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INTRODUCTION |
Following treatment with chemotherapy agents or exposure to
cellular stress such as heat shock or ionizing radiation, malignant cells can respond by undergoing apoptosis or necrosis, or may be
resistant to treatment. The failure of malignant cells to undergo cell
death in response to chemotherapy is a major cause of treatment failure
(1), and, in many cases, chemoresistance is associated with aberrant
expression of the proteins involved in the activation and regulation of
apoptosis (1-3). Consequently, several therapeutic strategies based on
modulating apoptotic pathways are currently in development (4, 5).
Apoptosis is characterized by well defined morphological and
biochemical changes, which are generally mediated by a family of
cysteine proteases, called caspases. There are at least two well
characterized molecular mechanisms leading to
caspase-dependent apoptosis, namely receptor-mediated and
mitochondrial pathways, which have been described in some detail in
recent reviews (1, 2). Both pathways lead to activation of caspase-3,
which ultimately results in fragmentation of chromosomal DNA and
proteolysis of selected nuclear proteins. The aim of the present study
is to investigate the role of nucleolin during apoptosis of leukemia cells treated with the chemotherapy agent, camptothecin, or irradiated with UV light, and to examine the relationship between changes in
nucleolin and effects on poly(ADP-ribose) polymerase-1
(PARP-1).1
Human nucleolin is a 707-amino acid protein consisting of an acidic
histone-like N terminus, a central domain containing four RNA binding
domains, and a C terminus that is rich in arginine and glycine (RGG
repeats). This multidomain structure reflects the diverse roles of
nucleolin in cell growth, proliferation, and death, which have been
recently highlighted in several excellent reviews (6-8). Nucleolin has
been implicated in many cellular processes, including transcription,
packing, and transport of ribosomal RNA, replication and recombination
of DNA, cell cycle progression, and apoptosis (6-14). Although
generally considered a predominantly nucleolar protein, nucleolin
appears to be very mobile and can also be present in the nucleoplasm
and cytoplasm and on the cell surface (9, 10, 15-19). In fact, there
are several reports describing redistribution of nucleolin within the
cell in response to a number of stimuli, including heat shock (9, 10),
mitosis (20), T cell activation (21), treatment with a
cyclin-dependent kinase inhibitor (22), and viral infection (23-25).
There is considerable interest in studying nucleolin function, not only
because it is involved in so many fundamental processes, but also
because of the significance of nucleolin expression in malignant cells.
Levels of nucleolin are positively correlated with cellular
proliferation (26) and high levels of silver-staining nucleolar
proteins (of which nucleolin is the major component) predict a poor
prognosis in many types of cancer (27). We have proposed nucleolin as a
novel target for therapeutic intervention, based on our finding that
G-rich oligonucleotides that bind to nucleolin protein can inhibit
proliferation and induce apoptosis in many cell lines derived from
solid tumors (28-30) and leukemias (31).
The levels and characteristics of PARP-1 protein have also been
examined in this study for two reasons. First, PARP-1 cleavage is a
characteristic feature of apoptosis and the timing of
apoptosis-associated events is often determined relative to the onset
of PARP-1 cleavage. Second, it has been reported that nucleolin can
form a complex with PARP-1 in B-cells (11) and in kidney cells (32),
suggesting that PARP-1 could potentially be involved in the regulation
of nucleolin function, or vice versa.
PARP-1 catalyzes the addition of poly(ADP-ribose) chains to a number of
nuclear proteins in response to DNA damage. It is clear that this
short-lived post-translational modification plays an important,
although incompletely defined, role in DNA damage response and
apoptosis (33, 34). PARP-1 enzymatic activity is dependent upon binding
to DNA strand breaks and is rapidly activated in response to cellular
stresses, such as heat shock, gamma radiation, exposure to carcinogens,
and treatment with chemotherapy agents. Synthesis of poly(ADP-ribose)
uses nicotinamide adenosine dinucleotide (NAD+), and cleavage of PARP-1
during apoptosis is thought to occur to conserve NAD+ and allow
production of ATP, which is needed for execution of apoptosis. This
cleavage appears to be a universal part of the apoptotic process, and
appearance of the 89- and 24-kDa proteolytic fragments of PARP-1 has
become one of the classical hallmarks of apoptosis (33, 34).
Here, we describe the effect of apoptosis in U937 leukemia cells on the
expression of nucleolin and PARP-1 proteins. Because nucleolin can
translocate between different cellular compartments, alterations in
nucleolin in both nuclear (containing the nucleoli and nucleoplasm) and
S-100 (containing the cytoplasm and plasma membrane) extracts have been evaluated.
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EXPERIMENTAL PROCEDURES |
Materials--
Camptothecin is an anti-neoplastic topoisomerase
I inhibitor, and 3-aminobenzamide (3-ABA) is an inhibitor of PARP-1.
