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J Biol Chem, Vol. 273, Issue 52, 34820-34827, December 25, 1998
From the Endonucleolytic DNA fragmentation is the common
end point and the prevailing indicator of apoptosis. We have identified
a 70-kDa endonuclease (NUC70) that is activated in drug-induced apoptosis of human hematopoietic cells. We purified NUC70 to
homogeneity and generated a rabbit polyclonal antibody to distinguish
it from previously identified nucleases. Biochemical characterization of isolated NUC70 demonstrates that it is
Ca2+/Mg2+-dependent and
active over a pH range of 6-8. When incubated with isolated HeLa
nuclei, NUC70 was capable of generating internucleosomal DNA
fragmentation. This endonucleolytic activity was inhibited by
Zn2+, aurintricarboxylic acid,
N-ethylmaleimide, spermine, and iodoacetamide. Western
immunoblots using the anti-NUC70 antibody and DNA-SDS-polyacrylamide gel electrophoresis assays indicate that NUC70 expression and activity
is restricted to human hematopoietic cells. No such activity was
detected in human epithelial cell lines or murine hematopoietic cells.
We also observed no difference in levels of NUC70 expression between
apoptotic and nonapoptotic cells, suggesting that activation of
NUC70 may be by posttranslational modification. We demonstrate that
NUC70 activity is diminished in cells pretreated with the caspase
inhibitors z-DEVD-fmk, z-VAD-fmk, and Z-CH2-Asp-DCB. Time course
studies of cytoplasmic and nuclear endonuclease activities during
apoptosis show that NUC70 is a cytoplasmic endonuclease that is
translocated to the nucleus after the initiation of apoptosis. We
confirmed this with immunostaining studies using anti-NUC70 antibody.
These results demonstrate that NUC70 is an endogenous cytoplasmic
endonuclease that is activated during apoptosis in a
caspase-dependent mechanism.
Apoptosis is a physiological process of orderly cell death that
occurs in response to a number of physiological, pathological, and
cytotoxic insults. Apoptotic cells are identified morphologically based
on compaction of chromatin against the nuclear membrane, condensation
of the cytoplasm, and nuclear and cytoplasmic blebbing to form
membrane-bound fragments that are phagocytosed by adjacent cells
(1-3). The morphological changes are paralleled by activation of a
number of complex biochemical effector pathways that contribute to
dissolution of cellular structural elements. One common end point of
apoptosis is the reduction of high molecular weight genomic DNA into
smaller oligonucleosomal fragments that can be effectively packaged.
Chromatin condensation has been shown to be dependent on DNA digestion.
The characteristic appearance of 180-200-base pair oligonucleosomal
ladders on agarose gels in cells undergoing apoptosis is in contrast to
the pattern of random DNA digestion occurring in the setting of
necrosis and denotes the importance of activation of specific
endonucleases in apoptosis, regardless of the stimulus for cell death
(4, 5).
Identification of apoptotic endonucleases has been limited by the low
abundance of these proteins and by poor understanding of the complex
patterns of their regulation. The first studies of apoptotic
endonucleases in glucocorticoid-induced apoptosis indicated that these
enzymes are likely constitutive and are activated by Ca2+
and inhibited by Zn2+ (6). Other studies in epithelial
cells demonstrated Ca2+-independent apoptotic DNA
degradation, suggesting that a family of apoptotic endonucleases exists
and may differ according to tissue type (7). Thus far, candidate
endonucleases have been identified with differing biochemical
characteristics, including pH and cation requirements as well as
pattern of tissue expression. Because only a few of these
endonucleases, caspase-activated nuclease and inhibitor of
caspase-activated DNase
(CAD)1 NUC18, and the 27-kDa
splenic endonuclease, have been purified or cloned (8, 9, 10, 11),
there is little information regarding their activation and interaction
with other effector components of the apoptotic cascade.
We have identified a 70-kDa apoptotic endonuclease activity in human
hematopoietic cells in modified DNA-SDS-PAGE renaturation assays that
is heat-stable and
Ca2+/Mg2+-dependent and the
activity of which is inhibited in the presence of inhibitors of
caspase. We now describe the purification and biochemical
characterization of NUC70 and demonstrate by immunohistochemistry that
it is a cytoplasmic protein that is translocated to the nucleus early
in apoptosis.
Reagents and Materials--
Vincristine, cycloheximide,
actinomycin D, VP-16, and chemicals and protease inhibitors for buffers
were obtained from Sigma. Proteinase K, DNase, and RNase were obtained
from Life Technologies. The caspase inhibitors z-DEVD-fmk, z-VAD-cmk,
and Z-CH2-Asp-DCB were obtained from Oncogene Research (Cambridge, MA).
Serine protease inhibitors TPCK and TLCK were obtained from Calbiochem
(La Jolla, CA). SDS-PAGE reagents and prestained molecular weight
markers were obtained from Bio-Rad (Hercules, CA), and gels were made according to the manufacturer's instructions, with the modifications mentioned below.
Cell Lines and Culture Conditions--
Human leukemia cell lines
Nalm-6 (pre-B leukemia), Molt-4 (acute lymphoblastic leukemia), CEM
(acute lymphoblastic leukemia), Hut-102 (Sezary leukemia), K562
(chronic myelogenous leukemia), and Jurkat (T-cell leukemia); murine
cell lines S49(thymoma) and P388 (lymphoid neoplasm); and human
epithelial cell lines A549 (lung adenocarcinoma), MCF7 (breast
adenocarcinoma), CACO2, and HT-29 (colon carcinoma) were
used in this study. Cells were obtained from ATCC (Manassas, VA). All
cells were grown in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 2 mM glutamine, penicillin (105
units/liter), and streptomycin (100 mg/liter) at 37 °C in 5% CO2. Cell culture media, sera, and antibiotics were
obtained from Life Technologies. Human CD34+ stem cells and mononuclear
cells were obtained from patients on protocols approved by the
Institutional Review Board.
