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INTRODUCTION |
One major active component released by cytotoxic natural killer
and T cells (CTLs)1 during
degranulation is perforin (1), which can damage cells through membrane
perturbation. The induction of target cell apoptosis and accompanying
DNA fragmentation and chromatin condensation additionally requires the
action of other granule components and in particular, serine proteases
termed granzymes (2). The most abundant granzyme in the mouse is
granzyme A (GrA, fragmentin-1), a ~60-kDa homodimer that, in contrast
to the monomeric granzyme B (GrB, fragmentin-2), is able to activate
interleukin-1
directly (3) and can cleave various
intracellular/extracellular proteins, including the thrombin receptor
(4) and the nuclear/nucleolar protein nucleolin (5). GrA and GrB differ
in their substrate specificities; although GrA cleaves at basic amino
acid residues, GrB is an aspase, cleaving at aspartic acid residues
(6).
Evidence for synergy between perforin and granzymes comes from a
variety of biochemical and cellular studies. Noncytotoxic rat
basophilic leukemia cells transfected to express perforin with GrA
and/or GrB become potent inducers of membrane damage and DNA
fragmentation in conjugated target cells (7, 8). Purified GrA or GrB in
combination with perforin are able to effect both the nuclear and the
nonnuclear changes of apoptosis, although GrA/perforin-mediated cell
killing exhibits slower kinetics (9, 10). Although CTLs from GrB gene
knock-out mice exhibit only severely delayed apoptotic capacity (11),
those from GrA knock-out mice do not, implying that GrA is not
indispensable for the induction of apoptosis in target cells by natural
killer cells and alloreactive CTL (12), although it is probably
important in inducing target cell apoptosis via a distinct mechanism
that is triggered after prolonged incubation of killer and target cells
and/or in cells that are relatively resistant to GrB (Ref. 11; see also
Refs. 10 and 13). GrA/GrB double knock-out mice are completely
defective in the induction of the nuclear changes associated with
apoptosis, whereas perforin-dependent target cell lysis is
unaffected (13). Because the trypsin-like substrate specificity of GrAs
is quite distinct from that of GrB, it must induce apoptosis by
cleaving either substrates different from those of GrB or at
tryptic sites within the natural substrates of GrB, this presumably
being the basis of the documented role of GrA in chromosome
degradation during apoptosis (9, 14).
We have recently shown that GrB can both enter the cell cytoplasm in
the absence of perforin and translocate from the cytoplasm to the
nucleus in target cells in the presence of perforin (15-17). Nuclear
targeting occurs before the nuclear events of apoptosis such as DNA
fragmentation (17), implying that nuclear translocation of granzymes
may constitute a means by which the apoptotic signal is communicated to
the nucleus; nuclear transport of GrB may bring it into contact with
potential substrates such as the DNA repair enzyme poly(ADP-ribose)
polymerase (PARP) and the catalytic subunit of
DNA-dependent protein kinase, both of which have been shown to be cleaved not only by caspases such as caspase-3, early in many
forms of apoptosis, but also in vitro (18, 19) as well as
in vivo (20) by GrB. On this basis, it has recently been proposed that GrB may bypass the requirement for active caspases in the
presence of viral caspase inhibitors (20). Our recent work (21) has
shown that GrA resembles GrB in terms of nuclear targeting in intact
cells in the presence of perforin as well as in in vitro and
in vivo nuclear transport assays, this apparently conserved
mechanism probably playing a role in contributing to the nuclear
changes associated with apoptosis.
Many forms of apoptotic cell death can be blocked by the proto-oncogene
bcl2 (B cell leukemia gene-2), a member of a family of
regulatory proteins involved in either promoting or inhibiting apoptosis. bcl2 expression is required for long term
survival of lymphoid cells (22), but following overexpression due to chromosomal translocation, can be oncogenic; unlike other oncogenes, however, it confers cell viability without promoting cell proliferation (23). Although bcl2 inhibits apoptosis induced by a variety of stimuli including cytotoxic drugs, tumor necrosis factor, and ionizing radiation, it does not confer general resistance to
CTL-induced apoptosis in myeloid cells (24-28). We have recently shown
that although BCL-2 does not confer resistance to apoptosis induced by
allogeneic CTL, natural killer cells, or whole cytolytic granules, it
can protect against apoptosis induced by purified perforin and GrB
(29).
