(Received for publication, November 21, 1995; and in revised form, January 22, 1996)
From the
The biochemical role of poly(ADP-ribosyl)ation on internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells. It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C, and cisplatin increased poly(ADP-ribosyl)ation of nuclear proteins, particularly histone H1. A poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 poly(ADP-ribosyl)ation in cells treated with the apoptosis inducers. When nuclear chromatin was made accessible to the exogenous nuclease in a permeabilized cell system, chromatin of UV-treated cells was more susceptible to micrococcal nuclease than the chromatin of control cells. Suppression of histone H1 poly(ADP-ribosyl)ation by 3-aminobenzamide reduced the micrococcal nuclease digestibility of internucleosomal chromatin in UV-treated cells. These results suggest that the poly(ADP-ribosyl)ation of histone H1 correlates with the internucleosomal DNA fragmentation during apoptosis mediated by DNA damaging agents. This suggestion is supported by the finding that xeroderma pigmentosum cells which are defective in introducing incision at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 poly(ADP-ribosyl)ation after UV irradiation. We propose that poly(ADP-ribosyl)ation of histone H1 protein in the early stage of apoptosis facilitates internucleosomal DNA fragmentation by increasing the susceptibility of chromatin to cellular endonuclease.
Apoptosis is an active form of cellular suicide, a process that
typically involves morphological changes such as the condensation of
chromatin into clumps, nuclear fragmentation, and packaging of nuclear
fragments into membrane-enclosed apoptotic bodies. These morphological
changes are usually accompanied by biochemical changes including
elevation of cytoplasmic Ca and internucleosomal DNA
fragmentation(1) . Many of the chemotherapeutic drugs and
radiation which cause DNA damage are known to induce
apoptosis(2, 3, 4, 5) . It is well
established that a variety of DNA damaging agents lower the cellular
NAD
content by raising the specific activity of
poly(ADP-ribose) polymerase (PARP) (
)(EC 2.4.2.30) that
results from the conformational changes which occur when the zinc
finger domains of the enzyme bind to the DNA strand
breaks(6, 7) . PARP is a nuclear enzyme which
transfers the ADP-ribose moiety of NAD
to nuclear
proteins including PARP itself and histones in cells(8) . In vitro studies have also revealed that topoisomerase
I(9) , RNA polymerase II(10) , DNA polymerase
(11) , DNA polymerase
(12) , and terminal
deoxynucleotidyl transferase (13) are the substrates of PARP.
Thus, poly(ADP-ribosyl)ation has been implicated in many biological
responses including DNA replication(14) , DNA excision
repair(15) , cell differentiation(16) , and tumor
promotion(17) .
In recent years, the role of
poly(ADP-ribosyl)ation in the cell death process has been discussed.
The decrease in cellular PARP activity during the course of
radiation-induced apoptosis in rat thymocytes was reported by
Nelipovich et al.(18) . Down-regulation of PARP
activity in cells undergoing apoptosis was supported by the finding
that C-nitroso compounds that inhibit PARP function provoke apoptosis
in cultured mammalian cells(19) . On the contrary, many studies
proposed that the activation of PARP contributes to the induction of
apoptosis. PARP activation was recognized during the cell death
processes induced by DNA damaging agents such as alkylating
agents(3) , HO
(20) ,
topoisomerase II inhibitors(21) , adriamycin(22) , and
x-ray(23) . Kaufmann et al. (24) attempted to
explain these controversial results by suggesting that early during the
course of apoptosis the activity of intact PARP is stimulated by DNA
strand breaks, but proteolytic cleavage decreases the activity of PARP
in the late course of apoptosis.
Poly(ADP-ribosyl)ation of nuclear proteins is one of the most dramatic post-translational modifications that occur during apoptosis. The potential correlation between poly(ADP-ribosyl)ation of nuclear proteins and apoptotic internucleosomal DNA fragmentation has been suggested(25, 26) . These studies have described the suppressive effect of PARP inhibitors on the generation of DNA fragmentation associated with apoptosis. However, the target protein of PARP during apoptosis or the biochemical mechanism by which poly(ADP-ribosyl)ation affects the internucleosomal DNA fragmentation has not been addressed. In the present study, a correlation was found between internucleosomal DNA fragmentation and poly(ADP-ribosyl)ation of histone H1 nuclear protein during apoptosis mediated by DNA damaging agents. We propose that the poly(ADP-ribosyl)ation of histone H1 that occurs early during the course of apoptosis facilitates oligonucleosomal DNA fragmen-tation by increasing the susceptibility of nuclear chromatin to cellular endonuclease.