For large scale purification of NUC70, Nalm-6 cells were grown in 500 ml of culture medium in roller bottles.
Assessment of Apoptosis by DNA Fragmentation--
Cells seeded
in 75-mm2 flasks at a semiconfluent density of 5 × 105 cells/ml. Cells were incubated in the presence or
absence 10 µM vincristine. At different time points,
cells were harvested by centrifugation (1000 × g for 5 min) and lysed in DNA lysis buffer (0.5% Triton X-100, 25 mM Tris-Cl, pH 7.4, 25 mM EDTA) for 30 min at
4 °C. Samples were divided into two aliquots. The first aliquot was
centrifuged at 12,000 × g for 30 min. DNA
concentration in the supernatant (fragmented DNA) and in the pellet
(chromatin) was determined using the diphenylamine colorimetric assay
for DNA as described by Gendimenico et al. (12). Apoptosis
was quantified by computing for % DNA fragmentation (fragmented
DNA/(fragmented DNA + pellet DNA)).
The second aliquot was processed for DNA extraction using 100 µg/ml proteinase K, 150 mM NaCl, and 0.2% (w/v) SDS and
incubated at 50 °C for 2 h. Nucleic acid was extracted twice
with phenol/chloroform and treated with 1 µg/ml DNase-free RNase A
for 1 h at 37 °C. DNA samples (10 µg/lane) were loaded on a
1.8% agarose gel (with 0.5 µg/ml ethidium bromide) and separated by
electrophoresis using 1× TBE buffer (89 mM Tris-Cl, 2 mM EDTA, pH 8.0, 89 mM boric acid). Gels were
run at 50 V for 5 h, viewed on a UV transilluminator, and photographed.
In experiments to determine the effects of protease inhibitors, cells
were pretreated with 50 µM inhibitors (TPCK, TLCK,
z-DEVD.-fmk, z-VAD-fmk, and z-CH2-Asp-DCB) for 1 h prior to
induction of apoptosis with 10 µM vincristine and then processed.
Preparation of Cell Lysates and Nuclear Extracts--
Total cell
lysates were prepared by resuspending 106 cells in protein
extraction buffer (0.5% (w/v) SDS, 50 mM Tris-Cl, pH 7.4, 1 mM PMSF, 2 mM EDTA, 0.5 µg/ml leupeptin,
and 1 mM B-mercaptoethanol) at 4 °C, vortexed, and
centrifuged at 10,000 × g for 30 min at 4 °C to
remove debris. Supernatant was collected and stored at
Nuclear extracts were prepared by the method of Caron-Leslie et
al. (13). Briefly, 107 cells were subjected to
hypotonic shock using ice-cold 1.5 mM MgCl2,
0.25% (v/v) Nonidet P-40, 1 mM PMSF, 2 mM
EDTA, 0.5 µg/ml leupeptin. Nuclei were harvested by centrifugation
(100 × g), and the resulting supernatant was collected
as cytoplasmic extracts. The nuclei pellet was washed twice with the
above buffer, and nuclear protein was extracted with 0.3 M
NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM PMSF, 2 mM EDTA, 0.5 µg/ml leupeptin at 4 °C for 1 h on a
moving platform. The chromatin material was pelleted by centrifugation
at 10,000 × g for 45 min at 4 °C. The supernatant (nuclear extract) was collected and stored at SDS-PAGE and Gel Nuclease Assay--
Total cell lysates were
normalized for protein concentration and nuclease activity was assayed
using the method of McGrew and Green (15). Cell lysates were analyzed
by SDS-PAGE gels (10%) loaded with 50 µg/ml high polymer calf thymus
DNA (Sigma) according to the method of Laemmli using the Bio-Rad
Mini-Protean II system. After electrophoresis, SDS was removed by
washing the gels in renaturation buffer (40 mM Tris-Cl, pH
9.0, 2 mM EDTA, 0.02% (w/v) sodium azide with DE-52
(Whatman). DE-52 was prepared by equilibrating 10 g of preswollen
DE-52 in 200 mM Tris-Cl, pH 9.0, for 1 h at room
temperature and washed three times with the same buffer before
resuspending the resin in the renaturation buffer at a concentration of
0.1 g/ml.
Gels were washed with two changes of the renaturation buffer for 30 min
on a moving platform and then incubated in renaturation buffer without
DE-52 for 60 min. After a brief rinse with distilled water, gels were
placed in activation buffer (40 mM Tris-Cl, pH 7.4, 0.02%
sodium azide, 5 mM MgCl2, 5 mM
CaCl2), and incubated for 12 h at 37 °C to allow
DNA hydrolysis. In experiments using different ion and pH conditions,
gels were cut into strips and treated in the same manner with
appropriate modifications of the activation buffer. The reaction was
stopped by washing the gels in 40 mM Tris-Cl, pH 7.4, 1 mM EDTA. The gels were stained with ethidium bromide (0.5 µg/ml) and visualized and photographed under UV light using the UVP
GDS 7500 gel documentation system (UVP Inc., Upland, CA). Images were
quantified by densitometry and analyzed using the National Institutes
of Health Image 1.61 program.
