
(
), or PBL-IL2 (
) cells (used as an internal control) were
co-incubated with U937 (A), K562 (B), or Jurkat (C) cells as targets.
Results are the mean ± standard deviation of three independent
experiments performed in triplicate. *P = 0.02 for E:T
ratio of 80:1 to 10:1.
G9
MoAb, a purified mouse anti-human PFN.