In the present study, we use confocal laser scanning microscopy (CLSM)
to assess the cellular uptake and intracellular distribution of
granzymes in bcl2-expressing mouse myeloid FDC-P1 and YAC-1 lymphoma cells. We show that perforin-dependent
redistribution and nuclear uptake of granzymes is strongly inhibited by
BCL-2, concomitant with greatly increased resistance to
granzyme-perforin-induced apoptosis and induction of DNA fragmentation
in particular. The clear implication is that nuclear targeting of
granzymes may participate directly in effecting the nuclear changes
associated with apoptosis.
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MATERIALS AND METHODS |
Chemicals and Reagents--
Fluorescein isothiocyanate (FITC)
was from Molecular Probes. Other reagents were from the sources
previously described (16, 17, 21, 29, 30).
Cell Culture--
Mouse FDC-P1 myeloid cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum and recombinant interleukin 3-containing culture supernatant (31) as described (17). Mouse YAC-1 lymphoma cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum as previously described (30). The bcl2-expressing FDC-P1 (FDbcl2 (23)) and YAC-1 (Ybcl2 (29)) cell lines, derived after
transfection, have been characterized previously with respect to the
lack of expression of functional BCL-2 protein and were cultured in
identical fashion to the parental lines.
Protein Purification and Labeling--
Human GrA, GrB, and
perforin were all purified as described previously (21, 32-35). GrA
and GrB were labeled with FITC as described, with less than 15% loss
of proteolytic activity (21, 30). Protein concentrations were
determined using the dye binding assay of Bradford (36) with bovine
serum albumin as a standard or by measuring absorbance at 280 nm in the
case of GrB (37).
Cellular Uptake and Distribution of Granzymes--
Whole cell
uptake and subcellular transport of labeled GrA or GrB were examined as
previously (17, 21). Cells were harvested in the logarithmic phase of
growth, washed, and resuspended (4 × 106 cells/ml) in
Hanks' buffered saline solution containing 10 mM Hepes, pH
7.2, 2 mM CaCl2, 0.4% bovine serum albumin,
and 0.1% (v/v) interleukin 3-containing culture supernatant. Cells (3 µl) were incubated for the specified times with an equal volume of perforin (100-1000 units/ml, final concentration) and/or FITC-GrA or
-GrB (0.25-10 µg/ml, final concentration) or a 20-kDa FITC-labeled dextran (Sigma). Perforin and GrA or GrB were diluted immediately before the assay in 10 mM Hepes, 150 mM NaCl, 1 mM EGTA, pH 7.2, and the whole mixture was pipetted onto a
glass slide, which was then sealed before incubating at 37 °C and
imaging of fluorescence using CLSM (16, 21, 30, 38) at various times.
The doses of perforin used produced <5% specific release of
51Cr from FDC-P1 cells (4-h assay at 37 °C).
Image Analysis--
Analysis of CLSM images using the NIH Image
1.49 public domain software and curve fitting were carried out as
described (16, 21, 30, 38). Results were expressed in terms of Fc/Fmed (cellular uptake: fluorescence quantitated in the cytoplasm (Fc) relative to fluorescence quantitated in the medium (Fmed) after subtraction of background fluorescence) and Fn/c (nuclear accumulation; fluorescence quantitated in the nucleus (Fn) relative to Fc after subtraction of background fluorescence), as previously (17, 21).
Apoptosis--
Apoptotic morphology was determined based on
visual criteria as previously (17, 21), the validity of which has been
confirmed using terminal deoxyribonucleotidyl transferase labeling of
DNA strand breaks with dUTP (TUNEL) analysis (see below) and annexin V
expression (17). To monitor nuclear apoptosis, cells undergoing DNA
fragmentation were quantitated using a TUNEL kit purchased from
Boehringer Mannheim (17). Cells (routinely 2000-5000) were analyzed
immediately on a cytofluorograph (FACScan, Becton Dickinson) (17).