Figure 1:
Internucleosomal DNA fragmentation
during apoptosis induced by DNA damaging agents. HL 60 cells were grown
to log phase in 100-mm culture dishes and treated with 250 J/m UV light (254 nm) (A), 10 µg/ml adriamycin (ADR) (B), 10 µg/ml mitomycin C (MMC) (C), or 10 µg/ml cisplatin (D). After incubation
for the indicated time periods, DNA was extracted and analyzed by 1.8%
agarose gel electrophoresis (lane 7 in A,
DNA-HindIII digest).
The change of PARP activity
during the course of UV-mediated apoptosis was shown in Fig. 2A. The cellular PARP activity, as measured by the
transfer of P-labeled ADP-ribose moieties from
NAD
to cellular proteins, was elevated immediately
after UV irradiation. The activity peaked at 2.5 h after UV irradiation
and declined thereafter. However, the apparent increase in the amount
of fragmented DNA was evident at 2.5 h after UV irradiation coinciding
with the time point when the maximal PARP activity was observed. The
observation of increased PARP activity before the commencement of DNA
fragmentation has led us to investigate the possible involvement of
PARP activation in the course of apoptotic DNA fragmentation. In Fig. 2B, we examined the effect of PARP inhibitors on
DNA fragmentation induced by UV irradiation. Dose-dependent inhibition
of UV-induced DNA fragmentation was observed with PARP inhibitors of
3-AB, nicotinamide, or thymidine. The internucleosomal DNA
fragmentation induced by the chemotherapeutic drugs as well as UV light
was completely inhibited by 3-AB, implicating a common requirement for
PARP activation in the course of apoptosis mediated at least by the
agents employed in this study (Fig. 3A). Interestingly,
the morphological changes of apoptosis were also prevented by 3-AB
treatment (Fig. 3B).
Figure 2:
A, time courses of PARP activity and DNA
fragmentation during UV-induced apoptosis. HL 60 cells were irradiated
with 250 J/m of 254 nm UV light. After incubating for
specified time periods, cells were harvested and divided into two
aliquots. Each aliquot was, respectively, measured for PARP activity
(
) and DNA fragmentation (
) as described under
``Experimental Procedures.'' Each data point represents the
mean value obtained from three separate experiments. B, effects of PARP inhibitors on DNA fragmentation induced by UV
irradiation. HL 60 cells were treated with 250 J/m
of UV
light and incubated for 3 h with various concentrations of 3-AB
(
), nicotinamide (
), or thymidine
(
).
Figure 3:
Effects of 3-AB on DNA fragmentation (A) and morphological characteristic of apoptosis (B)
induced by DNA damaging agents. Agarose gel electrophoresis of DNA
extracted from control cells (lane 2) and from cells treated
with 5 mM 3-AB for 15 h (lane 3).
DNA-HindIII digest was used as molecular weight markers (lane 1). HL 60 cells were irradiated with 250 J/m
of UV light, and incubated for 3 h with (+) or without
(-) 5 mM 3-AB. Cells were treated with 10 µg/ml
adriamycin (ADR) for 9 h with (+) or without(-) 5
mM 3-AB. Cells were treated with 10 µg/ml mitomycin C (MMC) for 15 h with (+) or without(-) 5 mM 3-AB. Cells were treated with 10 µg/ml cisplatin for 10 h with
(+) or without(-) 5 mM 3-AB. Cells were incubated
for the time periods necessary to initiate DNA fragmentation (see Fig. 1). Cell morphology was determined by phase contrast
microscopy (
320).
Figure 4:
Poly(ADP-ribosyl)ation of histone H1
during UV-induced apoptosis. HL 60 cells were irradiated with 250
J/m of UV light and incubated for 0, 1, 2, or 3 h (lanes 1-4, respectively). The
P-labeled
ADP-ribose moiety of NAD
was attached to cellular
proteins in a permeabilized cell system. Histone H1 was
extracted(32) , analyzed by 15% SDS-PAGE (A), and
subjected to autoradiography (B). Total cellular proteins were
also prepared at 3 h after UV irradiation by washing 2 times with
phosphate-buffered saline and sonication for 3 min with W-385 sonicator
(Heat systems-Ultrasonics), and were examined for
poly(ADP-ribosyl)ation (lane 5).
Figure 5:
Poly(ADP-ribosyl)ation of histone H1
during apoptosis mediated by UV and chemotherapeutic drugs. Cells were
treated with 250 J/m of UV light, 10 µg/ml adriamycin (ADR), 10 µg/ml mitomycin C (MMC), or 10
µg/ml cisplatin and incubated for 3, 9, 15, or 10 h, respectively,
in the presence (+) or absence(-) of 5 mM 3-AB.
