From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
The Acid Deoxyribonuclease of Neutrophils: A Possible Participant in
Apoptosis-Associated Genome Destruction
By R o b e r t a A. Gottlieb, Heidi A. Giesing, Robert L. Engler, and B e r n a r d M. Babior
Human neutrophils are terminally differentiated cells that
spontaneously undergo apoptosis in tissue culture.
Apoptosis in these cells can be delayed by culture in the
presence of granulocyte colony-stimulating factor or other
inflammatory mediators. Neutrophils were found to contain
an acid endonuclease that appeared to be responsible for
the internucleosomal DNA cleavage that accompanies
apoptosis. As measured by a plasmid nicking assay, this
endonuclease had a molecular weight (M,) of 35,000, a pH
optimum of 5.5, and a threshold for activity of pH 6.6 to 6.8.
It was weakly inhibited by divalent cations (Ca’’, Mg2+,and
Zn2+)and more strongly inhibited by aurintricarboxylic acid
N
EUTROPHILS ARE terminally differentiated cells with
a life span of hours to days. I n culture, they undergo
programmed cell death or apoptosis, an event characterized
among otherthings by the condensation of chromatin and the
digestion of genomic DNA intooligonucleosomal fragments
(manifested in apoptotic cells as altered nuclear morphology
and the appearance of nucleosomal ladders on gel electrophoresisof DNA),whereasenergy stores andmembrane
integrity remain for the most part intact.’ In the absence of
cytokines, apoptosis occurs in 30% to 50% of the cells by
24 hours. Apoptosis in neutrophils can be delayed, but not
prevented, by cytokines such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), interleukin- 1 (IL- I), or lipopolysaccharide.*.’
Apoptosis-associated DNA degradation in other systems
has been attributed to any of several endonucleases, including a calcium- and magnesium-dependentendonuclease: the
calcium-dependent enzyme DNaseI,’ and an acidic endonuclease similar oridentical to DNase IL6 In the present report
we characterize an acidendonuclease as theonlyDNase
detectable in mature human neutrophils and show that, when
the intracellular pH of neutrophils is lowered into the range
inwhichthisnucleaseis
active, internucleosomal DNA
cleavage is seen.
and N-bromosuccinimide. DNA from neutrophils treated
with nigericin in buffers of defined pH displayed nucleosomal ladders whose prominence varied with pH ina manner
that paralleled the pH dependence of the plasmid cleavage
assays, consistent with internucleosomal DNA cleavage by
the acid endonuclease. We have previously shown that neutrophils undergo acidification to a pH value as low as 6.0
during apoptosis; we suggest that this endonuclease may
be responsible for the DNA cleavage seen in apoptotic neutrophils.
0 1995 by The American Societyof Hematology.
MATERIALS AND METHODS
Isolation ofneutrophils. After obtaining informed consent from
the volunteer donors, bloodwas drawn from the donors. Neutrophils
were isolated by dextran sedimentation, hypotonic lysis, and Ficoll
centrifugation, as described previously.’
P reparation of nuclear extracts. Purified neutrophils were incubated with 2.7 mmol/L diisopropylfluorophosphate(DFP) on ice for
20minutesand then disruptedbyhypotoniclysis
in 1.5 mmol/L
MgCI2/10 mmolL Tris (pH 7.4). The nuclear pellet obtained after
sedimentation at 2,0008 (2 minutes at 20°C) was washed once with
TMS (10 mmol/L Tris [pH 7.4111.5 mmol/L MgC1,/0.24 mom sucrose). To remove the remaining persistently adherent granules, nuclei were incubated on ice for 15 minutes in TMS containing 0. l %
Triton X-100, weresedimentedat 2,000g for 1 minute,andwere
washed three times at 20°C with TMS containing no detergent. The
resulting nuclear pellet was resuspended in nuclear extract buffer
(20 mmol/L Tris[pH 7.5]/0.4 mol/L NaCU1.5 mmoVL MgCld1
mmol/L DTT/25% [voVvol] glycerol) and incubated for 30 minutes
at 4°C withgentleshaking.Aftersedimentation
at 17,00Og, the
supernatant (nuclear extract) was stored at -70°C until use.