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RESULTS |
Perforin-dependent Nuclear Uptake of Granzymes by
Intact Cells--
We have previously shown that purified GrA and GrB
can localize in the nucleus of intact rat hepatoma and FDC-P1 mouse
myeloid cells in the presence of sublytic concentrations of purified
perforin (16, 17, 21), this nuclear accumulation occurring before the
nuclear and cell membrane changes of apoptosis. Because bcl2 expression affords protection against apoptosis in the case of purified
GrB and perforin (29), we decided to investigate granzyme subcellular
transport in bcl2-expressing FDC-P1 and YAC-1 cell lines
using FITC-labeled granzymes and CLSM. Untransfected FDC-P1 and YAC-1
cells both showed perforin-dependent nuclear uptake of
either GrA or GrB whereby two populations of cells became rapidly evident as observed previously (17, 21); those showing accumulation of
granzymes in the nucleus (Fig. 1,
left panels and not shown) that subsequently underwent
apoptosis ("preapoptotic" cells) based on visual criteria (17) and
those that did not accumulate granzymes and did not undergo apoptosis
("non-apoptotic" cells).

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Fig. 1.
Visualization of the uptake and nuclear
accumulation of granzyme A or B by FDC-P1 (left
panels) and FDbcl2 cells (right panels) in
the presence of perforin. Cells were exposed to FITC-GrA or
FITC-GrB in the presence of perforin at 37 °C as indicated, and
fluorescence was visualized at 60 min using CLSM (see Figs. 2-4 for
quantitative data).
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BCL-2 Blocks Perforin-dependent Translocation of
Granzymes to the Nucleus--
Quantitative analysis of the cellular
and nuclear uptake of FITC-labeled granzymes in the absence or presence
of perforin indicated significant uptake of GrA and GrB into the
cytoplasm, but not nuclear accumulation, in both wild type FDC-P1 and
YAC-1 cells in the absence of perforin (Figs.
2, left panel and not shown).
In the presence of perforin, both granzymes were taken up by the cells
to a greater extent and accumulated strongly in the nucleus, with
maximum levels of accumulation in the nucleus around 2-fold those in
the cytoplasm (Fig. 2, left panel). Granzyme uptake in the
absence of perforin when bcl2 was expressed in the cells
appeared not to be significantly different to that in the parental
lines (Fig. 2, right panel, and see also Figs.
3 and 4,
middle panels). However, when the
bcl2-transfected cells were additionally exposed to
perforin, the rapid redistribution of perforin from the cytoplasm to
the nucleus and the pronounced increase in granzyme uptake into the
cell were clearly not evident, maximum levels of nuclear accumulation
not exceeding 1.0 (Fig. 2 and see below). A control molecule (a 20-kDa
FITC-labeled dextran) did not enter cells in the presence of perforin,
indicating that the effect of perforin with respect to granzymes was
specific. Measurements of granzyme uptake (with or without perforin) in YAC-1/Ybcl2 cells at 15 and ~60 min showed a pattern of granzyme subcellular distribution identical to that seen with FDC-P1/FDbcl2 (Figs. 3 and 4; data not shown).

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Fig. 2.
Uptake and nuclear accumulation of granzyme A
or B or a 20-kDa dextran by FDC-P1 cells expressing (FDbcl2,
right panels) or not expressing (FDC-P1, left
panels) bcl2 in the absence or presence of
perforin. Cells were exposed to FITC-GrA, FITC-GrB, or a 20-kDa
FITC-labeled dextran in the absence or presence of perforin at 37 °C
as indicated and visualized at various times using CLSM. Cells with
apoptotic morphology (preapoptotic) as opposed to nonapoptotic were
distinguished as described under "Results" (Ref. 17; see Fig. 3 for
quantitation thereof), and image analysis was performed to quantitate
either cellular (Fc/Fmed, top panels) and nuclear (Fn/c,
bottom panels) uptake (see "Materials and Methods") for
the different cell populations. Results are averaged from at least
three separate experiments, each individual measurement representing at
least five separate measurements for each of Fc, Fmed, Fn, and
autofluorescence, where the S.E. was not greater than 7.2% the value
of the mean.
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Fig. 3.