Both were purchased from Sigma, dissolved in Me2SO,
and diluted with phosphate-buffered saline (PBS) to give stock
solutions in 0.5% Me2SO. Anti-nucleolin antibody (mouse
monoclonal IgG1), anti-PARP-1 antibody (mouse monoclonal
IgG2A), and normal mouse IgG were from Santa Cruz
Biotechnology (Santa Cruz, CA), and anti-poly(ADP-ribose) (rabbit
polyclonal) was from Calbiochem (San Diego, CA). Protein A used for
immunoprecipitation was purchased from Sigma. Secondary antibodies
(anti-mouse or anti-rabbit linked to horseradish peroxidase) were
purchased from Santa Cruz Biotechnology. Fluorescein
isothiocyanate-conjugated secondary antibodies (Alexa-488) were
purchased from Molecular Probes (Eugene, OR).
Cell Culture and Treatment--
U937 cells (human myeloid
leukemia) were grown in suspension in RPMI 1640 medium supplemented
with 10% heat-inactivated (20 min at 65 °C) fetal bovine serum
(FBS), 100 units/ml penicillin, 100 µg/ml streptomycin at 37 °C
with 5% CO2.
For UV irradiation, cells were plated at 5 × 105
cells/ml in dishes (60 mm diameter) or flat-bottomed six-well plates.
The cells were irradiated with UV light by placing the plate (without a
lid) directly in a Stratagene UV Stratalinker and irradiating for the
time indicated. Cells were then replaced in the incubator at 37 °C
for the times shown. For camptothecin treatment, exponentially growing
U937 cells were incubated with camptothecin at the concentration shown
for various times (2, 4, 8, and 24 h). Where indicated, 3-ABA (1 mM final concentration) was added to cells 30 min prior to
treatment (and not removed).
Cell Growth Assay (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl
Tetrazolium Bromide (MTT))--
Untreated and irradiated cells were
plated at 2 × 104/ml in a 96-well plate. Viable cells
were assessed using the MTT assay (35) at 48 h after irradiation.
Samples were plated in triplicate, and the error
bars (Fig. 3D) represent the standard error of
the data.
DNA Fragmentation--
DNA fragmentation was detected using a
previously reported method (36). Briefly, cells were collected by
centrifugation and resuspended in PBS containing 5 mM
MgCl2. Cells were lysed by overnight incubation at 37 °C
in a buffer containing 0.1% SDS and 1.5 mg/ml proteinase K. Samples
were extracted twice with an equal volume of phenol/chloroform/isoamyl
alcohol (25:24:1), the aqueous layer was transferred to a new tube, and
DNA was precipitated with ethanol and digested with 400 µg/ml RNase
A. Electrophoresis was performed on 1% agarose gels, stained with
ethidium bromide.
Immunofluorescence Staining of Nucleolin--
Cells were
collected by centrifugation, washed twice with PBS, and placed on glass
slides using a cytospinner. Samples were fixed in 4% paraformaldehyde
in PBS for 15 min at room temperature, and then permeabilized with
0.2% Triton X-100 in PBS for 10 min. After two washes with PBS,
nonspecific binding of antibody was blocked by a 1-h incubation at room
temperature with 5% normal goat serum in PBS. After three washes with
PBS, slides were incubated in primary antibody (1:100 anti-nucleolin
antibody in blocking buffer) for 1 h at room temperature and then
washed three times in PBS. Slides were incubated with
Alexa-488-conjugated anti-mouse (diluted 1:500 in blocking buffer) for
1 h at room temperature, washed three times in PBS, then observed
using an Olympus BX60F fluorescence microscope and photographed using
an Olympus DP10 camera. To stain the small bodies from the culture
medium, cells were cultured in serum-free medium, irradiated with UV
light, and then returned to the incubator. At the indicated time, cells were pelleted by centrifugation and medium was collected and placed onto glass slides using a cytospinner. Slides were stained using the
same procedure described for cells.
TUNEL Staining--
Slides containing the small bodies from the
medium of apoptotic cells were prepared as described above. Slides were
washed with PBS and incubated in permeabilization solution (0.1%
Triton X-100, 0.1% sodium citrate) for 2 min on ice, washed twice with PBS, and dried. Samples were stained as previously described (29). Briefly, 50 µl of TUNEL reaction mixture (Roche) was added to each
sample and slides were incubated in the dark in a humidified chamber
for 60 min at 37 °C, then washed three times with PBS and observed
as above.
Preparation of Cellular Extracts and Protein from Cell Culture
Medium--
At the appropriate time after treatment, cells were
harvested and washed twice with cold PBS, and nuclear and S-100
extracts were prepared according to the method of Coqueret et
al. (37). Briefly, 100 µl of ice-cold extraction buffer B (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) was added to the cells. After
three cycles of freeze-thaw, S-100 extracts were recovered as
supernatant following centrifugation at 12,000 × g for
1 min, and pellets (nuclei) were resuspended in 40 µl of buffer C (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM KCl, 0.2 mM EDTA, 25% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Following 30 min of incubation at 4 °C,
insoluble material was precipitated by centrifugation at 12,000 × g for 5 min and nuclear extracts were collected as
supernatant. Extracts were either used immediately or stored at
80 °C.
For preparation of proteins from the cell culture medium, the medium
was first replaced with serum-free medium and cells were irradiated as
described above. At the appropriate time, samples were centrifuged at
300 × g for 10 min to pellet intact cells and
supernatant was collected. The medium was filtered (syringe filters
with polyvinylidene difluoride membranes, Whatman) where indicated, and
proteins present in the medium were concentrated using Centricon YM-30
(Millipore, Bedford, MA) according the instructions of the manufacturer.