PlasmidDNAdigestionassay.
Substrate(supercoiled pCAT2
plasmid) was prepared by CsCl purification. One microliter of nuclear extract or 10 @Lof column fraction was incubated at 37°C for
1 hour with 100 ng supercoiled plasmid DNA in APT buffer ( I O
mmol/L sodium acetate/lO mmol/L potassium phosphate/lO mmol/
L Tris) at pH 5.5 or as indicated.‘ The total reaction volume was
50 pL. Proteins in some extractscausedaggregationofplasmid
of the 1 hourofincubation,sodium
DNA;therefore,attheend
dodecyl
sulfate
(SDS;
1
%)
and
EDTA ( I 0 mmol/L) were added.
From the Division of Biochemistry, Department of Molecular and
Samples were then assayedfornicking by measuring loss of SUExperimentalMedicine, The ScrippsResearchInstitute, La Jolla,
1% agarose gel toseparate
CA; andthe Research Service, Departmentof Veterans Affairs Medi- percoiling,usingelectrophoresisona
supercoiled from nicked and linearized DNA and ethidium bromide
cal Center, San Diego, CA.
staining to visualize the plasmids.
Submitted January 9, 1995; accepted May 22, 1995.
Fast proteinliquidchromatograph(FPLC)gel,filtration.
NuSupported in part by US Public Health Services Grants No. AIclear
or
whole
cell
extracts
were
subjected
to
FPLC
gel
filtration
on
24227, AI-30742, AI-28479, and RR-00833 and by the Department
aPharmacia(Uppsala,Sweden)Superdex75column
( I O X 300
of Veterans’ Affairs. R.A.G. was supported by a Howard Hughes
mm) in APT-saline buffer ( I O mmoVL sodium acetate/lO m m o K
Medical Institute Physician Postdoctoral Fellowship and a Minority
potassium phosphate/lO mmol/L Tris [pH 7.5]/100 mmol/L NaC1).
Scientist Development Award from
the American Heart Association.
Chromatography
was performed at 0.4 mUmin, collecting 0.5 mL
Address reprint requests to Roberta A. Gottlieb, MD, Division of
Biochemistty, Departmentof Molecular and Experimental Medicine, fractions. Fractions were assayed for endonuclease activity and the
molecular weight inferred by comparison to elution profiles of moThe Scripps Research Institute, La Jolla, CA 92037.
lecular weight standards (Sigma, St Louis, MO).
The publication costs of this article were defrayed
in part by page
DNAisolationfromneutrophils.
Neutrophilswereculturedat
chargepayment. This article must thereforebeherebymarked
I o6 cells/mL in phosphate-buffered saline (PBS) with 0 . 1 % glucose
“advertisement” in accordance with I8 U.S.C. section 1734 solely to
and 0.25% autologous plasma for the times designated. Harvested
indicate this fact.
cells were washed twice in PBS and resuspended in lysis buffer ( 1 0
0 1995 by The American Society of Hematology.
mmol/L Tris [pH 7.5]/10mmol/L NaC1/20 mmol/L EDTA11 % SDS).
0006-4971/95/8606-0020$3.00/0
241 4
Blood, Vol 86, No 6 (September 15), 1995: pp 2414-2418
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ACID DEOXYRIBONUCLEASE OF NEUTROPHILS
After adding proteinase K (0.5 mg/mL), the samples were incubated
at 55°C for intervals ranging from 3 hours to overnight. DNA was
extracted once with phenol:chloroform:isoamyl alcohol, (24:24:1)
and once with ch1oroform:isoamyl alcohol, ( 2 4 I ) and was then precipitated with 1/20 v01 of 3 mol/L sodium acetate and 2 v01 of 100%
ethanol at -20°C for I hour. The precipitated DNA was sedimented
for I5 minutes at 17,OOOg and then resuspended in TE buffer (10
mmol/L Tris [pH 7.5]/1 mmol/L EDTA). Five-microgram portions
of the DNA from each sample were resolved by electrophoresis on
a 2% agarose gel containing 0.5 pg/mL ethidium bromide.