Endonuclease Purification--
The purification methods used
were adapted from Chow and Fraser (16) in their isolation of
exo-endonucleases from Neurospora crassa. Briefly,
109 Nalm-6 apoptotic cells were lysed in SDS-PAGE loading
buffer and separated using preparative electrophoresis (Model 491 Prep Cell, Bio-Rad) attached to a peristaltic pump (Model P1, Amersham Pharmacia Biotech) and fraction collector (Waters, Bedford, MA). Fractions with activity as determined by cleavage of intact HeLa nuclei
were pooled; simultaneously, fractions were concentrated and SDS was
dialyzed off using equilibration buffer (20 mM Tris-Cl, pH
7.4, 5 mM EDTA, 1 mM PMSF) on Amicon (Beverly,
MA) microconcentrators with a 30-kDa cut-off. The consolidated sample
was loaded onto a SP-Sephadex C25 (Amersham Pharmacia Biotech) column
equilibrated with the equilibration buffer and eluted with a NaCl
(0.1-0.5 M) step gradient. Fractions with endonuclease
activity were pooled and reconcentrated in equilibration buffer with 50 mM NaCl. The sample was then loaded onto a double-stranded
DNA-agarose column (Amersham Pharmacia Biotech) and eluted in a manner
similar to that described above.
Endonuclease activity was detected in the fractions by concentrating 50 µl on a Speed-Vac Plus lyophilizer (Savant Instruments Inc.
Farmingdale, NY) and assayed on DNA-SDS-PAGE gel as described previously. Fractions containing activity were quantified by measuring the ability to generate DNA laddering with isolated nuclei (6). One
unit of enzyme activity was defined as fragmentation of 25% of the DNA
from intact HeLa nuclei in 5 min in the presence of 5 mM
Ca2+ and 5 mM Mg2+ at 37 °C.
Fragmented DNA was quantified using the diphenylamine DNA colorimetric
assay (12) and expressed as a percentage of the total nuclear DNA.
Specific activity was expressed as activity units per mg of protein.
Protein concentration was determined by the Bradford method (14).
Biochemical characterization was performed using 4 units of enzyme
incubated at different pH, ions and recognized nuclease inhibitors.
Relative activity is expressed by quantifying experimental activity as
a percentage of the standard nuclei assay described above.
Antibody Production--
About 1 mg of purified NUC70 was
obtained from the above purification scheme, bypassing the ion-exchange
procedure (SP-Sephadex column). Fractions from the double-stranded
DNA-cellulose column were lyophilized using Speed-Vac Plus. Polyclonal
antibody (rabbit antiserum) production was subcontracted to
Zymed Laboratories Inc. (South San Francisco, CA) for
their proprietary accelerated procedure (PolyQuick).
Western Blotting--
Cell lysates from apoptotic and
nonapoptotic Nalm-6 cells, were separated on 10% SDS-PAGE gels and
electrotransferred onto polyvinylidene difluoride (Millipore, Bedford,
MA) for 1 h at 4 °C using 100 V. Membranes were blocked with
5% nonfat milk, PBST (0.1% Tween-20, 10 mM phosphate
buffer, pH 7.4, 2.7 mM potassium chloride, 137 mM sodium chloride) for 1 h followed by incubation with anti-NUC70 antibody (dilution, 1:5000) for another 1 h at room temperature. The blots were washed three times with PBST and
incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) and then developed using chemiluminescence (NEN Life Science Products). Images were developed on
Kodak X-AR film.
Immunoprecipitation--
Nalm-6 cells (107 cells)
were washed with PBS (10 mM phosphate buffer, pH 7.4, 2.7 mM potassium chloride, 137 mM sodium chloride) and lysed with the addition of 1ml of immunoprecipitation buffer (1%
Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, l0
mM Tris-Cl, pH 7.4, 1 mM EDTA, 0.2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) under
nondenaturing conditions and incubation for 1 h with constant agitation on a vortex mixer. Insoluble material was removed by centrifugation at 14,000 × g. Supernatant was collected and
precleared with 10 µl of protein A/G-agarose (Santa Cruz
Biotechnology, Santa Cruz, CA) for 1 h. Samples were centrifuged
(14,000 × g) for 5 min, and the resulting supernatant
was collected and incubated with 10 µg of anti-NUC70 antibody on a
rocking platform for 12-16 h. Immunoprecipitates were collected by
centrifugation (5,000 × g) and washed three times with
immunoprecipitation buffer. All procedures were performed at 4 °C.
Pellets were resuspended in SDS-PAGE loading buffer and analyzed for
endonuclease activity on 10% DNA-SDS-PAGE gels.
Immunostaining and Fluorescence Microscopy--
Nalm-6 cells
were exposed to 10 µg/ml etoposide for 8 h to induce apoptosis.
Cells were immobilized onto glass slides by cytospin (Shandon,
Pittsburgh, PA), fixed and permeabilized by immersion in
methanol/acetone (1:1) for 10 min at A 70-kDa Endonuclease Is Exclusively Activated in Hematopoietic
Cells--
To identify the size and activity of putative apoptotic
endonucleases, we fractionated whole cell lysates from apoptotic cells on DNA-SDS-PAGE gels under denaturing conditions using a technique adapted from McGrew and Green (15). After electrophoresis, the gels
were exposed to alkaline pH to renature proteins in the presence of
DEAE to remove SDS efficiently and allow restoration of enzymatic activities with minimum denaturation and degradation. Previous studies
using a similar method have solely utilized the passive method of
renaturation of Rosenthal and Lacks (17). Endonuclease activity was
defined as negative staining in the gels treated with ethidium bromide
and visualized under ultraviolet light.