Immunoblot Analysis--
The concentration of extracted proteins
was determined using the Bio-Rad DC protein assay kit. Samples (10 µg) were incubated in sodium dodecyl sulfate (SDS)-loading buffer
(100 mM Tris-HCl, pH 6.8, 200 mM
dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) at
65 °C for 15 min, and separated on 10% (for nucleolin detection) or
8% (for PARP-1) polyacrylamide-SDS gels, followed by electroblotting
to polyvinylidene difluoride membranes (Bio-Rad). After blocking
nonspecific binding sites for 1 h in 5% nonfat dried milk in PBST
(0.1% Tween 20 in PBS), the membrane was incubated for 1 h at
room temperature or overnight at 4 °C with primary antibody (1:1000
anti-nucleolin or anti-PARP-1 in PBS-T). After three washes in PBST,
the membrane was incubated with horseradish peroxidase-conjugated goat
anti-mouse antibody for 45 min at room temperature, washed three times
in PBST, and detected using enhanced chemiluminescence (ECL kit from
Amersham Biosciences). Equal gel loading and transfer of proteins were
confirmed by staining membranes with India ink (28).
Immunoprecipitation--
Nuclear extracts were prepared from
untreated cells or following UV irradiation. Immunoprecipitations were
performed by incubating 200 µg of extract with 2 µg of PARP-1
antibody for 1 h at 4 °C, followed by addition of Protein
A-agarose conjugate (20 µl) and overnight incubation at 4 °C on a
rotator. Control immunoprecipitations were performed with normal mouse
IgG in place of primary antibody. The agarose beads were precipitated
(centrifugation at 1300 × g for 5 min) and washed four
times with radioimmunoprecipitation assay buffer (PBS, 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 1 mM sodium fluoride, 10 mg/ml phenylmethylsulfonyl
fluoride, 2 µM aprotinin, 100 mM sodium
orthovanadate). They were resuspended in SDS-loading buffer, boiled for
3 min, and loaded on SDS-polyacrylamide gels. Immunoblot analysis was
performed using nucleolin and PARP-1 antibody as primary antibody as
described above. To analyze poly(ADP-ribosyl)ation of nucleolin,
nucleolin antibody was used for immunoprecipitation, and
anti-poly(ADP-ribose) polyclonal antibody (1:2000) was used in
immunoblot analysis.
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RESULTS |
Alterations of Nucleolin and PARP-1 Proteins after UV-induced
Apoptosis--
Before investigating alterations in protein levels, we
confirmed that apoptosis was occurring in treated U937 cells using a
DNA fragmentation assay (36). In this assay, apoptosis is indicated by
the appearance of a DNA "ladder," which is produced by endonuclease
cleavage of chromosomal DNA into nucleosomal fragments. Fig.
1 shows that apoptosis could be clearly
detected within 2 h following treatment of cells by UV
irradiation for 30 s or incubation with the chemotherapy
agent, camptothecin at 10 µM final concentration.

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Fig. 1.
UV irradiation and camptothecin-induced
fragmentation (nucleosomal ladder) of genomic DNA in U937 leukemia
cells, indicative of apoptosis. Samples were derived from
untreated cells (lane 6), UV-irradiated cells at
2, 4, or 24 h after irradiation for 30 s (lanes
1-3), or cells that were treated with 10 µM
camptothecin for 4 or 24 h (lanes 4 and
5).
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To examine apoptosis-induced changes in nucleolin and PARP-1, cells
were irradiated with UV, and protein extracts were collected at
different time points after irradiation. Equal amounts of protein fractions were examined and consisted of nuclear extracts (soluble nuclear proteins) or S-100 extracts (soluble proteins from the plasma
membrane, cytosol and non-nuclear organelles). Our previous studies
(28) suggest that the majority of the nucleolin present in the S-100
fraction is derived from the plasma membrane. Fig. 2 (lane 1,
panels A-C) shows that untreated U937 cells
contained high basal levels of nucleolin in both S-100 and nuclear
fractions, and of PARP-1 in nuclear extracts. PARP-1 existed
predominantly as the full-length product (116 kDa) and nucleolin
migrated on SDS-polyacrylamide gels as an ~110-kDa band. An
additional minor band was sometimes observed in the S-100 extracts
blotted for nucleolin, and the significance of this band is not known,
but the mobility of the major S-100 nucleolin band corresponded to that
of the nuclear fraction. Following irradiation with UV light for
30 s (equivalent to ~30 J/m2), a profound decrease
in the levels of S-100 nucleolin was observed (Fig. 2A),
such that by 24 h this band was almost undetectable. Apoptosis
also resulted in decreased levels of nuclear nucleolin (Fig.
2B). These nuclear changes were less pronounced than in the
S-100 fraction, but occurred more rapidly and were already obvious by
2 h after irradiation. By 72 h after irradiation, levels of
nuclear nucleolin had returned to base-line levels. Clearly, apoptosis-induced alterations in nuclear and S-100 nucleolin did not
occur in parallel. It is possible that apoptosis causes some of the
nuclear nucleolin to relocate outside of the nucleus or vice
versa, but because the nuclear and S-100 nucleolin bands are
indistinguishable, we were not able to investigate this possibility by
this method.