In situ nick end labeling (ISEL). Cells were labeled using a
modification of the method of Wijsman et al? Briefly, neutrophils
were fixed for 5 minutes with 4% formaldehyde in PBS, air-dried
on glass slides, and then rinsed and incubated with a reaction mix
containing 0.05 pmol/L biotin-14-dATP, 5 pmol/L dlTP, 5 prnoll
L dCTP, and 5 pmol/L dGTP in ISEL buffer (50 mmol/L Tris [pH
7.5315 mmol/L MgCI2/0.01% bovine serum albumin [BSAIIKlenow
fragment [ 10 U/mL]) for 30 minutes at room temperature. Slides
were then rinsed with PBS, incubated for 5 minutes at room temperature with 0.28% periodic acid, rinsed again in PBS, incubated for 1
minute with 1 mmol/L 3-aminotriazole containing 0.3 mmol/L H202
to block endogenous peroxidases, rinsed with PBS, incubated with
PBS containing 5% BSA for 5 minutes, and rinsed in PBS. Cells then
were incubated with streptavidin-horseradish peroxidase ( I :1,ooO;
Kirkegaard and Perry Laboratory, Gaithersburg, MD) for 30 minutes
at 37°C and rinsed twice in Tris-buffered saline (l40 mmol/L NaCI,
IO mmol/L Tris). Color was developed with Enhance Black (diaminobenzidine/OsO,; Kirkegaard and Perry) according to the manufacturer's instructions. Eosin-Y was used as a counterstain.
241 5
DH 5.0
4.5
5.5
6.5
6.0
7.0
oc
sc
Ctrl 7.5
Ca"
Mg" Both
5.5
5.5+EDTA
sc
RESULTS
Properties of theneutrophilendonuclease.
Nuclear extracts prepared from freshly purified neutrophils were assayed for their ability to cleave plasmid DNA under a variety
of conditions (Fig I). At neutral pH, neutrophils lacked endonuclease activity bothin the presence and the absence of
divalent cations. Under the conditions of the cell-free assay,
activity began to appear as weak single-strand nicking at pH
6.4, becoming progressively more evident as the pH in the
incubation mixtures was decreased (Fig I, panels 1 and 2).
This acidic endonuclease activity was independent of divalent cations, because it was seen in the absence of added
divalent cations and in the presence of 10 mmol/L EDTA
(Fig I , third panel). (The extent of digestion varied in the
three experiments shown in Fig 1 because different preparations of endonuclease were used in each of the experiments.)
With longer incubation times, higher enzyme concentrations,
or lower pH, supercoiled plasmid DNA was digested into
progressively smaller fragments. It is not clear whether this
is due to multiple single-strand nicks or to double-strand
breaks as well. Spleen acidic DNase is known to exhibit
both types of activity?
This cation-independent acidic endonuclease activity was
found to be verywidely distributed. Cells in which this
activity was sought included cardiomyocytes, HeLa cells,
HL-60 cells, Jurkat cells, and C127 cells, a mammary epithelial line. Figure 2 shows that the acidic DNase was present
in cardiomyocytes, C127 cells, and Jurkats, but that, unlike
neutrophils, these cells also contained one or more cationdependent DNases that were active at neutral pH. Acid endo-
Fig 1. Endonuclease activity: pH and ion dependence. Nuclear extracts were incubated with supercoiled plasmid DNA at the indicated
pH or with other additives as indicated. Samples were resolved on
a 1% agarose gel. Endonuclease activity is indicated by the conversion of supercoiled (SCI plasmid DNA to open circle (OCI, linearized
form, and smaller fragments (disappearance of DNA). Panels 1 and
2 show the pH dependency of t h e nuclease activity, whereas panel
3 shows t h e activity depends only on low pH and not on divalent
cations.
nuclease activity was also found in HL-60 cells and HeLa
cells and showed a similar molecular weight (M,) and pH
dependency (data not shown). In no cell type examined was
the acidic endonuclease absent.