Kinetics of uptake and nuclear accumulation
of granzyme A (A) or B (B) or a
20-kDa dextran by FDC-P1 cells expressing (FDbcl2, right
panels) or not expressing (FDC-P1, left
panels) bcl2 in the absence or presence of
perforin. Incubations were performed as described in the legend to
Fig. 2, and the results from image analysis are plotted against time.
Results are averaged from at least three separate experiments, each
individual measurement representing at least five separate measurements
for each of Fc, Fmed, Fn, and autofluorescence, where the S.E. was not
greater than 9.2% the value of the mean.
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Fig. 4.
Kinetics of uptake and nuclear accumulation
of granzyme A (A) or B (B) or a
20-kDa dextran by YAC-1 cells expressing (Ybcl2, right
panels) or not expressing (YAC-1, left
panels) bcl2 in the absence or presence of
perforin. Incubations and analysis were performed as described in
the legend to Fig. 3. Results are averaged from at least two separate
experiments, each individual measurement representing at least five
separate measurements for each of Fc, Fmed, Fn, and autofluorescence,
where the S.E. was not greater than 9.8% the value of the mean.
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Perforin/Granzyme-induced Apoptosis Is Blocked by BCL-2--
We
quantitated apoptotic cell death induced by GrA and GrB in the absence
or presence of perforin in both cell lines and in their
bcl2-expressing variants. As demonstrated previously for GrB
(29), BCL-2 almost totally blocked killing in response to both GrB and
GrA applied with perforin (Figs. 3 and 4, top panels). Whereas 60-90% of wild type cells underwent apoptosis over the period
of study (up to 150 min), bcl2-transfected cells exhibited less than 10% apoptosis (29). The kinetics of nuclear uptake and
apoptosis induction by GrA (Figs. 3A and 4A,
left panels) were slower than for GrB (Figs. 3B
and 4B, left panels (21)), and YAC-1 cells
exhibited slower cellular and nuclear uptake of granzymes compared with
FDC-P1 cells (compare Figs. 3A and 4A and
3B and 4B). Thus, the onset of apoptosis
correlated with the kinetics of granzyme redistribution; the slower
nuclear targeting of GrA was reflected in slower apoptosis compared
with the same concentrations of GrB, whereas YAC-1 cells showed slower
rates of apoptotic death and subcellular redistribution of both
granzymes, compared with FDC-P1 cells.
BCL-2 Prevention of Granzyme Nuclear Uptake Correlates with
Protection against DNA Fragmentation--
To determine whether
the nuclear events of granzyme/perforin-induced apoptosis
were specifically prevented in bcl2-expressing cells
coincident with the inhibition of granzyme nuclear translocation, bcl2-expressing and nonexpressing FDC-P1 and YAC-1 (data not
shown) cells were exposed to perforin and GrB, and DNA breakdown during apoptosis was examined by TUNEL (Fig. 5).
Neither perforin nor GrB alone induced DNA fragmentation in the cell
population above background levels (~3%) in any of the lines. The
combination of both reagents induced a strong response in terms of DNA
fragmentation in the FDC-P1 cells (maximally up to 70%) but had no
significant effect on bcl2-expressing cells (<10% TUNEL
positive cells) (Fig. 5). Comparable results were obtained for the
YAC-1 lines (not shown). It was concluded that bcl2
expression prevented the nuclear events of apoptosis induced by
perforin and GrB, presumably as a consequence of the inhibition of
GrB nuclear translocation.

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Fig. 5.
Lack of DNA fragmentation in
bcl2-expressing FDC-P1 cells in response to GrB and
perforin. FDC-P1 and FDbcl2 cells were incubated in the presence
of different concentrations of perforin, GrB, or both as indicated for
75 (A) or 60 min (B) at 37 °C before TUNEL
staining and cytofluorography. The percentages of TUNEL positive cells
(indicative of DNA fragmentation) are indicated. A and
B represent two separate experiments from a series of 6 similar experiments (see also Ref. 29). U, units.