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Fig. 2.
Expression of nucleolin and PARP-1 proteins
in U937 cells after apoptosis was induced by UV irradiation. The
upper three immunoblots show nucleolin
in S-100 extracts (A), nucleolin in nuclear extracts
(B), and PARP-1 in nuclear extracts (C) at
various times following 30 s of UV irradiation. The
lower three immunoblots show nucleolin
in S-100 extracts (D), nucleolin in nuclear extracts
(E), and PARP-1 in nuclear extracts (F) at 8 h following UV irradiation for various times.
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Presumably, UV irradiation also caused activation of PARP-1, because
cleavage was induced within 2 h following treatment (Fig. 2C). Although UV does not generally induce DNA strand breaks
directly, it can lead to both DNA damage (thymine dimers) and the
production of free radicals. The presence of either intracellular free
radicals or unrepaired DNA damage could initiate the apoptotic cascade (38), and PARP-1 is likely activated in response to DNA fragmentation. In any case, PARP-1 cleavage preceded the disappearance of S-100 nucleolin by several hours. On the other hand, the inhibition of
nuclear nucleolin levels appears to occur roughly in parallel with
cleavage of PARP-1.
The overall intensity of PARP-1 bands was significantly increased in
apoptotic cells relative to untreated cells. This could be the result
of increased PARP-1 expression or possibly to an enhanced affinity of
the monoclonal antibody for the 89-kDa fragment. The reappearance of
PARP-1 and nucleolin at 48-72 h after irradiation was most likely
caused by proliferation of cells that survived treatment.
In addition, the dose dependences of the alterations in nucleolin and
PARP-1 were examined. Panels D and E
of Fig. 2 show that there was a clear dose-dependent
decrease in nucleolin levels in both the nuclear and S-100 extracts of
cells at 8h following irradiation with UV for 5, 10, 30, or 45 s
(equivalent to ~5, 10, 30, and 45 J/m2, respectively).
Induction of PARP-1 cleavage apparently had a lower threshold, and
complete cleavage to the 89-kDa fragment was achieved with only 5 s of irradiation (Fig. 2, panel F).
Effect of PARP-1 Inhibitor, 3-ABA--
To investigate whether
there was a direct relationship between PARP-1 and UV-induced changes
in nucleolin, similar experiments were carried out in the absence or
presence of a PARP inhibitor, 3-ABA. This compound blocks the enzymatic
activity of PARP-1 (and also other ADP-ribosylation enzymes), probably
by competing for the substrate (NAD+) binding site (34).
Fig. 3 shows the results of these
experiments and suggests that 3-ABA can prevent the loss of nuclear
nucleolin (Fig. 3B) and drastically inhibit the
disappearance of S-100 nucleolin (Fig. 3A) following 30 s of UV irradiation. 3-ABA could also partially inhibit PARP cleavage
(Fig. 3C). Because PARP-1 inhibition can both increase cell
death or decrease it, depending on conditions (39), it was important to
establish that the effect of 3-ABA in abrogating changes in nucleolin
was not simply caused by prevention of apoptosis. Therefore, we next
determined the ability of 3-ABA to inhibit cell death caused by UV
irradiation of various doses. Irradiation of cells with increasing
amounts of UV was found to lead to dose-dependent induction
of cell death, as assessed by the MTT assay (data not shown). Although
the presence of 3-ABA could significantly protect cells from lower
doses of UV (5 or 10 s of irradiation, data not shown), at the
dose in question (30 s of irradiation), 3-ABA had only minimal effect
on UV-induced cell death (Fig. 3D). Therefore, this small
degree of protection from apoptosis is unlikely to explain the dramatic
inhibitory effects on nucleolin alterations. In addition, under these
conditions, the presence of 3-ABA had no effect on DNA fragmentation
because the appearance of the DNA ladder following agarose gel
electrophoresis was unaffected (data not shown), confirming that
apoptosis is still occurring.

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Fig. 3.
Effect of PARP-1 inhibitor 3-ABA on
alterations in nucleolin and PARP-1 expression, and on cell
proliferation, following UV-induced apoptosis. Immunoblots show
nucleolin in S-100 extracts (A), nucleolin in nuclear
extracts (B), PARP-1 in nuclear extracts (C), and
MTT assay indicates cell viability (D).
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Although the inhibition of PARP-1 activity by 3-ABA is well
established, the observation that PARP-1 cleavage was inhibited by
3-ABA was somewhat surprising. Proteolytic cleavage of PARP-1 during
apoptosis is carried out by caspase-3, whose activity is not known to
depend on ADP-ribosylation. This unexpected effect of 3-ABA may be
related to its inhibition of another enzyme apart from PARP-1, either
as a result of a direct effect on the enzyme, or because of the
prevention of its ADP-ribosylation. However, it is interesting that
other independent reports have also suggested that 3-ABA can inhibit or
delay caspase-3 activation (40, 41).