To determine if this endonuclease was similar to DNase
11, it was tested for sensitivity to a panel of agents known to
inhibit DNase I1 (Fig 3).'.'' Like DNase 11, the endonuclease
activity was inhibited by aurintricarboxylic acid (100 pg/
mL) and N-bromosuccinimide (1 mmol/L) but not by iodoacetamide (10 mmol/L). Not surprisingly, the neutrophil enzyme was inactivated by boiling and by treatment with proteinase K (data not shown). However, it was moderately
resistant to limited trypsinolysis.
To estimatethemolecularweight
oftheDNase,nuclear
extracts were subjected to FPLC gel filtration on a Superdex75 column andcolumnfractionswereassayed
as described
above (Fig 4). Peak activity waq detected in fractions cornsponding to an estimated M, of 35,000. Furthermore, the M,
was the same whether the chromatography was conducted at
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2416
GOTTLIEB ET AL
pH 5.5 or pH 7.5, and enzyme rechromatographed at the two
different pH values showed similar properties in that ( I ) the
M, was unchanged and(2) both preparations were active at pH
5.5 but inactive at pH 7.5. Similarly, preincubation at pH 5.5
before chromatography at pH 7.5 (or vice versa) did not alter
the M, or pH dependency of the enzyme. These results suggest
that the activity observed at pH 5.5 was not due to the acidinduced conversion of a proenzyme to a catalytically active
species but was an inherent property of the enzyme itself.
In neutrophils, the apoptosis program appears be
to independent of both gene expression and protein synthesis, because it
is activated bytheprotein synthesis inhibitor cycloheximide,
as indicated by the loss of very high molecular weight material
and the prominence of the nucleosomal ladder in DNA from
cycloheximide-treatedneutrophils a5 comparedwith
DNA
from neutrophils cultured for 24 hours without cycloheximide
(Fig 5). Consistent with a role for the acidic endonuclease in
apoptosis, the enzyme was present in freshly isolated neutrophils as well as in aged neutrophils, and its activity in these
cells was not affected by cycloheximide (Fig 6).
Digestion of chromatin in whole cells depends on pH.
To determine if the digestion of nuclear chromatin depended
on pH, DNA was extracted and analyzed after overnight
incubation of purified neutrophils at 37°C with the proton
ionophore nigericin ( I 0 pmol/L) in a high-potassium buffer
(120 mmol/L KCI, 30' mmol/L potassium phosphate, 5
"
5.5 7.5 Ca** Mg" Both
oc
sc
oc
sc
oc
sc
Fig 2. DNase activity from various cell types. Nuclear extracts
were prepared from cells by lysis in 10 mmol/L Tris, 1.5 mmollL
MgCI., 0.5% NP-40 (pH 7.41 and extraction in nuclear extract buffer.
Activity was assayed by plasmid DNA digestion at pH 5.5 or pH 7.5
with the indicated ions 15 mmol/LI. Samples were resolved on a 1%
agarose gel. Activity is indicated by the conversion of supercoiled
(SCIto open circle (OC) and subsequent complete digestion of DNA.
The amount of endonuclease activity in different cell extracts was
not standardized.