Using this method on time and dose-dependent studies on
apoptotic Nalm-6 cells, we detected a previously described
constitutively active endonucleolytic activity that appeared as a
triplet with an apparent molecular mass of 40-50 kDa in untreated
cells (Fig. 1B, lane 1) and
that was unchanged or only slightly increased when cells were induced
to undergo apoptosis (18, 19). In addition, we identified an
endonuclease activity (NUC70) that was present only in minute amounts
in untreated cells and that increased 10-50-fold as measured by
densitometric analysis of DNA-SDS-PAGE gel (Fig. 1B) in
apoptotic cells. This increase in the 70-kDa activity was concordant
with the appearance of DNA fragmentation (Fig. 1A). The low
level of basal expression in the untreated cells (lane 2)
likely results from spontaneous apoptosis, which occurs in 1-5% of
cells in culture as detected in our diphenylamine assays. The fact that
the whole cell lysates examined in these experiments were prepared
under denaturing and reducing conditions indicating that the renatured
NUC70 activity was heat resistant and likely represented a single
protein species.
We next attempted to identify NUC70 activity in cells of different
lineages. Apoptosis was induced in the epithelial cell lines MCF-7,
T47D, CACO-2, HT-29, HeLa, and A549 using vincristine or VP-16 and in
the hematopoietic cell lines Molt 4, CEM, Hut 102, Hut 78, K562,
Jurkat, and Raji using vincristine, dexamethasone, and, for Jurkat
cells, anti-CD95-antibody to induce Fas-mediated apoptosis. We found
induction of NUC70 activity in all hematopoietic cell lines examined
irrespective of the method used to induce apoptosis. Interestingly, no
endonucleolytic activity representing a 70-kDa endonuclease was
identified in any of the epithelial cell lines tested under conditions
where apoptosis was induced as measured by DNA fragmentation, but the
previously described 40-50-kDa endonuclease activity was identified in
these cells (Fig. 2, A-D).
This 40-50-kDa activity was also present in the hematopoietic cell
lines. These data suggest that NUC70 activity is restricted to cells of
hematopoietic lineage, whereas the 40-50-kDa endonucleases appear to
be ubiquitously expressed.
We next examined normal CD34+ human hematopoietic progenitor cells
obtained from CD34+ selected peripheral blood stem cell collections and
lymphocytes from Ficoll-separated peripheral blood from a patient with
chronic lymphocytic leukemia. We found induction of NUC70 activity and
a 50-kDa endonuclease when apoptosis was induced by exposure to 5 µM Fludarabine (Fig. 3). At
24 h, 40- and 90-kDa endonuclease activities were also observed in
CD34+ cells. When we examined murine hematopoietic cell lines,
including P388, S49, and 2E8, we detected a 40-kDa endonucleolytic
activity and several smaller sized endonucleases but were unable to
detect NUC70 activity when apoptosis was induced, suggesting that the murine analog of NUC70 may have a different molecular mass.
Because it has been proposed that mycoplasma endonucleases may
interfere with endonuclease renaturation assays (20, 21), we assayed
for mycoplasma infection in our cells by reverse
transcription-polymerase chain reaction (ATCC) and treated cells with
ciprofloxacin (Bayer) for 2 weeks (22). The treatment did not affect
the kinetics of NUC70 activation during apoptosis, and transfer of
conditioned media from NUC70 positive Nalm-6 cells onto NUC70 negative
cell lines (MCF-7 and A549) did not result in expression of NUC70. In
cell lines that were positive for mycoplasma, we detected varying increases in the 40-50-kDa endonucleases described above (data not
shown). Nalm-6 cells from which NUC70 was isolated and purified were
mycoplasma negative by reverse transcription-polymerase chain reaction.
Isolation of NUC70--
We based our initial strategy for
isolating NUC70 on its size under reducing conditions that indicate
that it is a single protein species. With the aid of preparative
electrophoresis apparatus (Bio-Rad model 491), we were able to
dramatically fractionate a whole cell lysate of 109
apoptotic Nalm-6 cells, separating the 70-kDa endonuclease from other
cellular endonucleases, including the previously described 40-50-kDa
endonuclease triplet, by assaying fractions on a DNA-SDS-PAGE gel.
Measurement of protein yield by Bradford assay (14) indicated that this
step allowed us to obtain an approximately 1000-fold purification of
NUC70. Unfortunately, we were unable to determine the amount of enzyme
activity in the initial sample due to the nonspecific DNase content.
To further isolate NUC70 from proteins of similar size, we separated
the pooled fractions containing NUC70 activity by ion-exchange chromatography (SP-Sephadex column) and affinity chromatography (DNA-agarose column). Our early attempts at purifying NUC70 indicated that NUC70 does not bind to DEAE column under neutral pH conditions. This distinguishes NUC70 from other Ca2+/Mg2+
endonucleases (11, 16). NUC70 activity elutes with 400 mM NaCl from SP-Sephadex column and with 200 mM NaCl from the
DNA-agarose column. Coomassie Blue staining of SDS-PAGE gels of the
fractions from these columns (Fig.
4A) showed a single band at 70 kDa, and incubation of these fractions with isolated HeLa nuclei
generated apoptotic DNA laddering (Fig. 4B).
Our purification scheme is summarized in Table
I. We estimate a 185-fold purification
from the second step. Because we could not measure endonuclease
activity in our first step, this is likely an underestimate, and
according to measurements of protein content by Bradford assay, we may
have a more than 1000-fold purification and an at least 100,000-fold
enhancement of endonuclease activity. Because the difference between
steps 3 and 4 is only 3-fold, we maximized our recovery by omitting
step 3 in subsequent purifications. This increased recovery provided
sufficient material for the biochemical characterization of NUC70 and
for antibody generation.