Because the major effect of 3-ABA is to inhibit the addition of
ADP-ribose chains to protein substrates, poly(ADP-ribosyl)ation of
nucleolin in response to apoptosis was also investigated. Nucleolin was
immunoprecipitated from nuclear extracts derived from untreated or
UV-irradiated U937 cells, and immunoblotted using an antibody to
poly(ADP)-ribose. In accord with a previous report examining HeLa cells
(42), we found that nucleolin was a substrate for poly(ADP-ribosyl)ation in U937 cells. Fig.
4 (A and B) shows
that there was a very low level of constitutive poly(ADP-ribosyl)ation of nucleolin in untreated cells, but in response to UV irradiation significant levels of poly(ADP-ribose) were transiently associated with
nucleolin. Poly(ADP-ribosyl)ation of nucleolin was detectable at high
levels at 2 h following irradiation (even though levels of total
nucleolin were low at this time point) but had disappeared by 4 h.
As expected, poly(ADP-ribosyl)ation of nucleolin was reduced when cells
were pre-incubated with 3-ABA.

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Fig. 4.
Immunoprecipitations (IP)
using nuclear extracts from untreated or UV-irradiated U937 cells.
To evaluate poly(ADP-ribosyl)ation of nucleolin, immunoprecipitations
were performed using a nucleolin antibody, and samples were analyzed by
immunoblotting for nucleolin (A) or poly(ADP-ribose)
(B). To determine whether nucleolin can interact with
PARP-1, immunoprecipitations were performed using anti-PARP-1 antibody
or control. Immunoblots of precipitated proteins were analyzed by
immunoblotting for nucleolin (C) or anti-PARP-1
(D).
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To further investigate the potential relationship between nucleolin and
PARP-1, immunoprecipitations of U937 nuclear proteins using a PARP-1
antibody were carried out to determine whether nucleolin and PARP-1
interact. Immunoprecipitated samples were electrophoresed on denaturing
SDS-polyacrylamide gels and analyzed by immunoblotting using antibodies
for nucleolin or PARP-1. Fig. 4C shows that nucleolin was
precipitated by a PARP-1 monoclonal antibody in both untreated and
UV-irradiated cells, but was not precipitated in the absence of PARP-1
antibody or control IgG. These data indicate that nucleolin is
associated with PARP-1 in both untreated and apoptotic cells, and it
can interact with both full-length and cleaved PARP-1 (Fig.
4D).
Alterations of Nucleolin and PARP-1 Proteins in Response to
Camptothecin Treatment--
To determine whether the phenomena we had
observed were specific to UV-irradiated cells or were a general feature
of apoptosis, we also examined protein changes in U937 cells treated
with the Topo I inhibitor, camptothecin. Fig.
5 shows that apoptosis induced by
camptothecin (10 µM final concentration) also caused a
disappearance of nucleolin from the S-100 fraction and a reduction in
the levels of nuclear nucleolin. However, these effects were less
pronounced, and occurred at later time points than for UV-irradiated
cells. Similarly, the response of PARP-1 cleavage was also slightly
delayed compared with the UV-treated cells, with only partial cleavage after 4 h (compare with Fig. 2C). In contrast to the
irradiated cells, pre-incubation with 3-ABA produced only a small
degree of protection from apoptosis-induced changes in nucleolin and PARP-1.

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Fig. 5.
Expression of nucleolin and PARP-1 proteins,
in the absence or presence of PARP-1 inhibitor (3-ABA), after apoptosis
was induced by incubation with camptothecin
(CPT). The upper three
immunoblots show nucleolin in S-100 extracts (A),
nucleolin in nuclear extracts (B), and PARP-1 in nuclear
extracts (C) when cells were incubated for various times
with 10 µM camptothecin. The lower
three immunoblots show nucleolin in S-100
extracts (D), nucleolin in nuclear extracts (E),
and PARP-1 in nuclear extracts (F) when cells were incubated
for 24 h in the presence of camptothecin at the concentration
shown.
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As in the UV-treated cells, the effect of camptothecin on nucleolin
alterations was found to be dose-dependent (Fig. 5,
panels D and E), and, again, the dose
required to induce cleavage of PARP-1 was lower than that required to
alter nucleolin (panel F).
Redistribution of Nucleolin in Cells Undergoing Apoptosis--
Our
data clearly indicate reductions in the levels of nucleolin in both the
nuclear and cytoplasm/plasma membrane fractions of apoptotic cells.
However, the fate of the nucleolin protein that "disappears" is not
clear. To investigate this further, we examined the nuclei of apoptotic
cells using immunofluorescent techniques to detect nucleolin. Fig.
6 shows that, following either camptothecin treatment (10 µM) or UV irradiation (30 s),
there was a dramatic redistribution of nucleolin in the apoptotic
nuclei. In untreated cells, nucleolin was located throughout the
nucleoplasm but was concentrated in the intensely stained nucleoli. In
contrast, in treated cells, distinct alterations in nucleolin staining
were observed. Some cells exhibited a "speckled" staining pattern, in which the nucleolin appeared to have left the nucleoli and redistributed throughout the nucleoplasm as small distinct foci (for
example, the cells highlighted at 2 h following UV or
camptothecin). In other cells, there was "perinuclear" staining,
where the nucleolin appeared to have migrated to the periphery of the
nuclei (for example, the cells highlighted at 4 h following UV or
camptothecin). Both types of staining patterns were observed at each
time point examined, and many cells exhibited staining pattern that was
intermediate between the speckled and perinuclear patterns.