1
2
3
4
5
6
7
8
9
oc
sc
Fig 3. Effect of inhibitorson endonuclease activity. Effect of inhibitors on endonuclease activity. DNase II (100ng from Sigma; lanes 2
through 5 ) or neutrophil nuclear extracts (1 pg; lanes 6 through 9)
were incubated with inhibitors for 15 minutes at room temperature
before the addition of supercoiled plasmid DNA. After digestion for
l hour at 37"C,samples were resolved on a 1% agarose gel.Inhibition
of endonuclease activity is indicated by preservation of supercoiled
plasmid DNA. Lane 1, DNA control; lanes2 and 6, no inhibitors; lanes
3 and 7,aurintricarboxylic acid (100pg/mL); lanes 4 and 8, iodoacetamide (10 mmol/L); lanes 5 and 9, N-bromosuccinimide (1 mmol/L).
mmol/L glucose) adjusted to defined pH (Fig 7). No DNA
fragmentation could be detected in neutrophils incubated at
pH 7.4, even though extensive laddering would have been
expected in neutrophils incubated overnight in the absence
of nigericin. This finding suggests that clamping the intracellular pH at 7.4 prevented endonuclease activation. Progressively more internucleosomal cleavage was noted as the pH
was decreased from 7.0 to 6.2. The observation that DNA
cleavage was seen atpH 7.0 in whole cells but not in the
cell-free assay could be explained but the greater duration
of digestion in the whole cell experiment and by the finding
that the intracellular pH in nigericin-treated cells analyzed
byflow cytometry in pH 7.0 buffer was 7.0 2 0.2 SD,
indicating that 2% of the cells showed pH values less than
6.6, which issufficient to permit some endonuclease activity.
However, in cells analyzed in pH 7.4 buffer, an intracellular
pH of 6.6 or lower occurred in less than 0.01% of the cells
(mean 7.40 2 0.15 SD), consistent with the absence of detectable DNA cleavage in these cells.
In a second series of experiments, neutrophils from two
donors were incubated in the presence of nigericin at pH
6.2, 6.6,7.0, or 7.4 for 30 and 60 minutes at 37°C. Portions
of each of the neutrophil suspensions were thenfixed,
mounted on glass slides, and processed for in situ nick end
labeling. Cells incubated at pH 5.8 and 6.2 were positive for
nicked DNA, whereas those incubated at pH 6.6 and 7.0
were negative at both the 30- and 60-minute time points (data
not shown). This finding showedagain that DNA cleavage in
neutrophils depended on a decrease in pH and showed that
the pH threshold for DNA cleavage in whole neutrophils
was similar to the threshold for DNA cleavage seen in the
digestion of plasmids by nuclear extracts from neutrophils.
DISCUSSION
The present study and earlier work by Lamers et al" indicate that the only DNase detectable in neutrophils is a cationindependent enzyme that is active only at pH values less than
6.6.* In its requirement for acid, its cation independence, its
* The trace amount of calicum- and magnesium-dependent endonuclease activity that was detected in some neutrophil preparations
probablyrepresentscontaminatingactivityfromlymphocytesand
monocytes.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2417
ACIDDEOXYRIBONUCLEASE OF NEUTROPHILS
Fraction# 17
Fig 4. FPLC gel filtration prothe
of
file
endonuclease. Column
fractionswereincubated
with
supercoiled plasmid DNA for 1
hour at 37°C at pH 5.5. Samples
were resolved on a 19'0 agarose
gel. Numbersdenotefraction
number. Arrows signify elution
of molecular weight standards.
Endonuclease activity
reis
flected
by the loss of supercoiled
plasmid DNA.
23
20 2221
19
18
24
26
28
I
iI
t
"0
t
t
Carbonic anhydrase
BSA
(66 kD)
pattern of inhibition, and its apparent molecular weight, this
DNase closely resembles enzymes of the class known collectively as DNase 11. The DNase I1 class of enzymes, which
have been known for over 40 years," are 35-kD proteins
that hydrolyze DNA in acidic solutions in the absence of
metal ions." A remarkable property of these enzymes is that
they produce oligonucleotides phosphorylated on the 3'-end
and not onthe 5'-end. The possible significance of this property is discussed below.