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DISCUSSION |
We have previously shown that GrB nuclear uptake
precedes the onset of apoptotic DNA fragmentation (17), implying a role of nuclear granzymes in the latter. In this study we establish for the
first time that bcl2 expression prevents
perforin-dependent nuclear translocation of granzymes in
two different cell lines, concomitant with protection against the
nuclear changes associated with granzyme/perforin-mediated apoptosis
and ultimately cell death. This strongly supports the idea that nuclear
translocation of granzymes plays a role in the nuclear events of
apoptosis, consistent with the recent demonstration that catalytic
subunit of DNA-dependent protein kinase and NuMA (nuclear
mitotic apparatus protein) are both cleaved by GrB in vivo
(20) as well as the fact that GrA/GrB knockout mice are completely
deficient in the CTL-mediated nuclear but not cytolytic apoptotic
response. Because our results here for cellular uptake (Figs. 2-4)
show that GrA and GrB enter bcl2-expressing cells to an
extent comparable with wild type in the absence of perforin and the
sensitivity of the bcl2-expressing cells to
perforin-mediated membrane damage appears to be normal (29), it can be
concluded that BCL-2 action is exerted at or downstream of the point of
synergy between perforin and granzymes. BCL-2 presumably targets an
essential component/step in the signaling pathway, a likely candidate
being caspase activation that we have recently shown to be required for
both granzyme nuclear translocation and induction of the nuclear events
of apoptosis (39); inhibition of caspase activation would block the
downstream events of granzyme nuclear translocation and subsequent
nuclear changes of apoptosis (17), including the cleavage of catalytic
subunit of DNA-dependent protein kinase and NuMA (38). The
Caenorhabditis elegans BCL-2 homolog CED-9 has been shown to
exert its effect at least in part by blocking activation by CED-4 of
CED-3, the homolog of the mammalian interleukin-1
-converting enzyme
(ICE) (40, 41). Interestingly, the nonnuclear events in
granzyme/perforin-induced cytolysis, although able to be blocked by
BCL-2 (29), appear to be caspase-independent (39, 42), suggesting that
BCL-2 also acts on an as yet uncharacterized caspase-independent
pathway that governs nonnuclear apoptotic phenomena; that the nucleus
is dispensable for apoptosis has been shown using enucleated
cytoplasts (43).
Of significance in the context of this study may be the fact that,
apart from being localized to the outer mitochondrial membrane and
endoplasmic reticulum, BCL-2 has been shown to be present in the
nuclear envelope (44-46). The complete lack of BCL-2 within the plasma
membrane is consistent with our observations that both granzyme uptake
(this study) and pore formation by perforin (this study and Ref. 29)
are unaffected by BCL-2, and its absence from endocytic vesicles argues
against the possibility that BCL-2 blocks perforin-induced release of
granzymes from encapsulated vesicles to the cytosol (33). Structural
homology between the BCL-2 family member BCL-XL and the
membrane translocation domain of some bacterial toxins presumed to form
a membrane pore (47) implies that BCL-2 may affect the transport of
proteins or ions across membranes. That perturbation of ionic
concentrations within the nuclear envelope can impair nuclear pore
complex function, and hence, nuclear protein import (48, 49) has been
reported, meaning that BCL-2 within the nuclear envelope could
conceivably perturb nuclear pore complex function, thereby blocking
granzyme passage into the nucleus directly. Significantly, BCL-2
antagonizes the transport of p53 and cyclin-dependent
kinases into the nucleus as well as that of cytochrome c
from mitochondria (50, 51), consistent with this possibility.
This study demonstrates that bcl2 expression blocks
perforin-dependent nuclear targeting of GrA and GrB in
intact cells and concomitantly confers protection against apoptosis and
its nuclear events in particular. The results here, consistent with
other studies (9, 10, 13, 21), indicate that GrB nuclear uptake and
induction of apoptosis were faster than for GrA, whereas YAC-1 cells
showed greater resistance to granzyme nuclear uptake and apoptosis than
FDC-P1 cells. Thus, in all cases, the rate of granzyme nuclear uptake
correlated well with the kinetics of the induction of apoptosis,
supporting the role of nuclear targeting of granzymes in apoptosis
(Refs. 16, 17, 21; see also Ref. 20). That perforin-dependent induction of apoptosis by both GrA and
GrB is inhibited by bcl2 expression is further evidence that
granzymes utilize a common and possibly unique nuclear transport
pathway (16, 21), strongly implying that nuclear targeting of granzymes plays a role in effecting the nuclear changes of apoptosis.