Counterstaining with propidium iodide (data not shown) was used to
confirm that both speckled and perinuclear nucleolin staining was
within the nuclei and not the cytoplasm. Using this approach, we also
observed the appearance of nucleolin-containing bodies at the periphery of the nucleus and in extranuclear regions (examples are indicated in
Fig. 6 by white arrows). Appearance of these
bodies was much more pronounced in cells irradiated with UV than in
camptothecin-treated cells.

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Fig. 6.
Immunofluorescence staining of permeabilized
U937 cells using a nucleolin monoclonal antibody. Selected
fields show typical images of untreated cells, or cells treated
with 10 µM camptothecin (CPT) or 30 s UV,
as indicated. Insets to the right (untreated and
camptothecin-treated) or left (UV-treated) of the
main panels show enlarged images of individual
cells from the region outlined in the main panel.
Counterstaining with propidium iodide (data not shown) indicates that
the nucleolin staining is predominantly within the nuclei. The
white arrows indicate small nucleolin-containing
bodies that appear to be extruded from apoptotic cells.
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It should be noted that, using this method, the portion of nucleolin
located in the plasma membrane cannot be observed because the cell
membrane is permeabilized by detergent and thus the nucleolin antibody
is internalized where it can only bind to nuclear and cytoplasmic
components. Additionally, consistent with the immunoblot data, the
overall intensity of nuclear nucleolin staining was significantly
decreased (relative to untreated cells) by UV irradiation or
camptothecin treatment at all time points examined. This differential intensity may not be evident in Fig. 6 because the images of treated cells have been enhanced for brightness to allow clear visualization of
the pattern of staining.
Identification of Nucleolin in Extracellular Apoptosis-induced
Bodies--
The overall disappearance of 110-kDa nucleolin is very
pronounced (Fig. 2) and could possibly be the result of proteolytic digestion of nucleolin (either fragmentation or complete degradation) or loss of nucleolin from the cell by secretion or extrusion. In the
previous experiments, we did not detect the appearance of nucleolin
cleavage products with either a monoclonal antibody (Figs. 2 and 3), or
a nucleolin polyclonal antibody (Santa Cruz, clone C-18; data not
shown). In light of our observation of nucleolin-containing bodies
(Fig. 6), we next investigated the possibility that nucleolin was shed
into the cell culture medium by examining proteins derived from the
medium of untreated and apoptotic cells. For these experiments, the
medium was first replaced by serum-free medium to minimize the presence
of nonspecific proteins. Immunoblot analysis (Fig. 7A) showed that almost no
nucleolin was detected in the culture medium of untreated cells (either
with or without 3-ABA incubation). However, after UV irradiation a
clear band corresponding to full-length nucleolin appeared in the
medium proteins, and the appearance of this band could be inhibited by
3-ABA. To determine whether this nucleolin was derived from soluble
protein or associated with cell-derived particles, the medium was
pre-filtered before blotting (Fig. 7B). Filtration with a
0.2-µm (data not shown) or 0.45-µm filter caused the loss of
nucleolin immunoreactivity, indicating that the nucleolin was not
secreted as soluble protein; instead, it was associated with particles
of a size greater than 0.45 µm. However, a significant amount of
nucleolin passed through a 0.8-µm filter, excluding the possibility
that the immunoreactivity was the result of the presence of intact
cells (which are >10 µm in diameter) that were not collected by
centrifugation. These apoptosis-induced particles are of a size
that is consistent with "apoptotic bodies" that are often observed
during apoptosis in cultured cells and in vivo (43-46).
These apoptotic bodies are commonly detected by TUNEL staining for
fragmented DNA (46); therefore, we prepared slides from the medium of
untreated or UV-irradiated U937 cells and analyzed them for TUNEL and
nucleolin immunoreactivity. The apoptosis-induced bodies (which were
clearly much smaller than intact cells) were found to specifically
appear following UV irradiation and stained strongly for both
nucleosomal DNA (TUNEL, Fig. 7C) and nucleolin (Fig.
7D). There was no staining using a control IgG or in the
absence of primary antibody (data not shown). To determine whether
nucleolin and DNA co-existed in the same particles, we carried out
double staining for nucleolin (green staining) and DNA (red staining,
propidium iodide). Some, but not all, of the apoptotic bodies that
stained positive for nucleolin also stained positive for the presence
of DNA, and an example is shown in the inset to Fig.
7D.

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Fig. 7.
Levels of nucleolin detected in the culture
medium of untreated and apoptotic U937 cells. Cells were cultured
in serum-free medium and then treated with UV light for 30 s, with
or without 3-ABA pre-incubation. Immunoblots show the presence of
nucleolin in the medium of cells at different times after UV
irradiation (A), and the size of the nucleolin-containing
particles was determined by pre-filtering the medium using filters with
pores of various sizes (B). The apoptosis-induced
extracellular bodies could also be detected by TUNEL staining
(C) or immunofluorescent staining for nucleolin
(D). The inset to panel D
shows that some of these particles contain both nucleolin
(anti-nucleolin staining, green) and DNA (propidium iodide
staining, red).