CON CHX
(29 W
(12.4 kD)
The very broad distribution of DNase 11-like activity has
been shown not only in the present study but in earlier work
as well. DNase I1 activity has beenisolatedfrom
several
tissues of diverse ontogeny (liver, spleen, and gastric mucosa) as well as from a number of cell types and cell lines
(HeLa, HL-60, cardiomyocytes, and C H 0 cells".".'4). The
breadth of its distribution suggests that DNase II is likely to
play a general and very important role in biologic systems.
We believe that this role involves the destruction of the
genome in cells undergoing apoptosis.
Objections can be raised to this idea. The endonuclease
generally regarded as responsible for DNA hydrolysis in
apoptosis is a Ca2'- and Mg"-dependent enzyme with a pH
optimum in the vicinity of ne~trality.~.'However, a DNase
with these properties could not be detected in neutrophils.
Furthermore, in neutrophils, calcium influxes actually delay
apoptosis,I5making a Ca2' and Mg*+-dependentendonuclease an unlikely participant in apoptosis a priori, at least in
these cells. Skepticism has also been expressed regarding
the participation of DNase I1 in apoptosis because of the
enzyme's requirement for a pH well less than7.0 for activity.
However, Barry et all6 showed that HL-60 cells undergoing
apoptosis acidified their cytoplasm to a level compatible with
the activity of enzymes of the DNase I1 class, and we obtained similar results with neutrophils. Finally, the unusual
nature of the cleavage reaction catalyzed by the DNase I1
enzymes, a reaction that, as discussed above, produces DNA
fragments phosphorylated on the 3' end and not on the 5'
end, could make the repair of nicks introduced into the genome by these enzymes a difficult matter, thereby facilitating
oc
sc
Fig 5. Effect of cycloheximide on DNA cleavage in neutrophils.
DNA was extracted from neutrophils cultured for 24 hours without
(lane 1) or with (lane 2) cycloheximide (100 pg/mL). DNA was extracted and resolved on 1.8% agarose gel.
t
Cytochromec
1
1
2
3
4
1
2
3
L-
4
1
Fig 6. Acid endonuclease activity from aged and from cycloheximide-treated neutrophils. Nuclear extracts were prepared from 24hour cultures of control
cells (lanes 1 through 4, left)and cells treated
with cycloheximide (100 pg/mL; lanes 1 through 4, right). Ten-fold
serial dilutions of these extracts were used in a plasmid digestion
assay.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
GOTTLIEB ET AL
2418
M 6.2 6.6 6.8 7.0 7.4
servations argue against the de novo synthesis of another
endonuclease (eg, a Ca2'- and Mg"-dependent DNase) during neutrophil apoptosis.
REFERENCES
Fig 7. Chromatin digestion depends onpH. Neutrophils were
treated with the proton ionophore nigericin in a high-potassium
buffer at theindicated pH and incubated at 37°C overnight. DNA was
obtained after proteinase K digestion and phenol extraction and 10
pg of DNA was resolved on a 1.8%agarose gel. M, 100-bp ladder.
the genomic demolition that is an almost universal feature
of cells undergoing apoptosis.
Brown et al" have suggested that two endonucleases participate in apoptosis, one generating infrequent nicks or double-strand breaks and the second producing the nucleosomal
fragments from the incised products of thefirst. Because
neutrophils possess only the acidic endonuclease, both early
nicking and later internucleosomal cleavage must be attributed to the activity of this enzyme. DNase I1 has been shown
to possess both activities.'
It could be postulated that the acidic endonuclease of neutrophils had nothing to do with apoptosis and that the neutrophil DNA was degraded by a different endonuclease that had
been synthesized as part of the apoptosis program. However,
nuclear extracts preparedfrom apoptotic neutrophils still
contained only the acid endonuclease. The findings that DNA
fragmentation could be prevented by clamping the intracellular pH greater than 7.0 and the recent reports that acidification accompanies apoptosis in neutrophil^'^ and other cells'6
suggest that the acid endonuclease (DNase 11) is responsible
for the DNA fragmentation of apoptosis. The protein synthesis inhibitor cycloheximide" precipitated apoptosis without
causing a decrease in endonuclease activity. Both these ob-
1. Ken JFR, Wyllie AH, Cume AR: Apoptosis: A basic biological phenomenom with wide ranging implications in tissue kinetics.