Histone H1 was extracted from cell samples and analyzed on 15% SDS-PAGE (A). Poly(ADP-ribosyl)ation of histone H1 was visualized by
autoradiography (B).
For
further insight into the correlation between poly(ADP-ribosyl)ation of
histone H1 and DNA fragmentation induced by DNA damaging agents, we
examined whether the correlation is still established in XP cells
(complementation group A, GM04312B), which are defective in introducing
incision at the site of DNA damage(38) . Complementation group
A is one of the most severe forms of this genetic disease(39) .
There are reports that XP cells are defective in the synthesis of
poly(ADP-ribose) in response to UV
irradiation(40, 41) . In contrast to HL 60 cells, the
internucleosomal DNA fragmentation (Fig. 6A) and
poly(ADP-ribosyl)ation of histone H1 (Fig. 6C) were
both abolished in XP cells treated with UV light ranging from 100 to
300 J/m. None of the morphological characteristics of
apoptotic cells were seen in XP cells after UV treatment (data not
shown).
Figure 6:
DNA fragmentation and histone H1
poly(ADP-ribosyl)ation after UV irradiation in HL 60 and GM04312B
cells. HL 60 and GM04312B cells were grown to log phase and irradiated
with UV light. After incubation for 3 h, cell preparation was divided
into two aliquots. One aliquot was prepared for agarose gel
electrophoresis (lane 1, DNA-HindIII digest; lanes 2-5, HL 60; lanes 6-9, GM04312B) (A). The other aliquot was prepared for the examination of
histone H1 poly(ADP-ribosyl)ation. Histone H1 was extracted and
analyzed by 15% SDS-PAGE. The mobility of commercially obtained histone
H1 was also analyzed (lane 5). After electrophoresis, the gel
was stained with Coomassie Blue (B) and exposed to x-ray film
for autoradiography (C).
Figure 7:
Reduced susceptibility of chromatin DNA to
micrococcal nuclease by 3-AB during apoptosis mediated by UV
irradiation. Micrococcal nuclease digestibility of nuclear chromatin
was examined in control HL 60 cells (), cells exposed to 5
mM 3-AB (
), cells treated with 250 J/m
of UV
(
), or in cells exposed to 250 J/m
of UV and 5 mM 3-AB (
). Cells were incubated for 2 h and were permeabilized
to allow the accessibility of their chromatin to micrococcal nuclease.
The measurement of DNA fragmentation was done using diphenylamine
reagent as described under ``Experimental Procedures.'' The
results of a typical experiment of three replicates is shown (A). DNA fragments were analyzed by 1.8% agarose gel
electrophoresis (B). Lane 5 is
DNA-HindIII digest, and the material shown at the bottom of
each lane indicates undigested RNA.
Our data suggest a correlation between internucleosomal DNA
fragmentation associated with apoptosis and poly(ADP-ribosyl)ation of
histone H1. It is well established that the free ends of DNA are strong
activators of PARP and that PARP activity is increased by DNA damaging
agents which introduce DNA strand breaks(6, 7) . Many
studies have described the relationship between PARP activity and the
cell death process. The consumption of NAD by PARP
activation was considered to be the main cause of cell death after DNA
damage(45) . Activated PARP cleaves NAD
into
nicotinamide and ADP-ribose resulting in a depletion of
NAD
. The reduction of the cellular NAD
level slows down glycolysis and other energy-generating reactions
leading to the depletion of cellular ATP. Accordingly, the activation
of PARP reduces the energy supply thereby slowing down cellular
metabolism including macromolecular synthesis. This in turn disturbs
cellular homeostasis resulting in an eventual cell death. However, the
requirement of energy-rich nucleotides differs between apoptosis and
necrosis. Unlike necrosis, apoptosis is an energy-requiring process
which needs macromolecular synthesis(46) , and the depletion of
intracellular ATP does not regularly precede the onset of irreversible
morphological changes that occur during apoptosis. Despite an increased
PARP activity during apoptosis, no massive depletion of NAD
and ATP occurred. For example, apoptotic internucleosomal DNA
fragmentation in L1210 cells occurred in 2 days after cisplatin
treatment, but the levels of NAD
and ATP were not
significantly decreased until 3 days after the treatment, suggesting
that the reduction of the cellular energy level was not the cause but
the result of cell death(47) .