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The appearance of the nucleolin-positive bodies was found to coincide
with the observation of nucleolin in the medium, the appearance of the
DNA ladder, and the loss of nuclear nucleolin, but preceded the loss of
the plasma membrane nucleolin. However, it was not possible to
determine whether the nucleolin in the apoptotic bodies originated from
the nuclei or the plasma membranes of apoptotic cells, because there is
no way to easily distinguish nuclear and plasma membrane nucleolin.
Studies are under way to further investigate the origin of nucleolin in
apoptotic bodies.
Relocalization of the 89-kDa PARP-1 Fragment--
Originally,
PARP-1 levels were determined only in nuclear extracts because no
PARP-1 was observed in S-100 extracts of untreated cells. Following our
observations that nucleolin translocation is blocked when the cleavage
of PARP-1 is abrogated (Fig. 3) and that nucleolin binds to PARP-1
full-length protein and its 89-kDa proteolytic fragment (Fig. 4), we
postulated that nucleolin may play a role in transporting cleaved
PARP-1 in apoptotic cells. To test the feasibility of this idea, the
presence of PARP-1 was re-evaluated in the S-100 extracts and cell
culture medium following UV treatment.
Fig. 8A shows that PARP-1 was
virtually undetectable in the S-100 extracts of untreated U937 cells
(lane 1). On the other hand, following induction
of apoptosis, the 89-kDa PARP-1 fragment (and a small amount of
full-length protein at 1 h) was clearly observed. The appearance
of this band was concurrent with reduction in the nuclear levels of
nucleolin (Fig. 2B). Fig. 8B shows that, in
response to UV irradiation or camptothecin treatment, the 89-kDa PARP-1
fragment was also extruded into the cell culture medium and was
associated with particles of a size similar to those containing nucleolin (compare with Fig. 7B). Therefore these results
are consistent with the hypothesis that nucleolin transports cleaved PARP-1 from the nucleus and translocates to the plasma membrane, where
both are packaged into apoptotic bodies. Bands corresponding to 89-kDa
PARP-1 and 110-kDa nucleolin could also be detected in the culture
medium following treatment of cells with 10 µM camptothecin (data not shown).

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Fig. 8.
Immunoblots showing levels of PARP-1 in the
S-100 fraction (A) and the culture medium
(B) of untreated and apoptotic U937 cells.
Some of the culture medium samples were pre-filtered, as
indicated.
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DISCUSSION |
There are a number of reasons why studying nucleolin function and
regulation is of interest. First, nucleolin is a fascinating protein
because of its multifunctionality and its ability to translocate within
the cell, yet the reasons for its ubiquitous behavior have not been
fully elucidated (6-8). Second, nucleolin is associated with many of
the processes that are dysfunctional in neoplastic cells
(proliferation, cell cycle control, apoptosis) and elevated levels
of nucleolin expression are generally related to malignancy (26, 27).
Third, we have identified nucleolin as the probable molecular target of
a class of non-antisense G-rich oligonucleotides that inhibit the
proliferation of tumor and leukemia cell lines, and therefore have
significant promise as novel therapeutic agents (28-31).
In this report, we have described alterations in the levels and
localization of nucleolin protein that occur in leukemia cells induced
to undergo apoptosis by UV-irradiation or treatment with the
topoisomerase I inhibitor, camptothecin. We found that induction of
apoptosis was accompanied by a rapid reduction in the levels of nuclear
nucleolin, followed several hours later by the disappearance of
nucleolin from the S-100 fraction containing plasma membrane nucleolin.
A distinct redistribution of nucleolin within the nucleus and the
appearance of extracellular apoptotic bodies containing nucleolin and
PARP-1 were also observed. By examining levels and localization of
nucleolin in relation to other apoptosis-induced changes, we have shown
that alterations in nucleolin occur very early in the apoptotic
process. For example, at 1 h following UV irradiation of cells
(for 30 s), nucleosomal fragmentation of DNA is barely detectable
on ethidium-stained agarose gels, and only ~50% of PARP-1 has been
cleaved. At the same time point, levels of nuclear nucleolin are
clearly reduced, a distinct alteration in the staining pattern of
nuclear nucleolin can be observed, and low levels of
nucleolin-containing apoptotic bodies can be detected (Figs. 4 and 6,
and data not shown). The fact that alterations in nucleolin are such an
early event may indicate that this protein plays an active role in the
initiation or progression of apoptosis, although further experiments
would be required to confirm this.
Previously, there have been several studies that have examined the role
of nucleolin in cell death. Martelli et al. (47-49) have
used light and electron microscopy to examine changes in nucleolar
proteins in HL60 leukemia cells treated with camptothecin (to induce
apoptosis) or ethanol (to induce necrosis). These authors reported a
redistribution (associated with fragmentation of nuclei) of nucleolin
in apoptotic cells but not necrotic cells, and found that nucleolin was
not degraded during apoptosis, in agreement with our data. In contrast
to our results and those of Martelli and colleagues, several other
reports suggest that nucleolin is proteolyzed in response to apoptosis.