Br J Cancer 26:239, 1972
2. Colotta F, Re F, Polentarutti N. Sozzani S , Mantovani A: Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 802012, 1992
3. LeeA,WhyteMKB,HaslettC:Inhibition
of apoptosisand
prolongation of neutrophil functional longevityby inflammatory mediators. J Leukoc Biol 54:283, 1993
4. Gaido ML, Cidlowski JA: Identification, purification,and characterization of a calcium-dependentendonuclease (NUC18) from
apoptotic rat thymocytes. J Biol Chem 266:18580, 1991
5. Peitsch MC, Polzar B, Stephan H, Crompton T, MacDonald
HR. Mannherz HG, Tschopp J: Characterization of the endogenous
deoxyribonucleaseinvolved in nuclear DNA degradationduring
apoptosis (programmed cell death). EMBO 12371, 1993
6 . Barry MA, Eastman A: Identification of deoxyribonuclease II
as an endonuclease involved in apoptosis. Arch Biochem Biophys
3W440, 1993
7. Markert M, Andrews PC, Babior BM: Measurement of 0;
production byhuman neutrophils. The preparation andassayof
NADPH oxidase-containing
particles
from
human
neutrophils.
MethodsEnzymol 105:358, 1984
8. Wijsman JH, Jonker RR. Keijzer R. Van De Velde CJH, Cornelisse C J , Van Dierendonck JH: A new method to detect apoptosis
in paraffin sections:In situ end-labelingof fragmented DNA. J Histochem Cytochem 41:7, 1993
9. Bernardi G, Griffe M: Studies on acid deoxyribonuclease. 11.
Isolationandcharacterization
of spleen-aciddeoxyribonuclease.
Biochemistry 3:1419, 1964
10. Liao T-H: The subunit structure and active site sequence of
porcine spleen deoxyribonuclease. J Biol Chem 260:10708, 1985
I l. Lamers MC, DeGroot ER, RoosD: Phagocytosis and degradation of DNA-anti-DNA complexes by human phagocytes. 1. Assay
conditions, quantitative aspectsand differences betweenhuman
blood monocytes and neutrophils. Eur J Immunol 11:757, 1981
12. Catcheside DG, Holmes B: The action of enzymes of chromosomes. Symp Soc Exp Biol 1:225, 1947
13. BrownDG,SunX-M,
Cohen GM: Dexamethasone-induced
apoptosis involvescleavage ofDNAto
largefragmentspriorto
internucleosomal fragmentation. J Biol Chem 268:3037, 1993
14. Gottlieb RA, Giesing HA, Zhu JY, Engler RL, Babior BM:
Cell acidificationin apoptosis. G-CSF delays programmed cell death
in neutrophils by upregulating the vacuolar H+-ATPase. Proc Natl
Acad Sci USA 925965, 1995
15. Whyte MKB, Hardwick SJ, Meagher LC, Savill JS, Haslett
C: Transient elevations of cytosolic free calcium retard subsequent
apoptosis in neutrophils in vitro. J Clin Invest 92446, 1993
16. BarryMA,ReynoldsJE,Eastman
A: Etoposide-induced
apoptosis in human HL-60cells is associated with intracellular acidification. Cancer Res 53:2349, 1993
17. Bearman SI, Schwarting GA, Kolodny EH, Babior BM:Incorporation of glucosamineby activated human neutrophils. A myeloperoxidase-mediated process. J Lab Clin Med 96:893, 1980
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1995 86: 2414-2418
The acid deoxyribonuclease of neutrophils: a possible participant in
apoptosis-associated genome destruction
RA Gottlieb, HA Giesing, RL Engler and BM Babior
Updated information and services can be found at:
http://www.bloodjournal.org/content/86/6/2414.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.