While the negative (18, 19) and positive (3, 20, 21, 22, 23) roles
of PARP activity on apoptosis have been independently presented, the
monitoring of cellular PARP activity during UV-induced apoptosis in HL
60 cells (Fig. 2A) suggests an explanation that
integrates the two conflicting interpretations. We observed a rapid
increase in PARP activity before the initiation of DNA fragmentation,
but with the commencement of internucleosomal DNA fragmentation, the
cellular level of PARP activity declined. This observation is supported
by the study of Kaufmann et al.(24) that reported
proteolytic cleavage of PARP in HL 60 cells undergoing apoptosis.
During the course of chemotherapy-induced apoptosis in human leukemia
cells, it was demonstrated that PARP was cleaved into a 25-kDa fragment
containing the DNA-binding domain and a 85-kDa fragment containing the
automodification and catalytic domains. Based on these observations,
they suggested that the initial consumption of NAD that ordinarily occurs early during the course of apoptosis is
attributed to the increase in the activity of intact PARP which is
stimulated by DNA strand breaks. However, proteolytic cleavage
decreases the activity of PARP and the 85-kDa fragment retains only
basal PARP activity necessary for the late course of apoptosis. More
recently, it was found that a protease of the CED-3/interleukin
converting enzyme family is responsible for the specific cleavage of
PARP during apoptosis(48, 49) . The involvement of
PARP activity in the induction of apoptosis mediated by DNA damaging
agents is reinforced by the data showing that the inhibitor of PARP
inhibits internucleosomal DNA fragmentation associated with apoptosis (Fig. 2B and Fig. 3A). Our data are
consistent with the report showing that 3-AB inhibits DNA fragmentation
associated with apoptosis in HL 60 cells at concentrations higher than
2 mM(26) . Other inhibitors of PARP were also observed
to inhibit DNA fragmentation in apoptotic cells induced by UV light (Fig. 2B), suggesting an obligate role of PARP activity
in the apoptotic DNA fragmentation.
In the present study, we
attempted to propose an explanation for the role of
poly(ADP-ribosyl)ation on the induction of apoptosis. Nuclear proteins
are poly(ADP-ribosyl)ated in order to modify their structures and
functions. We have shown an increase of histone H1
poly(ADP-ribosyl)ation in HL 60 cells by apoptosis inducers ( Fig. 4and Fig. 5). The poly(ADP-ribosyl)ation of histone
H1 was measured in permeabilized cells which allow rapid access of P-labeled NAD
to nuclei. It is, however,
unlikely that the permeabilization process affects any undue effects on
the poly(ADP-ribosyl)ation reaction, since the process is generally
used to measure the nucleotide polymerizing reactions under near
physiological conditions(50, 51) . Electron
microscopic studies showed that the permeabilized cells retained intact
morphology throughout the processing and incubation(52) . The
link between histone H1 poly(ADP-ribosyl)ation and induction of
apoptosis is reinforced by data in Fig. 6showing GM04312B
cells, which are unable to poly(ADP-ribosyl)ate histone H1 and are also
lacking the ability to generate internucleosomal DNA fragmentation.
Among the known acceptors of poly(ADP-ribose), histone H1, a very basic
protein located within the space of internucleosomal DNA region,
particularly plays an important role in the formation and stabilization
of the highly ordered solenoid structure of native chromatin (reviewed
in (53) ). Internucleosomal DNA and bound histone H1 are hidden
in the interior of the solenoid structure(42, 43) . It
is thus conceivable that the change of polarity of the basic portions
of histone H1 by the association of highly negative poly(ADP-ribose)
may reduce their affinity to the associated DNA and consequently affect
the stability of solenoid structure. Evidence has been accumulated
showing the correlation between the change of chromatin structure and
poly(ADP-ribosyl)ation of histone H1. Electron microscopic studies have
revealed that the solenoid chromatin structure was relaxed by the
poly(ADP-ribosyl)ation of histone H1 protein exposing internucleosomal
DNA regions from the interior of the
structure(54, 55) . De Murcia et al. (56) further demonstrated that the degradation of ADP-ribose
units on poly(ADP-ribosyl)ated histone H1 by poly(ADP-ribose)
glycohydrolase restored the solenoid chromatin structure. The change of
chromatin superstructure by poly(ADP-ribosyl)ation of histone H1 might
explain the internucleosomal DNA fragmentation that occurs during
apoptosis of which the mechanism requires the accessibility of
endonuclease to internucleosomal DNA. The data in Fig. 7indicate the elevated level of micrococcal nuclease
susceptibility of chromatin DNA during apoptosis. It was also shown
that the micrococcal nuclease susceptibility was inhibited by the
treatment of PARP inhibitor.