Biochemical Properties of NUC70--
We further characterized
isolated NUC70 by determining its optimum cation requirements and pH
using the HeLa nuclei assay. As shown in Fig.
5A, NUC70 activity is optimum
in the presence of both Ca2+ (2 mM) and
Mg2+ (5 mM). However, these cations at higher
concentrations inhibit NUC70 activity (Ca2+,
IC50 = 6.5 mM; Mg2+,
IC50 = 9 mM). Experiments in which
Ca2+ or Mg2+ was used alone showed diminished
NUC70 activity. Mn2+ and Co2+ did not stimulate
NUC70 activity (Fig. 5B), and Zn2+ inhibited
even at low concentrations (IC50 = 0.25 mM).
Assessment of NUC70 at different pH conditions indicated that NUC70
functions over a broad pH range. Optimal activity was seen at pH
7.0-7.2; however, 50% activity was observed at pH 5.7-7.0 and pH
7.5-8.0 (Fig. 5C).
We next examined the effect of identified nuclease inhibitors (16) on
NUC70 activity. Endonuclease activity was inhibited by
aurintricarboxylic acid (IC50 = 0.3 mM),
spermine (IC50 = 2 mM),
N-ethylmaleimide (IC50 = 0.6 mM),
and iodoacetamide (IC50 = 0.5 mM) (Fig.
4D). G-actin, an inhibitor DNase I, did not affect NUC70
endonuclease activity.
Characterization of Polyclonal Antibody to
NUC70--
Enzyme-linked immunosorbent assay analysis of the antiserum
raised against NUC70 showed that dilutions up to 10,000-fold could identify the 70-kDa endonuclease protein in hematopoietic cells and on
Western blots at a dilution of 1:5,000-1:10,000, suggesting that our
polyclonal antibody preparation has high avidity. Further, anti-NUC70
antibody was capable of immunoprecipitating the 70-kDa endonuclease
activity, as shown in Fig. 6.
Western Blot Analysis Using Polyclonal Anti-NUC70
Antibody--
Immunoblots of untreated and apoptotic Nalm-6 cells
demonstrate that the polyclonal anti-NUC70 antibody recognizes both the inactive and active forms of the protein, as shown in Fig.
7B. A replica immunoblot
showing PARP cleavage confirms that the cells underwent apoptosis (Fig.
7A). NUC70 expression was noted in all hematopoietic cell
lines tested but not in epithelial cell lines, confirming our earlier
observation that NUC70 endonuclease activity was confined to cells of
hematopoietic lineage (Fig. 8).
Anti-NUC70 antibody detects a 50-kDa protein in epithelial cell lines
and minimally cross-reacts with a similar protein in murine cell lines. These cross-reacting species may be homologues of NUC70.
Translocation of NUC70 from the Cytoplasm to the Nucleus during
Apoptosis--
In previous studies, apoptotic endonucleases were
identified primarily in nuclear extracts, even though some of these
endonucleases, such as DNase II and DNase I, have extranuclear
locations. Our initial studies used whole cell extracts. To identify
the cellular localization of NUC70 activity, we prepared nuclear and
cytoplasmic extracts from the Nalm-6 cells using standard techniques
and measured endonuclease activity in the presence of
Ca2+/Mg2+. As shown in Fig.
9A, NUC70 activity was
initially confined to the cytoplasmic fraction. After induction of
apopotosis, the NUC70 activity in the nuclear extracts increased
significantly by 6 h along with cytoplasmic activity. By 24 h
after induction of apoptosis, the activity in the cytoplasm decreased
with a concordant marked increase in nuclear activity. We further
extended our cellular localization studies with anti-NUC70 antibody
immunostain on Nalm-6 cells exposed to l0 µg/ml etoposide for 8 h to induce apoptosis. Using a DAPI counterstain (Fig. 9B, panel
2) to identify apoptotic nuclei, immunoreactivity in the
nonapoptotic cells was found to be perinuclear and associated with
string-like cytoskeletal and vesicular structures in the cytoplasm.
These suggest that, like DNase I, NUC70 is localized to the endoplasmic
reticulum (string-like structures), the Golgi apparatus (heavy
perinuclear stain), and perhaps secretory vesicles (Fig. 9B,
panel 1).
In apoptotic cells, in which cytoplasmic shrinkage was evident,
anti-NUC70 antibody stained the apoptotic nucleus heavily but appeared
to be excluded from condensed DNA, as opposed to DAPI, which
preferentially stained the condensed chromatin. These suggest that
NUC70 binds open loops of DNA but not chromatin bodies. Although a
formal time course was not performed, it is clear that NUC70 was
cytoplasmic in nonapoptotic cells and was translocated to the nucleus
after induction of apoptosis, suggesting that NUC70 may be initially
present in its inactive form in the cytoplasm and then, when activated,
is translocated to the nucleus during the progression of apoptosis.
NUC70 Activity Is Modulated by Caspase Activity--
To determine
whether NUC70 activity, like p40 CAD, is modulated by caspase-mediated
events occurring early in apoptosis, we examined NUC70 activity in the
presence of inhibitors of caspase. When Nalm-6 cells were pretreated
with the caspase inhibitors (50 µM) z-DEVD-fmk,
z-VAD-fmk, and z-CH2-Asp DCB prior to vincristine-induced apoptosis,
there was no evidence of DNA fragmentation and likewise no activation
of NUC70, as shown in Fig. 3. DNA fragmentation was observed in cells
pretreated with the serine protease inhibitors TPCK and TLCK, which do
not block vincristine induced apoptosis in Nalm-6 cells, and, as shown
in Fig. 10, NUC70 activity was induced at a level similar to that of the cells exposed in the absence of
inhibitor. Interestingly, the endonucleolytic activity of the 40-50-kDa nucleases did not vary significantly in these inhibition experiments. These data suggest that activation of NUC70 is dependent on upstream events involving cysteine proteolytic events and that either modifications of associated regulatory effectors or of NUC70
itself by proteolytic events occurring early in apoptosis may be
involved in the modulation of NUC70 activity.