Brockstedt et al. (14) used two-dimensional electrophoresis
to identify nucleolin as a protein that was cleaved in response to
anti-IgM antibody-mediated apoptosis in a Burkitt's lymphoma cell
line. Morimoto et al. (50) described the proteolysis of a
110-kDa silver-stained nucleolar protein (presumed to be nucleolin) to
give a 80-kDa fragment when human salivary gland or oral carcinoma
cells were treated with okadaic acid. Finally, Pasternack et
al. (51) reported that nucleolin was a substrate (in
vitro) for cleavage by granzyme A, an apoptosis-associated
protease secreted by cytotoxic T lymphocytes. The seemingly
contradictory findings regarding apoptosis-induced cleavage of
nucleolin could potentially be caused by cell death occurring via
different mechanisms. Although the precise apoptotic pathways for each
treatment are far from clear, it seems reasonable to expect that DNA
damage (such as UV irradiation or camptothecin treatment) could
activate a different pathway from anti-IgM, which binds to B-cell
surface receptors (14), or okadaic acid, which is known to up-regulate
Fas receptor (52).
The apoptosis-induced changes in nucleolin levels that we have
described appear to depend in some way upon PARP-1, inasmuch as an
inhibitor of PARP-1 (3-ABA) can repress them. Further studies will be
required to determine whether this is related to poly(ADP-ribosyl)ation of nucleolin by PARP-1, or the binding of nucleolin to the 89-kDa fragment of PARP-1 (or perhaps both). In accord with our observations, previous reports have also described redistribution of 89-kDa PARP-1 in
response to apoptosis (53-55). Indeed, PARP-1 can be localized in the
nucleolus under some circumstances (53-56), and there is some evidence
to suggest that the nucleolus is central to the process of apoptosis
and the earliest site of caspase-mediated proteolysis (57, 58).
Another novel result of our study is the observation that nucleolin
appears to be shed from apoptotic cells in the form of small bodies.
Kerr et al. (43) first described apoptotic bodies in 1972, and since then several others types of apoptosis-associated particles,
including nucleolar-derived structures, have been reported (59, 60).
Apoptotic bodies are thought to be derived from collapsing nuclei,
which are transported to the plasma membrane and released into the
extracellular space, where they are normally cleared by phagocytosis
(61). Although apoptotic bodies have not been extensively
characterized, they are known to contain several components including
DNA or RNA, plasma membrane components, and nuclear matrix proteins
(49, 60).
The presence of nucleolin in apoptotic bodies has a number of clinical
implications. In healthy individuals, apoptotic bodies are engulfed by
macrophages or neighboring cells, and cleared from the circulation.
However, under conditions of excessive apoptosis, apoptotic bodies may
be released into the circulation (61) and could potentially be detected
in plasma or serum. This excessive apoptosis can be caused by a number
of conditions including inflammation, autoimmune disease, ischemic
injury, and cancer. There is evidence that the presence of apoptotic
cells or bodies can be an important diagnostic or prognostic marker for
several types of cancer (45, 62-64) and detection of circulating
apoptotic material (e.g. nucleosomes or nucleic acids) in
serum has been proposed as a non-invasive method to detect the presence
of malignancy or to evaluate therapeutic response in cancer patients
receiving chemotherapy or radiation (65, 66). Nucleolin may be a
particularly useful marker for apoptosis in these applications because
tumor-derived apoptotic bodies are expected to be rich in
nucleolin. The ability of apoptotic bodies to deliver their contents to
the cells that engulf them is also an interesting property, which has
been linked to the horizontal transfer of oncogenes (67, 68) and
exploited as a drug delivery mechanism (69).
Our data also have implications for some autoimmune diseases, which are
thought to develop, at least in part, because of defects in apoptotic
responses (2, 61). Autoimmune diseases are characterized by the
development of autoreactive T-cells and B-cells against self-antigens,
and there is mounting evidence that this response could be triggered by
exposure of the immune system to excessive amounts of intracellular
materials from apoptotic cells (70-73). The appearance of nucleolin
autoantibodies is one of the hallmarks of the autoimmune disease
systemic lupus erythematosus (SLE), and these are in fact some of the
earliest autoantibodies to develop as the disease progresses (74, 75).
Our observation that apoptotic bodies containing nucleolin are rapidly
shed from cells undergoing apoptosis suggests a possible
explanation for the early appearance of nucleolin autoantibodies in
SLE. Patients with SLE also have a high frequency of PARP-1
autoantibodies (76).
In conclusion, our studies have identified nucleolin as an important
component of the apoptotic pathway in leukemia cells. Although we have
not yet elucidated its precise role in this complex process, our
results indicate that nucleolin may be involved in the processing of a
proteolytic fragment of PARP-1. Furthermore, the distinct nuclear
redistribution of nucleolin, its disappearance from the plasma
membrane, and its presence in apoptotic bodies may be useful markers to
detect apoptosis in experimental and whole animal systems.