The generation of internucleosomal DNA fragmentation has been
widely used as an indicator of chemical or physiologically induced apoptosis and various endonucleases of molecular masses that range from
18 to 97 kDa have been proposed to catalyze this event (reviewed extensively by Khodarev et al. (23) and Walker and Sikorska (24)). We have identified and purified a 70-kDa endonuclease (NUC70)
the expression of which is confined to cells of hematopoietic lineage.
NUC70 is a neutral
Ca2+/Mg2+-dependent endonuclease
that is biochemically distinct from previously reported endonucleases
primarily based on its cellular localization, mechanisms of activation,
and tissue distribution.
Whereas most of apoptotic endonucleases reported had previously been
described in the context of nuclear extracts, recently several
cytoplasmic apoptotic have been identified. DNase II, which is
activated by acidification occurring in early apoptosis, is
compartmentalized in lysosomes (25, 26), and DNase I, when overexpressed in COS cells, was localized to the Golgi, endoplasmic reticulum, and perinuclear area (27). Takeuji et al. (28) have demonstrated a Ca2+-dependent endonuclease
activity in cytoplasmic fractions that is enhanced in the nucleus
during apoptosis, and Fraser et al. (29) identified a mobile
95-kDa endo-exonuclease and a bound 55-kDa endo-exonuclease activity in
endoplasmic reticulum/cytosolic fractions that was liberated by trypsin
to 18- and 46-kDa fragments capable of translocating to the nucleus in
apoptotic cells (29). Using antibodies to a 31-kDa endo-exonuclease of
N. crassa, Fraser et al. (29) identified a number
of immunoreactive species in CEM and MOLT-4 cells, including a
63-67-kDa species with minor endonuclease activity that was localized
to both the cytosol and nuclear extracts, the activity of barely
increased after induction of apoptosis. Translocation of 60- and 80-kDa
cytoplasmic endonucleases associated with acylphosphatase isoenzymes
into the nucleus during apoptosis in Jurkat and K562 cells has also
been described (30).
Our data suggest that existing pools of NUC70 protein are activated
during apoptosis, because Coomassie Blue stains and Western blot for
NUC70 showed no increase in 70-kDa protein species, whereas there was a
50-fold increase in activity after induction of apoptosis. The
induction of activity occurred within 6 h after drug treatment of
the cells even in the presence of cycloheximide. Our immunofluorescence staining experiments demonstrate that NUC70 is localized in the cytoplasmic compartments and translocates into the nucleus during apoptosis. NUC70 was activated in a caspase-dependent
manner in that its activity was abrogated in the presence of caspase
inhibitors. It is unclear whether caspases directly cleave NUC70 or
whether its activity is regulated by an endogenous inhibitor or
activator, the activity of is modulated by a caspase-mediated event. In
our immunoprecipitation studies, the 70-kDa protein is a distinct species and not a complex. NUC70, which is purified from apoptotic cells, was activated and capable of cleaving DNA in intact HeLa nuclei
without co-incubation with cytoplasmic factors. Because the amount of
NUC70 protein did not change significantly after induction of apoptosis
as observed on our Western immunoblots, the activation is likely by
posttranslational modification.
NUC70 is most closely related to the recently identified CAD and DNase
I. The 40-kDa CAD endonuclease is complexed with inhibitor of CAD in
the cytoplasm and is activated during apoptosis when inhibitor of CAD
is cleaved by caspase-3, thereby releasing CAD to be translocated into
the nucleus (9). Similarly, its human homologue, caspase-activate
nuclease, is complexed with DNA fragmentation factor, a 45-kDa
inhibitor that is cleaved by a caspase-mediated event (8). DNase I is
also associated in its inactive form with a cytoplasmic inhibitor,
G-actin. Caspase digestion of G-actin has been demonstrated as a
mechanism of activating DNase I (31).
It is unlikely that NUC70 is a homologue of CAD or DNase I, given its
biochemical properties. CAD, a 40-kDa protein, is
Mg2+-dependent, whereas NUC70 requires both
Ca2+ and Mg2+ for activity. DNase I has a mass
of 31 kDa and exhibits activity not only with
Ca2+/Mg2+ but also with
Mg2+/Mn2+ (32). Nuc70 is inactive in the
presence of Mn2+, Zn2+, or Co2+.
G-actin inhibits DNase I but not NUC70 activity. NUC70, however, can be
inhibited by other previously identified nuclease inhibitors, such as
aurintricarboxylic acid, N-ethylmaleimide, spermine, and iodoacetamide (33).
Because it is active at neutral pH, NUC70 is unlike the 31-kDa acidic
cytoplasmic endonuclease DNase II, which is active at pH <6.5 (25), or
the 35-kDa acidic endonuclease (34). Both endonucleases have no cation
requirements for activity. Intracellular acidification has been shown
to occur in drug-induced apoptosis of human leukemia cell lines HL-60
and Jurkat (35), and this alteration in pH activates DNase II and
possibly other acidic endonucleases, such as a recently reported 45-kDa
Ca2+-dependent endonuclease (36). Our results
show that NUC70 is active at pH 6-8 but has only 60-70% activity at
pH 6-6.5. Other Ca2+/Mg2+-dependent endonucleases
that can function in an acidic milieu include DNase Other 70-kDa endonuclease activities have been identified, but
none has been characterized. Kawabata et al. (39) have
reported a 65-70-kDa Mg2+- or
Mn2+-dependent cytosolic endonuclease that is
capable of internucleosomal DNA fragmentation from human myeloid
leukemia cells (39). This enzyme, however, was not isolated from
apoptotic cells, and its activity during the progression of apoptosis
remains to be elucidated. Another
Ca2+/Mg2+-dependent 70-kDa
endonuclease is up-regulated in the nucleus of cervical carcinoma cells
induced to undergo apoptosis by irradiation (40). The nature of this
endonuclease is less well understood because it was assayed under
native conditions, and its ability to digest genomic DNA was not demonstrated.
Our studies confirm that NUC70 activity is unlikely to be related to
mycoplasma contamination. Experiments using ciprofloxacin treatment of
NUC70 expressing cell lines demonstrated that NUC70 activity was not
altered. Incubation of NUC70 negative epithelial cell lines with
conditioned media from NUC70 positive hematopoietic cells did not
result in the appearance of NUC70 activity in these cells. Furthermore,
we detected NUC70 activity in freshly isolated CD34+ hematopoeitic
progenitor cells and freshly harvested lymphocytes from a patient with
chronic lymphocytic leukemia. According to Bendjennat et al.
(21), the likelihood of mycoplasma infection in these primary cells is
less than 2%.
The significance of multiple endonucleases that can digest genomic DNA
during apoptosis is intriguing. We and others previously attributed
this diversity to the need for tissue-specific death effector pathways.
Our results show that NUC70 expression is restricted to human
hematopoietic cells. Nishikawa et al. (41) also confirm tissue specific endonuclease expression by demonstrating the
diversification of DNase The redundancy of candidate apoptotic endonucleases also upholds the
importance of DNA degradation in apoptosis. Early studies have
suggested that genomic DNA fragmentation is a dispensable process in
induced apoptosis of a subclones of virally transformed fibroblasts
(42), in enucleated fibrosarcoma cells (43), and most recently in
studies of overexpression of inhibitor of CAD in Jurkat cells (44).
These studies demonstrated that in the absence of DNA digestion,
cytoplasmic apoptotic events progressed, thereby calling to question
the relevance of DNA digestion to the apoptotic process. The role of
apoptotic endonucleases to digest genomic DNA during apoptosis in
vivo, however, may be significant. In necrotic cell death, for
example, in which genomic digestion is limited, inflammatory response
occurs due to release of proteases, nucleases, and DNA into the
extracellular milieu. Although controversial, it has been speculated
that this uncontrolled release of cellular contents may contribute to
the development of chronic autoimmune diseases. In malignant diseases,
the consequence of necrotic cell death is best manifested in tumor
lysis syndrome, an oncologic emergency. It is therefore likely that
endonuclease function is crucial in the maintenance of homeostasis in
the organism, and further identification and characterization of these
ultimate effectors of apoptosis will be worthwhile.
*
This work was supported by a grant from the Wendy Will Case
Cancer Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by a Boston University School of Medicine Graduate
Student Research Fellowship.
**
To whom correspondence should be addressed: Tufts New England
Medical Center, Hematology-Oncology, 750 Washington St., Boston, MA 02111. Tel.: 617-636-5145; Fax: 617-636-2342.
The abbreviations used are:
CAD, caspase-activated DNase; PAGE, polyacrylamide gel electrophoresis; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline.
Isolation and Characterization of NUC70, a Cytoplasmic,
Hematopoietic Apoptotic Endonuclease*
§¶,
, and**
Department of Hematology-Oncology, Developmental Therapeutics Program,
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
70 °C.
70 °C. Protein concentrations were determined by the method of Bradford (14) using
bovine serum albumin as standard. Both cytoplasmic and nuclear extracts
were prepared from cells during apoptosis at different time points and
analyzed using the DNA-SDS-PAGE method described below.
20 °C and air-dried. Slides
were blocked with 1% bovine serum albumin in PBS for 10 min, incubated
with anti-NUC70 antibody (dilution, 1:500) for 1 h, and probed
with goat anti-rabbit IgG-fluorescein isothiocyanate (Sigma) together
with 1 µg/ml 4',6-diamidino-2-phenylindole stain for 1 h. Slides
were washed with PBS between buffer changes and mounted in glycerol.
Immunofluorescent staining was viewed using a Nikon Optiphot 2 fluorescence microscope (Nikon Instruments, Melville, NY) equipped with
mercury lamp and excitation filters for UV(365 nM) and
fluorescein isothiocyanate (470-490 nM). Images were
recorded using a Nikon photographic attachment (MicroFlex-UFX-DX), on
Kodak ASA200 color film. Figures were scanned using Microtek ScanMaker IIXE and assembled using Adobe Photoshop.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (46K):
[in a new window]
Fig. 1.
Time- and dose-dependent
induction of NUC70 endonuclease. A, agarose gel
electrophoresis of DNA from Nalm-6 cells treated with 10 µM vincristine for 0, 12, 24, and 48 h (lanes
2-5, respectively), or with 0, 0.5, 1, 10, and 20 µM vincristine for 24 h (lanes 6-10,
respectively). Panel B shows corresponding renatured
DNA-SDS-PAGE gel.
View larger version (52K):
[in a new window]
Fig. 2.
Expression of NUC70 in hematopoietic and
epithelial cells. Representative gels demonstrating DNA
fragmentation (A and B) and endonuclease activity
(C and D). Panel A represents Nalm-6
(lanes 1-4) and CEM (lanes 5-8) cells, and
panel B represents A549 (lanes 1-4) and MCF-7
(lanes 5-8) cells. All cells were treated with 10 µM vincristine for 0 (lanes 1 and
5), 12 (lanes 2 and 6), 24 (lanes 3 and 7), or 48 (lanes 4 and
8) h.
View larger version (87K):
[in a new window]
Fig. 3.
NUC70 activity in CD34+ human hematopoietic
progenitor cells and fresh leukemia cells from a patient with chronic
lymphocytic leukemia. DNA-SDS-PAGE gels of whole cell
lysates from CD34-selected cells (A) and Ficoll-separated
lymphocytes (B) exposed to 5 µM Fludarabine at
different time points. Lane 1, untreated cells; lane
2, 12 h postexposure; lane 3, 24 h;
lane 4, 48 h.
View larger version (46K):
[in a new window]
Fig. 4.
Analysis of purified NUC70. Fractions
isolated from preparative electrophoresis were further purified by
SP-Sephadex and DNA cellulose columns and enodnuclease activity
determined by incubation with isolated HeLa nuclei. A,
Coomassie Blue stain; lane 1, whole cell extract; lane
2, eluted sample from SP-Sephadex; lane 3, eluted
sample from DNA-cellulose column. B, ethidium
bromide-stained 2% agarose gel. Lane 1, untreated HeLa
nuclei; lane 2, treated with SP-Sephadex elutriate;
lane 3, with DNA cellulose elutriate.
Purification scheme
View larger version (21K):
[in a new window]
Fig. 5.
Biochemical Properties of NUC70.
A, effect of Ca2+ and Mg2+ on NUC70
activity. Experiments show NUC70 with Ca2+ and
Mg2+ together under varying conditions ( 
and 
) or
Ca2+ (
) and Mg2+ (
) alone. B,
effect of Zn2+, Mn2+, and Co2+ on
NUC70 activity. Experiments show different concentrations of
Co2+ (), Mn2+ (), and Zn2+
(
) together 2 mM Ca2+/5 mM
Mg2+. Filled symbols represent the NUC70
incubated with the ions alone. C, pH requirement of
NUC70. D, inhibition of NUC70 by previously identified
inhibitors of nucleases. NUC70 activity with G-actin (), spermine
(), N-ethylmaleimide (), iodoacetamide (
), and
aurintricarboxylic acid (), determined by measuring DNA digestion as
described.
View larger version (58K):
[in a new window]
Fig. 6.
Immunoprecipitation of NUC70 with rabbit
polyclonal antibody and analysis of immunoprecipitates on DNA-SDS-PAGE
for endonuclease activity. Lane 1, molecular mass
markers; lane 2, with preimmune serum; lane 3, with anti-NUC70 serum.
View larger version (72K):
[in a new window]
Fig. 7.
Expression of NUC70 in nonapoptotic and
apoptotic Nalm-6 cells by Western immunoblot analysis.
A, Western immunoblot using anti-PARP antibody;
B, Western immunoblot using Anti-NUC70 antibody;
C, Coomassie Blue stain of polyvinylidene difluoride paper.
Co, untreated Nalm-6 cells; Tr, Nalm-6 cells
treated with 10 µM vincristine for 24 h.
View larger version (10K):
[in a new window]
Fig. 8.
Expression of NUC70 in cell lines by Western
immunoblot analysis of whole cell lysates. Lane 1, Nalm-6; lane 2, CEM; lane 3, Molt-4; lane
4, K562; lane 5, A549; lane 6, MCF-7;
lane 7, P388; and lane 8, S49.
View larger version (63K):
[in a new window]
Fig. 9.
Cellular localization of NUC70.
A, densitometric analysis of DNA-SDS-PAGE gels of
fractionated Nalm-6 cells after exposure to 10 µM
vincristine for varying times: cytoplasmic and nuclear fractions.
B, fluorescence microscopy of anti-NUC70 and anti-rabbit
IgG-fluorescein isothiocyanate conjugate immunostained Nalm-6 cells
exposed to 10 µg/ml etoposide for 8 h to induce apoptosis.
Panel 1, anti-NUC70 antibody; panel 2, counterstain with DAPI.
View larger version (54K):
[in a new window]
Fig. 10.
Inhibition of NUC70 in presence of caspase
inhibitors. Nalm-6 cells were preincubated in the presence of 50 µM z-DEVD-fmk, z-VAD-fmk, z-Asp-CH2-DCB, and the serine
protease inhibitors TPCK and TLCK prior to exposure to 10 µM vincristine. Whole cell lysates were run on
DNA-SDS-PAGE gels and renatured as described under "Experimental
Procedures."
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(37) and the
p97 endonuclease (38).
into two forms with different tissue
distributions during metamorphosis of Xenopus: a 38-kDa form
found in erythroblasts/erythrocytes and hepatocytes and a 36-kDa form
found in lymphocytes and macrophages (41).
Ca2+/Mg2+-dependent
endonucleases that are highly expressed in spleen, liver, kidney,
and thymus, and less highly expressed in lung, brain heart, or testis,
have also been described (7). Lineage-specific expression of
endonuclease activity may likely confer specificity not only to
sensitivity of cells to external apoptotic stimuli but also to
selection events in the course of normal development. The fact that
NUC70 activity is observed as one of the predominant apoptotic
endonucleases in CD34+ hematopoietic progenitor cells suggests that its
activity may be relevant in early hematopoietic maturation.
FOOTNOTES
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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